Rheumatoid Arthritis (Current Directions in Autoimmunity)

Rheumatoid Arthritis .. ............................ Current Directions in Autoimmunit...

Author: J. J. Goronzy | Cornelia M. Weyand (Editors)

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............................ Rheumatoid Arthritis

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Current Directions in Autoimmunity Vol. 3

Series Editor

A.N. Theofilopoulos, La Jolla, Calif.

............................

Rheumatoid Arthritis

Volume Editors

J.J. Goronzy, Rochester, Minn. C.M. Weyand, Rochester, Minn.

46 figures, 1 in color, and 14 tables, 2001

Current Directions in Autoimmunity

............................ J.J. Goronzy C.M. Weyand Department of Internal Medicine, Division of Rheumatology, Mayo Foundation, Rochester, Minn.

Library of Congress Cataloging-in-Publication Data Rheumatoid arthritis / editors, J.J. Goronzy, C.M. Weyand. p. cm. – (Current directions in autoimmunity; vol. 3) Includes bibliographical references and index. ISBN 3805571208 1. Rheumatoid arthritis – Pathogenesis. I. Goronzy, J.J. II. Series. RC933.R422 2000 616.7227–dc21 00–048164

Bibliographic Indices. This publication is listed in bibliographic services, including Current ContentsÔ and Index Medicus. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Ó Copyright 2001 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel ISSN 1422–2132 ISBN 3–8055–7120–8

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Contents

VII Prologue Goronzy, J.J.; Weyand, C.M. (Rochester, Minn.) 1 Susceptibility Genes in Rheumatoid Arthritis Corne´lis, F. for ECRAF (Evry) 17 Elucidation of Pathways Leading to Rheumatoid Arthritis by Genetic

Analysis of Animal Models ˚ .; Olofsson, P.; Lu, S. (Lund) Holmdahl, R.; Bockermann, R.; Jirholt, J.; Johansson, A

36 Structural Basis for the HLA-DR Association of Rheumatoid Arthritis Sinigaglia, F. (Milan); Nagy, Z.A. (Munich) 51 T Cell Repertoire Formation and Molecular Mimicry in Rheumatoid

Arthritis Prakken, B.J.; Carson, D.A. (La Jolla, Calif.); Albani, S. (La Jolla, Calif./Catania)

64 Exploring the Pathogenesis of Rheumatoid Arthritis in Transgenic and

Mutant Mice Cope, A.P. (London)

94 Molecular Mimicry and Lyme Arthritis Gross, D.; Huber, B.T.; Steere, A.C. (Boston, Mass.) 112 T Cell Homeostasis and Autoreactivity in Rheumatoid Arthritis Goronzy, J.J.; Weyand, C.M. (Rochester, Minn.) 133 Leukocyte Homing to Synovium Patel, D.D.; Haynes, B.F. (Durham, N.C.) 168 Lymphoid Microstructures in Rheumatoid Synovitis Weyand, C.M.; Braun, A.; Takemura, S.; Goronzy, J.J. (Rochester, Minn.)

188 The Role of TNFa and IL-1 in Rheumatoid Arthritis Feldmann, M.; Brennan, F.M.; Foxwell, B.M.J.; Maini, R.N. (London) 200 Innate Response Cytokines in Inflammatory Synovitis:

A Role for Interleukin-15 McInnes, I.B.; Leung, B.P. (Glasgow)

216 Regulation of Apoptosis of Synovial Fibroblasts Mountz, J.D.; Zhang, H.-G. (Birmingham, Ala.) 240 Biologics in the Treatment of Rheumatoid Arthritis:

Mechanisms of Action Kavanaugh, A.; Lipsky, P. (Dallas, Tex.)

274 Author Index 275 Subject Index

Contents

VI

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Prologue

In 1947, a technician working with Dr. H. Rose and Dr. C. Ragan discovered that her own serum caused agglutination. She had rheumatoid arthritis, and subsequent studies demonstrated that the agglutinins were complexes of IgM and IgG. Investigations of the agglutinating autoantibodies, rheumatoid factors, eventually led to the discovery of antibody structure and diversity and a Nobel Prize for Dr. G. Edelmann. A case can be made that studying rheumatoid arthritis has not only been instrumental in the development of academic rheumatology but has been a driving force for immunology in general. During the last decades, investigation into rheumatoid arthritis has benefited from the enormous growth in understanding principal functions of the immune system and the explosion in the knowledge of molecules involved in regulating normal and pathologic immune responses. Despite this progress, the disease has defied a unifying concept and new challenges and perspectives have emerged. Once again, it is becoming evident that research of autoimmune diseases is not a one-way street but a dialectic process between basic science and disease studies. We have learned that rheumatoid arthritis is not a single mechanism-single molecule-single gene disease. The chapters commissioned in this volume illustrate the many facets of disease pathogenesis. Major progress has been made in the structural characterization of the MHC genes, which are the strongest genetic risk factor for the disease. How HLA molecules influence disease mechanisms in rheumatoid arthritis continues to be a productive forum of scientific controversy, and varying positions on this issue can be found in different chapters of this volume. The unifying paradigm that rheumatoid arthritis is an antigen-specific, HLA-restricted autoimmune disease is being challenged from different directions, providing a unique opportunity to broaden the horizon on how HLA polymorphisms shape immunity. Aberrations in the innate immune system, so far not considered critical in autoimmunity, have found increasing attention. TNF-a-directed

VII

and not antigen-directed therapy has emerged as the most impressive therapeutic advance in managing rheumatoid arthritis. A good argument can be made that the identification of additional genetic risk factors will modify our understanding of the chronic destructive inflammation underlying rheumatoid arthritis. Progress can be expected from using genetic markers to define the biological heterogeneity of the disease, the most immediate problem confronting physicians caring for patients with rheumatoid arthritis. A view into the future, inspired by the comprehensive work presented in this volume, predicts that researchers studying rheumatoid arthritis will continue to make rapid progress in identifying relevant components to the disease process. More importantly, they will likely benefit from emerging new computational tools that allow for the analysis of complex systems. Rather than a disease of a single cellular or molecular component, rheumatoid arthritis is emerging as a disease at the level of a complex biological organization that includes the specific immune repertoires, the innate immune system, and the differences in tissue susceptibility. We thank the series editor, Dr. Theofilopoulos who took the initiative to start this series, and the authors who dedicated their time and expertise to this project. J.J. Goronzy, MD C.M. Weyand, MD

Prologue

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Goronzy JJ, Weyand CM (eds): Rheumatoid Arthritis. Curr Dir Autoimmun. Basel, Karger, 2001, vol 3, pp 1–16

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Susceptibility Genes in Rheumatoid Arthritis François Corne´lis for ECRAF 1 ECRAF-University Paris 7, GENOPOLE, Evry, France

Rheumatoid arthritis (RA) involves genetic factors and, therefore, susceptibility genes are involved in the initial mechanisms, from which follows the complex disease that is RA. The full understanding of RA pathophysiology must include the mechanisms through which those genetic factors predispose to RA. The complexity of RA biology is such that genetics would be of great help to identify those main biological mechanisms within the many abnormalities that can be observed. Even though the choice of therapeutic targets from the observed biological abnormalities, such as the excess of tumor necrosis factor-a (TNF-a) production, has recently proved to be effective [1], a definitive treatment can be expected only when targeting the initial mechanisms. The fast development of basic research in genetics, in particular the sequencing of the human genome, raises the hope that such discovery would come soon for RA. In this chapter, the state of knowledge on RA genetics is reviewed, the current paradigm to search for genetic factor of multifactorial diseases is explained and its application to identify new RA genes, to which every laboratory can contribute, is described.

1 ECRAF, the European Consortium on Rheumatoid Arthritis Families, was initiated with funding from the European Commission (BIOMED2) by T. Bardin, D. Charron, F. Corne´lis (coordinator), S. Faure´, D. Kuntz, M. Martinez, J.F. Prudhomme, J. Weissenbach (France); R. Westhovens, J. Dequeker (Belgium); A. Balsa, D. Pascuale-Salcedo (Spain); M. Spyropoulou, C. Stavropoulos (Greece); P. Migliorini, S. Bombardieri (Italy); P. Barrera, L. Van de Putte (The Netherlands), H. Alves, A. Lopes-Vaz (Portugal).

The Genetic Predisposition to RA Is Only Partially Explained by HLA RA Affects Predisposed Individuals There is familial aggregation in RA: the prevalence of the disease is increased in the first-degree relatives of RA patients, with a sibling occurrence risk estimated between 2.8 and 12.1% [reviewed in 2]. Compared to the prevalence in the general population estimated to be about 0.8% in most populations [3], this provides a prevalence ratio, the parameter ‘lambda s’ used as a global measure of familial factors [4], between 3 and 15. The lack of precision of this estimation reflects the need for new genetic epidemiology studies, taking into account the updated diagnosis criteria for RA [5], with both general and family prevalence measured in the same study population. If unequivocal genetic inferences from familial aggregation studies are unwarranted, since the common environment shared by relatives may explain disease clustering in families, twin studies confirm indirect evidence for RA genetic factors. The concordance rate for monozygotic twins is 3.5–5.2 times higher than the concordance rate for same-sex dizygotic twins [6–8]. The global monozygote twin concordance estimated range is 12–32% [6–8]. These data provide strong evidence for the implication of genetic susceptibility in RA as a risk factor, with about 1/5 genetically susceptible individuals developing the disease (estimated from a monozygote twin concordance rate of 20%). This leads to an estimation of 4% of the general population (5¶0.8%) predisposed to the disease, out of which 1/5 would have RA triggered by as yet unidentified noninherited factor(s). HLA Is Involved in RA Genetics The existence of HLA-encoded susceptibility was detected in population studies [9] and confirmed by family studies [reviewed in 10, 11]. Initially recognized in seropositive Caucasoid RA patients as the Dw4 group in the mixed lymphocyte reaction [9, 12], and then as the serologic specificities DR4 and DR1 [13, 14], this association has been refined at the molecular level: the HLA-DRB1 alleles implicated, mostly HLA-DRB1*0101, *0102, *0401, *0404, *0405, 0408 and *1001, were found to contain a homologous amino acid motif in the third hypervariable region of the HLA-DR b-chain, the ‘shared epitope’ [15]. This shared epitope, associated with RA in almost all populations investigated, is present in 70–90% of Caucasoid patients, conferring a relative risk for RA of 6–12 in most Caucasian populations [15, 16]. The association might be more pronounced in seropositive, erosive forms with extra-articular features than in seronegative and mild cases of RA [17–21]. It was suggested that particular combinations of HLA-DRB1 alleles influence disease expression: the frequency of the genotype DRB1*0401/*0404 was

Corne´lis for ECRAF

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found increased in severe and nodular RA [22], whereas the homozygous DRB1*0401 genotype was associated with the presence of extra-articular involvement [23, 24]. However, these data need to be confirmed and prospective studies are particularly warranted to evaluate the diagnostic and prognostic value of HLA-DRB1 typing in defined clinical situations. The nature of the molecular mechanisms mediating HLA susceptibility in RA is not known. This shared epitope might be directly implicated in antigen binding, as reviewed by Sinigaglia and Nagy [25]. Alternatively, the shared motif could mediate RA susceptibility because it is itself potentially immunogenic for T cells [26–28]. It has been proposed that maternal noninherited antigen might predispose to RA [29], although this could not be confirmed by other groups [30, 31]. Apart from HLA-DRB1, there is growing evidence that the HLA contribution involves other HLA genes [32–35]. For instance, polymorphisms of TNFA gene, coding for the proinflammatory cytokine TNF, might be implicated [36–40]. This would be of particular importance, given the role of TNF in RA and the efficacy of anti-TNF treatment [1]. The recent report of the full sequence of the HLA region should help in defining the full HLA component [41]. The HLA locus provides only part of the genetic predisposition to RA, as it accounts for about one third of the familial clustering of the disease [2, 10, 42]. Therefore, non-HLA genes are involved simultaneously in RA susceptibility and it is likely that several combinations of genetic factors could contribute, at least differing by the presence or absence of the known HLADRB1 alleles. Non-HLA Genes Contribute to RA Susceptibility Sex is a major genetic factor, as RA affects predominantly females (sex ratio 1:3). However, sex could influence mainly the penetrance of the disease (the proportion of genetically susceptible individuals developing the disease). There is no genetic epidemiology evidence for the presence of RA genes on the X chromosome [43]. The favorable influence of pregnancy on the course of the disease and the possible long-acting protective effect of parity and of oral contraceptive use suggest the implication of sex hormones [44, 45]. The number and relative importance of genes conferring susceptibility to RA cannot be reliably predicted, as no validated model is known. Genetic epidemiology studies suggest that there would be a major gene and other genes [46, 47]. It is likely that many genes could be involved, interacting to predispose to RA and influence the outcome. Because of the probable role of T cells in RA [26, 48, 49], T cell receptor (TCR) genes have been considered as major candidate genes. Allelic coding

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variations of V segments from the a-chain of the TCR might be involved in RA susceptibility, providing a small risk of subgroups of patients [50, 51]. As could be expected for small relative risks provided by common alleles, linkage studies were negative for that gene [52–54]. Definite proof must await replication by other groups, confirmation by family-based association studies and stronger statistical evidence. Many other candidate genes have been investigated, but none has yet been definitely implicated, as none has been widely replicated, confirmed by family studies and supported by strong enough statistical evidence [reviewed in 11]. The genes involved in RA predisposition might be shared with other autoimmune diseases, as suggested by the increased prevalence in RA patients and their relatives of autoimmune thyroiditis and insulin-dependent diabetes mellitus (IDDM) [55–57]. The considerable advances in the field of IDDM genetics could soon provide IDDM genetic factors to test for a role in RA [58], and RA genes will have to be tested in IDDM and other autoimmune diseases. RA animal models could also provide candidate genes to be tested, in particular collagen-induced arthritis in rats, for which a number of susceptibility loci have been identified [59, 60]. Recent major advances in the knowledge of murine genomes, including comparative gene maps for rat, mouse and human, will facilitate this search [61].

The Search for New RA Genes Follows the Current Paradigm for Multifactorial Diseases The search for multifactorial diseases genes, following the example of IDDM [58], uses two approaches, which are now combined: the investigation of candidate genes and the systematic approach. The Candidate Gene Approach Starting from what is known from the pathophysiology of the disease, educated guesses can be made for a given gene, such as the insulin gene in diabetes, which proved to be a genetic factor to IDDM [62, 63]. This requires searching for common alleles of the genes and testing of those for association with the disease. To consider that an association finding demonstrates a new genetic factor, the following criteria should be met: (1) follow-up studies should provide a replication of the finding in an independent data set from the same population, (2) family-based association tests, such as the transmission disequilibrium test (TDT) or haplotype relative risk [64, 65], which avoid the major drawback of imperfect matching for cases and controls, should confirm


4

the finding and (3) the combined analysis should reach the significance threshold of at least p00.000001, if not p00.00000001 [54, 66]. Once the association is established, linkage disequilibrium should help identifying the causal polymorphism, which should then be investigated biologically to unravel the underlying mechanism. The limiting factors of this approach are disease understanding and gene function knowledge to define candidate genes, the availability of gene polymorphisms and the size and heterogeneity of the DNA sample available to test the hypotheses. The list of candidate genes increases quickly with our knowledge of the human genome, as well as the identification of gene polymorphisms, although a systematic investigation of exons and regulatory elements for polymorphisms is still required for most genes. The desired DNA sample for the TDT test would be on the order of 1,000 families from as homogeneous an origin as possible [54, 66]. The Systematic Approach The genome-wide linkage analysis is of paramount importance, not only to define loci where linkage can be demonstrated, which must contain susceptibility genes, but also to describe loci with suggestive linkage, where the probability of harboring RA genes is increased. Putative loci are identified through genome scans and further investigated by linkage disequilibrium mapping. Genome Scan. Using the affected sib-pair (ASP) analysis, a genome-wide analysis is performed, as was first applied to IDDM, confirming the involvement of the HLA locus and detecting a number of potential linkages [67, 68]. Follow-up studies are then necessary to test potential linkages for replication. The combined analysis should reach a significant threshold of at least p00.0001, if not p00.000025, and linkage evidence should be reported by at least two independent groups before a new locus is considered demonstrated [54, 69]. The limiting factors for this strategy are the number of families studied and the presence of parents for the ASP, the spacing and informativity of the markers and the analysis method, which need to be improved to detect interacting loci. A number of 100 ASP is recommended to start and the replication set should be larger than the scan sample, keeping in mind that detection of true loci as well as replication might be challenging [54, 66, 70]. Inclusion of unaffected sibs and software improvements help to handle the absence of parents. A panel of 3-cM-spaced microsatellite markers would provide all linkage information and the development of new methods of analysis, such as those based on artificial neutral networks, should soon allow for detection of interacting loci [71].

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Linkage Disequilibrium Mapping. At a susceptibility locus, association studies are performed systematically with microsatellite or single nucleotide polymorphisms, the availability of which is rapidly increasing. Indeed, it should become feasible in the next few years to perform genome-wide association studies [54]. Once found, an association must be confirmed, in a similar way as indicated in the candidate gene approach. The specific limitation is that this approach relies on the hypothesis of a detectable association in the sample studied, which cannot be assured a priori. Moreover, the region to be reached has no precise limits, as no boundaries can be defined: the disease gene is only likely to lie within the region of nominal linkage (delimited by markers with linkage p00.05). As no definite success has been registered so far with this latter approach, a combination of both approaches appears desirable, if not necessary. Combination of Both Approaches Obviously, candidate genes within susceptibility loci are major candidates. Moreover, as every susceptibility gene cannot be expected to have its existence demonstrated by definite linkage, linkage information should be used to guide candidate gene analysis: candidate genes within suggested loci should deserve particular attention. Finally, and most importantly, linkage data should be consistent with the associations found and could provide insight into the pathophysiological mechanisms. (1) Analysis for a given subset of the disease, such as the most aggressive forms, could reveal specific loci, harboring genes involved specifically in the subset of the disease. Conversely, when a candidate gene is putatively involved in such aggressive forms, increased linkage evidence for that subset would bring further support to the hypothesis. (2) Taking into account a given locus for the analysis might reveal other loci harboring genes interacting specifically with gene(s) of that locus, to predispose to the disease. Conversely, when a candidate gene is putatively involved through the interaction with a known susceptibility gene, increased linkage evidence when taking into account the putative interaction would bring further support to the hypothesis.

RA Is Being Investigated by Both Approaches Several Groups Apply the Systematic Approach This approach requires a massive effort with large funding and is therefore undertaken by a small number of teams. The prerequisite is the DNA collection of suitable families.


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RA Family DNA Is Being Collected ASP Families. Large collections of families with at least 2 RA sibs are being established in the UK [72], continental Europe with the European Consortium on Rheumatoid Arthritis Families (ECRAF) [42], Japan [73] and the USA with the North American Rheumatoid Arthritis Consortium (NARAC) [74]. Apart from Japan, which has a homogeneous population and for which the sample size was of 40 families only, DNA of about 400 families has been collected by each of the three other groups, which aim at a sample size of up to 1,000 ASP families. The clinical features and the HLA-DRB1 typing of RA cases from such families showed no peculiarities, supporting the view that the genetic factors revealed in ASP families would apply to all RA patients [75–77]. TDT Families. Because the mean age of onset of RA is within the 5th decade, only 15% of ASP families have both parents and a specific effort is needed to collect DNA for a large number of RA families with both parents. Such families are required to use association tests like the TDT with a maximum power and reliability. The specific collection effort has been initiated by the UK group and by ECRAF, which leads the way with DNA for more than 500 families, of which 400 from France, aiming at 1,000 French families and 100 families for each other country. Apart from a younger age of onset of RA in patients from such families, no differences were found in clinical parameters or for the HLA-DRB1 typing with RA in ASP families, supporting the view that the genetic factors revealed in ASP families could be identified in TDT families and would apply to all RA patients [78]. RA Genome Scans Are Being Performed ECRAF reported the first genome scan for RA with a 12-cM spacing on 90 French ASP families and made publicly available the raw data on the web [42] (www.genopole.com). The HLA locus was confirmed and 26 regions with suggested linkage evidence (p00.05) were listed (www.genopole.com). This scan is being refined down to a 3-cM spacing, more than 1,000 of the 1,211 markers having been genotyped [79]. Preliminary analysis of 400 additional markers showed 11 new suggested loci (p00.05) [80]. Taking into account the clinical and genetic heterogeneity, as described above, leads to the identification of additional suggested loci. Most importantly, such a refined linkage study provides valuable information for any candidate gene, as well as for any combination of genes [81]. The study of additional European families showed replication evidence for loci on chromosomes 1, 3 and 18, the analysis of all families yielding global linkage evidence in the range of 0.0010p00.0001 [42, 82, 83]. Moreover, a suggestion of interaction between HLA and the putative chromosome 1 locus

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was observed, as well as between the putative chromosome 3 and chromosome 18 loci (linkage evidence was stronger at chromosome 1 in HLA-identical ASP, and at chromosome 18 in chromosome 3 locus-identical ASP, respectively, compared to other ASP). Interestingly, those regions on chromosome 3 and 18 have been implicated in IDDM, although identification of a common association is required to put forward the hypothesis of a common autoimmunity gene [42, 84]. Following the criteria mentioned above, these loci should be considered as only suggestive, as long as linkage evidence is not reported by other groups and the statistical evidence strengthened. The Japanese group reported genome scan results on 40 families with an 11-cM spacing [73]. Surprisingly, linkage evidence for the HLA region was only suggested while 3 regions with significant linkage were reported: on chromosome 1 close to the region reported by ECRAF, on chromosome 8 where no linkage evidence was found by ECRAF and on chromosome X, in a region where ECRAF observed nominal evidence. Compared with the ECRAF findings, chromosome 3 showed no linkage evidence in the Japanese families and chromosome 18 showed only a trend, below nominal linkage evidence. Of course, comparisons between Europe and Japan might be limited by the ethnic differences, even though the HLA-DRB1 association is found in both populations. NARAC presented recently preliminary results of a genome scan in 158 families, confirming the HLA locus and listing 21 suggested loci, out of which 1 lies close to the chromosome 18 locus of ECRAF and 5 were close to loci only suggested by ECRAF on chromosomes 1, 5, 6, 12 and 16 [85]. The UK groups have reported so far confirmed linkage for HLA and suggested linkage at candidate gene loci [86–88]. The publication of these NARAC and UK genome scans performed on Caucasian populations will allow for comparisons with the ECRAF results, from which consistent loci are expected to be identified and the list of suggested loci be enlarged. The availability of the human genome sequence will allow for the precise localization of the genetic markers studied, facilitating the choice of candidate genes. Linkage Disequilibrium Mapping and Candidate Gene Studies Are Being Conducted at the Loci Identified Through Genome Scans The systematic approach for disequilibrium mapping is just being initiated. Within the chromosome 1 region reported by ECRAF and the Japanese group, the most obvious candidates are TNFR2, coding for the p75 receptor of TNF-a and DR3, coding for the death receptor 3 involved in apoptosis, proposed as an RA gene by the Japanese group [89]. At the ECRAF putative chromosome 3 locus, candidate genes are CD80 and CD86, coding for cell surface molecules implicated in the regulation of T cell recognition. Although


8

no obvious candidate genes were found at the chromosome 8 Japanese locus or at the chromosome 18 ECRAF locus, a new RA gene has been suggested by the Japanese group at its chromosome X locus, DBL, coding for the diffuse B cell lymphoma proto-oncogene involved in cell growth [90]. Every Laboratory Can Contribute to the Candidate Gene Approach RA Genetics Is Likely to Involve Many Genes, Interacting in Various Combinations From the complexity of RA biology, it can be expected that many genes could contribute to RA susceptibility and outcome. Moreover, the identification of functional relationship between molecules suggests potential interactions. For instance, given the biological synergy of TNF-a and IL-1 [1], it is plausible that alleles that would increase the production of each of those cytokines would contribute synergistically to the RA phenotype. Preliminary analysis of ECRAF linkage data would support this hypothesis, as the IL-1 gene lies in a suggested locus and the HLA-identical ASP (which are TNFAidentical as TNFA lies in the HLA region) show stronger linkage evidence than other ASP [91]. The suggestion of an association between IL-1 polymorphisms and destructive RA in HLA-defined subgroups of patients is particularly interesting in this regard [92, 93]. Numerous hypotheses could be elaborated, especially from laboratories studying the complex functional interplay of molecules potentially involved in RA. The limiting factor for many scientists to test interesting hypotheses is the availability of the suitable material in a technical setting dedicated to candidate gene studies where time and cost could be optimized. Candidate Gene Studies Should Extend Outside of Loci Identified Through Linkage. Ressearch groups involved in genome-wide searches focus on the RA genes that are easiest to detect through linkage analysis. However, it should be made clear that linkage evidence does not reliably predict the strength of the underlying genetic factor (the strength of the association with RA), which depends on the population frequency of the disease allele and the model of inheritance. To illustrate this, in a given arbitrary model, a similar linkage evidence would be observed for a disease allele present on 80% of chromosomes of the general population and for a disease allele providing a twice smaller risk, but present on 50% of chromosomes (there would be more informative parents in ASP families in the second case, compensating for the weaker association) [54]. Therefore, it is clear that linkage evidence might reveal relatively weak RA genes, as HLA-DRB1, and most importantly, that major RA genes might be overlooked by the approach. This would be most apparent for interacting genes, the contribution of which would depend on the contribution of other gene(s): as long as the interaction, unknown a priori, is not

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taken into account, the linkage analysis would have little power to detect either gene. For that reason, association studies being much more powerful than linkage studies [54], candidate gene studies should be performed regardless of the linkage data. This is illustrated by investigation of the IL-1 locus, where no evidence for linkage was found by the UK group and only a suggestion was reported by ECRAF [87, 91], whereas interesting results were reported by association studies [92, 93]. Those linkage data would be, however, of paramount importance to check for consistency with the biological hypothesis. For example, when an association is proposed for a clinical subgroup of patients, linkage at that locus should be stronger for that subgroup, as illustrated above for the IL-1 locus. A Common Resource for RA Association Studies Is Available at GENOPOLE To optimize the use of its DNA collection, the laboratory coordinating ECRAF at GENOPOLE is opened to any group wishing to test a hypothesis. A common resource of 100 TDT RA Caucasian families, with homogenous background (the 4 grandparents are French), is available with the clinical data at GENOPOLE. Additional TDT families of the same population are available for replication tests. Moreover, the genotypes performed for other candidate genes on the same resource are available to refine the analysis, in particular to test for interaction between specific alleles for different genes. Refined linkage data on the same population are available to help in interpreting the results of association studies. As was done for the first genome scan, the genotype for all hypotheses tested will be made available on our web site (www.genopole.com) as soon as the results are published. In that way, any group could contribute to the analysis, in particular to the search for complex gene interactions, as new hypotheses arise from the rapid progress of human biology.

Conclusion and Perspectives While the sequence of the human genome and the description of its allelic polymorphism are being revealed, the search for RA susceptibility genes is now intensified, with every laboratory invited to contribute to this search. Within the next few years, all the more rapidly since more scientists will be involved, new genetic factors predisposing to RA should be discovered. The first clinical evaluation will be for their diagnostic and prognostic value, as well as for their ability to predict therapeutic responses to established treatments. Some genetic factors might be more involved in the modulation


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of the severity of the disease than in its predisposition per se. A very large DNA sample should help provide answers to most questions and specific DNA samples will be desirable to provide precise answers, such as those of prospective studies or of therapeutic assays. All major ethnic groups will have to be investigated, providing opportunities to refine the understanding of the pathophysiology. From this new understanding of the initial mechanism, therapeutic targets could be chosen in order to cure the disease. Genetic epidemiology studies will have to refine the measures on which to rely for evaluating the use of genetic factors to predict the risk of disease in asymptomatic individuals, especially in relatives of patients. The use of the genetic factors should help in identifying the remaining factors, probably environmental, which trigger the disease in susceptible individuals. The eradication of the disease might then be possible.

Acknowledgments Funded by the Association de Recherche sur la Polyarthrite, Association Française des Polyarthritiques, Socie´te´ Française de Rhumatologie, Fondation de la Recherche Me´dicale and Conseil Re´gionale d’lle de France.

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Shiozawa S, Hayashi S, Tsukamoto Y, Goko H, Kawasaki H, Wada T, Shimizu K, Yasuda N, Kamatani N, Takasugi K, Tanaka Y, Shiozawa K, Imura S: Identification of the gene loci that predisopose to rheumatoid arthritis. Int Immunol 1998;10:1891–1895. Seldin M, Amos C, Ward R, Gregersen P: The genetics revolution and the assault on rheumatoid arthritis. Arthritis Rheum 1999;42:1071–1079. Corne´lis F, Fritz P, Prud’homme J, Liote´ F, Weissenbach J, Kuntz D, Bardin T: DNA resource of affected sib pair families for genetic studies of rheumatoid arthritis in genome scan families (abstract). Arthritis Rheum 1997;40(suppl):S155. Barrera P, Radstake T, Albers J, van Riel P, van de Putte L, for ECRAF: Familial aggregation of rheumatoid arthritis in The Netherlands: A cross-sectional hospital-based survey. Rheumatology 1999;38:415–422. Balsa A, Barrera P, Westhovens R, Alves H, Maenaut K, Pascual-Salcedo D, Corne´lis F, Bardin T, Riente L, Radstake T, de Almeida G, Lepage V, Stavropoulos C, Alves H, Spaepen M, Martinez M, Alibert O, Prudhomme J, Migliorini P, Fritz P, for ECRAF: Clinical and immunogenetic characteristics of European multicase rheumatoid arthritis families. Submitted. Westhovens R, for ECRAF: Comparison of clinical and immunogenetic characterisitcs of rheumatoid arthritis patients of affected sib pair families and single case families (abstract). Arthritis Rheum 1999;42:S134. Moindrault S, for ECRAF: A 3 cM pannel of microsatellite for the human genome (abstract 1364). Am J Hum Genet 1998;63:A237. Corne´lis F, for ECRAF: Genome-wide search for rheumatoid arthritis genes (abstract). Ann Rheum Dis, in press. Corne´lis F, for ECRAF: Loci interaction providing susceptibility to rheumatoid arthritis, the most common autoimmune disease (abstract). Am J Hum Genet 1999,65:A59. Corne´lis F, for ECRAF: New susceptibility locus for rheumatoid arthritis on chromosome 1 (abstract 1649). Am J Hum Genet 1998;63:A286. Caponi L, Martinez M, Corne´lis F, for ECRAF: Combination of new loci providing susceptibility to the most frequent autoimmune disease, rheumatoid arthritis (abstract). Eur J Hum Genet 1999; 7(suppl):130. Merriman T, Eaves I, Twells R, Merriman M, Danoy P, Muxworthy C, Hunter K, Cox R, Cucca F, McKinney P, Shield J, Baum J, Tuomilehto J, Tuomilehto-Wolf E, Ionesco-Tirgoviste C, Joner G, Thorsby E, Undlien D, Pociot F, Nerup J, Ronningen K, Bain S, Todd J: Transmission of haplotypes of microsatellite markers rather than single marker alleles in the mapping of a putative type 1 diabetes susceptibility gene (IDDM6). Hum Mol Genet 1998;7:517–524. Jawaheer D, Costello T, Amos C, Monteiro J, Seldin M, Criswell L, Bridges S, Schroeder H, Pisetsky D, Kastner D, Wilder R, Pope R, Clegg D, Ward R, Albani S, Nelson J, Wener M, Callaha L, Pincus T, Gregersen P: Analysis of affected sibling pairs with rheumatoid arthritis: The North American Rheumatoid Arthritis Consortium (abstract). Am J Hum Genet 1999;(suppl 65): A276. John S, Marlow A, Hajeer A, Ollier W, Silman A, Worthington J: Linkage and association studies of the natural resistance associated macrophage protein 1 (NRAMP1) locus in rheumatoid arthritis. J Rheumatol 1997;24:452–457. John S, Myerscough A, Marlow A, Hajeer A, Silman A, Ollier W, Worthington J: Linkage of cytokine genes to rheumatoid arthritis. Evidence of genetic heterogeneity. Ann Rheum Dis 1998; 57:361–365. John S, Myerscough A, Eyre S, Roby P, Hajeer A, Silman A, Ollier W, Worthington J: Linkage of a marker in intron D of the estrogen synthase locus to rheumatoid arthritis. Arthritis Rheum 1999; 42:1617–1620. Konishi Y, Mukae N, Hayashi S, Yamamoto E, Komai K, Kawasaki H, Miura Y, Shiozawa K, Shiozawa S: Death receptor 3 as a candidate for rheumatoid arthritis disease gene RA1 (abstract). Arthritis Rheum 1999;42:S392. Komai K, Hikasa M, Kawasaki H, Murata M, Konishi Y, Kitagawa M, Shiozawa K, Shiozawa S: Identification of 3 deletion mutant of DBL protooncogene as rheumatoid arthritis disease gene RA3 (abstract). Arthritis Rheum 1999;42:S392.

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François Corne´lis, ECRAF-University of Paris 7, GENOPOLE, 2, rue Gaston Cre´mieux, CP 5727, F–91057 Evry Cedex (France) Tel. +33 1 60 87 45 70, Fax +33 1 60 87 45 71, E-Mail [email protected]


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Elucidation of Pathways Leading to Rheumatoid Arthritis by Genetic Analysis of Animal Models ˚ sa Johansson, Rikard Holmdahl, Robert Bockermann, Johan Jirholt, A Peter Olofsson, Shemin Lu Section for Medical Inflammation Research, Lund University, Sweden

Genetic Influence on Rheumatoid Arthritis Rheumatoid arthritis (RA) is not one single disease entity but rather a syndrome that is described by a number of clinical criteria of signs and symptoms (table 1). These criteria reflect an erosive inflammatory and chronic attack of peripheral joints. A number of systemic effects, such as skin nodules, vasculitis and autoimmune abnormalities, for example, the production of rheumatoid factors (RF) or antibodies to cartilage proteins, may also occur. This is apparently not an uncommon way for the body to respond because a relatively large proportion of the human population develops RA. The disease affects approximately 1% of the Caucasian population and afflicts other ethnic populations to a variable degree. The incidence is, however, difficult to assess for many reasons as has been discussed in other recent reviews [1–3]. One reason is the difficulty in defining the disease in different populations; another obstacle is that the onset of disease may occur at any age. Having taken these uncertainties into account, the disease seems to occur in every ethnic population studied at a significant frequency. There is a clear and well-documented sex difference for the susceptibility to RA, which is 3–4 times more common in females than in males. It is most likely that this difference depends on both hormonal and genetic factors. The complexity is demonstrated in that pregnancy tends to protect from disease whereas relapses are often seen postpartum, indicating a protective effect by female sex hormones [4]. The incidence in females tend to decrease with age and an influence by estrogen treatment has been discussed [5, 6]. Besides sex hormone effects, it has, however,

Table 1. Comparison of animal models of arthritis and RA by the use of the American College of Rheumatology (ACR) criteria and other characteristics RA

AIA

OIA

PIA

CIA

ACR criteria for the classification of RA Morning stiffness Arthritis of at least 3 areas (PIP-MCP, wrist, elbow, knee, ankle, PIP-MTP) for more than 6 weeks Arthritis of hand joints for more than 6 weeks Symmetric arthritis Rheumatoid nodules Serum RF Radiographic changes of erosions on hand films 4 criteria fulfilled

+ +

ND –

ND –

ND +

ND +

+ + + + + +

– – ND – + –

– + – ND – –

+ + ND + + +

+ – – +(IgG) + +

Other characteristics Chronic relapsing disease course MHC association Serum anti-CII antibodies Spine (tail) affections Ankylosis/enthesopathy

+ + +/– +/– –

– + – +/– +

– + – – –

+ + – +/– +/–

+/– + + +/– +/–

Fulfilment of 4 out of the 7 criteria is required for a diagnosis of RA. AIA>Mycobacteria cell wall adjuvant-induced arthritis; CIA>collagen-induced arthritis; ND>not determined; OIA>oil-induced arthritis; PIA>pristane-induced arthritis; PIP>proximal interphalangeal; MCP>metacarpophalangeal; MTP>metatarsophalangeal.

been difficult to isolate specific environmental factors although considerable efforts have been made. The problems in assessing disease frequency and the environmental influence on RA make it difficult to define a possible genetic contribution. One classical approach is to determine the occurrence of disease in monozygotic twins. A large number of such studies have been performed and, not surprisingly, indicate quite a variable prevalence, but, nevertheless, estimate a concordance rate between 12 and 30%. A meta-analytic value has been suggested to be around 15%. Another classical genetic estimate is to determine the ks value, i.e. the disease prevalence among siblings (2–4%) divided by the disease prevalence in the population (0.2–1.2%). This also gives extremely variable values from 2 to 20 but is most likely around 10. Taken together, these findings suggest that there is a significant genetic contribution to RA. One of the contributing genetic factors has been clearly documented to be the major histocompatibility complex (MHC) denoted HLA in humans. The

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MHC region encodes a series of cell surface receptors of crucial importance for the activity of the immune system. The MHC class I and class II receptors bind antigenic peptides presented to T lymphocytes, which enables them to give rise to an antigen-specific immune response. The MHC class II genes are highly polymorphic, explaining the fact that the specific immune responsiveness varies between individuals. In fact, a comparative analysis of all MHC class II alleles associated with RA indicates that they share a structure (denoted the shared epitope) located in the peptide binding pocket [7]. This has supported the argument that RA could be an inflammatory autoimmune disease. Hypothetically, T cells recognize peptides derived from specific tissues and respond to them as if they were of foreign or infectious origin. A T cell-directed inflammatory response to the cartilaginous joints ensues. The hypothetical autoantigenic peptides have, however, not been isolated. But DR4 alleles are common in the general population and consequently the vast majority of carriers do not develop RA. Thus, a search for other genes as well as environmental factors is needed to understand the etiology and pathogenesis of RA. The search for predisposing genes for the development of RA is, however, hampered by several factors. Firstly, RA is phenotypically heterogeneous. There are many different clinical subtypes, some of which have been more distinctly defined (such as Felty’s syndrome) but in many cases it is difficult to distinguish RA from other arthritides. These different variants are most likely caused by different genotypes and environmental factors. Secondly, there is most likely a pronounced genetic heterogeneity in RA. In other words, an identical clinical picture could be caused by different sets of genes in different individuals. Thirdly, RA is a polygenic disease. The disease is associated with many different genes in a single individual. Many of the disease-causing genes are most likely of low penetrance but act in concert and/or interact with each other. Fourthly, RA is a multifactorial disease. Unknown environmental factors are likely to play an important role and stochastic effects cannot be excluded. These factors have made it difficult to identify the genes or even gene regions associated with disease. Linkage analysis of sibling pairs or multicase families have so far not led to a significant identification of any non-MHC loci associated with disease.

Animal Models A shortcut in the genetic analysis of RA is provided by animal models – both for direct comparisons with the human diseases and, maybe more importantly, for the understanding of the mechanisms in the pathogenesis. Genetic mapping of inbred line crosses is generally much simpler than genetic

Elucidation of Pathways Leading to Rheumatoid Arthritis

19

mapping of outbred populations. In crosses between inbred lines there are only two alleles that segregate at each locus. In inbred line crosses the linkage phase is known, which is generally not the case in outbred populations, meaning that both phases have to be evaluated for each parent-offspring pair. For studies of RA several animal models have been developed. Obviously, none of them will be identical to the human disease but it is likely that the diseases in the animal models involve one or several of the pathways leading to RA. In fact, each animal model might use unique pathways also involved in certain of the different RA subtypes. Therefore, a multitude of different well-characterized models will be a valuable asset for the genetic analysis. A list of animal models and their characteristic features in comparison with RA is depicted in tables 1 and 2. In consequence with the possibility of a variety of different pathways leading to RA in humans there are several ways to induce arthritis in animals. We will review here only models in which genetic linkage results have been reported (an overview of rat strains used and the loci found so far in mice and rats are depicted in table 3). Adjuvant Arthritis Models Mycobacterium Cell Wall-Induced Arthritis. The first animal model for RA was the so-called adjuvant arthritis induced in rats after injection of mycobacteria cell walls suspended in mineral oil, a component of complete Freund’s adjuvant (CFA) [8]. Surprisingly, only rats (and not mice or primates) have been shown to develop arthritis after mycobacteria challenge [9], although it has been reported that joint-related granuloma formation has occurred in humans treated with BCG [10]. CFA is a potent adjuvant that not only induces immune activation but also granulomatous inflammation in many organs, for example, the spleen, liver, bone marrow, skin and eyes, and causes profound inflammation in peripheral joints [8]. The adjuvantinduced disease is severe but self-limited and the rat recovers within a few months. Both the mycobacteria cell wall and the oil are most likely disseminated throughout the body and engulfed by tissue macrophages and other cells. In the process leading to arthritis, T cells are essential because the disease can be transferred by T cells and abrogated by in vivo deletion of T cells [11, 12]. The specificity of such T cells has, however, not been reproducibly demonstrated, although some possibilities have been suggested including bacterial structures, cross-reactive self-components and heat shock proteins [13, 14]. While a role for heat shock proteins in the induction of the disease has not been confirmed, they seem to play an important regulatory role for the development of arthritis [15]. In the search for the minimal arthritogenic structure in mycobacterium it was observed that one of the essential structural


20

Table 2. Arthritis susceptibility of some inbred rat strains used in experiments aimed to determine the genetic influence Strain

MHC Origin

Animal models AIA

OIA

PIA

aCIA

hCIA

DA

av1

Inbred

+++++ +++ severe, mild, acute acute

+++++ severe, chronic

+++++ severe, chronic/acute

++++ severe, acute

DA.1N

n

Inbred

+++++ – severe, acute

ND

+

+

E3

u

Inbred

ND

–

–

–

ND

LEW.1F f

MHCcongenic (LEW)

ND

–

+++++ severe, chronic

+

ND

LEW

Inbred

++++ severe, acute

–

+++

+

++++ severe, acute

LEW.1A a

MHCcongenic (LEW)

++++ severe, acute

–

+++ severe, chronic/acute

++++ severe, chronic/acute

++++ severe, acute

F344

l

Inbred

+

–

ND

ND

++

BN

n

Inbred

+++ severe, acute

–

ND

–

–

l

The grading is evaluated from several published reports [18, 23, 38–40, 44, 59, 61, 62, 88–90] as well as our unpublished data. Roughly: +++++>90–100%; +>0–10%; –>0% (resistant). AIA>Mycobacteria cell wall adjuvant-induced arthritis; aCIA>CIA induced with autologous CII; hCIA>CIA induced with heterologous CII; ND>not determined.

elements of the mycobacterium peptidoglycan, muramyl dipeptide, could induce arthritis [16]. Interestingly, T cells do not recognize these structures but they have potent adjuvant capacities, indicating that they stimulate antigenpresenting cells or innate immune recognition. Whatever the cause of the inflammatory disease triggered by mycobacterium in oil is, it cannot explain why joints, specifically, are inflamed, as in RA, because the mycobacteriainduced disease is systemic.


21

Table 3. Quantitative trait loci associated with arthritis in mouse and rat models QTL Chr Position Significance Closest cM locus Mouse loci Cia1 17

Transgenic

Cia2 Cia3 Cia4 Cia5 Cia6 Cia7

2 6 2 3 6 7

34 48.7 34 39.7 33.5 50

Cia8

10

30

Rat loci Aia1 20 Aia2 4 Aia3 4 Pia1 20

1 51 73 1

Pia2 Pia3 Pia4 Pia5 Pia6 Oia1 Oia2 Oia3 Cia1 Cia2 Cia3 Cia4 Cia5 Cia6 Cia7 Cia8

43 82 31 73 29 1 109 91 1 82 73 71 95 77–88 85 53

4 6 12 4 14 20 4 10 20 1 4 7 10 8 2 7

Lod>6.34 Lod>3.19 Lod>3.19 Lod>4.12 Lod>4.7 Suggestive (Lod>2.8) Suggestive (Lod>3.3) Lod>18 Lod>5.8 Lod>3.9 Congenic, Lod>3.3 Lod>3.9 Lod>4.5 Lod>8.4 Lod>4.5 Lod>4.9 Congenic p>10Ö13 p>10Ö6 Congenic Lod>5.0 Lod>4.8 Lod>4.3 Lod>4.9 Suggestive Lod>4.6 Lod>5.1

Strains susceptible

resistant

disease

Reference No.

MHC class II C3H.NB (Aq) Ab gene D2Mit61 DBA/1j D6Mit10 DBA/1j D2Mit61 DBA/1j D3Mit72 B10.RIII D6Mit19 DBA/1j D7Mit34 DBA/1j

C3H.NB

CIA

67

SWR/J SWR/J SWR/J RIIIS/J B10.Q1 B10.Q1

CIA CIA CIA CIA CIA CIA

78, 80 80 80 86 87 87

D8Mit205

DBA/1j

B10.Q1

CIA

87

tnfa D4Mgh15 D4Arb24

DA DA DA LEW.1F, DA DA DA DA DA DA DA DA DA DA, LEW.1A F344 DA DA DA DA DA DA

F344 F344 F344 LEW, DXEC E3 E3 E3 E3 E3 DA.1N LEW.1AV1 LEW.1AV1 LEW, F344 DA F344 F344 F344 F344 ACI F344

AIA AIA AIA PIA

18 18 18 23

PIA PIA PIA PIA PIA OIA OIA OIA CIA CIA CIA CIA CIA CIA CIA CIA

38 38 38 38 38 39, 40 41 41 44, 61 61 61 61 61, 62 92 62 93

D4Mgh14 D6Wox5 D12Wox14 D4Wox20 D14Csna D4Mgh10 D4Mgh10 D10Mgh1 D1Arb36 D4Arb24 D7Arb5 D10Arb22 – Cpb1 D7Rat26

Chromosome positions have been taken from the mouse genome database (MGD) website http:// www.informatics.jax.org/ [91] for mouse markers or http://www.nih.gov/niams/scientific/ratgbase/index.htm and http://ratmap/gen.gu.se/default.html for rat markers. Lod>Logarithmic odds ratio; Chr>chromosome; QTL>quantitative trait locus; AIA>adjuvant-induced arthritis. 1 B10.Q is less susceptible than DBA/1j and regarded as relatively resistant.


22

A genetic analysis of the classical mycobacteria-induced arthritis has been performed using F2 crosses between resistant and susceptible strains. In a very early report Battisto et al. [17] found that the MHC haplotype RT1av1 was dominantly associated with disease susceptibility using a cross between the DA and F344 rat strains. This was more recently confirmed by Wilder and coworkers [18] who analyzed a complete genome scan of an F2 cross between DA and F344 and who denoted the locus as Aia1. Weaker contributions were seen from two loci on chromosome 4 (Aia2 and Aia3). The strong influence by the MHC region is surprising considering the earlier difficulties in isolating a specific antigenic response in the model. If the Aia1 locus contributes with the MHC class II gene, it would be of great interest to determine its pathogenic role because an antigenic peptide derived from mycobacteria cell walls, or from the targeted joints, has so far not been isolated. Mineral Oil-Induced Arthritis, Avridine-Induced Arthritis and Pristane-Induced Arthritis. The induction of arthritis in rats was found to be not only dependent on the mycobacteria cell walls, but also on the oil into which the mycobacteria fragments were suspended. Interestingly, some oils were found to support the induction of arthritis whereas others did not [19]. Many years later it was noted that the oil component of CFA was in fact arthritogenic itself depending on the rat strain used [20, 21]. It was also found that adjuvant compounds such as avridine and pristane (2,6,10,14-tetramethylpentadecane), which bear no relationship to bacteria cell walls, were highly effective in inducing arthritis in rats [22, 23]. These adjuvant compounds produce inflammation confined to the joints. They offer principally different experimental models for RA than the earlier commonly used ‘adjuvant arthritis’ or mycobacteria in oil-induced arthritis (OIA) models because they are not directly related to an infectious organism. Mineral OIA [20, 21], avridine-induced arthritis (AvIA) [22] and pristaneinduced arthritis (PIA) in the rat [23] share many common features and differ mainly in the severity and chronicity of the disease. They are induced with adjuvant compounds lacking immunogenic capacity, i.e. no specific immune responses are elicited towards them after injection. Instead they are rapidly and widely spread throughout the body after a single subcutaneous injection, and penetrate through cell membranes into cells. After a delay of at least 1 or 2 weeks arthritis suddenly ensues. The arthritis appears in the peripheral joints, with a similar distribution as seen in RA, and is mainly symmetrical. Occasionally other joints are involved but systemic manifestations in other tissues have so far not been reported. In certain rat strains, especially in the AvIA and PIA models, the arthritis develops along a chronically relapsing disease course [23, 24]. Surprisingly, no immune response to cartilage proteins has been observed although RF are present in the serum.


23

A role for cartilage proteins in regulating disease activity is possible because the disease can be prevented and, in fact, therapeutically ameliorated by nasal vaccination with various cartilage proteins [25]. Another way to both prevent and therapeutically decrease disease severity at an already established phase is by blocking ab T cells [23, 24]. Together with the observation that the chronic disease course is associated with the MHC region, this suggests activation of T cells that recognize cartilage proteins. However, such T cells have not been observed and T cell transfer of the disease has so far failed to identify antigen-specific T cells [26, 27]. The inducing agents all have small molecular structures that are unable to bind to MHC class II molecules or to be recognized by T cells. A role for environmental infectious agents is not likely because no difference in disease susceptibility was seen in germ-free rats, although only conventional rats respond to heat shock proteins [28]. There is no evidence for recognition by lymphocyte receptors or receptors involved in the innate immune system. Surprisingly, some of the arthritogenic adjuvants are in fact components already present in the body before injection. For example, pristane is a derivative of the phytol tail of chlorophyll and is normally ingested by all mammals including laboratory rats [29]. Pristane is taken up through the intestine and spread throughout the body. In fact, an endogenous component of cell membranes, squalene, is also arthritogenic [30]. The arthritogenic adjuvants seem to share the capacity to penetrate into cells where they could change membrane fluidity and modulate transcriptional regulation [31, 32]. The injection route and doses are critical, i.e. it is most likely of critical importance which cell is first activated and to what extent. Although the subcutaneous injection of pristane is artifactual, in that it has no apparent resemblance to a naturally occurring environmental effect, the ensuing development of chronic arthritis has a striking resemblance to RA. Several weeks after the injection of pristane, arthritis in peripheral joints suddenly appears whereafter the disease spreads to other peripheral joints and often develops an active chronic relapsing disease course. The inflammation is restricted to the peripheral joints but occasionally affects other more centrally located joints or other tissues. As in RA, the joints are usually symmetrically affected. The triggered joint erosions can be quantified by measuring the serum levels of cartilage oligomeric matrix protein (COMP), which is increased in rats with chronic arthritis, and correlates to the clinical severity [33]. In RA, serum levels of COMP serve as a prognostic marker where high levels in the early stage of disease indicate a rapid progression and joint destruction [34]. In similarity with RA, IgM RF are produced in rats after injection of pristane. RFs also evolve in the collagen-induced arthritis (CIA) model but only as detected by a diffusion-in-gel ELISA method, which most likely reflects self-associated


24

IgG RF [35–37]. However, in contrast to RA and CIA, it has not been possible to detect antibodies to type II collagen in sera from rats with PIA. As for RA, there is a genetic predisposition to develop PIA. DA rats are highly susceptible, LEW rats are intermediately susceptible, and E3 rats are resistant. Studies using MHC-congenic LEW rat strains have shown that the MHC locus (designated Pia1) contributes to the genetic predisposition [23]. The most susceptible haplotype was the RT1f, which is also associated with the genetic control of AvIA [24] and type XI CIA. Interestingly, a clear association was only seen with the chronic development of the disease whereas the days to arthritis onset or incidence were not significantly associated. However, the influence by MHC cannot explain the dramatic difference in susceptibility to PIA between DA and E3 because the MHC haplotypes of both strains are equally permissive on the LEW background. Instead there is a major non-MHC genetic influence determining the difference in PIA susceptibility between the DA and E3 strains. PIA is clearly a polygenic disease, as there is a variable penetrance of the disease in a series of recombinant inbred strains made from DA and E3 rats [23, 38]. The DA strain is highly susceptible to PIA and develops disease with a high incidence, pronounced joint erosions, high clinical severity and with a chronic relapsing disease course. In contrast, the E3 rats are resistant. Among recombinant inbred strains between DA and E3, the DXEA rats have a mild disease with a high incidence, the DXEB have a more severe and chronic disease but with a lower incidence and the DXEC strain is resistant [23]. This demonstrates that different genes control the different disease phenotypes. F1 rats were susceptible to chronic PIA, but with a delayed onset and a reduced incidence and severity. Still, both parental and F1 rats showed a considerable variation in disease penetrance and disease course, indicating environmental and stochastic influence even in a situation in which every attempt is made to minimize such effects. In genetically segregated F2 populations the development of disease was only slightly more variable but still useful for genetic analysis. Such analyses have revealed genetic regions specifically controlling different phases of the disease, such as onset (Pia2, Pia3), severity (Pia4) and chronic active arthritis (Pia1, Pia5, Pia6). In the OIA model, three loci have been identified that control the development of acute arthritis in MHC-congenic DA and LEW strains (Oia1) [39, 40] and in a cross between DA and LEW.1AV1 strains (Oia2 and Oia3) [41]. The finding that different subphenotypes of the arthritis pathogenesis – such as disease onset, joint erosions, severity and chronicity – are controlled by different genes may be of critical importance for a deeper understanding of pathogenic mechanisms in RA. Temporary joint affections in conjunction with infections or unknown causes are common in humans and do not neces-


25

sarily lead to RA. The specific events leading to RA are more likely to be connected with further progression, or self-perpetuation, of the disease. Such pathogenic events are most likely associated with different sets of genes combined with certain environmental factors, resulting in a very complex pathogenesis of the disease. With an experimental model, in which genetic and environmental factors are better controlled, the various pathogenic mechanisms controlling specific phenotypes of the disease could be elucidated. The loci associated with the chronicity of arthritis (Pia1, Pia5 and Pia6) were most likely associated with a separate pathogenic mechanism because no linkage was found with early events of arthritis. Interestingly, the major locus controlling arthritis severity in the (E3¶DA)F2 cross, Pia4, was also associated with increased COMP levels in serum, suggesting that the gene is in fact associated with erosions of cartilage. COMP is a marker for severe erosive arthritis in human RA [34, 42] and correlates strongly with arthritis severity in the rat [33]. An interesting observation using the MHC congenic strains was the linkage of chronic arthritis to the a, u and f haplotypes of the RT1 region, the Pia1 locus. A similar association between MHC and more chronic arthritis has been suggested to occur also in RA [43]. The observation may cast some light on the pathogenesis of chronic PIA because both the a, u and the f haplotype are associated with an arthritogenic response to various autologous cartilage proteins, such as type II collagen (CII), type IX collagen, type XI collagen and COMP [44, 45, unpubl. data]. It is also notable that the linkage to MHC in the PIA model is far weaker than the linkage on mycobacteriainduced arthritis or in the CIA model (see below), indicating different mechanisms or genes. Cartilage Protein-Induced Arthritis Most proteins that are more or less restricted to cartilage will induce arthritis if used for immunization together with an appropriate adjuvant in selected mouse or rat strains. Arthritis is caused by a specific immune attack on cartilage in peripheral joints. Confirmed reports of arthritogenic cartilage proteins include: CII, leading to the most commonly used arthritis model in mice and rats, CIA [46], aggrecan, the major cartilage proteoglycan induces arthritis in certain Balb/c substrains, called proteoglycan-induced arthritis [47], type XI collagen induces arthritis in rats [48] and COMP induces arthritis in rats [45]. These various models have different characteristics and genetics but the CIA induced with CII is still the most commonly used model and is a prototype of cartilage protein-induced disease. Immunization with the major collagen restricted to cartilage, leads to an autoimmune response and, as a consequence, sudden onset of severe arthritis [recently reviewed in 49]. Although it is neces-


26

sary to emulsify the CII in adjuvant, such as mineral oil, the disease can be distinguished from the various forms of adjuvant arthritis [46, 50, 51]. The disease characteristics of the CIA vary considerably depending on the experimental animal species and on whether the CII used is of self or nonself origin. The use of mycobacteria supplement in the adjuvant oil is not necessary for induction of CIA although in the mouse the addition of mycobacteria cell walls will increase the arthritis susceptibility. In both rats and mice immunized with heterologous CII, a severe, erosive polyarthritis develops 2–3 weeks after immunization. The inflammation usually subsides within 3–4 weeks although in certain strains a few animals may develop a chronic relapsing disease. The disease is critically dependent on both a strong T- and B-cell response to CII [52–54] and a significant part of the inflammatory attack on the joints is most likely mediated by pathogenic antibodies [55, 56]. These CII-specific antibodies bind to the cartilage surface, fix complement, attract neutrophilic granulocytes and activate macrophages. The disease induced with homologous CII in both rats and mice is not as easily inducible but once started it is as severe; however it tends to be more chronic than the disease induced with heterologous CII [44, 57]. The pathogenic events in the chronic disease phase are largely unknown but are most likely dependent on both autoreactive B- and T-cell activity. Genetic Control of CIA in Rats Early observations using the CIA model in both mice and rats induced with heterologous CII indicated a role for the MHC region [58, 59]. It was later found that the MHC association of CIA induced with homologous CII was even more limited to certain haplotypes. Immunization of rats with homologous (rat) CII leading to arthritis development is associated with MHC class II genes, with the av1 haplotype as the most permissible, u, f and l intermediate and n resistant as shown by analysis of both MHC congenic LEW strains and F2 gene segregation experiments using DA and LEW strains [44]. Recombinant inbred strains further localized this genetic influence to the MHC class II region and sequence comparison suggested the DQ homologue as also shown in the mouse (see below) [44, 60]. In a seminal study, Remmers et al. [61] determined the contribution by genes outside MHC in an F2 cross between DA and F344 induced with bovine CII and found several highly significant loci on chromosome 1 (Cia2), 4 (Cia3), 7 (Cia4) and 10 (Cia5) [61]. Because this cross was polymorphic at the MHC region at chromosome 20, a linkage confirming earlier results was found and denoted Cia1. Interestingly, the subtrait associated with Cia1 was incidence whereas the subtrait associated with Aia1, identified using the same type of cross, was severity rather than incidence [18]. This difference suggests that the MHC region has a different


27

influence on CIA than on mycobacteria-induced arthritis. One possible interpretation is that the MHC control of CIA induced with heterologous CII controls the initial priming of immune response to a heterologous peptide whereas in the adjuvant-induced arthritis model the MHC region is associated with amplification of the secondary immune recognition of mycobacterial or joint antigens. In an F2 cross between DA and ACI rats, which share the RT1av1 MHC region, induced with rat CII an additional locus on chromosome 2 associated with arthritis severity (Cia7) was identified [62]. No or only weak associations with the loci earlier identified in the DA¶F344 cross induced with heterologous CII were found, however. The most likely explanation is a pronounced genetic heterogeneity of the control of arthritis such as CIA. Interestingly, some of the loci controlling CIA in the rat correspond to loci controlling OIA and CIA and some also to loci identified in the mouse (see below). The Cia5 locus on chromosome 10 corresponds to the Oia3 locus identified in a DA¶LEW F2 cross. The Cia3 locus on chromsome 4 is located in the vicinity of the Pia5 locus controlling PIA and the Aia2 locus in mycobacteria-induced arthritis defined in a rat cross that also involved the DA strain [18, 61]. In addition, a locus associated with lymphopenia, diabetes and thyroiditis (Lyp/Iddm1) [63, 64] has been found in crosses involving the BB rat, suggesting a general autoimmune disease gene. Furthermore, it is homologous to the Cia6 locus in the mouse. However, this could also be coincidental, especially since the analyzed phenotypes are somewhat different. For example, in the analysis of the CIA model, the rats were immunized with bovine CII that induced a severe disease without chronicity whereas in PIA the linkage was seen only with late chronic disease. It is possible that the close correspondence of loci found in different models is due to the common use of the highly susceptible DA strain in most of the crosses. It can be anticipated that many more loci will be found when other strain crosses are used. A comparative analysis of the genetic control of the different models and the various subtraits they control will most likely give valuable information on specific pathways used in each model that leads to arthritis. Genetic Control of CIA in Mice There is considerable variability in the susceptibility to CIA among inbred mouse strains. A major factor is the MHC dependence. CIA induced with both heterologous and homologous CII is most strongly associated with the H-2q and H-2r haplotypes although most other haplotypes such as b, s, d and p are not totally resistant to disease induced with heterologous CII [58, 65, 66]. Of interest is that it has been possible to further map the MHC association of CIA to the genes coding for the MHC class II A molecule, the DQ homo-


28

logue in the mouse [67]. Moreover, the immunodominant peptide derived from the CII molecule, which binds to the arthritis-associated q variant of the A (Aq) molecule, has been defined as positions 256 and 270 of CII [68, 69]. This is a glycopeptide with an oligosaccharide pointing towards the T cell receptor and is recognized by many of the CII-reactive T cells [70]. Interestingly, the peptide binding pocket of the Aq molecule is very similar to that of the DR4 (DRB1*0401/DRA) and DR1 molecules, which are associated with RA [71]. Furthermore, mice transgenically expressing DR4 or DR1 are susceptible to CIA and respond to a peptide from the same CII region [72–74, recently reviewed in 75]. This finding provides a model for studies of RA by displaying some critical structural similarities to the human disease. There is also an influence by many other so far unknown genes outside MHC. A number of spontaneously occurring gene mutations in inbred strains have given some clues to the pathogenesis of CIA. A functional gene for complement (C5) is important [76–78], but not absolutely necessary [79], for the development of CIA. The deletion of the C5 gene is most likely responsible for the locus observed in crosses involving the C5-deficient SWR strains denoted Cia2 and Cia4 [78, 80]. In addition, spontaneous mutations affecting B-cell function have been shown to lead to resistance to CIA [81, 82]. A search for non-MHC loci in crosses between resistant and susceptible strains has turned out to be difficult due to the pronounced influence by environmental factors such as stress, behavior, light and sex hormones [recently reviewed in 6, 49]. These factors are difficult to control and in fact certain strains may spontaneoulsly develop clinical arthritis that is seen histopathologically as a healing response with pronounced enthesopathy and ankylosis [83–85]. This spontaneous disease is not immune-mediated and, therefore, different from CIA, but it will confuse evaluation of CIA, especially if DBA/1 males are used. Nevertheless, with these factors in mind, it has been possible to determine some contributing loci in certain mouse crosses induced with heterologous CII. In an F2 cross between the highly susceptible B10.RIII and the resistant RIIIS/J strains, a locus was found on chromosome 3 that was associated with arthritis severity [86]. This locus was first denoted mcia2 but later changed to Cia5. Interestingly, the Cia5 locus colocalizes with Eae3, a locus associated with chronic EAE, a model for multiple sclerosis, indicating that these models may share some pathways leading to disease. From the results in this cross it seems that the role of the extensive deletion of the tcrb locus in the RIIIS/J strain has been overestimated because no linkage was found to this region. In another cross between the highly susceptible DBA/1 and the intermediate susceptible B10.Q strain, a significant locus was identified on chromosome 6 (Cia6) associated with arthritis severity [87]. Interestingly, together with two additional loci that were found, on chromosome 7 and 10 (Cia7 and


29

Cia8), all of the loci corresponded to earlier identified loci in the rat. In addition, the Cia5 locus identified in the B10.RIII¶RIIIS/J cross corresponds to the Cia7 locus identified in the rat. Although the localization of the gene, or genes, is far from certain, it suggests that the development of arthritis in the two species share important pathways. This gives hope to comparing, finding and analyzing the role of genes in RA because the identified loci are possibly located at gene clusters controlling critical pathways leading to arthritis, irrespective on how the disease is induced and in which species.

Identification of Genes and Their Pathogenic Role Another advantage of using animal models is that the identification of the responsible genes will be straightforward, although time-consuming. It has been possible with MHC class II genes and will be possible with other genes controlling arthritis in animal models. There is, however, no space to describe these procedures. In short, there are three steps to reach the goal: isolation of loci by backcross breeding using speed congenic techniques and the identification of subphenotypes associated with the disease, identification and sequence analysis of candidate genes, and establishment of transgenic mice to confirm that the gene controls the described trait. This is a process that will consume time and considerable effort. During this work we will, however, be able to learn a great deal of the role of the genes we are searching for. The difficulties described that operate in the control of RA in humans will most likely also be seen in the genetic control of animal models but it may in this case be possible to more or less neutralize them. A major obstacle will be the low penetrance of each gene. It is likely that each trait, or subtrait, will be controlled by more than one gene. These could be clustered but can also interact over distance. Thus, it is apparent that we need to isolate sets of genes rather than single genes. We expect neither the animal models, nor RA, to be controlled by a few genes. Rather, it is more likely that sets of genes will vary between each animal cross, or each family with RA. Gene regions like MHC, which seem to have a major importance in most settings, might not generally reflect how genes will control arthritis. Although similar types of gene regions could be found, such as regions involving cytokine genes, apoptosis genes, genes controlling immune cell costimulation or the NK cell receptor region, it is also likely that the genetic heterogeneity is more pronounced. Thus, the most important use of animal models will probably be to control the genetic background to understand the major pathways leading to arthritis, rather than isolating specific genes corresponding to human RA.


30

Acknowledgments The work was supported by grants from the Swedish Medical Research Council, the Swed¨ sterlunds ish Rheumatism Association, the King Gustaf V’s 80-year fund, and the Kock and O foundations and the European Union (BMH4-CT97-2522 and ERBBIO4CT960562).

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Cannon GW, Woods ML, Clayton F, Griffiths MM: Induction of arthritis in DA rats by incomplete Freund’s adjuvant. J Rheumatol 1993;20:7–11. Lorentzen JC, Glaser A, Jacobsson L, Galli J, Fakhrai-Rad H, Klareskog L, Luthman H: Identification of rat susceptibility loci for adjuvant-oil induced arthritis. Proc Natl Acad Sci USA 1998;95: 6383–6387. Forslind K, Eberhardt K, Jonsson A, Saxne T: Increased serum concentrations of cartilage oligomeric matrix protein. A prognostic marker in early rheumatoid arthritis. Br J Rheumatol 1992;31:593– 598. Wordsworth P, Pile K, Buckely J, Lanchbury J, Ollier B, Lathrop M, Bell J: HLA heterozygosity contributes to susceptibility to rheumatoid arthritis. Am J Hum Genet 1992;51:585–591. Holmdahl R, Vingsbo C, Hedrich H, Karlsson M, Kvick C, Goldschmidt TJ, Gustafsson K: Homologous collagen-induced arthritis in rats and mice is associated with structurally different major histocompatibility complex DQ-like molecules. Eur J Immunol 1992;22:419–424. Carlseń S, Hansson AS, Olsson H, Heinega˚rd D, Holmdahl R: Cartilage oligomeric matrix protein (COMP)-induced arthritis in rats. Clin Exp Immunol 1998;114:477–484. Trentham DE, Townes AS, Kang AH: Autoimmunity to type II collagen: An experimental model of arthritis. J Exp Med 1977;146:857–868. Glant TT, Mikecz K, Arzoumanian A, Poole AR: Proteoglycan-induced arthritis in Balb/c mice. Arthritis Rheum 1987;30:201–212. Cremer MA, Ye XJ, Terato K, Owens SW, Seyer JM, Kang AH: Type XI collagen-induced arthritis in the Lewis rat. Characterization of cellular and humoral immune responses to native types XI, V and II collagen and constituent alpha-chains. J Immunol 1994;153:824–832. Holmdahl R, Carlsen S, Mikulowska A, Vestberg M, Brunsberg U, Hansson A-S, Sundvall M, Jansson L, Pettersson U: Genetic analysis of murine models for rheumatoid arthritis; in Adolpho KW (ed): Human Genome Methods. New York, CRC Press, 1998, pp 215–238. Cremer MA, Stuart JM, Townes AS, Kang AH: Collagen-induced polyarthritis in rats: A study of native type II collagen for adjuvant activity. J Immunol 1980;124:2912–2918. Holmdahl R, Kvick C: Vaccination and genetic experiments demonstrate that adjuvant oil induced arthritis and homologous type II collagen induced arthritis in the same rat strain are different diseases. Clin Exp Immunol 1992;88:96–100. Goldschmidt TJ, Holmdahl R: Anti-T cell receptor antibody treatment of rats with established autologous collagen-induced arthritis. Suppression of arthritis without reduction of anti-type II collagen autoantibody levels. Eur J Immunol 1991;21:1327–1330. Svensson L, Jirholt J, Holmdahl R, Jansson L: B cell-deficient mice do not develop type II collageninduced arthritis (CIA). Clin Exp Immunol 1998;111:521–526. ˚ , Vestberg M, Holmdahl R: Collagen-induced arthritis development requires Corthay A, Johansson A alpha beta T cells but not gamma delta T cells: Studies with T cell-deficient (TCR mutant) mice. Int Immunol 1999;11:1065–1073. Stuart JM, Cremer MA, Townes AS, Kang AH: Type II collagen induced arthritis in rats. Passive transfer with serum and evidence that IgG anticollagen antibodies can cause arthritis. J Exp Med 1982;155:1–16. Stuart JM, Dixon FJ: Serum transfer of collagen induced arthritis in mice. J Exp Med 1983;158: 378–392. Holmdahl R, Jansson L, Larsson E, Rubin K, Klareskog L: Homologous type II collagen induces chronic and progressive arthritis in mice. Arthritis Rheum 1986;29:106–113. Wooley PH, Luthra HS, Stuart JM, David CS: Type II collagen-induced arthritis in mice. I. Major histocompatibility complex (I region) linkage and antibody correlates. J Exp Med 1981;154:688–700. Griffiths M: Immunogenetics of collagen-induced arthritis in rats. Int Rev Immunol 1988;4:1–15. Vestberg M, Brunsberg U, Bergsteinsdottir K, Karlsson M, Gustafsson K, Wedekind D, Hedrich H, Holmdahl R: Limited polymorphism in the first domain of the rat MHC class II RT1-D molecule. Immunogenetics 1998;48:344–349. Remmers EF, Longman RE, Du Y, O’Hare A, Cannon GW, Griffiths MM, Wilder RL: A genome scan localizes five non-MHC loci controlling collagen-induced arthritis in rats. Nat Genet 1996;14: 82–85.


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Gulko PS, Kawahito Y, Remmers EF, Reese VR, Wang J, Dracheva SV, Ge L, Longman RE, Shepard JS, Cannon GW, Sawitzke AD, Wilder RL, Griffiths MM: Identification of a new nonmajor histocompatibility complex genetic locus on chromosome 2 that controls disease severity in collageninduced arthritis in rats. Arthritis Rheum 1998;41:2122–2131. Jacob HJ, Pettersson A, Wilson D, Mao Y, Lernmark A, Lander ES: Genetic dissection of autoimmune type I diabetes in the BB rat. Nat Genet 1992;2:56–60. Pettersson A, Wilson D, Daniels T, Tobin S, Jacob HJ, Lander ES, Lernmark A: Thyroiditis in the BB rat is associated with lymphopenia but occurs independently of diabetes. J Autoimmun 1995; 8:493–505. Courtenay JS, Dallman MJ, Dayan AD, Martin A, Mosedal B: Immunization against heterologous type II collagen induces arthritis in mice. Nature 1980;283:666–667. Holmdahl R, Jansson L, Andersson M, Larsson E: Immunogenetics of type II collagen autoimmunity and susceptibility to collagen arthritis. Immunology 1988;65:305–310. ¨ hrlund-Richter L, Pettersson S, Mattsson Brunsberg U, Gustafsson K, Jansson L, Michae¨lsson E, A R, Holmdahl R: Expression of a transgenic class II Ab gene confers susceptibility to collageninduced arthritis. Eur J Immunol 1994;24:1698–1702. Michae¨lsson E, Andersson M, Engstro¨m A, Holmdahl R: Identification of an immunodominant type-II collagen peptide recognized by T cells in H-2q mice: Self tolerance at the level of determinant selection. Eur J Immunol 1992;22:1819–1825. Brand DD, Myers LK, Terato K, Whittington KB, Stuart JM, Kang AH, Rosloniec EF: Characterization of the T cell determinants in the induction of autoimmune arthritis by bovine alpha 1(II)CB11 in H-2q mice. J Immunol 1994;152:3088–3097. Corthay A, Ba¨cklund J, Broddefalk J, Michae¨lsson E, Goldschmidt TJ, Kihlberg J, Holmdahl R: Epitope glycosylation plays a critical role for T cell recognition of type II collagen in collageninduced arthritis. Eur J Immunol 1998;28:2580–2590. Fugger L, Rothbard JB, Sonderstrup-McDevitt G: Specificity of an HLA-DRB1*0401-restricted T cell response to type II collagen. Eur J Immunol 1996;26:928–933. Rosloniec EF, Brand DD, Myers LK, Whittington KB, Gumanovskaya M, Zaller DM, Woods A, Altmann DM, Stuart JM, Kang AH: An HLA-DR1 transgene confers susceptibility to collageninduced arthritis elicited with human type II collagen. J Exp Med 1997;185:1113–1122. Andersson EC, Hansen BE, Jacobsen H, Madsen LS, Andersen CB, Engberg J, Rothbard JB, So¨nderstrup-McDevitt G, Malmstro¨m V, Holmdahl R, Svejgaard A, Fugger L: Definition of MHC and T cell receptor contacts in the HLA-DR4 restricted immunodominant epitope in type II collagen and characterization of collagen-induced arthritis in HLA-DR4 and human CD4 transgenic mice. Proc Natl Acad Sci USA 1998;95:7574–7579. Rosloniec EF, Brand DD, Myers LK, Esaki Y, Whittington KB, Zaller DM, Woods A, Stuart JM, Kang AH: Induction of autoimmune arthritis in HLA-DR4 (DRB1*0401) transgenic mice by immunization with human and bovine type II collagen. J Immunol 1998;160:2573–2578. Holmdahl R, Andersson EC, Andersen CB, Svejgaard A, Fugger L: Transgenic mouse models of rheumatoid arthritis. Immunol Rev 1999;169:161–173. Watson WC, Townes AS: Genetic susceptibility to murine collagen II autoimmune arthritis. Proposed relationship to the IgG2 autoantibody subclass response, complement C5, major histocompatibility complex (MHC) and non-MHC loci. J Exp Med 1985;162:1878–1891. Spinella DG, Jeffers JR, Reife RA, Stuart JM: The role of C5 and T-cell receptor Vb genes in susceptibility to collagen induced arthritis. Immunogenetics 1991;34:23–27. Mori L, de Libero G: Genetic control of susceptibility to collagen-induced arthritis in T cell receptor b-chain transgenic mice. Arthritis Rheum 1998;41:256–262. Andersson M, Goldschmidt TJ, Michae¨lsson E, Larsson A, Holmdahl R: T cell receptor Vb haplotype and complement C5 play no significant role for the resistance to collagen induced arthritis in the SWR mouse. Immunology 1991;73:191–196. McIndoe RA, Bohlman B, Chi E, Schuster E, Lindhardt M, Hood L: Localization of non-Mhc collagen-induced arthritis susceptibility loci in DBA/1j mice. Proc Natl Acad Sci USA 1999;96:2210–2214. Jansson L, Holmdahl R: Genes on the X chromosome affect development of collagen-induced arthritis in mice. Clin Exp Immunol 1993;94:459–465.


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Jansson L, Holmdahl R: The Y chromosome-linked ‘autoimmune accelerating’ yaa gene suppresses collagen-induced arthritis. Eur J Immunol 1994;24:1213–1217. Holmdahl R, Jansson L, Andersson M, Jonsson R: Genetic, hormonal and behavioral influence on spontaneously developing arthritis in normal mice. Clin Exp Immunol 1992;88:467–472. Corthay A, Hansson A, Holmdahl R: Spontaneous arthritis in DBA/1 mouse is characterized by enthesopathy that is not dependent on T cells. Arthritis Rheum Submitted. Weinreich S, Capkova J, Hoebe-Hewryk B, Boog C, Ivanyi P: Grouped caging predisposes male mice to ankylosing enthesopathy. Ann Rheum Dis 1996;55:645–647. Jirholt J, Cook A, Emahazion T, Sundvall M, Jansson L, Nordquist N, Pettersson U, Holmdahl R: Genetic linkage analysis of collagen-induced arthritis in the mouse. Eur J Immunol 1998;28: 3321–3328. Yang HT, Jirholt J, Svensson L, Sundvall M, Jansson L, Pettersson U, Holmdahl R: Identification of genes controlling collagen-induced arthritis in mice: Striking homology with susceptibility loci previously identified in the rat. J Immunol 1999;163:2916–2921. Wilder RL, Griffiths MM, Remmers EF, Cannon GW, Caspi RR, Kawahito Y, Gulko PS, Longman RE, Dracheva SV, Du Y, Sun SH, Wang J, Shepard JS, Joe B, Ge L, Chen S, Chang L, Hoffman J, Silver PB, Reese VR: Localization in rats of genetic loci regulating susceptibility to experimental erosive arthritis and related autoimmune diseases. Transplant Proc 1999;31:1585–1588. Dracheva SV, Remmers EF, Gulko PS, Kawahito Y, Longman RE, Reese VR, Cannon GW, Griffiths MM, Wilder RL: Identification of a new quantitative trait locus on chromosome 7 controlling disease severity of collagen-induced arthritis in rats. Immunogenetics 1999;49:787–791. Blake JA, Richardson JE, Davisson MT, Eppig JT: The Mouse Genome Database (MGD): Genetic and genomic information about the laboratory mouse. Nucleic Acids Res 1999;27:95–98.

Rikard Holmdahl, Medical Inflammation Research, So¨lvegatan 19, Lund University, S–223 62 Lund (Sweden) Tel. +46 46 222 4607, Fax +46 46 222 3110, E-Mail [email protected], Homepage http://net.inflam.lu.se/


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Structural Basis for the HLA-DR Association of Rheumatoid Arthritis Francesco Sinigaglia a, Zoltan A. Nagy b a b

Roche Milano Ricerche, Milan, Italy; Genome Pharmaceuticals Corporation, Martinsried/Munich, Germany

HLA class II proteins bind peptide fragments derived from protein antigens and display them on the surface of antigen-presenting cells for interaction with the antigen receptors of helper T lymphocytes. The HLA class II loci encoding these proteins are extremely polymorphic. The allelic variations between HLA class II molecules account for a differential ability to bind antigenic peptides, ‘resulting in individualization’ of immune responses. Polymorphism of HLA class II genes also seems to play a major role in autoimmunity. HLA typing of large groups of patients with various autoimmune diseases has revealed that certain HLA alleles occur at significantly higher frequencies in patients than in the general population. Susceptibility to rheumatoid arthritis (RA) is specifically associated with the class II MHC alleles HLA-DRB1*0101, 0401, 0404 and 0405 in several ethnic groups [1–4]. Altogether, ?90% of rheumatoid factor-positive RA patients carry one of these susceptibility alleles [5]. However, a discordance of D50% among monozygotic twins also indicates a strong influence of environmental factors on the disease. The effect of DRB1 locus on RA is manifested in different ways: first, the disease association shows ethnic preference for one or the other allele [3, 4, 6], second, DRB1*0401 is associated with more severe forms of RA than other alleles [7], and third, homozygosity for a susceptibility allele or combinations of two susceptibility alleles is associated with more severe chronic forms of the disease than a single allele or with a juvenile disease onset [8, 9]. The latter findings indicate that the DRB1 locus can control both initiation and progression of the disease. In addition to disease susceptibility, protection from RA also appears to be DRB1-associated: certain

alleles, such as 0402, 0701, 11011 and 1501, occur at a decreased frequency in RA patients, and heterozygosity with a susceptibility allele leads to less severe arthritis than the presence of two nonprotective alleles [10, 11]. To translate this multifaceted genetic linkage into actual disease mechanisms has been one of the most challenging tasks for autoimmunity research, and although many aspects have been clarified, several questions still wait to be answered. It was recognized more than a decade ago that the DRb chains encoded by RA-linked DRB1 alleles, although exhibiting polymorphic differences, all share a stretch of identical or almost identical amino acid sequence at positions 67–74, known as the ‘shared epitope’ [5, 12]. Since autoimmunity has been regarded to be central in the pathogenesis of RA, it was hypothesized that the shared epitope could impose disease linkage on the respective DR molecules by at least two different mechanisms: first, by selecting the relevant autoantigenic peptides for presentation, and second, by selecting the appropriate autoreactive T cell specificities during ontogeny. Indeed, the three-dimensional structure of DR molecules [13–16] has revealed that the shared epitope is located in the center of the a-helix flanking one side of the peptide binding groove, i.e. ‘strategically positioned’ to be able to interact with both peptide and T cell receptor (TCR). Here we will summarize pertinent information on HLA-DR-peptide interactions, discuss the role of the DRb67–74 region in determining peptide binding specificity, and outline models to explain how the latter may account for the MHC-association of RA. The practical implications of peptide-DR interactions for immunotherapy will also be covered in brief.

Peptide-HLA-DR Molecule Interaction HLA-DR molecules are heterodimers consisting of a noncovalently associated a- and b-chain, both inserted into the cell membrane. The three-dimensional structures of several DR molecules have been determined by X-ray crystallography [13–16]. The class II peptide binding site is formed by the membrane-distal domains of both class II chains, each contributing one a-helix and four b-strands. The b-sheet floors and helical walls define a groove of suitable dimensions for occupancy by 9 amino acid residues. The class II cleft is open at both ends, and consequently, it allows peptides to extend, and enables the binding of peptides with different lengths, typically consisting of 12–24 residues. Class II molecules enter into two types of interaction with peptides. First, conserved side chains of the binding site form a network of hydrogen bonds

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with main chain atoms of the peptide [14]. This network is thus independent of the peptide sequence. The interacting conserved residues are distributed along the binding groove so that the peptide main chain is kept in close contact with the MHC cleft and in an extended conformation. Furthermore, the hydrogen bonds induce a torsion into the peptide main chain, determining thereby the angle of peptide side chains to point toward the corresponding binding pockets. Second, side chains of MHC proteins make contact with side chains of the peptide. Some of these interactions can increase the overall binding affinity [17–19] whereas others reduce it [20–22]. These peptide sequence-dependent interactions are due to the irregular surface of the MHC cleft. Namely, MHC side chains form pockets or ridges in the binding site, causing strong preferences for interaction with particular amino acid side chains. Most pockets in the MHC groove contain polymorphic residues resulting in distinct size and physical-chemical characteristics of pockets in different MHC allotypes. Certain side chains of the ligands interact with the pockets and increase the overall binding affinity (anchor residues), whereas others interfere with pocket residues and reduce binding (inhibitory residues). Thus, the side chain-side chain interactions determine the peptide sequence specificity or ‘peptide-binding motifs’ for each MHC protein.

Class II Binding Motifs The interaction of peptide side chains with pockets of the MHC cleft imposes sequence requirements on the peptides that can bind. These requirements are summarized in peptide-binding motifs [17, 23]. A breakthrough for the analysis of MHC-binding motifs was the characterization of large, MHCselected peptide pools, which permitted to establish rules for peptide binding to MHC molecules [24, 25]. For class II HLA-DR molecules, motifs were identified by the analysis of large peptide pools selected from M13 bacteriophage peptide display libraries [19, 22, 25] comprised of several million random peptides. Sequence analysis of the DNA encoding the displayed peptides led to the identification of class II anchors [reviewed in 26]. The results were confirmed by pool sequencing of peptides eluted from class II molecules. The class II motifs generally consist of 4 anchor positions at fixed distances reflecting the architecture of the DR groove, which has 4 major pockets to accept side chains at relative positions 1, 4, 6 and 9 of the peptide [14]. An important finding was the identification of conserved anchor residues, i.e. anchors found in each of the HLA-DRselected peptide pools as well as allele-specific anchor residues. For example,

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most of the DRB1*0101-, DRB1*0401- and DRB1*1101-selected peptide pools were found to have aromatic and aliphatic amino acid residues at anchor position 1 and 4, respectively, while strong allele-specific amino acid preferences were identified at position 6: Ala and Gly for DRB1*0101, Ser and Thr for DRB1*0401, and Arg and Lys for DRB1*1101 [19]. These results provided a molecular basis for both the promiscuity and the specificity of peptide recognition by HLA-DR molecules. Thus, peptides with conserved anchor residues at positions 1 and 4, and small Ala residues at position 6 should bind, at least with intermediate affinity, to several HLA-DR alleles. Alternatively, DRB1*1101 ligands with a conserved anchor at position 1 and a large, positively charged Arg at position 6 will not bind to DRB1*0101 and DRB1*0401, because Arg will not be accommodated by the pocket 6 of the latter two molecules [19]. Increasingly complex HLA class II motifs were identified by varying the conditions used to elute bacteriophage from the DR molecules [27]. For example, a ‘low-pH’ wash step prior to elution increased the stringency of peptide selection, and resulted in the identification of secondary anchors at positions 2, 3 and 7 [27]. Based on these findings it was possible to design short peptides in which 6 of the 7 residues were anchors, and thus bound very tightly to DR molecules. Such peptides provided the basis for the design of nonpeptidic MHC blockers [27, 28].

Quantitative Matrices and the Computational Prediction of HLA-DR-Peptide Binding X-ray crystallographic and functional studies have indicated that different peptides bound to HLA-DR molecules essentially have an identical conformation [14, 16]. This observation implies that each amino acid residue in a peptide sequence contributes to the affinity of binding almost independently of the neighboring residues [22, 27, 29, 30]. It was thus possible to characterize HLADR pocket specificities (‘pocket profiles’) by substituting the corresponding peptide position with all natural amino acids and quantifying their effects on binding [22]. The sum of all pocket profiles of a given HLA-DR allele was then defined as a ‘quantitative matrix’. Since the pocket profiles turned out to be nearly independent of the remaining DR cleft, a small database of profiles was sufficient to generate a large number of binding matrices predicting the binding specificity of the majority of allelic HLA-DR variants. These matrices were incorporated into a software program (TEPITOPE) capable of predicting T cell epitopes from natural protein sequences binding to any desired HLA-DR molecule (see fig. 1) [31, 32].


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Fig. 1. Prediction of selective peptides in human CII with TEPITOPE. The predicted region corresponds to a dominant T cell epitope in bovine collagen identified in DRB1*0401 transgenic mice [60; from 31, with permission].

Peptide-Binding Specificities and RA-Associated HLA Class II Molecules In an attempt to analyze the role of DRb-67–74 ‘shared epitope’ on HLA-DR peptide-binding specificity, we compared the pocket specificity profiles of RA-associated DR4 subtypes with profiles of nonassociated DR molecules [33]. Striking differences, especially in the specificity of pocket 4, were identified between DR4 subtypes which are associated with RA and those which are not. For example, peptides with negatively charged residues at position 4 bound to RA-associated DRB1*0401 or DRB1*0404 molecules, but not to the nonassociated DRB1*0402 molecule; the reverse was true for peptides with positively charged residues at position 4 (see fig. 2). Sitedirected mutagenesis demonstrated that the charge of the DRb71 residue (positive in DRB1*0401 and negative in DRB1*0402) was responsible for most of these effects [33]. Similar conclusions were reached by the pooled peptide sequencing technique [34] and by selected single-substitution experiments [35]. Altogether, these results demonstrated a striking correlation between binding specificity and disease association, thus supporting the hypothesis that selective binding of autoantigenic peptides is the mechanism underlying HLA association in RA.

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Fig. 2. Computer model of the DRB1*0402 molecule with bound peptide. DRb residues 67, 70 and 71 distinguishing the DRB1*0402 molecule from the RA-linked DR4 molecules are shown in yellow [from 33, with permission].

How to Translate the HLA-DR Association of RA into Disease Mechanisms? The Concept of Selective Autoantigen Presentation The role of the b67–74 region in peptide binding has been largely clarified by the studies of Hammer et al. [33], as summarized above. It is now clear that the basic residue (Lys or Arg) at DRb-p71 of RA susceptibility-associated DR molecules confers a charge selectivity of peptide binding by favoring acidic and disfavoring basic residues at peptide p4. Importantly, the DRB1*0402 molecule, which is associated with protection rather than susceptibility, has the acidic residues Asp-b70 and Glu-b71, and exhibits the opposite charge preference in peptide binding. These data suggest that the association of RA


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with certain DRB1 alleles could be explained by a pathogenic T cell response to autoantigenic peptides which have a negative charge at p4, and are, therefore, presented selectively by DR molecules encoded by the susceptibility alleles. Conversely, the DRb chains with a negative charge at p70–71 cannot present the pathogenic peptides, and thus mediate protection from the disease. Although the autoantigens involved in RA remain somewhat elusive, several joint cartilage proteins have peptide sequences, which can selectively bind to RA-associated DR molecules due to an acidic residue at p4 [33]. One of these peptides, type II collagen (CII) 1168–1180, has been crystallized together with an RA-associated DR molecule (DRA*0101, DRB1*0401), and the interaction between Lys DRb71 and Asp at peptide p4 via a salt bridge has been proven [15]. Interestingly, the opposite charge preference of DRB1*0402 has also been linked to the presentation of a pathogenic self peptide involved in the 0402-associated autoimmune disease, pemphigus vulgaris [36]. Additional aspects of the MHC association of RA are also readily explained by selective autoantigen presentation. For example, the finding that DRB1*0101, 0405 and 0401/0404, respectively, dominate the RA association in distinct ethnic groups [3, 4, 6] can reflect the presentation of different autoantigenic peptides by the respective DR allotypes. Namely, DRB1*0101 differs from 0401 at 9 amino acid positions, and 0405 from 0401 at 1 important position (b57) of the DRb1 domain outside the 67–74 region [37], and these substitutions cause significant changes in the fine specificity of peptide binding [25, 38], although the preference for a negative charge at p4 remains unaltered. It is also possible that the disease is triggered by different pathogens in the distinct ethnic groups, which results in cross-reactivity to different self peptides. Alternatively, the ethnic-related allele preference can simply reflect the fact that the RA-associated alleles are usually also prevalent in the respective healthy populations. The influence of a particular allele on disease severity [7] can indicate that certain RA-associated DR molecules have a higher affinity for the pathogenic self peptides than others, or present more pathogenic peptides than the others. That two RA-associated alleles are associated with more severe disease than one [8, 9] appears to be a gene dosage effect, which can be explained by the presentation of larger amounts of autoantigenic peptide in the presence of two appropriate presenting molecules. The phenomenon of protective alleles [10, 11] is explained by the inability of the protecting DR molecules to present the autoantigenic peptides, and the disease-mitigating effect in susceptible/protecting DRB1 heterozygotes can also be a gene dosage effect with the underlying mechanism outlined above. The remaining major question of the selective presentation concept is the following: why are peptides with a negative charge in the middle necessary for the development of RA? The peptide-binding motifs of RA-associated

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DR molecules alone cannot provide an answer. Namely, not only do these molecules select for a negative and against a positive charge, but their p4 pocket also readily accepts uncharged hydrophobic residues (Met, Val, Leu) [19, 25]. Altogether, the binding motif of RA-associated DR molecules with hydrophobic anchors at p1, 4 and 7, and a small residue at p6 [19, 25] is rather ‘generic’, and therefore, in any protein, the potential binding frames having this motif would outnumber those with a negative charge at p4. Thus autoantigenic peptides without charge would occur with a higher probability than those with a negative charge. This point is also relevant to the ‘spreading’ of autoimmunity from one epitope to another, or from one autoantigen to another [39, 40], in which case later stages of the disease would be less and less likely to be driven by peptides with charge at p4, and consequently, the DRassociation of late-occurring pathological forms [7, 8] could not be explained by selective presentation. Thus, although a T cell response to self peptides selectively presented by RA-associated DR molecules appears to be a feasible explanation for MHC-association of the disease, additional mechanisms are required to explain how such a response arises, and how it is maintained in the face of a possible competition with excess autoantigenic peptides that can bind in an unselective manner. Some immunological mechanisms that could account for a sustained response to negatively charged peptides include the following. (1) Processing of the relevant autoantigen(s) preferentially yields peptides with a negative charge. At first sight, this proposition may appear somewhat far fetched. However, the sequence of CII [41], for example, reveals a number of acidic residues, often in clusters, separated by long stretches of uncharged residues, an arrangement which would be compatible with differential processing. Although the acidic residues being in the middle of peptides are not likely to influence proteolytic cleavage directly, the basic residues often coclustered with them could serve as protease recognition sites. Alternatively, the negative charge may play a role in the unfolding of the protein during processing, and thereby making the respective sequences accessible for proteases. This mechanism thus implies that processing itself is the major limitation to epitope spreading. (2) Autoimmunity to a self peptide with negative charge is induced by a pathogen via cross-reactivity. In view of the importance of environmental factors in the disease, the idea of cross-reactive induction by pathogenic microorganisms has long been considered [42]. This hypothesis is attractive because the low probability of chance cross-reactivity reduces the number of relevant self peptides to a minimum. Thus, even in the presence of other self peptides, the autoreactive T cell response will only be directed to the cross-reactive ones. A well-documented example for this mechanism is chronic


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Lyme arthritis that is induced by Borrelia burgdorferi and maintained by a cross-reactive anti-self response to LFA-1 [43]. In this instance, the disease is triggered by the pathogen and maintained by reactivity to virtually a single self peptide, a scenario in contrast to epitope spreading. It is also conceivable that chronic infections can continuously stimulate a cross-reactive response, i.e. long-persisting pathogens can be responsible for both initiation and progression of the disease, and the latter would thus maintain the same MHC association throughout. (3) Maintenance of the initiating response by immunodominance. Under this hypothesis, the initiating T cell response to negatively charged peptide(s), which may be caused by differential processing or cross-reactivity (propositions 1 or 2), will remain dominant throughout the disease due to immunological mechanisms. For example, mechanisms that ensure preferential stimulation of memory T cells upon antigenic challenge [44–46] could account for a dominance of the initial pathogenic response. (4) Interaction of the autoreactive TCR with the peptide-MHC complex requires a negative charge in the peptide. The question here is the following: how can an acidic side chain buried in the p4 pocket of the binding site, and thus not accessible for the TCR, influence the TCR-contacting surface of the peptide-MHC complex? Crystallographic studies provide a clue as to how this may happen [14–16]. In the DR4/CII crystal structure [15], Lys-b71 forms hydrogen bonds with both the Asp-p4 side chain and the main chain carbonyl oxygen of p5. The Lys-b71 residue, therefore, points into the binding site, whereas Gln-b70 points out of the groove toward the T cell. In the DR1/HA complex [14], Arg-b71 has the same interactions as Lys-b71 in DR4, but Glnb70 points down into the pocket to contact the side chain of p4-Gln. Thus, the positively charged b71 side chain is involved in peptide binding in both cases, and is unaccessible for T cell recognition. However, the side chain position of the neighboring b70 residue differs depending on whether the peptide p4 is occupied by a negatively charged or an uncharged residue: it points up in the former and down in the latter case, thus causing a change in the interface toward the TCR. The side chain of p4 can, in addition, alter the location of the b-chain a-helix. In the DR3-CLIP structure [16], the small side chain of Ala-p4 leads to a somewhat collapsed p4 pocket, and as a consequence, the a-helical residues are moved closer to the peptide. In the DR1/HA and DR4/CII structures [14, 15], p4 is occupied by the larger Gln and Asp side chains, respectively, which causes a relative expansion of the p4 pocket, and a consequent dislocation of the a-helical region. Interestingly, the b65–74 region is not exactly superimposable even in the latter two crystal structures. Altogether, the p4 side chain has an indirect but rather substantial effect on the shape of the

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peptide-MHC complex, and therefore, possibly also on the selection of complementary TCR surfaces. All four propositions above are experimentally testable, and since they are not mutually exclusive, the involvement of one or more of them in the disease process can be proven. The DRb-Chain as a Peptide Donor An alternative hypothesis to explain MHC association of RA has been put forward by David and colleagues [47]. Their concept is largely based on experimental data from collagen-induced arthritis (CIA) in human class II MHC-transgenic mice as a model for RA [47–49]. These authors have observed that HLA-DQ8 transgenic mice readily develop CIA in the absence of murine class II molecules, indicating that DQ8 can present arthritogenic peptides of collagen [50]. Since DQ8 is in linkage disequilibrium with most RA-associated DR4 alleles, this finding has raised the possibility that the actual antigenpresenting molecule in RA may be DQ, and thus the mechanism by which DR molecules determine disease linkage is different from antigen presentation. To investigate the latter, the authors produced synthetic peptides from the third hypervariable region (HV3) of DRb-chains, encompassing the 67–74 region, and immunized DQ8 transgenic mice with these peptides [48]. The results have demonstrated that DQ8 mice can mount a T cell response to all peptide sequences derived from protective DR molecules, but they fail to respond to peptides from RA susceptibility-associated DR molecules. Thus the pattern of responsiveness correlates with disease linkage. Subsequently, they have shown that coimmunization of DQ8 mice with collagen and a peptide from the protective DRB1*0402 molecule results in reduced severity and decreased incidence of disease [49]. The DRB1*0402 peptide-mediated protection has been shown to be due to a peptide-specific, noninflammatory T cell response that downregulates the collagen-specific proinflammatory response [49]. Based on these findings, the authors have outlined the following scenario to explain the role of MHC class II in the pathogenesis of RA. Several DQ molecules, including DQ8, can present arthritogenic antigens, and thus predispose individuals carrying these molecules to RA. In individuals expressing protective DR molecules, HV3 peptides will arise by processing; these peptides will bind to the predisposing DQ molecules and induce a protective T cell response. In individuals carrying RA susceptibility DR types, the HV3 peptides resulting from processing cannot bind to DQ, and the lack of a protective response will allow the disease to develop. Basically, this hypothesis is also a selective presentation concept, but in contrast to the previous one, it ascribes selectivity to DQ molecules, and DR


45

molecules serve as donors of the peptide sequences to be bound in a selective manner. Since little is known at present about the rules of peptide binding to DQ molecules [51, 52], the structural basis of their proposed selectivity remains unexplained. Under this hypothesis it appears almost mandatory that the relevant autoantigen in RA be CII, and that the protective effect be dominant over the disease. For these assumptions there is no compelling evidence at present. Furthermore, Rosloniec et al. [53] have shown that DR transgenic mice can also develop CIA. Nevertheless, the experimental support of the hypothesis leaves little doubt that DQ molecules can present CII, and thus, that the DQ locus could control susceptibility to arthritis. Certain forms of arthritis, for example, juvenile chronic arthritis [54] and perhaps also Felty’s syndrome [55], are indeed DQ-associated. The selective presentation and the peptide donor hypotheses are often viewed as mutually exclusive, and this is certainly the case as long as RA is considered to be a single disease. However, it is possible that different forms of the disease exist with different pathomechanisms, and thus, a single model may turn out to be insufficient to describe RA as a whole.

Prospects for Immunointervention Detailed knowledge of HLA class II peptide-binding specificity might have interesting applications for immunointervention in autoimmune diseases. One strategy ought to be directed toward the development of MHC-specific antagonists that block the peptide-binding site of disease-linked class II MHC molecules. The principle is based on the mode of peptide binding by HLADR molecules, which requires specific side chains at anchor positions but permits a large variety of side chains at nonanchor positions. Thus, the replacement of autoantigenic peptides by a ligand, having the same binding motif but differing at nonanchor positions, should prevent the activation of autoimmune T cells, thereby interrupting the disease process. The advantage of this strategy is that it is applicable without specific knowledge of the diseaseinducing autoantigen(s). Since different class II molecules exhibit different peptide-binding specificities, the antagonist is expected to be selective, i.e. to bind to the disease-linked class II molecules only. Thus, a considerable part of the host’s antigen-presenting capacity would remain intact, and consequently patients would not be severely immunocompromised. The pharmacological utility of this mechanism was explored a decade ago [56], and the principle was proven in animal models of autoimmune disease [57, 58]. However, peptidebased class II MHC antagonists were not sufficiently active in vivo [59], probably because of inherent pharmacological limitations of peptides, such

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as protease sensitivity, poor bioavailability and pharmacokinetics. To overcome these difficulties, peptidomimetic ligands were designed for RA-associated HLA-DR molecules [28]. The mimetic design has been based on a heptapeptide that can bind to all RA-associated HLA-DR molecules with high affinity. A computer model was then constructed by replacing the hemagglutinin 307–319 peptides in the DR1 crystal structure [14] with the lead peptide. Nonpeptidic substituents were selected on the basis of their capability to replace one or two amino acids of the lead peptide in the model. A number of substitutions found suitable by computer-aided design were then synthesized and incorporated into the lead peptide. Specific combinations of such mimetic blocks resulted in cathepsin-resistant compounds that were several hundred to thousand times more potent inhibitors of antigen presentation than the best peptidic antagonists [28]. Compounds of this type represent promising candidates for therapeutic use as MHC-selective antagonists of antigen presentation in the treatment of RA as well as other MHC class II-associated autoimmune diseases. The peptide donor concept of David and colleagues [50] can also have therapeutic implications. Namely, the HV3 peptides from RA-protective DR molecules appear to induce a T cell response that downregulates the proinflammatory response causing RA [49]. It is thus conceivable that such HV3 peptides could be used as a vaccine to prevent the disease.

Concluding Remarks A striking characteristic of certain autoimmune diseases is the increased frequency of specific HLA class II alleles in affected individuals. Moreover, as demonstrated in RA, alleles positively associated with the disease share unique amino acid residues in their hypervariable regions that confer a capability of selective peptide binding. Although there is much to be learned about the pathogenesis of autoimmune diseases, it is generally believed that diseaseassociated HLA class II molecules can bind and present autoantigenic peptides to T cells, and thus, selective antigen presentation could be a feasible mechanism for the HLA class II association of disease. The structural definition of HLA class II molecules combined with new computer programs that predict amino acid sequences of self protein binding to HLA molecules has led to effective ways of identifying peptides selective for disease-associated molecules. Such peptide sequences may represent a step forward in our understanding of autoimmune disease.


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6 7

8 9 10 11 12

13 14

15

16 17 18

19 20

21

22

McMichael AJ, Sasazuki T, McDevitt HO, Payne RO: Increased frequency of HLA-Cw3 and HLADw4 in rheumatoid arthritis. Arthritis Rheum 1977;20:1037–1042. Stastny P: Association of the B-cell alloantigen DRw4 with rheumatoid arthritis. N Engl J Med 1978;298:869–871. Ohta N, Nishimura YK, Tanimoto K, Horiuchi Y, Abe C, Shiokawa Y, et al: Association between HLA and Japanese patients with rheumatoid arthritis. Hum Immunol 1982;5:123–132. Schiff B, Mizrachi Y, Orgad S, Yaron M, Gazit E: Association of HLA-Aw31 and HLA-DR1 with adult rheumatoid arthritis. Ann Rheum Dis 1982;41:403–404. Nepom GT, Byers P, Seyfried C, Healey LA, Wilske KR, Stage D, et al: HLA genes associated with rheumatoid arthritis. Identification of susceptibility alleles using specific oligonucleotide probes. Arthritis Rheum 1989;32:15–21. Feldmann M, Brennan FM, Maini RN: Rheumatoid arthritis. Cell 1996;85:307–310. Lanchbury JS, Jaeger EE, Sansom DM, Hall MA, Wordsworth P, Stedeford J, et al: Strong primary selection for the Dw4 subtype of DR4 accounts for the HLA-DQw7 association with Felty’s syndrome. Hum Immunol 1991;32:56–64. Wordsworth P, Pile KD, Buckely JD, Lanchbury JS, Ollier B, Lathrop M, et al: HLA heterozygosity contributes to susceptibility to rheumatoid arthritis. Am J Hum Genet 1992;51:585–591. Nepom BS: The role of the major histocompatibility complex in autoimmunity. Clin Immunol Immunopathol 1993;67:S50–55. Deighton CM, Cavanagh G, Rigby AS, Lloyd HL, Walker DJ: Both inherited HLA-haplotypes are important in the predisposition to rheumatoid arthritis. Br J Rheumatol 1993;32:893–898 Weyand CM, Hicok KC, Conn DL, Goronzy JJ: The influence of HLA-DRB1 genes on disease severity in rheumatoid arthritis. Ann Intern Med 1992;117:801–806. Gregersen PK, Silver J, Winchester RJ: The shared epitope hypothesis. An approach to understanding the molecular genetics of susceptibility to rheumatoid arthritis. Arthritis Rheum 1987;30:1205– 1213. Brown JH, Jardetzky TS, Gorga JC, Stern LJ, Urban RG, Strominger JL, et al: The three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature 1993;364:33–39. Stern LJ, Brown JH, Jardetzky TS, Gorga JC, Urban RG, Strominger JL, et al: Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide. Nature 1994;368:215–221. Dessen A, Lawrence CM, Cupo S, Zaller DM, Wiley DC: X-ray crystal structure of HLA-DR4 (DRA*0101, DRB1*0401) complexed with a peptide from human collagen II. Immunity 1997;7: 473–481. Ghosh P, Amaya M, Mellins E, Wiley DC: The structure of an intermediate in class II MHC maturation: CLIP bound to HLA-DR3. Nature 1995;378:457–462. Sette A, Buus S, Colon S, Smith JA, Miles C, Grey HM: Structural characteristics of an antigen required for its interaction with Ia and recognition by T cells. Nature 1987;328:395–399. Jardetzky TS, Gorga JC, Busch R, Rothbard JB, Strominger JL, Wiley DC: Peptide binding to HLA-DR1: A peptide with most residues substituted to alanine retains MHC binding. EMBO J 1990;9:1797–1804. Hammer J, Valsasnini P, Tolba K, Bolin D, Higelin J, Takacs B, et al: Promiscuous and allelespecific anchors in HLA-DR binding peptides. Cell 1993;74:197–203. Boehncke WH, Takeshita T, Pendleton CD, Houghten RA, Sadegh N, Racioppi S, et al: The importance of dominant negative effects of amino acid side chain substitution in peptide-MHC molecule interactions and T cell recognition. J Immunol 1993;150:331–341. Sette A, Sidney J, Oseroff C, del Guercio M-F, Southwood S, Arrhenius T, et al: HLA DR4w4 binding motifs illustrate the biochemical basis of degeneracy and specificity in peptide-DR interactions. J Immunol 1993;151:3163–3170. Hammer J, Bono E, Gallazzi F, Belunis C, Nagy Z, Sinigaglia F: Precise prediction of major histocompatibility complex class II peptide interaction based on peptide side chain scanning. J Exp Med 1994;180:2353–2358.

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Byrne JA, Butler JL, Cooper MD: Differential activation requirements for virgin and memory T cells. J Immunol 1988;141:3249–3257. Sanders ME, Makgoba MW, June CH, Young HA, Shaw S: Enhanced responsiveness of human memory T cells to CD2 and CD3 receptor-mediated activation. Eur J Immunol 1989;19:803–808. Falcioni F, Shah H, Vidovic D, Morimoto C, Belunis C, Bolin D, et al: Influence of CD26 and integrins on the antigen sensitivity of human memory T cells. Hum Immunol 1996;50:79–90. Nabozny GH, Baisch JM, Cheng S, Cosgrove D, Griffiths MM, Luthra HS, et al: HLA-DQ8 transgenic mice are highly susceptible to collagen-induced arthritis: A novel model for human polyarthritis. J Exp Med 1996;183:27–37. Zanelli E, Krco CJ, Baisch JM, Cheng S, David CS: Immune response of HLA-DQ8 transgenic mice to peptides from the third hypervariable region of HLA-DRB1 correlates with predisposition to rheumatoid arthritis. Proc Natl Acad Sci USA 1996;93:1814–1819. Das P, Bradley DS, Geluk A, Griffiths MM, Luthra HS, David CS: An HLA-DRB1*0402 derived peptide (HV3 65-79) prevents collagen-induced arthritis in HLA-DQ8 transgenic mice. Hum Immunol 1999;60:575–582. Zanelli E, Gonzalez-Gay MA, David CS: Could HLA-DRB1 be the protective locus in rheumatoid arthritis? Immunol Today 1995;16:274–278. Johansen BH, Buus S, Vartdal F, Viken H, Eriksen JA, Thorsby E, et al: Binding of peptides to HLA-DQ molecules: Peptide binding properties of the disease-associated HLA-DQ(alpha 1*0501, beta 1*0201) molecule. Int Immunol 1994;6:453–461. Raddrizzani L, Sturniolo T, Guenot J, Bono E, Gallazzi F, Nagy ZA, et al: Different modes of peptide interaction enable HLA-DQ and HLA-DR molecules to bind diverse peptide repertoires. J Immunol 1997;159:703–711. Rosloniec EF, Brand DD, Myers LK, Whittington KB, Gumanovskaya M, Zaller DM, et al: An HLA-DR1 transgene confers susceptibility to collagen-induced arthritis elicited with human type II collagen. J Exp Med 1997;185:1113–1122. Scholz S, Albert ED: Immunogenetic aspects of juvenile chronic arthritis. Clin Exp Rheumatol 1993;11(suppl 9):S37–S41. Sansom DM, Bidwell JL, Maddison PJ, Campion G, Klouda PT, Bradley BA: HLA DQ alpha and DQ beta restriction fragment length polymorphisms associated with Felty’s syndrome and DR4-positive rheumatoid arthritis. Hum Immunol 1987;19:269–278. Adorini L, Muller S, Cardinaux F, Lehmann PV, Falcioni F, Nagy ZA: In vivo competition between self peptides and foreign antigens in T cell activation. Nature 1988;334:623–625. Sakai K, Zamvil SS, Mitchell DJ, Hodgkinson S, Rothbard JB, Steinman L: Prevention of experimental encephalomyelitis with peptides that block interaction of T cells with major histocompatibility complex proteins. Proc Natl Acad Sci USA 1989;86:9470–9474. Hurtenbach U, Lier E, Adorini L, Nagy ZA: Prevention of autoimmune diabetes in non-obese diabetic mice by treatment with a class II major histocompatibility complex-blocking peptide. J Exp Med 1993;177:1499–1504. Ishioka GY, Adorini L, Guery JC, Gaeta FC, LaFond R, Alexander J, et al: Failure to demonstrate long-lived MHC saturation both in vitro and in vivo. Implications for therapeutic potential of MHC-blocking peptides. J Immunol 1994;152:4310–4319. Fugger L, Rothbard J, Sonderstrup-McDevitt G: Specificity of an HLA-DRB1*0401 restricted T cell response to type II collagen. Eur J Immunol 1996;26:928–933.

Francesco Sinigaglia, Roche Milano Ricerche, Via Olgettina 58, I–20132 Milano (Italy) Tel. +39 02 288 4805, Fax +39 02 215 3203, E-Mail [email protected]

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T Cell Repertoire Formation and Molecular Mimicry in Rheumatoid Arthritis Berent J. Prakken a, Dennis A. Carson b, Salvatore Albani a, c Departments of Pediatrics and b Medicine, University of California San Diego, La Jolla, Calif., USA; c Androclus Therapeutics, Catania, Italy a

During millions of years of evolution, the human immune system has evolved together with microorganisms in the environment, leading to a dynamic and delicate balance of cohabitation. The healthy immune system consists of a complex network of interacting antigen-specific and nonspecific cells, capable of fulfilling a triad of functions, namely recognition of the danger, action and memory. T cells are central in the regulation of the immune system and mediate specific cellular immune responses. A mature T cell recognizes an antigen presented to it as peptide fragments bound to major histocompatibility complex (MHC) molecules on an antigen-presenting cell (APC) in the context of different costimulatory molecules [1, 2]. T cells originate from progenitor cells in the bone marrow, and migrate to the thymus where they differentiate into mature effector T cells, after undergoing positive selection. One of the most intriguing characteristics of the immune system is its specificity; when threatened by a potential dangerous invading microorganism, the immune system mounts a specific immune response tailored to eliminating the challenge, without causing harm to itself. For years it was thought that the immune system primarily achieves this by differentiating between self and nonself. According to this theory, all self-reactive cells are deleted when undergoing thymic selection, resulting in an adult immune system that does not react to any self antigen, and that recognizes and eliminates any nonself structure, such as foreign antigens. However, with the growing knowledge of immunology, this theory has become more and more unsatisfactory.

First, it does not explain why in perfectly healthy individuals autoantibodies and self-reacting T cells can be detected without any symptom of autoimmunity. Second, it also does not explain why the healthy immune system does not react aggressively to the enormous variety of foreign antigens presented to it on the mucosal surfaces of the gastrointestinal and respiratory tract. Third, certain sites of the body, such as the central nervous system, are so-called immunologically privileged sites, where allogeneic or even xenogeneic transplanted tissues are not rejected [3]. Finally, the self/nonself paradigm does not provide an explanation of how the immune system is capable of identifying millions of different peptide sequences from self proteins as being self and thus unharmful, and at the same time will react strongly to almost identical peptide sequences from invading pathogens. A new concept has been proposed which provides, perhaps, more satisfactory explanations for these issues [4, 5]. This concept suggests that the immune system does not merely differentiate between self and nonself. The decision whether a lymphocyte is activated depends not solely on the recognition of a foreign antigen. Instead, the immune response must be considered as the outcome of a complex interaction of a lymphocyte with an antigen presented by an APC in the context of costimulation. The quality and intensity of the response is heavily affected by the local environment in which the immune response takes place. The driving force in this concept is not the question of self and nonself, but whether a type of reaction is dangerous to the integrity of the individual. Zinkernagel [6] extended this concept by stating that antigen localization, and transport via migrating cells will determine whether mature T cells will be induced to react and for how long. This view of the immune system provides more satisfactory explanations for the issues raised above and, even more, leads to more insight in the formation of the T cell repertoire and its relationship to the development of autoimmunity.

Recognition of Self Is the Driving Force in Thymic Selection During the process of differentiation in the thymus from naı¨ve into mature T cells, T cells bearing a T cell receptor (TCR) with a high affinity for a self antigen presented to in the thymus are eliminated [7]. Only a small minority of thymocytes differentiates into mature T cells; the majority of thymocytes reactive with self-MHC peptide complexes are actively eliminated during thymic selection [8]. This process of negative thymic selection leads to socalled central tolerance. However, this negative selection is not absolute. For several reasons autoreactive T cells can escape thymic selection and become part of the mature T cell repertoire [9]. For instance, if the self antigen is not

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expressed in the thymus, but in a sequestered site, T cells with specificity for this self antigen will not be eliminated. In this context, it has been suggested that resistance to an organ-specific autoimmune disease may be partly regulated by thymic expression of the relevant autoantigen. The level of expression of this autoantigen may actually correlate with the ability to establish central tolerance to this autoantigen [10]. Also, a self antigen can escape central tolerance induction when the avidity of the TCR for the self peptide-MHC is very low, leading to positive selection. Ultimately, however, all cells which survive negative selection may be considered self-reactive, since their selecting antigen(s) is a self-derived one. Recognition of self, under this standpoint, must be regarded a physiological and essential process of the healthy immune system.

Thymic Selection Is a Dynamic and Adaptable Process Recent findings have made clear that the process of thymic selection of the T cell repertoire is far more complicated than assumed before [11]. These new developments have important implications for understanding the origin of autoimmune diseases such as rheumatoid arthritis (RA), as will be discussed below. The mature T cell repertoire is highly diverse and is armed to respond to the enormous amount of exogenous antigens it may encounter. The selection of the T cell repertoire takes place in the thymus through contact of thymocytes with self peptide-MHC complexes [12]. This process is far more promiscuous than assumed before. First of all, the selection of thymocytes by certain MHCpeptide complexes in the thymus does not necessarily lead to mature T cells with the same specificity [13, 14]. Second, it has been shown in transgenic systems that selection of thymocytes by even a single peptide-MHC ligand can lead to the induction of a diverse, though not complete, T cell repertoire [13, 15]. However, it is still controversial to what extent peptides are required for selection of the mature T cell repertore. In contrast to some of the earlier studies, a recent study in a transgenic system demonstrated that specific recognition of a divers panel of peptides is crucial for the generation of a complete T cell repertoire [16]. It is essential that mature T cells, after being selected on a self peptideMHC complex in the thymus, can actively respond to a foreign antigen and at the same time be unresponsive to the corresponding self peptide [12]. The structural basis for this dual capacity of mature T cells has been established, revealing that the same TCR can actually recognize different, yet homologous peptides in the context of the same MHC [17]. This dual capacity of a T cell has important implications for autoimmunity, since it provides a molecular basis for antigenic mimicry. The physiological milieu, in which the mature

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T cell recognizes the foreign antigen, will ultimately determine the outcome of the immune response, namely peripheral tolerance of activation. Most of the knowledge on this topic has been obtained in elegantly designed transgenic system, which, however, may not necessarily fully emulate the complex and variable series of events which occur during thymic selection. In this respect, recent progresses in immunology, particularly the possibility of tracking antigen-specific T cells, have opened new avenues to explore thymic selection in a more physiological context.

A Self-MHC-Derived Peptide Contributes to Positive Selection of T Cells Which Are Cross-Reactive with a Homologous Foreign Antigen Ia52 is a naturally processed peptide derived from the IE-a chain which can be presented in the embryonic thymus of BALB/c mice. We employed this peptide to evaluate its capability to affect T cell selection in fetal thymic organ cultures of BALB/c mice. The main hypothesis for this study was that increased availability of this peptide would affect positive selection of T cells with the capability of reacting against homologous foreign antigens, thus providing experimental proof to the theory exposed above. Based on structural similarities, we identified as the foreign homologous peptide, Haemophilus influenzae isoleucyl tRNA transferase 15–31 (Hi15). To identify antigen-specific T cells in this nontransgenic system, we employed a novel assay (T cell capture, TCC). In this method, MHC class II loaded with the relevant peptide is inserted into fluorescent-labeled liposomes, to create artificial APC. The MHC/peptide complex is tagged to enable identification on a FACS machine or by microscopy. These artificial APC can activate T cells with the proper restriction and specificity. When bound to T cells, the resulting complexes can be visualized by flow-cytometric analysis. Using the T cell capture we were able to show that the IEd-derived peptide Ia52 can enable positive thymic selection of a diverse population of T cells with different specificities. Within this polyclonal population of T cells whose fate in thymic selection was determined by Ia52, we could identify a subpopulation of Ia52specific cells that displayed cross-reactivity with Hi15. This cross-reactive population was narrowly oligoclonal, insofar as it used a highly restricted, though not identical, set of TCR genes, distinct from the wide polyclonal population positively selected by Ia52. These Ia52–Hi15 cross-reactive cells can be found in the periphery and are functionally competent. This model may very well reflect the physiological genesis and maintenance of a response to a foreign antigen. An MHC-derived self peptide such as Ia52 may first play a pivotal role during the selection of T cells in the thymus. The same peptide is then continuously available in the periphery, and it may sustain


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the maintenance of the same repertoire in the adult, through processes of recognition and peripheral tolerance. The activation in the periphery of crossreactive T cells will be largely determined by the environment, such as the presence or absence of costimulatory signals and cytokine production, and the relative concentration of the relevant antigens. An encounter with the foreign counterpart of the selecting self peptide can result in a dramatic change in the quality of the response, which is needed to eliminate a foreign invader (fig. 1). Under certain conditions, this finely tuned mechanism based on molecular mimicry between a self peptide and a foreign antigen may get out of control and thus harbor the risk of developing autoimmunity through antigenic mimicry. It is proposed that this may especially be the case of MHC-derived self peptides [18, 19]. First, because MHC-derived peptides are prominent in shaping the T cell repertoire in the thymus [20] and, second, because they can act as antigens themselves and can be presented by professional APC. Indeed, generation of T cells specific for foreign antigens through MHC mimicry may very well be one of the events, which contribute to antigen-specific proinflammatory pathways relevant to pathogenesis of RA.

Rheumatoid Arthritis RA is a systemic autoimmune disease characterized by chronic synovial inflammation [21–23]. Although the etiology of the disease is still unknown, it is commonly accepted that initiation of proinflammatory responses may depend on both environmental and genetic factors [24, 25]. In the initiating phases of RA, immune-mediated events based on autoreactive T cells most likely play a pivotal role [25–31]. These abnormal responses may target, most likely, more than one antigen. Hence, identification of antigens, which may be correlated to abnormal T cell responses, together with the discovery of genetic factors, which influence such immune responses, is one of the avenues of development of current research.

Environmental Factors: Heat Shock Proteins Are a Group of the Antigenic Proteins in Autoimmune Arthritis One of the best studied groups of antigens that triggers T cell responses in RA is composed by the family of heat shock proteins (HSP) [32, 33]. HSP are highly conserved proteins, which are essential for cell function [34, 35]. They are overexpressed during inflammatory conditions, and they are recognized by the immune system [36]. Cross-recognition between human and microbial HSP


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Table 1. HLA and susceptibility in RA [adapted from 1, 51]. DRB1 allele

HLA DR4 molecule

Prevalence in RA %

*0401 *0404 *0101

DR4, Dw4 DR4, Dw14 DR1, Dw11

50 30 24

has been described in several different systems [37]. HSP represent a good model to study how cross-recognition of homologous epitopes of either self or microbial origin may influence the genesis and control of proinflammatory pathways [38–40]. This approach may also provide a model for understanding the delicate balance between regulatory and proinflammatory responses. HSP are, in fact, the triggering antigen for the adjuvant model of arthritis in rats [41]. This disease can be successfully treated by acting on the regulatory network of T cells which cross-recognize self and microbial HSP peptides [42–45]. In human arthritis, juvenile RA as well as RA [32, 46, 47], HSP both of exogenous and self origin are targets of immune responses. Depending on the local environment, these self HSP cross-reactive cells can be related to autoimmune inflammation or, conversely, modulate it [48, 49]. This makes HSP highly attractive candidates to attempt to actively modulate T cell-mediated autoimmunity in RA and juvenile RA [50]. Among HSP the Escherichia coli HSP dnaJ holds some peculiar characteristics. Indeed, it shares with those HLA alleles, which are prominently associated with RA, a five-amino acid stretch called ‘shared epitope’. dnaJ, as an HSP and due to this homology, is a model to study the role that cross-recognition of highly represented self and microbial antigens may play in the pathogenesis of autoimmune arthritis.

Genetic Factors: ‘Shared Epitope’ in RA As mentioned above, one of the most striking features of RA is its association with certain HLA alleles. RA is, indeed, strongly associated with certain HLA class II alleles, in Caucasians namely DRB1*0401, DRB1*0404 and DRB1*0101 [51, 52]. The sequence that distinguishes them from non-RA susceptibility HLA-DR alleles is the so-called ‘shared epitope’, a conserved amino acid sequence (LLEQRRAA or LLEQKRAA) that is located at positions 67–74 on the b-chain of the relevant MHC class II molecule [53, 54] (table 1). More importantly, the presence of the shared epitope is predictive


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of progressive, more serious disease and individuals that are homozygous for this gene are at a higher risk. The effect that the presence of the shared epitope has on immune pathogenesis of arthritis is not clear yet. Several different models have been proposed, which emphasize the potential role of this sequence either as a factor favoring presentation of ‘arthritogenic’ peptides, or as a direct binding site for proinflammatory T cells. Our approach has proposed to rather look at the shared epitope as one of the possibly many peptides, which may affect the composition of the T cell repertoire in RA. Considering the genetic inheritability of the shared epitope, and the fact that it is also present on common environmental agents, our approach may be effective in conciliating genetic and environmental factors into a single pathogenic model.

The Multistep Mimicry Hypothesis Our model [19] takes into account the availability of shared epitope peptides of self origin both in the thymus and in the periphery. In the thymus, self peptides expressing the shared epitope are available for recognition by the immature T cells in the cortical epithelium. Low affinity interactions between specialized thymic APC presenting shared epitope peptides and immature T cells may shape the T cell repertoire, by allowing the maturation of T cells with a potential inherent self reactivity. In the periphery, a pool comprising these cells is maintained by the same self HLA-derived selecting peptide (fig. 1). The same cells may cross-react at higher affinity in the periphery with homologous, albeit not identical, peptides of microbial origin containing the shared epitope. This interaction, in the proper milieu, such as in conditions where adequate moieties of the interacting molecules are present, and proinflammatory and costimulatory stimuli are available, may lead to the genesis of proinflammatory responses. These responses may be reverberating, both due to the availability of self peptides locally, and to the presence of non-antigen-specific proinflammatory stimuli. Hence, immune recognition of an antigen can lead, given the proper T cell repertoire and a proinflammatory milieu, to a cascade of events eventually leading to epitope spreading and chronic inflammation.

Microbial Peptides Activate Shared Epitope-Specific T and Induce Production of Proinflammatory Cytokine Cells in RA Patients: A Dangerous Aberration from Physiological Homeostasis? Several different microorganisms, including common human pathogens such as E. coli and the Epstein-Barr virus (EBV), encompass a cassette contain-


57

Fig. 1. Multistep molecular mimicry and T cell repertoire.

ing the shared epitope sequence in the context of immunologically relevant proteins, such as HSP, as in the case of E. coli, Brucella ovis and Lactobacillus lactis, or external envelope proteins, such as the EBV protein gp110. Epitope mapping studies have shown that peptides from these foreign antigens which contain the shared epitope sequence at the N-terminal are presented by HLA class II alleles and trigger proliferative T cell responses in patients with early diagnosed RA. Interestingly, these responses seem to be an early immunological event in the natural history of the disease, insofar as they cannot be detected easily in patients with long-standing diseases. Therapy with DMARDs, particularly methotrexate and cyclosporin, abolishes this reactivity as well. Remarkably, T cell proliferation is correlated, in these patients, to


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production of proinflammatory cytokines such as IFN-c and TNF-a. Hence, recognition of these foreign peptides containing the shared epitope in a situation where such foreign proteins are available to the immune system seems to be related to the same mechanisms of production of proinflammatory cytokines in autoimmune arthritis. This reactivity is not self-limiting such as it is found in physiological responses to foreign antigens, but it perpetuates itself. It seems, therefore, that the main aberration from physiology of these responses is their stubborn persistence in the initial stages of the disease.

Self Shared Epitope Peptides Shape and Maintain a Repertoire of T Cells Cross-Reactive with Foreign Homologues One of the interesting aspects of this model is that it is a based on the interplay between two groups of peptides, one of self and the other of foreign origin. The experiments detailed below are based on a set of two peptides: dnaJP1 is the E. coli dnaJ HSP-derived peptide that we found immunogenic in RA patients. This peptide (QKRAAYDQYGHAAFE) is the one which has shown the main antigenicity among those screened. The S1 peptide (QKRAAVDTYCRHNYG) is the dnaJP1 homologous, self HLA-derived peptide. As mentioned above, we have proposed [19, 55, 56] that these two peptides are recognized in RA patients by cross-reactive T cells. Recognition of S1 may be responsible for homeostasis of T cell populations that are activated upon encounter at higher affinity of the homologous dnaJP1. This concept requires proof of existing S1/dnaJP1 cross-reactive T cells. T Cells from Patients with RA Are Expanded by Incubation with dnaJP1 and Can Be Identified by TCC We have previously described in published work [55, 56] that T cells from patients with early RA proliferate when incubated in vitro with dnaJP1. We have correlated this response to generation of proinflammatory stimuli, which could be part of the disease process. By applying TCC, we can now identify peptide-specific T cells. This approach is crucial to demonstrating that the same population of T cells which recognize the HLA-derived peptide is presumably maintained by its weak interaction and it is then activated by its cross-reactivity with the microbial peptides. T Cells from RA Patients Cross-Reactive for dnaJP1 and its HLA Homologue S1 Can Be Identified and Isolated by TCC We raised short-term T cell lines from PBMC of RA patients. The percent of dnaJP1-specific CD3+ T cells present in unmanipulated PBMC samples


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varied between 0.1 and 0.5% in repeated experiments. Incubation with the relevant peptide significantly increases the number of specific T cells, up to 14% of CD3+ cells. We then sorted by TCC cells specific for either dnaJP1 or S1 peptide-extracted mRNA and found a partial overlap in TCR usage, particularly Vb 14. These experiments demonstrate that dnaJP1-specific T cells cross-recognize the HLA-derived homologue S1. Importantly, T cells proliferate and produce proinflammatory cytokines only when incubated with dnaJP1.

Conclusion Current evolution of research in RA clearly identifies components of the pathogenic process, which are shared with the normal physiology of the immune response. One of such aspects is the dynamic interaction which T cells have with a network of homologous, but not identical, peptides. These peptides can be of both self and foreign origin and can play diverse, and sometimes contradictory, roles in the maintenance, activation and control of T cells. Peptides containing the shared epitope provide one of the known examples for such network. The study of their role as one of the possibly many proinflammatory antigenic systems involved in pathogenesis of RA provides also a model to understand better the function and regulatory mechanisms of the normal immune system. It is clear that autoimmunity can be regarded as an aberration of physiological processes. In RA, the genetic association with HLA DR alleles points at a genetic predisposition and underlines that the susceptibility to the disease finds its origin in the selection of the T cell repertoire in the thymus. It is of great importance to understand better the physiology of processes of positive selection of potentially autoreactive cells in the thymus, their maintenance in the periphery, and the control of inflammatory processes. The outcome of the immune response in the periphery is determined by the interaction of the T cell and the antigen presented by the APC on its MHC. In this interaction, a variety of conditions, such as the amount of antigen available, affinity between MHC peptide and TCR, the length of the interaction and the expression of costimulatory molecules, will eventually determine the fate of the T cell. The main avenue for the future will be to pursue the study of these networks in all their aspects and at the level of a single antigen-specific T cell. To achieve this, we must obtain beter tools, such as the TCC, to study in depth an immune response. It will be a major goal to implement these tools in future studies attempting then antigen-specific modulation in autoimmunity.


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Acknowledgments We thank Dr. Rodrigo Samodal for expert technical assistance and support. Berent J. Prakken was supported by the ‘Ter Meulenfonds’ of the Royal Netherlands Academy of Arts and Sciences and by the Dutch Rheumatoid Arthritis Foundation. This work is supported in part by grants AR40770, AR44850, AI37232 and AR41897 from the National Institutes of Health.

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Prakken ABJ, Van Eden W, Rijkers GT, Kuis W, Toebes EA, de Graeff-Meeder ER, van der Zee R, Zegers BJM: Autoreactivity to human heat-shock protein 60 predicts disease remission in oligoarticular juvenile rheumatoid arthritis. Arthritis Rheum 1996;39:1826–1832. Prakken ABJ, van Hoeij MJ, Kuis W, Kavelaars A, Heijnen CJ, Scholtens E, de Kleer IM, Rijkers GT, Van Eden W: T-cell reactivity to human hsp60 in oligoarticular juvenile chronic arthritis is associated with a favorable prognosis and the generation of regulatory cytokines in the inflamed joint. Immunol Lett 1997;57:139–142. Van Eden W, van der Zee R, Paul AGA, Prakken BJ, Wendling U, Anderton SM, Wauben MHM: Do heat shock proteins control the balance of T-cell regulation in inflammatory diseases? Immunol Today 1998;19:303–307. Klippe JH, Dieppe PA: Rheumatology, ed 2. London, Mosby, 1998. Nepom GT, Byers P, Seyfried C, Healey LA, Wilske KR, Stage D, Nepom BS: HLA genes associated with rheumatoid arthritis. Arthritis Rheum 1989;32:15–21. Penzotti JE, Nepom GT, Lybrand TP: Use of T cell receptor/HLA-DRB1*04 molecular modeling to predict site-specific interactions for the DR shared epitope associated with rheumatoid arthritis. Arthritis Rheum 1997;40:1316–1326. Hammer J, Galazzi F, Bono E, Karr RW, Guenot J, Valsasnini P, Nagy ZA, Sinigaglia F: Peptide binding specificity of HLA-DR4 molecules: Correlation with rheumatoid arthritis association. J Exp Med 1995;181:1847–1855. Albani S, Keystone EC, Nelson JL, Ollier WER, La Cava A, Montemayor AC, Weber DA, Montecucco C, Martini A, Carson DA: Positive selection in autoimmunity: Abnormal immune responses to a bacterial dnaJ antigenic determinant in patients with early rheumatoid arthritis. Nat Med 1995;1:448–452. La Cava A, Nelson JL, Ollier WER, MacGregor A, Keystone EC, Thorne JC, Scavuli JF, Berry CB, Carson DA, Albani S: Genetic bias in immune responses to a cassette shared by different microorganisms in patients with rheumatoid arthritis. J Clin Invest 1997;100:658–663.

Dr. Salvatore Albani, Department of Pediatrics, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0663 (USA) Tel. +1 858 534 0394, Fax +1 858 534 5399, E-Mail [email protected]


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Exploring the Pathogenesis of Rheumatoid Arthritis in Transgenic and Mutant Mice Andrew P. Cope The Kennedy Institute of Rheumatology, Hammersmith, London, UK

Animal models of arthritis have contributed significantly to our understanding of the pathogenic mechanisms of rheumatoid arthritis (RA). They have also provided useful tools for evaluating novel therapies. For many years these models have underscored concepts of immune-mediated arthritis broadly based upon lymphocyte responses to cartilage antigens. In mice, CD4+ T lymphocyte responses are restricted to the MHC class II molecule I-A, the murine homologue of HLA-DQ, rather than to I-E, the homologue of HLADR, which has been linked to RA susceptibility in man. This concept has remained unchallenged, largely through the absence of robust and relevant spontaneous models of arthritis, and until recently, such models have not addressed directly issues central to the disease process in man. Questions of particular interest relate to studies of the functional basis for the associations between RA and HLA class II molecules, how immune-mediated provocations lead to autoreactivity, and the molecular basis of cytokine dysregulation in vivo. Neither have these models been able to reconcile the paradoxical finding of activated T cells at sites of chronic inflammation which are hyporesponsive to T cell receptor (TCR) stimulation. Accordingly, doubts as to the contribution of T cells to the disease process persist. This review attempts to highlight how a series of well-characterised transgenic mouse models have helped dissect in depth the pathogenic mechanisms of inflammatory arthritis in ways that would not be possible in man. In the context of the evolution of the disease process, I shall discuss the progression across specific immunopathological checkpoints which I believe may play an important role during different stages of the inflammatory process; the function

of genes encoded within the MHC will feature prominently. I will begin with models that have provided insight into the molecular nature of antigenic peptide epitopes presented by RA-associated HLA-DR4 molecules to T cells in vivo, and which have explored how broad flexibility of TCR recognition affords immunodominance and protective immunity at the expense of reactivity to self antigens. In this context, the contribution of systemic autoreactivity to self-peptide/self-MHC complexes to the development of joint specific autoimmunity will also be discussed in the light of exciting new data emerging from a TCR transgenic model of spontaneous arthritis. I will then focus on mouse models that have shed light on the molecular basis of cytokine dysregulation, and how a shift from antigen-dependent to antigen-independent T cell effector responses may perpetuate the chronic inflammatory process. With the results of recently published data in mind, I will conclude by speculating about possible mechanisms through which genes carried by the non-susceptible host could influence progression across each checkpoint. It is anticipated that the knowledge gained from this transgenic approach will provide a working framework for the development of new therapeutic strategies in the future.

Exploring HLA-DR4-Restricted T Cell Responses in vivo The development by a number of laboratories of transgenic mice expressing functional HLA class II molecules has provided a unique opportunity to explore the function of disease-associated HLA class II molecules in vivo, in a way that has not been previously possible in human subjects [1–7]. Comprehensive reviews published recently have detailed the experimental approaches, highlighting the constraints of such models, and discussing how these limitations have been resolved [8–12]. For some transgenic lines, optimal interactions between mouse CD4+ T cells and the human HLA-peptide complex has been achieved through the introduction of the human CD4 minigene on a genetic background that is also deficient for murine MHC class II molecules [1, 13–16]. But even for mice carrying homozygous HLA-DR4/human CD4 transgenes, in addition to the I-Ab null mutation, the efficiency of interactions between human class II molecules and mouse antigen-processing machinery can only be assumed. Despite this, HLA transgenic mice have been enormously useful for probing in vivo the molecular nature of T cell responses restricted to human class II molecules. What Do HLA-DR4-Restricted T Cells Really See? As a first step, it was necessary to study in detail a CD4+ T cell response specific for cognate antigen that was restricted to the HLA-DRB1*04 allele

Transgenic Mouse Models of Arthritis

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encoding HLA-DRab1*0401, and to characterise the immunogenic epitopes presented by these HLA-DR4 molecules in vivo. This allele was chosen because of its strong associations with RA [17a, 17b]. To this end, it was necessary to generate a repertoire of CD4+ T cells whose TCR had been shaped by HLADR4 during thymic maturation. This was only achieved after a detailed analysis of the peripheral CD4+ T cell compartment in transgenic mice expressing different genotypes [15]. For example, while I-Ab0/0 murine MHC class II deficient mice have =0.5% mature CD4+ T cells in the peripheral blood [13], introduction of a single copy of the HLA-DR4 transgene onto this mouse class II-deficient background increased this level to around 4%. After crossing to human CD4 transgenic mice the peripheral CD4 compartment increased further to 13%, while homozygous HLA-DR4/human CD4 transgenic, I-Ab0/0 mice had peripheral mouse CD4+ T cell numbers approaching those observed in mice expressing endogenous I-A molecules (D25–30%). These observations, combined with the fact that HLA-DR4 had been shown to alter the repertoire of selected Vb TCR in I-A expressing mice [1], demonstrated that human HLA-DR4 can shape both quantitatively and qualitatively the TCR repertoire in mice. It also provided a molecular framework for exploring in depth the specificity of mature CD4+ T cells that had previously undergone maturation and selection through cognate interactions with self-peptide/HLADR4 complexes in the thymus of transgenic mice. T cell epitopes of the human cartilage antigen HCgp-39 were defined [16]. Although HCgp-39 is expressed in many tissues, it is produced in abundance by chondrocytes, and protein can easily be detected in synovial membrane, synovial fluid and serum [18–22]. The finding that mRNA transcripts are not detectable in healthy cartilage explants, but can be induced in tissue from arthritic joints, as well as by proinflammatoy cytokines in vitro, made this an attractive candidate antigen for study [19, 20, 23]. Through the generation of immortalised T cell hybridomas using an experimental approach outlined in figure 1, the specificities of literally thousands of T cell responses could be evaluated. From more than 250 HCgp-39-specific responses, nine immunogenic epitopes were identified with frequencies of responses of T cells to specific peptide ranging from =1% to as high as 35% of the total response [16] (table 1), indicating that this was a sensitive as well as specific method for studying HLA-DR4-restricted T cells responses in vivo. Epitopes 100–115, 262–277 and 322–337 contributed around 80% of the total peptide specificity. More than 95% of all antigen-specific responses were restricted to HLA-DR4, the remainder being to the DRa/I-Eb cross-species heterodimer which is also expressed in these mice, albeit at very low levels. All responses could be blocked with anti-DR monoclonal antibodies, and each epitope identified was subsequently found to be processed and presented efficiently by human

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Fig. 1. Schematic for the generation of HLA-DR4-restricted T cells specific for human autoantigens. Once immunogenicity of a candidate autoantigen is established, draining lymph node T cells from immunised mice are restimulated in vitro and used for the generation of T cell hybridomas by fusing with TCRab –/– BW5147 cells. Selected T cells are subsequently tested for reactivity to native antigen presented by transgenic splenic APC. Positive clones can then be tested on pools of synthetic peptides spanning the entire molecule, and the core epitope determined using N- and C-terminal truncated variant peptides. IFA>Incomplete Freund’s adjuvant.

DRab1*0401 expressing APC. In a pilot study designed to validate the relevance of HCgp-39 epitopes defined in mice, all immunogenic epitopes of HCgp39 were capable of eliciting peripheral blood T cell proliferative responses in at least some patients with RA carrying HLA-DRB1*04 alleles encoding the shared epitope consensus sequence [16]. Further analysis of immunogenic epitopes of HCgp-39 was of interest in several respects. Firstly, while there was a definite trend for immunodominant epitopes to bind more strongly to DRab1*0401, responses to the strongest DRab1*0401 binder, peptide 259–271 (IC50 D30 nM ) were found at very low frequency (table 1). On the other hand, a significant number of T cells were found to respond to other peptides which bound DRab1*0401 weakly, with


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Table 1. Characteristics of immunogenic epitopes of HCgp-39 recognised by HLADR4-restricted T cells in transgenic mice Peptide residue

Putative core motif relative position 1 2 3 4 5 6 7 8 9

40–55 100–115 160–175 197–211 259–271 262–277 280–295 322–337 334–349

F F F F F F F Y Y

P S I I G T T D L

D K K S R L K D K

A I E I S A E Q D

L A A M F S A E R

D S Q T T S G S Q

R N P Y L E T V L

F T G D A T L K A

L Q K F S G A S G

Frequency of DR4restricted hybridomas (n>250)

Relative binding to DRab1*0401

8 18 1 6 1 35 3 26 2

+/– +++ + + ++++ +++ ++ ++ ++

IC50 as low as 50 lM. Several other peptides identified as strong DRab1*0401 binders were not immunogenic at all in this model. Secondly, the amino acid sequences of DRab1*0401 epitopes revealed an amino acid motif that would be predicted to bind to most HLA-DR molecules [24, 25]. Surprisingly, there were no other features of this motif distinguishing these immunogenic epitopes from peptides likely to be immunogenic in the context of other HLA-DR molecules [25]. In particular, none of the immunodominant epitopes of HCgp39 carried negatively charged residues at P4, a characteristic predicted to favour stronger binding to DRab1*0401 [26]. This is not to say that such peptides could not be identified using this experimental approach. Indeed, three minor epitopes of HCgp-39 (160–175, 280–295 and 334–349, comprising D6% of all T cell specificities detected) carried Asp or Glu at this position (table 1), and immunodominant epitopes of pancreatic b islet cell autoantigens carrying Asp or Glu at P4 have now been reported using an identical experimental approach [15, 27]. For the majority of T cells, responses were specific for individual HCgp-39 peptides, and no responses were observed following stimulation with different peptide ligands. Small changes in the peptide sequence also had dramatic effects on T cell reactivity. For example, no T cells specific for immunodominant epitopes of the human HCgp-39 could be identified that also responded to the murine peptide homologues of the same epitope, despite relatively conservative amino acid substitutions for some of these peptide homologues [Cope and Sønderstrup, unpubl. data]. Further restriction was imposed by HLA-DR4

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Table 2. Sequence polymorphisms of DRb chain alleles: consensus residues permissive for RA (67–74b) – L L E Q/R R/K R A A DR allele

B1*0401 B1*0401 B1*0402 B1*0403 B1*0404 B1*0405 B1*0408 B1*1402 B1*1001

Amino acid positions in DRb chain diversity regions first

second

third

9 11 13

26 28 30 37

57 67 70 71 74 86

W E E E E E E E E

L F F F F F F F L

D D D D D S D D D

L V V V V V V S V

F H H H H H H S F

E D D D D D D E E

C Y Y Y Y Y Y Y R

S Y Y Y Y Y Y N Y

L L I L L L L L L

Q Q Q Q Q Q Q Q R

R K E R R R R K R

A A A E A A A A A

G G V V V G G G G

RA

+ ++ – – ++ + + + +

molecules, since the majority of T cells recognised peptides when presented by DRab1*0401, but not by a panel of APC carrying closely related HLADRB1 alleles (table 2, fig. 2a). One exception to this, was a subset of clones recognising peptide 100–115 presented by DRab1*0401 and *0404, but not by DRab1*0402 or *0403 molecules (fig. 2b) [16]. While this result appears to reflect some degree of flexibility of TCR recognition, the single amino acid difference at b74 (Ala to Glu) that distinguishes DRab1*0404 from *0403 (table 2) provides evidence of considerable stringency at the level of TCR recognition of HLA-DR4 molecules. This approach has made it possible to define precisely for HLA-DR4restricted T cells the immunodominant epitopes of a cartilage antigen of potential importance in RA, and as such defined a molecular checkpoint of cartilage antigen peptide recognition by TCR. In this model, TCR recognition appeared to be highly specific for a large number of clones, since strict dependence on specific peptide for reactivity was demonstrated, and TCR triggering did not occur in the absence of specific peptide. However, as will be discussed below, new experimental approaches suggest that seemingly monospecific T cells such as these are also highly cross-reactive. Immunisation with HCgp-39 Generates Reactivity to Self-Peptide/ Self-MHC Complexes While screening for positive responses to HCgp-39, a subset of hybrids were found to be highly reactive to transgenic but not non-transgenic splenic


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a

b

Fig. 2. Specificity of HCgp-39-specific T cells. Hybridomas were stimulated with specific peptide in the presence of a panel of EBV-transformed B cells expressing a range of HLADRB1 alleles (see table 2 for sequence differences). Results, expressed as stimulation indices derived from IL-2 production, are shown for clone 2B1 which recognises peptide 322–337 only when presented by DRab1*0401 (a), and for clone 1G5 which sees peptide 100–115 presented by both DRab1*0401 and *0404 (b ).

APC in the absence of exogenous antigen [16, Cope and Sønderstrup, unpubl. data]. In many cases the magnitude of response was as pronounced, if not stronger than those responses observed to native antigen or peptide. Similar responses were observed when DRab1*0401 expressing EBV-transformed B cells were used as APC, and all anti-self responses could be completely inhibited by anti-DR monoclonal antibodies, confirming that these responses were directed to complexes of self peptide and HLA-DR4. Detailed analysis of cross-reactive T cells exhibiting strong responses to DRab1*0401 in the absence of exogenous antigen revealed flexibility of TCR recognition at multiple levels. While all cross-reactive T cells responded robustly to APC in the absence of exogenous antigen, activation of a subset of clones was further enhanced by the addition of HCgp-39. To characterise this response in more detail, hybrids were stimulated with DRab1*0401 expressing APC plus a panel of peptides derived from HCgp-39 (fig. 3a). Surprisingly, enhanced responses to three or more different HCgp-39 peptides were observed over and above the strong responses observed by T cells stimulated with APC alone, despite repeated subcloning of T cells. These so-called intramolecular peptide mimics, while carrying predicted HLA-DR binding motifs, carried sequences which were quite distinct from each other at the amino acid level (table 1). Secondly, these clones had a tendency to respond in the absence of exogenous

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a

b

Fig. 3. Flexibility of TCR recognition by HCgp-39-specific T cells. a T cells were stimulated with APC plus HCgp-39 peptides or native antigen; note high background counts ?100,000 IL-2 fluorescence units in the absence of antigen. b T cells were stimulated with a panel of DRB1-expressing APC in the absence of exogenous antigen. Data are shown for clones 4A10 and 1A2, both specific for peptide 262–277. Results are expressed as arbitrary IL-2 fluorescence units.

antigen to APC expressing different HLA-DRB1 alleles that encoded the shared epitope consensus sequence b67–74. Responses to DRab1*0401 and to *0405 APC were particularly vigorous (fig. 3b). Despite this, responses to shared epitope-negative HLA-DR4 molecules, including DRab1*0402 and *0403, were never observed. This analysis identified a highly cross-reactive population of TCR capable of recognising multiple, diverse sets of self and/ or HCgp-39 peptides presented by DRab1*0401 or closely related molecules, and provided evidence for MHC-biased TCR recognition, where DRb consensus residues are critical recognition determinants for cross-reactive TCR. The Nepom laboratory have reported studies of HLA-DR4-restricted T cell responses which are especially relevant here. They identified a high frequency of Asp residues at position 30 of complementarity determining region (CDR) 1 of TCRVB sequences derived from RA synovial joint T cells, and suggested that charge complementarity may indeed occur at the level of pocket 4 of HLA-DR4 molecules, not between peptide and residue 71 of the DRb chain as previously predicted, but between the DRb chain and the TCR [28]. This finding would be entirely compatible with a scenario where, at least for antigens such as HCgp-39, immunodominant peptides lack negative charges at P4; the presence of a salt bridge between TCR and DRb chain as


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well as between DRb chain and peptide would be rather unlikely. Within this cross-reactive molecular framework, there may exist yet further flexibility. For example, TCR recognition of the peptide/MHC configuration does not discriminate contact residues contributed by peptide or the DRb chain a helix [29]. Thus, in the context of DRab1*0404 it was shown that TCR can see a complex which includes a charged amino acid regardless of whether this residue is in the peptide or the DRb chain. A rubella peptide, with a Glu residue at P5, could be efficiently presented by HLA-DR4 molecules that carry Ala at b74 (see table 2). When the peptide P5 position was changed to Val, and HLADR4 molecules carrying Glu at b74 were used to present, TCR recognition was not compromised [29]. These simple but elegant experiments illustrate structural complementarity, where a TCR sees not peptide residues and MHC residues, but a tertiary configuration derived from a complex of MHC and peptide. While molecular models have been instructive [30], crystals of HLA/ peptide with or without specific TCR are beginning to provide further molecular insight into the diversity and flexibility of these critical interactions [31–35]. In summary, an investigation of antigen-specific T cell responses in a transgenic mouse model has revealed the nature of peptide epitopes of a human cartilage antigen presented by disease-associated HLA-DR4 molecules. Great diversity and flexibility of TCR recognition can be detected early in the evolution of the immune response, and cross-reactivity to self-peptide/selfMHC complexes is common following immunisation with cognate antigen. For many responses, MHC-biased recognition by TCR predominates. While the fact that the repertoire of TCR is shaped on self-peptide/self-MHC complexes during thymic maturation provides the most plausible explanation for this, the model also implies that cross-reactivity of peripheral T cells is universal. Thus, it should be possible to demonstrate broad self-reactivity for all TCR, including populations of peptide-specific T cells that are apparently ‘monospecific’, seemingly lacking the broad flexibility of other T cell subsets. Since immune responses to foreign antigens are generated from the same pool of potentially self-reactive TCR, it follows from this that immunisation of HLA-DR4 transgenic mice with proteins derived from foreign pathogens would activate and expand populations of autoreactive T cells. Self-Reactivity following Immune Responses to Foreign Pathogens In collaboration with the Kamradt laboratory, this hypothesis has been tested directly by repeating the immunisation strategy, this time substituting HCgp-39 with OspA, the outer surface protein A derived from the tick-borne spirochete Borrelia burgdorferi, the causative organism of Lyme disease [36]. This was a relevant model antigen to study because T cell responses to OspA have been implicated in chronic treatment-resistant Lyme arthritis, which is

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Fig. 4. Schematic for demonstrating cross-reactivity of OspA-specific T cells to selfpeptide mimics. Candidate peptide mimics were identified by sequential substitution analysis of each amino acid residue of the core epitope, or by database search for sequence alignment. Peptide mimics were identified, and the best fit peptides synthesised and tested on panels of OspA-specific T cell hybridomas.

also associated with HLA-DR4 [37–40]. Large numbers of HLA-DR4-restricted, OspA-specific T cell hybridomas were generated, and the immunodominant epitopes identified as before [41, Maier and Kamradt, unpubl. data]. Two strategies were employed for identifying self peptide mimics of immunodominant OspA epitopes capable of stimulating OspA-specific T cell hybridomas. An example is shown in figure 4 for OspA peptide 235–246. In the first, the Swissprotein/TREMBL database was used to define mimics of OspA peptides based on conventional sequence alignment. In the second, a systematic amino acid analysis was undertaken substituting each residue of the core nine-mer OspA epitope with all naturally occurring amino acids and testing responses of OspA peptide-specific T cells to these synthetic derivative peptides. On the basis of the amino acid substitutions permissive for T cell responsiveness, a structural ‘supertope’ motif was defined and used to scan the same database. Mimics were identified and synthesised prior to testing on a panel of OspA-specific T cell hybridomas. The results were remarkable in several respects. When responses to the ‘best fit’ peptides identified by either alignment or substitution analyses were com-


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pared on the panel of T cells that responded to the original peptide, by far the majority of reactivity was observed with peptide mimics fulfilling the supermotif. Intriguingly, many of these peptide mimics, derived from either mouse or human proteins, bore little or no resemblance to the wild-type sequence. Indeed, for some there existed no homologies at any residue. By contrast, few if any peptides identified by conventional sequence alignment stimulated OspA-specific T cells. This lack of reactivity was analagous to that observed for human HCgp-39specific T cells when tested on the closely homologous, but not identical mouse peptides. These findings support data from other laboratories [42–44], and predict that multiple cross-reactive self ligands could be identified for immunodominant epitopes of autoantigens and any protein derived from infectious pathogens. They provide direct evidence that T cells apparently highly specific for a given peptide ligand are also broadly cross-reactive to self peptide ligands, and illustrate that recognition of MHC/peptide complexes by TCR is degenerate [44]. The model also indicates that progression across two immunopathological checkpoints, namely T cell activation by cognate antigen and expansion of crossreactive clones, is almost simultaneous. Immunodominance and Autoreactivity Are Inextricably Linked These data provide a direct link between protective immunity and the development of self-reactivity, a concept based on the fundamental observation that the repertoire of peripheral TCR is generated on self-peptide/self-MHC complexes during T cell development in the thymus. What is the biological significance of such extensive cross-reactivity? The presence of cross-reactivity within the TCR repertoire could provide for immunological memory and immunodominance. For example, it has been shown in mutant mice that in the absence of peripheral complexes of self-peptide/self-MHC, the survival of peripheral T cells selected on MHC molecules expressed as transgenes exclusively in the thymus is reduced [45]. The survival curves suggest a peripheral selection process of ‘tickling’ of TCR to maintain clonal populations of primed T cells in the periphery. This is analogous to that described for Ig receptors expressed on B cells in the periphery [46]. In transgenic mice expressing MHC molecules covalently linked to a single peptide [47], or in H-2Ma-deficient mice, where the majority of complexes express MHC and the invariant chain peptide CLIP [48, 49], it has been possible to demonstrate a surprisingly broad repertoire of TCR. These T cells see peptide sequences unrelated to the selecting peptide in the thymus [50]. The evidence also suggests that recognition by TCR is more MHC-biased because T cells maturing in thymuses expressing a single MHC/peptide ligand react frequently with foreign MHC [50]. These mice are not obviously compromised, as might be expected in an immune system where a single peptide antigen is recognised by a single antigen receptor.

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This suggests that complexes of self-peptide/self-MHC can sustain a frequency of cross-reactive TCR sufficient to afford protective immunity to foreign pathogens. If, as has been suggested, a single TCR could recognise up to 106 peptide/ MHC complexes [51], it is conceivable that the generation and rate of productive and protective immunity to multiple foreign antigens could be met through the activation and efficient expansion of large numbers of cross-reactive T cells. If, in addition, TCR can undergo conformational changes at the interface with MHC/peptide, this could add a further level of flexibility [52]. The extent of cross-reactive TCR recognition observed in these mouse models could also account for the massive expansions (up to 44% of all CD8+ cells) of peptidespecific TCR in the periphery of patients with acute EBV infection visualised with HLA-peptide tetramers [53]. According to this model, the capacity for protective immunity brings with it the potential for enhanced autoreactivity. If the precursor frequency of such cells is indeed critical, it is not difficult to see how repeated exposure to foreign antigens could herald the onset of wild, unrestrained T cell reactivity and autoimmunity in the susceptible host. What is the evidence that T cell crossreactivity precedes the onset of autoimmune disease, and how may this reactivity induce inflammatory disease?

Is Systemic Autoreactivity Sufficient to Induce Joint-Specific Autoimmunity? While studying thymic selection of I-Ak-restricted CD4+ T cells by I-Ab molecules in a TCR (KRN) transgenic mouse model, Mathis and colleagues [54] noted that mice expressing this transgenic TCR but carrying I-Ag7 from NOD as well as or instead of I-Ak developed with complete penetrance a spontaneous, chronic, progressive inflammatory arthritis from 3–4 weeks of age. There were many features resembling human RA. Joint disease was symmetrical, with gradation of severity from proximal to distal joints, and led to deformity and functional disability. Involvement of distal interphalangeal joints and the axial skeleton was also noted, but to a lesser degree, while hip joints were spared. Serial analyses of histological sections from synovial joints revealed fibrin deposits, oedema and neovascularisation, extensive synovitis comprising nodules and follicles of inflammatory infiltrating cells, and the formation of pannus and erosion of cartilage-bearing chrondrocytes with pyknotic nuclei. By several weeks evidence of remodelling and fibrosis was quite pronounced. Intriguingly, by the time that inflammation was established, macrophages were the predominant cell type in inflamed synovium (with neutrophils predominating in synovial fluid), while T and B cells were sparse.


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Detailed dissection of the immunopathological features of this spontaneous arthritis model revealed several important characteristics. First and foremost, multiple crosses to mice expressing different H-2 revealed an absolute requirement for I-Ag7 for the disease to manifest itself [54]. Consistent with this was the propensity for TCR (KRN) transgenic T cells to proliferate to APC-expressing I-Ag7 in the absence of exogenous peptide, but not to the intensity that these same T cells responded to RNase peptide presented by I-Ak. The kinetics of disease onset together with the histological features and phenotyping of the cellular infiltrates supported the contention that an inflammatory arthritis could arise as a result of sustained recognition of systemic self-peptide/self-MHC complexes by a significant proportion T cells. Furthermore, this T cell-dependent arthritic disease occurred despite clear evidence of tolerance at multiple levels, as demonstrated by efficient thymic clonal deletion for the first 3 weeks of life, downregulation of the KRN TCR presumably because of upregulation of endogenous TCR chains and incomplete allelic exclusion, lower than expected numbers of peripheral CD4+ T cells, and the presence of T cells hyporesponsive to cognate self-antigen/ MHC complexes. Within the confines of animal models of autoimmunity, these elegant studies confirm unequivocally that systemic activation of cross-reactive T cells by self-peptide/self-MHC complexes is sufficient for autoimmunity to peripheral tissues, in this case the synovial joint. Since details of the model were first published, more recent experiments have demonstrated some of the parameters within this cross-reactive framework that influence arthritis development. Firstly, activation of KRN transgenic T cells per se is not sufficient for disease. When mice carrying an endogenous retroviral superantigen that selectively stimulates the transgenic Vb6 (in this case mtv7 encoded by mls-1a expressed in the DBA/2 genetic strain) are crossed to KRN transgenic mice, the progeny do not develop arthritis [55]. Nor do mice carrying a mutant allele of I-Ak (Aak69 Abk) which also stimulates the transgenic TCR [55]. In both experiments strong negative selection was found to be dominant, as determined by significant reductions in the expression of Vb6-positive T cells. Secondly, both intact CD40/CD40 ligand interactions and a complete repertoire of immunoglobulin genes are required for full expression of the disease phenotype, indicating that T-B cell-linked activation is essential [56]. Finally, arthritogenic activity was found to reside in the serum IgG fraction based upon the finding that arthritis can be passively transferred within several days of multiple injections of serum from arthritic mice [56]. Using pooled IgG from arthritic mice, the Mathis laboratory have heroically eluted from pooled NP40 tissue extracts a 60-kD protein which, following tryptic digestion, HPLC purification and sequencing by Edman degradation, was found to have sequence identitiy with an enzyme

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of the glycolytic pathway glucose-6-phosphate isomerase (GPI) [Matsumoto and Mathis, unpubl. data]. This cytoplasmic protein is not expressed exclusively in synovial joints, but is expressed ubiquitously. With recombinant protein it was also possible to confirm significant B cell reactivity using serum from arthritic mice. Indeed, injections of serum depleted of GPI reactivity did not induce arthritis. More importantly, strong T cell reactivity to GPI was observed when NOD splenocytes (I-Ag7 ) are used as APC to stimulate KRN transgenic, but not non-transgenic T cells. What Does This Model Tell Us about the Development of Joint-Specific Autoimmunity? The data favour the following sequence of events. Firstly, sufficient levels of systemic antigen expressed and presented by I-Ag7 must exist to activate a relatively large number of specific T cells. This provides the initial T celldependent antigenic drive, facilitated by ‘leaky tolerance’. Antigen activated T cells provide B cell help through CD40/CD40 ligand interactions, and populations of B cells expressing this antigen receptor expand preferentially. At this point, there may be further presentation of systemic antigen if expression persists, by B cells carrying the specific Ig receptor, which itself may facilitate internalisation and processing of antigen. The effector phase is antibodydependent (and largely T cell-independent), involving tissue destruction through processes of immune complex formation, complement activation and activation of inflammatory cells through Fc receptors; a cascade of proinflammatory cytokines would ensue. Why does systemic autoreactivity target synovial joints? One simple explanation could be molecular mimicry of systemic antigen with unknown jointspecific antigens. On the other hand, the unusual expression and cellular localisation of GPI in joint tissues cannot be excluded. Subsequently accumulation and/ or impaired clearance of immune complexes could contribute, perhaps facilitated by the rather unique blood supply and unusual anatomical properties of synovial joints [57]. In man, this process could be amplified further under circumstances where multiple systemic antigens are implicated. Detectable levels in serum, and preferential expression in inflamed joints would make HCgp-39 another good candidate [18, 21, 22]. The KRN model also raises questions about the immunopathological contribution of I-Ag7. Perhaps the instability of this particular subtype of I-A molecules promotes ‘leaky tolerance’, favouring permissive binding of peptides, and cross-reactivity above and beyond a critical threshold [58–60a]. Regardless of the precise mechanisms, we can conclude from this model that the predominant functional contribution of MHC to arthritis susceptibility is to establish a high frequency of potentially autoreactive T cells in a compartment where self antigen and MHC are presented at high density.


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The Effector Phase of Inflammatory Arthritis Much attention has focussed on the role of cytokines in the pathogenic events that herald the onset of cartilage destruction and bone erosion [60b, 60c]. If cytokine drive resulting from persistent antigenic stimulation is central to the chronic phase of disease progression, and a common pathway for perpetuating chronic inflammatory responses, then it should be possible to reproduce inflammatory disease by overexpressing proinflammatory cytokines in vivo, and bypassing upstream antigen-driven pathways. Indeed, studies of transgenic and mutant mice have shed considerable light on processes through which proinflammatory cytokines might contribute to the chronic phase of inflammation and target organ damage. I will illustrate this, by way of example, with mouse models that have revealed striking facets of the biology of the prototypic proinflammatory cytokine TNF. Unravelling the Molecular Basis of TNF Gene Expression in Inflammatory Arthritis There exist multiple and complex mechanisms which regulate TNF biosynthesis and responsiveness. While factors such as nuclear factors kappa B and activator of T cells appear to contribute to transcriptional control of TNF gene expression [61, 62], studies of TNF gene regulation in stromal cell lines that do not produce TNF have implicated the 3-untranslated region (UTR) as playing a dominant role in translational repression of TNF transcripts under non-stimulated conditions [63, 64]. In cells of the myeloid lineage, which do produce TNF, it is thought that the stress-activated protein kinase pathway leads to translational activation of TNF mRNA through reiterative adenosineuracil-rich pentanucleotide elements (ARE) in the 3UTR of TNF [65, 66]. The critical function of ARE appears to be strongly associated with the regulation of mRNA stability, but the role of this element in TNF gene regulation in vivo necessitated further elucidation. Initial evidence for the role of translational repression of TNF mRNA in chronic inflammation in general, and arthritis in particular, came from mice expressing human TNF as a transgene [67]. While transgenic mice expressing a wild-type human TNF transgene are completely healthy, mice expressing a similar transgene but with a modified 3’UTR element (in which ARE are absent) develop a spontaneous chronic inflammatory arthritis that could be completely blocked if repeated injections of neutralising monoclonal antibodies to human TNF were administered soon after birth. To test unequivocally whether ARE were directly implicated in the chronic inflammatory process, the Kollias laboratory generated mice expressing a ‘knock-in’ mutated murine TNF locus where a 69-base pair fragment encompassing the TNF

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gene ARE had been deleted [68]. Mice homozygous for TNFDARE had such severe inflammatory disease of joints and intestine at an early age that animals had to be crossed to TNF-deficient mice to generate a monoallelic system devoid of the wild-type allele (TNFDARE/–) that allowed more detailed study over time. The findings were of interest in several respects. Firstly, significant levels of TNF could be detected in sera from homozygous TNFDARE or TNFDARE/– mice, and spontaneous production detected in cultures of thyoglycollateelicited peritoneal macrophages or bone marrow derived macrophages from the same mice. The absence of ARE also had an enhancing effect on LPSinduced TNF protein and mRNA due to a 4-fold increase in the half-life of mutant mRNA, while transcription and pre-mRNA processing were unaffected. Secondly, inhibition of TNF secretion by a specific p38/SAPK inhibitor was not observed in macrophages from TNFDARE/– mice, indicating a requirement for ARE in p38/SAPK-mediated activation of TNF translation. Thirdly, expression of mutant mRNA in non-haemopoietic cells such as synovial or lung fibroblasts revealed both spontaneous and LPS-induced production of TNF, while fibroblasts from mice expressing wild-type TNF mRNA produced no TNF protein, confirming that ARE are important repressor elements of translation in these cell types under non-stimulated conditions. It is tempting to speculate that the effect on fibroblasts (e.g. proliferation and matrix degradation) could provide one important clue as to why chronic TNF overexpression selectively targets inflammatory responses to synovial joints. Finally, these aberrations of TNF mRNA steady state decay and translation led to the development of severe, chronic symmetrical inflammatory polyarthritis from 5 weeks of age, with the development of pannus, bone erosion and cartilage destruction. Interestingly, autoantibodies such as serum IgG and IgM rheumatoid factors were detectable, but were not pathogenic, because when TNFDARE/– mice were crossed to RAG-1-deficient mice, the arthritic process was not perturbed. That these specific ARE are important for TNF regulation in vivo is supported by recent data demonstrating that through binding mRNA, the zinc finger-containing protein tristetraprolin is associated with negative regulation of TNF mRNA stability [69]. Consistent with this, tristetraprolin-deficient mice develop a chronic inflammatory syndrome, including an erosive arthritis, which can be completely prevented with anti-TNF monoclonal antibodies [70]. These findings suggest that overproduction of TNF by macrophages and synovial fibroblasts is sufficient for progression to joint-specific pathology. They define another immunopathological checkpoint that could be essential for disease development, and a common pathway for many different models of inflammatory arthritis.


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Fig. 5. Possible mechanisms through which hyporesponsive T cells could contribute to the pathogenesis of chronic inflammatory arthritis. Hyporesponsive T cells from inflamed synovial joints possess unique characteristics (left), many of which can be reproduced by chronic TNF exposure in vitro or in vivo. While these could have protective effects on disease, impaired T cell activation could have downstream consequences (right) which promote and perpetuate the chronic inflammatory response.

Chronic TNF Induces Reversible, Non-Deletional T Cell Hyporesponsiveness Some years ago I began studying the reciprocal effects of inflammation on immunity, and in the context of chronic inflammatory diseases, such as RA, tried to set about asking how the chronic inflammatory process itself could influence the effector functions of autoreactive T cells. I designed experiments to address whether the inflammatory milieu promotes autoreactivity either directly, or indirectly perhaps by impairing regulatory or suppressor mechanisms. Using transgenic mouse models it was possible to probe directly the effects of the chronic inflammatory process on the adaptive immune system. I was particularly interested to address how sustained TNF signalling in vivo might influence cognate immunity, and the progression of autoreactive T cell responses, since at that time, the possibility that TNF could be implicated in the chronic phase of RA pathogenesis was just beginning to emerge. Preliminary experiments revealed that chronic exposure of T cells to TNF generated T cells in vitro or in vivo with characteristics rather similar to those of RA synovial T cells from inflamed joints (fig. 5, on the left). Chronic TNF exposure was examined in vivo in several ways [71]. Firstly, HNT-TCR

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Table 3. TNFa induces non-deletional and reversible T cell hyporesponsiveness in vitro and in vivo Chronic exposure of T cells to TNF Suppresses proliferation and cytokine production Efects are dose- and time-dependent Observed at non-toxic concentrations of TNF IL-2 and mitogen responses are spared Are inducible over days Rapid reversibility occurs in vitro and in vivo on withdrawing TNF Chronic TNF blockade Restores T cell proliferative responses in vitro and in vivo Enhances cytokine responses Effects are detectable under physiological conditions Effects are more pronounced in chronic inflammatory disease

transgenic mice expressing rearranged TCRa and b cDNA encoding TCR specific for an influenza haemagglutinin peptide in the context of I-Ad were injected repeatedly with TNF for weeks at a time, and peptide responses of lymph node or splenic T cells examined. In parallel experiments, groups of mice received TNF-neutralising antibodies or control antibodies. Secondly, HNT-TCR transgenic mice were crossed to the hTNF-globin transgenic mice described above so that peptide-specific responses could be compared in single TCR transgenic, and arthritic double TCR¶hTNF-globin transgenic mice. Regardless of the means of chronic TNF exposure, the results were clear (table 3). Exposure to endogenous chronic TNF at ‘physiological’ levels, or to TNF overexpressed by exogenous administration or through transgene overexpression led to suppression by up to 70% of T cell responses, as determined by proliferative and cytokine responses to peptide, while mitogen- or IL-2proliferative responses were spared. These data were confirmed in in vitro cultures of transgenic T cells stimulated with specific peptide in the presence or absence of TNF/anti-TNF. No evidence of cytotoxicity has been demonstrated with concentrations of TNF sufficient to suppress T cell function; indeed, in vitro models have demonstrated rapid reversibility of T cell hyporesponsiveness on withdrawal of exogenous TNF. In TCR ¶ human TNF-globin double transgenic mice it was possible to demonstrate that hyporesponsiveness could be detected before the onset of clinical inflammation [71]. This suggested that the dominant effects of TNF in vivo are to suppress rather than enhance T cell responses, and inferred that TNF could play an important role in the evolution of antigen-specific responses.


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Parallel studies of T cells from TCR transgenic mice on Balb/c (favouring Th2 responses) and B10.D2 (Th1) background strains, which both carry H-2d but differ at other loci, confirmed that the suppressive effects of TNF did not appear to discriminate between Th1 or Th2 cytokine responses [71]. Nevertheless, profound reduction of peptide-specific IL-4 and IL-10 production from transgenic T cells from Balb/c following chronic TNF exposure provided evidence that chronic inflammation could perturb the function of immunoregulatory T cell subsets, thereby promoting and perpetuating the disease progresses. Chronic TNF Uncouples Proximal TCR Signal Transduction Pathways To be able to explore this effect in depth, it became a priority to understand the mechanisms for suppression of T cell activation by TNF. The most significant finding was the demonstration that chronic TNF uncouples proximal TCR signalling pathways [71]. Thus, intracellular mobilisation of calcium following peptide stimulation is attenuated by prolonged TNF exposure, indicating that proximal pathways are likely to be uncoupled. A phenotype of impaired TCR signals has been thoroughly documented in RA synovial T cells [72]. Downregulation of TCRf chain expression and recruitment and phosphorylation of p36 LAT have also been implicated [72, 73]. Chronic TNF is probably just one of many factors that are capable of uncoupling TCR signals. Reactive oxygen species have also been implicated [74]. The data also provided a crucial link between chronic inflammation and immune suppression (fig. 5). What are the functional implications of T cell hyporesponsiveness? On the one hand, suppression could reflect an adaptive response to inflammatory signals (or ‘danger’!) that leads to downregulation of multiple cross-reactive T cell responses. This could be interpreted as a protective mechanism for the host. Certainly, exacerbation of some disease models in TNF-deficient mice and the effects of TNF injections in murine models of autoimmunity would seem to favour this view; I have reviewed these data recently [75]. An alternative hypothesis is that T cell hyporesponsiveness, due to an acquired proximal defect of TCR signal transduction pathways, could lead to attenuation of multiple TCR-specific responses including activation-induced cell death, production of immunoregulatory cytokines (the absence of which could profoundly influence the inflammatory process), and protective immunity to foreign pathogens (fig. 5). The consistent and rapid improvement in T cell responsiveness observed in RA patients as disease remits is consistent with such a model [76]. Nevertheless, data to suggest that restoring T cell responsiveness in anyway contributes directly to the resolution of inflammation is, at best, indirect. However, one has to consider the possibility that, through

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Fig. 6. A proposed role for T cells in the pathogenesis of chronic inflammation. Antigen drive may predominate during the early phase of inflammatory responses. More T cells could be recruited through bystander activation, or by stimulation with self antigens released from inflamed tissues. As the inflammatory process progresses, chronic cytokine production induces profound T cell hyporesponsiveness. These hyporesponsive T cells function as effector cells and sustain the chronic inflammatory process through antigen-independent mechanisms (see fig. 5). It is proposed that by reversing T cell hyporesponsiveness, antigen-dependent responses that serve to regulate the inflammatory process (e.g. immunoregulatory cytokines) are restored.

restoring T cell ‘tone’, the immune system is somehow reset, regaining the regulatory functions capable of suppressing the inflammatory process [75]. Cytokines rather than Antigen May Drive T Cell Effector Responses in Chronic Inflammation Since TCR hyporesponsiveness in chronic inflammation correlates with disease severity and/or chronicity, it is difficult to reconcile the long-held view that T cell effector responses in inflamed joints are antigen-driven. Rather they suggest that during the chronic phase it is the cytokine milieu that sustains and maintains the population of T cells. According to this model there is a gradual decline of antigen responsiveness as cytokine drive increases (fig. 6). Antigen drive and cytokine drive may be inextricably linked, with antigen responsiveness initiating the cytokine cascade as an early event, and subsequent hyporesponsiveness being due, at least in part, to sustained cytokine drive.


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In reality, the reversibility of T cell hyporesponsiveness, together with the recruitment of T cells stimulated recently with antigen, means that there is probably a dynamic equilibrium in joints between antigen- and cytokine-driven T cells, the balance of which depends on the severity and chronicity of the inflammatory process. T cells in inflamed joints that are poor responders to antigen, what role if any could they play in chronic inflammation? Are they merely passive bystanders? One possible explanation for this paradox lies in the observation that depletion of T cells from synovial T cell cultures significantly reduces the expression of TNF [Brennan et al., unpubl. data]. Furthermore, T cells propagated with a cocktail of cytokines in the absence of antigen are potent stimulators of proinflammatory cytokine production by macrophages through direct cognate cell-to-cell interactions which are MHC/antigenindependent. Macrophage activation can be reproduced with T cell membrane preparations [77, 78]. Lymphocyte activation gene-3 (LAG-3) is one cell surface molecule that could be implicated in this response [79]. In the context of chronic inflammation, persistence of this cognate interaction could occur through shifts in the balance of T cell recruitment and survival. Firstly, cytokine drive may be sufficient to recruit and activate bystander T cells in an antigen-independent fashion as they traffic through synovium. Secondly, synovial joint T cells from RA patients fail to undergo apoptosis [80], and could exert effector functions through their very persistence in joints. Interestingly, in the KRN spontaneous arthritis model, T cells are hyporesponsive to TCR ligation but continue to contribute to the disease process in the later stages of disease through mechanisms that are not understood [56]. Perhaps in this model, B cell help is upregulated through cell surface antigens expressed on T cells whose expression is maintained by chronic cytokine stimulation rather than through TCR signals. What these data imply is that hyporesponsive synovial T cells become potent effector cells, through cytokine-dependent, antigen-independent signal transduction pathways. Investigating precisely what role hyporesponsive T cells play in the chronic phase of inflammation has long been a priority in many laboratories. Strategies designed to study the effects of restoring TCR signalling defects would be highly informative.

Predicting the Progression through Disease Checkpoints in the Non-Susceptible Host The discussion above proposes that there exists a series of checkpoints that are implicated in the development and progression of the inflammatory

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Fig. 7. Immunopathological checkpoints implicated in the pathogenesis of chronic inflammatory arthritis. Activation and subsequent expansion of autoreactive T cells leads to cell-to-cell interactions that initiate a cascade of effector responses. According to this model, immunoregulatory mechanisms may influence activation, expansion and/or effector responses.

response (fig. 7). These checkpoints provide a theoretical framework for predicting how the immune system of an RA-non-susceptible host could function in ways that would attenuate the potential for sustained inflammatory responses in synovial joints. Is progression through these checkpoints completely inhibited, or merely retarded? The generation of transgenic mice expressing an allele of HLA-DRB1*04 that is not associated with RA (DRab1*0402), and which has been proposed to exert a protective effect on disease development and/or severity, has made it possible to begin to address these issues directly [16]. In molecular terms, they also provided an experimental approach which would identify the immunological consequences of the 4 amino acid differences encoded by the DRB1 chain that distinguish DRab1*0401 and *0402 at codons 86, 69, 70 and 71 (table 2). Using this strategy, each checkpoint could be evaluated by asking (1) whether antigen processing and presentation to CD4+ T cells was different, (2) whether the frequency of anti-self-reactivity of antigen-specific T cells differed, (3) whether there was evidence of an attenuated effector response in T cells from transgenic mice carrying the non-associated DR*0402 genotype, and (4) what immunoregulatory or tolerance mechanisms were in evidence.


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Do HLA-DRab1*0402 Molecules Present Different Epitopes to CD4+ T Cells in vivo? Using HCgp-39 as the model antigen, immunisation of HLA-DR4 transgenic lines expressing DRab1*0402 revealed that this molecule, which differs from DRab1*0401 by only 4 amino acids, presents quite distinct sets of immunodominant peptides of HCgp-39 to CD4+ T cells in vivo [16]. As observed for DRab1*0401 epitopes, these peptides did not carry the expected charged residues at P4. Indeed, the sequence of peptide 298–313, which accounted for ?50% of DRab1*0402-restricted T cell responses, carries a prototypic DRab1*0401 motif, and, using the algorithm of Hammer et al. [26], would have been predicted to be one of the major immunodominant DRab1*0401 epitopes. This raises important questions regarding the selection of epitopes for presentation by DRab1*0401 and *0402 molecules, under circumstances where neither the binding, nor the motif of immunogenic epitopes can adequately account for the differences. The answer is still not known, but preliminary data would suggest that for most of the immunodominant epitopes HLA-DM and pH dependence for peptide dissociation from DRab1*0401 and *0402 may be important determinants of the stability and half-life of such complexes, and influence substantially whether such complexes reach the cell surface for presentation to T cells [Hall and Sønderstrup, unpubl. data]. These data provided clear evidence that non-associated alleles of HLADRB1 can profoundly influence the molecular nature of the interactions between TCR and peptide/MHC complexes. Other possible mechanisms have been proposed. For example, Zanelli et al. [81, 82] favour a model, based on the different contributions of I-A (susceptible) and I-E (non-susceptible) to collagen-induced arthritis, where HLA-DQ presents arthritogenic peptides to T cells, and HLA-DR influences the repertoire of responding T cells. According to this intriguing model, disease protection is based upon the capacity for RA-associated DQ molecules, such as DQA1*0301/ DQB1*0302, to present DRb chain-derived peptides. In the context of DRab1*0401 and *0402, it was shown that HLA-DQA1*0301/DQB1*0302 could present peptides derived from DRb1*0402 but not *0401 molecules [82]. The model proposes that lack of responses to DRab1*0401 peptides might influence thymic-positive selection of protective Th2 clones. Using HLA-DR¶DQ double transgenic mice it should now be possible to explore this model further. Is the Frequency of Anti-Self-Reactive T Cells Different in HLA-DRab1*0402 Transgenic Mice after Immunisation with Cognate Antigen? This is not an easy question to address, especially as the nature of stimulating antigen could influence specific reactivity. However, a simple quantitative compar-

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ison of the proportion of responses of T cell hybridomas to HLA-DR4-expressing APC in the absence of exogenous antigen was undertaken to address this. In DRab1*0401 transgenic mice, it was found that approximately 17.5% of all T cell hybridomas generated from the draining lymph nodes of HCgp-39-immunised mice responded significantly to transgenic spleen or DRab1*0401-expressing EBV-transformed B cells in the absence of antigen. For DRab1*0402-restricted T cell responses, the frequency of anti-self-reactivity was 9.5% [Cope and Sønderstrup, unpubl. data]. Since MHC recognition is an intrinsic property of TCR genes, regardless of the genotype, the finding of significant cross-reactivity in DRab1*0402 mice at the outset of the response is not surprising at all. Nevertheless, there is certainly a suggestion that differences could exist. However, the readout for cross-reactivity used here is rather crude, and so the precise significance of these differences needs further evaluation. A more detailed kinetic analysis of cross-reactivity studied in both transgenic lines over longer periods of time might provide a more definitive answer. Since collagen-induced arthritis can be induced in DRab1*0401 transgenic mice [6, 7], an alternative approach would be to compare arthritis susceptibility in the two transgenic lines using a variety of self antigens. In these experiments, B cell responses could also be examined in depth. Is the Cytokine Effector Response Different for HLA-DRab1*0402-Restricted T Cells? The effects of HLA-DRab1*0402 on this particular checkpoint were addressed by comparing cytokine expression of draining lymph node T cells from DRab1*0401 and *0402 mice immunised with HCgp-39, following restimulation with native antigen or the relevant pool of immunodominant epitopes. The results were interesting in several respects. While proliferative responses were no different for DRab1*0401- and *0402-restricted responses, HCgp-39-specific lymph node T cells from DRab1*0402 transgenic mice produced significantly less IFNc [16]. On the other hand, DRab1*0402 T cells specific for OspA could make abundant IFNc, indicating that DRab1*0402 mice do not have an intrinsic defect of IFNc production. These data provided the first real clues that one of the consequences of DRab1*0402 presenting different peptides is to induce a very different profile of proinflammatory cytokine production. The finding of substantially reduced TNFa levels in DRab1*0402 cultures is also consistent with this model [16]. According to this data, the proinflammatory cascade might be attenuated in the non-susceptible host carrying genes not associated with RA. Are There Differences in Immunoregulation of the T Cell Response? The definitive answer to this question is not known. However, there are some suggestions from the results of experiments outlined above, which suggest that


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DRab1*0402 responses may differ in this respect. Firstly, chronic as well as short-term stimulation of DRab1*0402 T cells led to dramatic reductions in IFNc production. No obvious upregulation of Th2 cytokines such as IL-4 and IL-10 was observed, indicating that this was, rather refreshingly, unlike any conventional Th1 to Th2 switch. Indeed, a complete lack of TNF in DRab1*0402 cultures was also noted, suggesting that a factor or factors are expressed in DRab1*0402 cultures which can attenuate these responses. Absence of TNF could be predicted to have several downstream consequences. On the one hand, it could uncouple the proinflammatory cytokine cascade. Potentially deleterious immunosuppressive effects of chronic TNF exposure on T cell function would also be attenuated. These functional differences are significant, and may be important, and are currently under intense investigation. This preliminary comparison of the function of T cells from DRab1*0401 and DRab1*0402 transgenic mice suggests that RA-non-associated HLA-DR4 molecules could contribute to the non-susceptible phenotype by more than one mechanism, including presentation of different sets of peptides to T cells, influencing the frequency of autoreactive T cells, and, through mechanisms that are yet to be defined, downregulating the production of proinflammatory cytokines.

Future Directions A new generation of mouse models of spontaneous arthritis have opened up new approaches to address some important old questions. It is clear that, for the reasons discussed above, therapeutic blockade of T cell autoreactivity could significantly impair host immunity since protective immunity is shaped on T cell responses to self-peptide/self-MHC molecules. Accordingly, exploring precisely how self-reactivity is kept in check, specifically under experimental conditions that resemble the non-susceptible host, has to be a priority. Another priority will be to understand how systemic autoreactivity leads to jointspecific autoimmunity. The KRN transgenic mouse model is an ideal vehicle for studying this rigorously. The precise pathogenic role and molecular characterisation of immune complexes can also be evaluated in this model. I predict that this knowledge will contribute significantly to our understanding of disease pathogenesis, and could well influence future prospects for therapy. On reflection, the promiscuity of MHC/peptide-TCR interactions suggests that peptidedirected immunotherapy could be more effective than previously supposed, since the target T cell population is likely to be far broader than was initially realised. By employing targeted therapeutic approaches such as this, perhaps in combination with agents such as TNF blockade, resetting the immune system to the benefit of patients could become a reality.

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Acknowledgments The author wishes to acknowledge the McDevitt laboratory where work presented here was carried out. Special thanks to Diane Mathis for communicating unpublished results, and to Thomas Kamradt for critical review of the manuscript. The author is a Wellcome Trust Senior Fellow in Clinical Science.

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Andrew P. Cope, The Kennedy Institute of Rheumatology, 1, Aspenlea Road, Hammersmith, London W6 8LH (UK) Tel. +44 181 383 4444, Fax +44 181 383 4499, E-Mail [email protected]


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Molecular Mimicry and Lyme Arthritis Dawn Gross, Brigitte T. Huber, Allen C. Steere Division of Rheumatology/Immunology (Medicine) and the Department of Pathology, Tufts University School of Medicine, New England Medical Center, Boston, Mass., USA

Lyme arthritis was recognized as a separate entity in 1976 because of geographic clustering of children in Lyme, Conn., who were thought to have juvenile rheumatoid arthritis [1]. It soon became apparent that Lyme disease was a complex, multisystem illness that affects primarily the skin, joints, nervous system and heart [2]. Lyme disease or Lyme borreliosis is now known to be caused by the tick-borne spirochete Borrelia burgdorferi sensu lato [3]. It is the most common vector-borne disease in the United States [4], and is also endemic in Europe and Asia. Arthritis is a prominent late manifestation of Lyme disease, particularly in the United States [5]. Although Lyme arthritis can usually be treated successfully with antibiotic therapy [6, 7], about 10% of patients have persistent arthritis for months or even several years after antibiotic treatment [8]. We have termed this disease course antibiotic treatment-resistant Lyme arthritis. In our experience, such patients have no detectable spirochetal DNA in synovial tissue or synovial fluid after antibiotic therapy [9, 10], suggesting that joint inflammation may persist after the apparent eradication of the spirochete from the joint. We propose a model of molecular mimicry affecting genetically susceptible individuals to explain this treatment-resistant course. The majority of patients with treatment-resistant Lyme arthritis have HLA-DRB1*0401, 0404, 0101 and 0102 alleles [11], the same alleles associated with the severity of rheumatoid arthritis [12–16]. In addition, the severity and duration of Lyme arthritis correlate with the cellular and humoral immune responses to outer surface protein A (OspA) of the spirochete [17–21]. Recently, we postulated that the immunodominant epitope of OspA presented in the context of the DRB1*0401 molecule may cross-react with human lymphocyte-function-associated antigen-1 (hLFA-1) [22], an adhesion molecule on T cells. We then showed that

synovial fluid T cells from 10 of 11 patients with treatment-resistant Lyme arthritis responded to spirochetal OspA or hLFA-1, and in most instances to both, whereas T cells from patients with other types of arthritis did not [22]. Molecular mimicry, leading to autoimmunity in the joint, would explain the persistence of joint inflammation in Lyme arthritis after the apparent eradication of the spirochete from the joint with antibiotic therapy.

Causative Agent B. burgdorferi sensu lato encompasses the three pathogenic species of Borrelia known to cause human Lyme borreliosis [23–25]. To date, all North American isolates have belonged to the first group, B. burgdorferi sensu stricto. All three groups have been found in Europe, but most isolates there have been group 2, Borrelia garinii, or group 3, Borrelia afzelii. Only the latter two groups have been found in Asia. These differences presumably account for certain regional variations in the clinical picture of Lyme borreliosis. The complete genome of a prototypic B. burgdorferi strain (B31) was recently sequenced [26]. The total genome was small (approximately 1.5 Mb); it included a linear chromosome of 950 kB along with 9 circular and 12 linear plasmids, which made up 40% of the genome. The organism had a minimal number of proteins with biosynthetic activity, and apparently depends on the host for much of its nutritional requirements. The remarkable aspect of the B. burgdorferi genome was the large number of sequences for predicted or known lipoproteins, inluding OspA–F. Some of these proteins are differentially expressed, and presumably help the spirochete to survive in markedly different arthropod and mammalian environments. They surely present a complex puzzle for the host immune system.

Disease Vector Lyme borreliosis is transmmitted by ticks of the Ixodes ricinus complex [27]. These ticks have larval, nymphal and adult stages. In the northeastern and midwestern United States, the tick vector is called Ixodes scapularis. The preferred host for the larval and nymphal stages of I. scapularis in these regions is white-footed mice, Peromyscus leucopus [28], and the preferred host for the adult stage is deer, Odocoileus virginianus [29]. The life cycle of the spirochete depends on horizontal transmission: from infected nymphs to mice in early summer, and in late summer from infected mice to larvae, which then molt to infected nymphs that begin the cycle again the following year [30].

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The infection is transmitted to humans primarily by nymphal ticks when they quest in May through July. Although adult ticks are often infected with the spirochete, they transmit the infection less frequently when they feed in the early fall. Prior to feeding, the spirochetes are located in the midgut of the tick [31]. Once the tick attaches to a host, the organisms undergo rapid growth, with a doubling time of nearly 4 h [32]. After approximately 24–48 h of attachment, the spirochetes begin to migrate to the tick’s salivary glands from which they are injected into the mammalian host. Thus, the tick must remain attached for 24–48 h for transmission to occur [33].

Clinical Picture After an incubation period of 3–32 days, the illness usually begins in summer with a characteristic expanding skin lesion, erythema migrans (stage 1), that occurs at the site of the tick bite [3, 34]. Within several days to weeks (stage 2), the spirochete may spread to many other sites, including to other skin sites, the nervous system, heart or joints. Months after the onset of illness (stage 3), within the context of strong cellular and humoral immune responses to B. burgdorferi, about 60% of untreated patients in the United States begin to experience intermittent attacks of joint swelling and pain, primarily in large joints, especially the knee [5]. However, both large and small joints may be affected, usually one or two joints at a time. Affected knees are commonly more swollen than painful. Attacks of arthritis generally last from a few weeks to months separated by periods of complete remission. The total number of patients who continue to have recurrent attacks of arthritis decreases by about 10–20% each year [5]. However, attacks of knee swelling sometimes become longer during the 2nd or 3rd year of illness. It is usually during this period that aproximately 10% of untreated patients develop chronic arthritis, defined as 1 year or more of continuous joint inflammation. The synovial histology in these patients, which shows synovial hypertrophy, vascular proliferation, fibrin deposition, and a heavy infiltration of mononuclear cells, is typical of that found in all of the chronic inflammatory arthritides, including rheumatoid arthritis [35, 36]. However, in Lyme arthritis, tests for rheumatoid factor and antinuclear antibodies are usually negative.

Antibiotic Treatment-Resistant Lyme Arthritis Most patients with Lyme arthritis can be treated successfully with appropriate antibiotic therapy [6, 7]. However, about 10% of patients have persistent

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arthritis, typically affecting one or both knees, for months to even several years after 2 months or more of oral doxycycline or 1 month or more of intravenous ceftriaxone therapy [8]. Although B. burgdorferi DNA can usually be detected in joint fluid by polymerase chain reaction prior to antibiotic therapy [9, 37], in our experience, the polymerase chain reaction results are negative in synovium and synovial fluid after antibiotic treatment of this duration [9, 10]. This suggests that Lyme arthritis may persist in certain individuals after the apparent eradication of the spirochete from the joint with antibiotic therapy. Thus, the question arises: why does arthritis persist in these patients after the causative agent has been eradicated?

HLA Association with Autoimmune Diseases Major histocompatibility complex (MHC) class II molecules play a critical role in the activation of the immune system. First, polymorphic amino acid residues on distinct class II proteins determine whether or not an individual peptide is bound and presented by a particular class II molecule displayed on an antigen-presenting cell (APC). Second, class II molecules regulate the developmental selection of T cell receptor (TCR) specificities in the thymus, thereby affecting the repertoire of T cells available to recognize foreign peptides. Given the fundamental role of MHC class II molecules in the development of an immune response, it is not surprising that they have been implicated in numerous forms of immune dysfunction, including autoimmunity [38]. The first indication that antibiotic treatment-resistant Lyme arthritis might have an autoimmune pathogenesis was a study of human lymphocyte antigen (HLA) alleles in patients with Lyme arthritis of brief, moderate or chronic duration. An increased frequency of the HLA-DR4 specificity, determined by serologic typing methods, was found in the patients with chronic arthritis [39]. In addition, the HLA-DR4 specificity was associated with a lack of response to antibiotic therapy. More recently, using contemporary molecular techniques, the majority of patients with chronic, treatmentresistant Lyme arthritis had HLA-DRB1*0401, 0404, 0101 and 0102 alleles [11]. These alleles, which have a similar shared sequence in the third hypervariable region of the HLA-DRB1 chain, are associated with the severity of rheumatoid arthritis [12–16]. This sequence may be found in at least 15 different DRB1 alleles. In rheumatoid arthritis, the cause of the disease and the critical antigen peptides are not known. However, in Lyme arthritis, it is possible to ask the question: what antigens are these class II molecules presenting?


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Treatment-Resistant Lyme Arthritis and Immunity to OspA Patients with untreated Lyme disease have a complex immune response to an increasing array of spirochetal proteins over a period of months to years [19, 40]. A number of membrane lipoproteins are differentially expressed, and presumably help the spirochete to survive in markedly different arthropod and mammalian environments as well as in different tissues in the mammalian host. OspA and OspB are expressed primarily in the midgut of the tick [31, 41]. These two closely related proteins are encoded by the same plasmid and share 56% sequence homology [42]. As the tick feeds and spirochetes migrate to the tick’s salivary glands, OspA and OspB are downregulated, and OspC is upregulated [43]. Even so, in some patients, an ephemeral antibody response may be found to OspA early in the infection, primarily of the IgM isotype [18]. Months to years later, approximately 70% of patients with Lyme arthritis develop an IgG antibody response to OspA and OspB, usually after several brief attacks of arthritis, near the beginning of prolonged episodes of arthritis [17, 19]. Furthermore, among patients with the HLA-DR4 specificity, the risk of treatment-resistant Lyme arthritis doubles when they generate an antiOspA antibody response [8]. The association of class II MHC molecules with treatment-resistant Lyme arthritis suggests that T cell reactivity is likely to be important in the persistence of synovial inflammation. As with antibody responses, T cell reactivity in Lyme disease is directed against multiple spirochetal antigens [44–46]. In addition, the response of synovial fluid lymphocytes to spirochetal antigens is generally greater than that of peripheral blood lymphocytes, presumably because of the concentration of antigen-activated cells in the inflamed joint. Antigen-specific synovial fluid lymphocytes and peripheral blood lymphocytes from Lyme arthritis patients secrete primarily the proinflammatory T helper (Th) 1 cytokine, IFN-c [21, 47–49]. In a study of 10 patients with Lyme arthritis, the severity of joint swelling correlated directly with the ratio of Th1 to Th2 cells in synovial fluid, such that the larger the effusion, the higher the ratio (fig. 1a) [49]. Moreover, patients with other forms of chronic inflammatory arthritis also showed a dominant Th1 response localized to synovial fluid (fig. 1b). Th1 cells also dominate in the local immune response in the synovial tissue of patients with Lyme arthritis [50, 51], as in patients with rheumatoid arthritis or other chronic inflammatory arthritides [47, 49]. We have recently shown that it is Th1 responses to OspA, and to a lesser degree to OspB, which correlate with the severity and duration of Lyme arthritis [21]. T cell lines from patients with treatment-resistant Lyme arthritis preferentially recognize OspA, whereas T cell lines from patients with treatment-responsive disease rarely have such reactivity [20]. Finally, OspA-reactive

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a

b Fig. 1. Th1 and Th2 profiles of mononuclear cells in peripheral blood (PBMC) and synovial fluid (SF) of patients with Lyme arthritis or other chronic inflammatory arthritides. The cells were pulsed with PMA/ionomycin plus menensin for 6 h, followed by intracellular cytokine staining and FACS analysis. a Pulsed CD4+ T cells from patients with Lyme arthritis showed a dominant Th1 response localized to SF. The SF-Th1:Th2 ratio correlated directly with the severity of the arthritis, as determined by the degree of knee swelling (r>0.67, p=0.05). b PMA/ionomycin-pulsed CD4+ T cells from patients with other types of chronic inflammatory arthritis also showed a dominant Th1 response localized to SF. Unpulsed PBMC and SF had background intracellular cytokine staining levels of =2% (data not shown). RA>Rheumatoid arthritis [from 49, with permission].


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Th1 cells may be detected in the synovial fluid of patients with treatmentresistant arthritis months to years after antibiotic treatment (fig. 2a), but not in the synovial fluid of patients with other chronic inflammatory arthritides (fig. 2b) [21, 49]. In initial epitope mapping studies, patients with Lyme arthritis recognized multiple epitopes of OspA [52, 53]. In addition, patients with treatment-resistant arthritis had OspA reactivity more frequently and recognized more epitopes of OspA than those with treatment-responsive arthritis [21]. Of 16 patients with treatment-resistant arthritis, 14 had responses to OspA peptides (usually 4 or 5 epitopes), whereas only 5 of the 16 patients with treatment-responsive arthritis had reactivity with these peptides (usually 1 or 2 epitopes). Patients with HLADRB1 alleles associated with treatment-resistant Lyme arthritis were more likely to react with an OspA peptide containing aa 154–173 and, to a lesser degree, with a peptide containing aa 214–233 than were patients with other alleles. However, from the study of patients, it was difficult to be certain of the immunodominant epitope of OspA presented in the context of the DRB1*0401 molecule.

LFA-1 as a Candidate Autoantigen in Treatment-Resistant Lyme Arthritis Using an algorithm designed by Hammer et al. [54], which predicts epitopes presented by DRB1*0401, the immunodominant epitope of OspA was predicted to be OspA165–173, with a secondary epitope at OspA237–244 [22]. These predictions were verified using murine class II –/– mice transgenic for a chimeric DRB1*0401 molecule capable of interacting with murine CD4 [22]. A search of the Genetics Computer Group gene bank for human proteins containing homologous sequences to OspA165–173 revealed only one sequence of human origin, LFA-1aL332–340, that was predicted to be presented by DRB1*0401 [22]. This peptide is located extracellularly in the region of LFA-1aL called the ‘interactive’ or ‘I-domain’, which mediates the binding of LFA-1 to its ligand, intracellular adhesion molecule-1 (ICAM-1) [55–58]. No human protein was identified that had sequence homology with the secondary epitope and 0401 binding activity. To test the hypothesis that LFA-1aL332–340 could act as a molecular mimic of OspA175–173, we tested patients with treatment-resistant Lyme arthritis for T cell reactivity with OspA, LFA-1 and the predicted immunodominant peptides (fig. 3). Of the 11 patients tested, 10 had Th1 responses in synovial fluid to OspA, LFA-1, or in most instances, with both, whereas patients with other forms of chronic inflammatory arthritis who had DRB1*0401 alleles did not respond to these proteins (fig. 3) [22]. This shows the need for exposure to B. burgdorferi to trigger the response.

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a

b Fig. 2. The Th1 and Th2 responses of OspA-specific T cells in peripheral blood (PBMC) or synovial fluid (SF) in patients with Lyme arthritis or other chronic inflammatory arthritides. The cells were stimulated with OspA for 48 h and then pulsed with anti-human CD3 and antihuman CD28 antibody plus mononesin for 5 h. Cells were then processed for intracellular cytokine staining and FACS analysis. a After restimulation with OspA at 48 h, a dominant Th1 response was seen in patients with Lyme arthritis, localized to SF. b 48 h restimulation with OspA showed no activation of SF cells in patients with other chronic inflammatory arthritides. Cells pulsed only with anti-hCD3 and anti-hCD28 for 5 h had background cytokine staining levels of =2% (data not shown). RA>Rheumatoid arthritis [from 49, with permission].


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Fig. 3. Responses of synovial fluid T cells to OspA or hLFA-1 in patients with treatmentresistant Lyme arthritis or other chronic inflammatory arthritides. 3¶105 synovial fluid cells were cultured with anti-CD3 hybridoma OKT3 supernatant, OspA (10 lg/ml), OspA164–173 (10 lg/ml) or hLFA-1 (70 ng/ml), or medium alone for 24 h, and reactivity was determined using an ElisaSpot assay. Values from medium alone were subtracted from those in wells containing antigen. Of the 11 patients with treatment-resistant Lyme arthritis, 10 had synovial fluid T cell reactivity with OspA or hLFA-1, or in most instances, with both, whereas patients with other chronic inflammatory arthritides did not. Due to technical limitations relating to the purification process, the concentration of hLFA-1 was 3 logs lower than the optimal concentration for OspA. When equimolar amounts of OspA and hLFA-1 were tested, reactivity with OspA was comparable to those for hLFA-1 (data not shown). RA>Rheumatoid arthritis [from 22, with permission].

Variations in OspA Sequences in European and Asian Strains In the United States, all pathogenic isolates of B. burgdorferi sensu lato, to date, have been a single species, B. burgdorferi sensu stricto [23], whereas B. garinii and B. afzelii most commonly cause this infection in Europe [23–25]. When we compared the sequences of the immunodominant epitope of OspA

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Table 1. Variations in the sequences of the immunodominant epitope of OspA among the 3 pathogenic species of B. burgdorferi sensu lato and the predicted binding of these sequences to the DR4 molecule Sequences

Total DR4-binding score

B. burgdorferi OspA165–173 Y V L E G T L T A Residue scores 2.1 1.0 0.4 0.5 0

(+) 6.5

#

F/L A/T L E G T L T A 0/–1 a 0/0 a

(+) 4.4/3.4

B. afzelii OspA Residue scores

F T L E G K V A N –2.3 0.5 0 –1.1

(–) 1.3

hLFA-1aL332–340 Residue scores

Y V I E G T S K Q 1.5 –0.4 0.9 0.7

(+) 7.3

B. garinii OspA160–168 Residue scores 165–173

The individual residue score and total scores were determined by the DR4 algorithm [54]. Scores are listed for aa residues disparate between peptides. a Variation in the first two aa of this 9mer is seen among a number of isolates; therefore, both are listed along with their respective binding scores.

among the 3 pathogenic species of B. burgdorferi sensu lato, we found that several residues are disparate (table 1). According to the prediction of the Hammer algorithm, the ability of the 0401 molecule to bind this epitope of B. garinii or B. afzelii is significantly less than that of B. burgdorferi or its molecular mimic, hLFA-1aL332–340 (table 1). These species-specific sequence differences may explain why treatment-resistant Lyme arthritis has only been reported with B. burgdorferi sensu stricto infection.

The Pathogenic Model We propose the following model to explain how an autoimmune response to LFA-1 may develop as a result of infection with B. burgdorferi (fig. 4). According to this hypothesis, B. burgdorferi, which downregulates OspA as it leaves the midgut of the tick, enters the host via a tick bite and disseminates to multipe tissues, including joints. Months later, an inflammatory response develops in joints dominated by Th1 IFN-c-producing cells [21, 47–49]. After several brief attacks of arthritis, the spirochete often expresses OspA again, accompanied by the recruitment of OspA-reactive T cells to affected joints [20, 21, 49].


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a

b

Fig. 4. Pathogenetic hypothesis to explain treatment-resistant Lyme arthritis. a OspAspecific T cells respond to OspA presented by macrophages and other APCs. This T cell response is aided by adhesion molecule interactions, including LFA-1 and ICAM-1, and by cytokines circulating in the localized inflammatory milieu of the joint. b Later, T cells that had been stimulated and expanded due to their recognition of a particular region of spirochetal OspA beging to respond to a nearly identical portion of hLFA-1. The combination of elevated LFA-1 expression on T cells and macrophages, as well as upregulation of class II MHC molecules on APCs may result in increased LFA-1 peptide presentation by macrophages and synoviocytes, which have processed either endogenous and/or phagocytosed LFA-1. In this way, T cell-supported inflammation may persist within the joint, even after the apparent eradication of the spirochete with antibiotic therapy.

Within the proinflammatory milieu of the joint, the high local concentration of IFN-c results in the upregulation of LFA-1 and its ligand ICAM-1 [55, 56, 59–65]. IFA-1 is most abundant on T and B cells, with highest expression on activated T lymphoblasts [56, 59–61]. Stimulation with IFN-c or lipopolysaccharide also induces LFA-1 expression on macrophages [56, 59, 60]. In addition, stimulation by IFN-c or B. burgdorferi upregulates the expression of ICAM-1 on synoviocytes, synovial fibroblasts, and endothelial cells [55, 56]. Moreover, IFN-c results in the increased expression of MHC class II molecules on local, professional and nonprofessional APCs [66]. This upregulation not only facilitates the margination and extravasation of T cells to the site of inflammation, but also promotes the adherence of localized T cells to connective tissue cells, thereby increasing the efficiency of T cell functions [56, 59, 60]. Enhanced antigen presentation, including class II presentation of endogenous self peptides, may occur during a chronic immune response [67, 68]. We speculate that the combination of elevated LFA-1 expression on T cells and macrophages, as well as upregulation of MHC class II molecules on APCs results in increased LFA-1 peptide presentation by macrophages and synoviocytes, which have processed either endogenous and/or phagocytosed

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LFA-1 [67, 68]. Hence, even after the elimination of spirochetes by antibiotic therapy, the OspA-primed T cells remain activated by stimulation with the cross-reactive epitope of LFA-1. The continued release of proinflammatory cytokines by these activated T cells and macrophages may result in tissue damage and joint destruction [69]. Several caveats should be stressed regarding the OspA/hLFA-1 hypothesis. First, some patients, even those with the appropriate genetic susceptibility, may not develop treatment-resistant arthritis because the spirochete may not upregulate OspA expression in the joint or because not enough time elapses prior to treatment for the development of this pathogenic response. Second, within this proinflammatory milieu, it is possible that other self peptides with less sequence homology than OspA and hLFA-1 may be stimulatory, even more stimulatory than hLFA-1. Thus, hLFA-1 may not be the only relevant autoantigen in treatment-resistant Lyme arthritis. Finally, other factors, such as genetically determined differences in cytokine responses, surely play a role. For example, patients who produce a dominant, proinflammatory response in the joint may be more likely to break tolerance, and they may be less likely to downregulate this response after the apparent eradication of the spirochete from the joint with antibiotic therapy.

Animal Models of Lyme Arthritis Animal models of Lyme disease have been developed in mice [70], hamsters [71], dogs [72] and nonhuman primates [73]. Particularly important has been the murine model of Lyme disease in C3H/HeJ mice, which develop severe, acute arthritis 2–4 weeks after inoculation with B. burgdorferi [70]. Despite the fact that spirochetes persist throughout the life of this inbred mouse strain, the arthritis resolves. Thus, this murine strain does not develop the equivalent of human, antibiotic-treatment-resistant arthritis in which the reverse scenario occurs: the arthritis persists despite the apparent eradication of the spirochete with antibiotic therapy. Comparison of infection in different inbred strains has shown the importance of regulation of the immune response in determining the severity of the arthritis [74–76]. C3H/HeJ mice develop severe arthritis when infected with B. burgdorferi, whereas BALB/c mice develop only mild arthritis. C3H/HeJ mice produce IFN-c (Th1 response) when infected with the spirochete, but still have a large burden of spirochetes in joints. In comparison, B. burgdorferiinfected BALB/c mice initially produce even higher levels of IFN-c, but they switch to IL-4 production (Th2 response). Thus, BALB/c mice appear to reduce the numbers of spirochetes initially with a Th1 response, and then


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dampen the inflammatory response of the subsequent arthritis with a Th2 response. Hamsters also develop only mild arthritis when infected with B. burgdorferi. However, after vaccination with formalin-inactivated spirochetes in adjuvant or rOspA vaccine, hamsters develop severe destructive arthritis if challenged with live B. burgdorferi sensu stricto before or after they have fully developed protective immunity [71, 77]. In addition, T cells taken from the lymph nodes of vaccinated hamsters confer susceptibility to severe destructive arthritis when they are transferred into naive hamsters and then challenged with viable B. burgdorferi. This is not true when naive recipients are infused with either normal T cells and challenged with viable spirochetes, or when they are infused with primed T cells and challenged with dead B. burgdorferi. These results suggest that in hamsters, immunization with OspA may amplify an arthritogenic T cell population which may be activated by B. burgdorferi infection. DR4-tg mice are not susceptible to treatment-resistant Lyme arthritis [78]. This is presumably because the sequence of hLFA-1aL332–340 has subtle differences from that of murine LFA-1aL332–340. Specifically, the p2 valine residue present in hLFA-1aL332–340 and OspA165–173 is altered in murine LFA-1aL332–340 to an alanine. Since p2 is predicted to influence the TCR contact [79], rather than MHC-peptide interaction, cross-reactivity may not develop between OspA and murine LFA, not as a result of insufficient DR4 binding, but rather as a result of distinct MHC-peptide-TCR interactions. Thus, to test the molecular mimicry hypothesis in a murine model, the mice will need to express not only human DRB1*0401, but also hLFA-1.

Implications beyond Lyme Disease These studies have implications beond Lyme disease. Before Lyme arthritis was described, patients with Lyme arthritis were often diagnosed with juvenile rheumatoid arthritis or rheumatoid arthritis [1], which are thought to be autoimmune diseases. After the clinical characterization of Lyme arthritis, the causative spirochetal agent of Lyme disease was identified proving the infectious etiology of the illness [80, 81]. With the implication of a specific immunogenetic susceptibility, spirochetal antigen and human autoantigen in treatmentresistant Lyme arthritis [22], a more complex picture emerges in which a spirochetal infection of the joints may trigger tissue-specific autoimmunity in genetically susceptible patients with the most severe and prolonged disease. This is the only form of human chronic inflammatory arthritis in which these factors are known.

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The emerging information about molecular mimicry in Lyme arthritis suggests that a very specific mechanism – cross-reactivity between a T cell epitope of OspA and hLFA-1 – may be the reason for the association of treatment-resistant Lyme arthritis with particular class II MHC alleles. Moreover, the immunogenetic susceptibility in treatment-resistant Lyme arthritis includes alleles associated with rheumatoid arthritis. Although the exact mechanism is surely different in rheumatoid arthritis, it will be important to determine why these two forms of chronic inflammatory arthritis share a similar immunogenetic susceptibility. It will also be important to learn whether the general mechanism presented here – infection-induced autoimmunity – has applicability to other chronic inflammatory arthritides.

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Croke CL, Munson EL, Lovrich SD, Christopherson JA, Remington MC, England DM, Callister SM, Schell RF: Occurrence of severe destructive Lyme arthritis in hamsters vaccinated with outer surface protein A and challenged with Borrelia burgdorferi. Infect Immun 2000;68;658–663. Feng S, Barthold SW, Bockenstedt LK, Zaller DM, Fikrig E: Lyme disease in human DR4Dw4transgenic mice. J Infect Dis 1995;172:286–289. Dessen A, Lawrence CM, Cupo S, Zaller DM, Wiley DC: X-ray crystal structure of HLA-DR4 (DRA*0101, DRB1*0401) complexed with a peptide from human collagen II. Immunity 1997;7: 473–481. Burgdorfer W, Barbour AG, Hayes SF, Benach JL, Grunwaldt E, Davis JP: Lyme disease – A tickborne spirochetosis? Science 1982;216:1317–1319. Steere AC, Grodzicki RL, Kornblatt AN, Craft JE, Barbour AG, Burgdorfer W, Schmid GP, Johnson E, Malawista SE: The spirochetal etiology of Lyme disease. N Engl J Med 1983;308: 733–740.

Dr. Allen C. Steere, New England Medical Center, #406, 750 Washington St., Boston MA 0211 (USA) Tel. +1 617 636 5951, Fax +1 617 636 4252, E-Mail [email protected]


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T Cell Homeostasis and Autoreactivity in Rheumatoid Arthritis Jo¨rg J. Goronzy, Cornelia M. Weyand Departments of Medicine/Rheumatology, and Immunology, Mayo Clinic and Foundation, Rochester, Minn., USA

Introduction Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects the synovial membrane and causes irreversible joint damage. The prevailing pathogenetic model of RA assumes an autoimmune mechanism. Considerable evidence exists that the disease is mediated or at least regulated by abnormal T cell responses. The most important and, so far, the only universally implicated disease risk genes are allelic polymorphisms of the HLADRB1 genes that serve as ligands for the T cell receptor (TCR) of CD4 T cells [1–3]. CD4 T cells are a major component of the inflammatory infiltrate in the rheumatoid synovium. In many patients, CD4 T cells and B cells in the synovial infiltrates are organized into lymphoid structures that resemble germinal centers [4, 5]. Clonally expanded T cell populations are easily detectable within synovial lesions and identical TCR sequences have been identified in distinct joints of the same patient, strongly suggesting that CD4 T cells recognize antigens in the synovium [6, 7]. In spite of this unequivocal evidence of antigen-driven T cell responses, it appears increasingly unlikely that RA is an antigen-specific autoimmune disease. In the strictest sense, autoimmunity is defined as the loss of tolerance and the generation of an adaptive immune response to an autoantigen. Such a single autoantigen has not been identified in RA. In contrast, recent data suggest that recognition of a multitude of self-antigens may be involved in the disease process. RA patients have global abnormalities in the peripheral T cell repertoire that are associated with increased T cell turnover and a contraction of repertoire diversity. These aberrations may not only reflect the recognition of multiple selfantigens but by themselves are likely to have a negative impact on peripheral

tolerance mechanisms [8]. The distortions in the global T cell repertoire have a clinical correlate in the multi-organ involvement of RA. In this model, the clinical dominance of joint manifestations in many RA patients would reflect defective tolerance mechanism and/or increased antigen presentation in the synovium rather than the presence of a joint-specific antigen. This review will summarize the qualitative and quantitative aberrations in the RA T cell compartment and discuss the models that may explain these repertoire abnormalities.

RA – A Disease of Systemic Autoimmunity with Predominant Joint Involvement RA differs from organ-specific autoimmune diseases, such as autoimmune thyroiditis or myasthenia gravis, in which there is evidence for a breakdown of tolerance to a single organ-specific autoantigen. The inflammation in RA targets the synovium, a poorly defined organ of fibroblast-like and macrophage-like synoviocytes. Cartilage is only affected if directly adjacent to the synovium, and cartilage destruction appears to be a consequence of synovial proliferation and cytokine production rather than of antigen-specific effector mechanisms such as autoantibodies and cytotoxic T cells. More importantly, the inflammatory manifestations of RA are not limited to the diarthrodial joints but can involve many different organ systems. Tenosynovitis and pleural/ pericardial inflammation are general features of the disease, even if they are not clinically apparent. Subcutaneous nodule formulation occurs in about one-third of RA patients; inflammation of the lacrimal glands leading to the sicca syndrome is commonly found in female patients. Even major organ involvement is not infrequent, largely accounting for the increased mortality in the RA. Interstitial lung disease, inflammatory blood vessel disease and involvement of the bone marrow can be part of the disease. Thus, RA is truly a systemic autoimmune disease, the manifestations of which are most evident in the unique environment of the synovium [8]. It is this systemic nature of RA that requires an explanation in terms how T cell responses could be dysregulated. Faultiness in mechanisms controlling central and/or peripheral T cell selection could provide an appealing model to explain the multi-organ targeting of RA [9, 10].

Defective Central Tolerance in Murine Models of RA Examples of T cell repertoire selection predisposing for autoimmune, in particular arthritic, conditions are evident from several murine models. Laufer

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et al. [11] have generated transgenic mice that allowed for positive thymic selection in the absence of negative selection. As a result, the mice developed autoimmune phenomena. Approximately 5% of the peripheral CD4 T cell repertoire were autoreactive, allowing for an estimate of the fraction of the positively selected T cells needed to be deleted to preserve peripheral tolerance. Aberrant repertoire selection as a disease mechanism is not limited to ‘artificial’ genetically engineered mouse models, but has a place in spontaneously occurring autoimmunity, such as in the most widely used autoimmune mouse strains, MRL-lpr and NOD. The MRL-lpr mouse develops, in addition to SLE-like disease manifestations, an erosive rheumatoid factor-positive arthritis that has features of RA. MHC-dependent as well as MHC-independent mechanisms appear to be involved. The NOD mouse is generally considered as a murine model of type I diabetes mellitus. However, a recent TCR transgenic mouse on the NOD background has been shown to develop rapidly progressive joint inflammation. H2-IAg7 mice transgenic for a TCR that recognizes a ubiquitously expressed autoantigen develop erosive synovitis in the absence of major systemic manifestations such as graft-versus-host disease. In both animal models, a defect in central tolerance contributes to the autoimmune phenomena. Manipulation of thymic selection by transferring a TCRb-chain transgene into the MRL background has resulted in the attenuation of all autoimmune manifestations [12]. More recently, Tolosa et al. [13] were able to show that decreasing glucocorticoid signaling in the thymus greatly diminished autoimmunity in MRL-lpr mice. Thymic glucocorticoids impair negative selection, thereby allowing the positive selection of T cells that recognize autoantigens with increased affinity. These data therefore suggest that defective central tolerance contributes to autoimmunity in these mice. Along the same lines, CD4 T cells from the NOD mouse have been described to include a high frequency of autoreactive clones that spontaneously proliferate in response to self-antigens [14]. This defect in negative selection appears to be mainly mediated by the H2-IAg7 molecule that is rather unstable and therefore less effective in forming high-affinity interactions necessary for thymic deletion of autoreactive T cells. In the arthritis model of the NOD mouse, the disease is associated with defective negative selection and the autoreactive TCR transgene is not deleted but initiates the arthritic inflammation [15]. Additional defects in positive selection may exist in the NOD mouse. ThomasVaslin et al. [16] have demonstrated that thymic epithelium of NOD mice transplanted into C57-BL/6 mice selects an autoreactive T cell repertoire that mediates pancreatic inflammation even in the H2-disparate host. The mechanism of this defect appears to lie in positive selection because the medullary thymus was derived from the C57-BL/6 strain and negative selection should have been intact.

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The T Cell Repertoire in RA RA is associated with an HLA-DRB1-encoded peptide sequence that is found in most HLA-DRB1*04 alleles but also some unrelated HLA-DRB1 alleles [1, 2]. In contrast to H2-IAg7, there is no evidence that the HLA-DR molecule has physicochemical properties that would render an HLA-peptide complex unstable and set it apart from other HLA-DRB1 molecules. A defect in negative selection along the same lines as has been proposed for H2-IAg7 is therefore unlikely. Albani et al. [17, 18] have proposed that RA patients have a defect in positive thymic selection. In their molecular mimicry model, RA patients preferentially select CD4 T cells on peptides derived from the disease-associated HLA-DR b-chains. These T cells cross-react on bacteriaderived dnaJ heatshock proteins, acquire memory function, and lead to enhanced autoreactivity. This model provides an elegant explanation for the observation that RA is associated with a peptide sequence that can be found in different HLA-DRB1 alleles rather than with a particular HLA-DR molecule. However, it does not explain the unique position of HLA-DRB1*04 among all disease-associated alleles and, in particular, not the finding that homozygosity of HLA-DRB1*04 alleles is associated with increased disease susceptibility and disease severity [19, 20]. Rather, this gene-dose effect suggests that the disease-associated HLA-DRB1 molecules are directly involved and that homozygosity favors the selection of lower affinity TCR that are relevant in the induction and progression of the disease [3]. While the HLA-DR4 molecule is likely to play an important role in the formation of the T cell repertoire in RA, current data suggest that a model focusing exclusively on the disease-associated HLA polymorphism is too narrow and that MHC-independent mechanisms are relevant as well. In the studies by Albani et al. [17], proliferative responses to the peptide sequence shared between HLA-DRB1 alleles and dnaJ heatshock proteins were found in RA patients but not in HLA-DRB1-matched control donors. Walser-Kuntz et al. [21] compared the repertoire of CD4+CD45RO– naive T cells in RA patients and HLADRB1-matched and unmatched control donors by determining TCR BV-BJ gene segment frequencies (fig. 1). HLA-DRB1*04 influenced the representation of BV-BJ gene segment combinations in a similar pattern in HLA-DRB1*04+ RA patients and HLA-DRB1*04+ control donors that could be distinguished from HLA-DRB1-disparate control donors. However, RA patients shared additional features in their repertoire of naive T cells that clearly distinguished them from HLA-DRB1-matched control donors, suggesting that variables in addition to the HLA-DRB1 allele shape a T cell repertoire characteristic of RA. These studies were done on naive T cells and it is therefore likely that the RA-specific findings reflect thymic or peripheral selection rather than peripheral T cell activa-


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Fig. 1. The TCR repertoire of naive CD4 T cells in RA. CD4+CD45RO– naive T cells were purified from 10 RA patients typing HLA-DRB1*01/04 (C) and from 21 normal donors typing either HLA-DRB1*03/07 (A) or HLA-DRB1*01/04 (B). The frequencies of TCR BV-BJ combinations were determined and the data were analyzed by cluster analysis. The RA patients formed an independent cluster distinct from HLA-DRB1–matched and unmatched healthy donors [reproduced with permission, 21].

tion and expansion of effector cells. They, however, do not allow any conclusions on whether negative selection is leaky as it is in the animal models and whether RA patients have a higher frequency of autoreactive cells.

Clonal Expansion of Autoreactive T Cells in RA Studies of the peripheral T cell repertoire have shown that RA patients frequently carry clonal T cell populations. These clones can expand to an impressive size and make up several percent of circulating lymphocytes, suggesting that the clonal sizes are in the order of 1¶109 to 1¶1010 lymphocytes. DerSimonian et al. [22] have described large clonal expansions of CD8 T cells in the circulation as well as the synovial tissue of RA patients that shared the usage of the TCR-AV12 gene segment. Such CD8 clones can also be found in healthy elderly individuals but they appear to be more frequent in RA

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patients. Clonal expansions of CD4 T cells are rarely observed in healthy individuals but are a common finding in RA patients [23]. It is possible that these clones arise in response to chronic stimulation with antigen. GonzalezQuintial et al. [24] and Rittner et al. [7] have compared the representation of expanded clones in paired samples of synovial tissue and peripheral blood of RA patients. These authors came to the conclusion that the clones are present in the synovial tissue, but are not selectively enriched, suggesting that the antigen is not selectively expressed in the joint. Schmidt et al. [25] have subsequently isolated clonally expanded CD4 T cell clones from the peripheral blood of patients with RA. These clones characteristically lacked the expression of the CD28 molecule and proliferated to autologous monocytes. CD8 T cell clones in RA patient also appear to recognize autoantigens; at least for one of these clones the antigen could be identified. This clone recognized a peptide derived from an HLA-DQ molecule in restriction to MHC class I [26]. Taken together, these data provide evidence that RA patients clonally expand CD4 and CD8 T cells and that at least some of these clones recognize autoantigens that are not specific for the synovium, such as peptides derived from selfMHC molecules. The clonal expansion of such autoreactive T cells could indeed indicate a defect in thymic selection.

T Cell Repertoire Contraction in RA Clonal expansion in response to antigen is one of the inherent features of the immune system and therefore not surprising. Under certain conditions, immunodomination is observed, i.e., a small number of T cells that respond to an immunodominant peptide of an antigen is preferentially expanded in an immune response. The mechanisms of such immunodomination are beginning to be understood [27]. However, immunodominance is usually not associated with such a clonal dominance as is seen in RA. As described above, studies of circulating T cells in RA patients have demonstrated that clonal expansion of autoreactive T cells is a frequent event and that each of these clones can comprise up to several percent of the total T cell repertoire [25, 26, 28]. In contrast, in a normal immune response homeostatic control mechanisms are in place to downsize the clonal expansion and to maintain the diversity of the repertoire. The question therefore arose whether these clonal expansions in RA are isolated events of autoreactive T cell clones that have not been deleted in the thymus or whether they reflect a global imbalance in T cell homeostasis. To address this possibility, Wagner et al. [29] have probed the diversity of the human CD4 T cell repertoire in RA patients and age-matched control individuals.


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In these studies, circulating CD4 T cells were arbitrarily chosen and their TCR b-chains were sequenced. Oligonucleotide probes corresponding to the junctional diversity of the TCR sequence were synthesized and used to determine the frequency of this TCR in the peripheral lymphocytes of the individual by limiting-dilution analysis. The experimental system had exquisite sensitivity and specificity, allowing for frequency determination in the range from 1 in 100 T cells to 1 in 50 million T cells. Healthy individuals displayed a highly diverse repertoire, with a median frequency of individual TCR b-chain sequences of 1 in 2.4¶107 CD4 T cells. In RA patients, the median TCR b-chain frequency was increased 10-fold. From these data, it can be estimated that a minimum of 23% of all CD4 T cells in a healthy individual had replicated, whereas this was increased to 77% in RA patients. This estimate suggested that it was not the minority, but the vast majority if not all, of the CD4 T cells that had contributed to the loss in diversity and the contraction of the repertoire. Patients with RA have a nearly normal ratio of naive-to-memory T cells as concluded from the expression of characteristic cell surface markers such as CD45RO and CD45RA. If nearly all T cells are involved, the loss in TCR diversity could not be limited to CD4 memory T cells but should also include naive T cells. Indeed, this was found to be the case when memory and naive CD4 T cells were separately analyzed. In patients and control donors, the median frequencies of arbitrarily picked TCR b-chain sequences were significantly higher in CD45RO+ (memory) than in CD45RO– (naive) T cells, consistent with the notion that this marker distinguishes naive and memory phenotypes. Compared to the control donors, RA patients had a less diverse repertoire of naive as well as memory cells, suggesting that the contraction in diversity resulted from an abnormality in central or peripheral T cell repertoire formation and was not a consequence of antigen recognition in the synovium (fig. 2). In further support of this interpretation, control patients with an antigen-induced chronic inflammatory disease such as hepatitis C expressed a diverse repertoire. How could repertoire contraction influence an autoimmune process such as RA? The current models of how the T cell repertoire shapes an immune response focus on the events on the single-cell level. T cells with low affinity to MHC/autoantigen complexes are positively selected on thymic epithelium cells because they may be useful in the recognition of exogenous antigen, and T cells with high avidity to self are deleted because they may be harmful [30–32]. In the tradition of Paul Ehrlich’s concept of the ‘horror autotoxicus’ it is assumed that the mere presence of autoreactive T cells is sufficient to create a risk for autorecognition and tissue damage. However, none of T cells in the T cell pool functions as an isolated individual. They are an integrated member of a complex population and

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Fig. 2. Diversity contraction of the naive and the memory T cell repertoire in RA. TCR b-chain sequences were arbitrarily selected from purified CD4+CD45RO+ memory and CD4+CD45RO– naive T cells of RA patients and age-matched control donors. The frequencies of these sequences in the respective T cell subsets were determined in a limiting dilution system using PCR and hybridization oligonucleotides, corresponding to polymorphisms in the N-D-N regions of the selected T cells. Compared to the control donors, RA patients had contracted repertoires of the naive and memory populations [reproduced with permission, 29].

compete for space and resources. Quantitative and dynamic aspects of the pool are instrumental in determining the function of the individual antigenspecific cell and the outcome of immune stimulation. Quantitative aspects of the immune system will affect whether an immune or autoimmune response remains below a critical threshold and is not sustained or gains momentum. It is well established that self-reactive T cells are present in the repertoire of healthy individuals but that control mechanisms are sufficient to keep them in check [33]. The efficacy of these control mechanisms depends on multiple factors, including the clonal frequencies of the responding T cell population. A self-sustaining T cell response is more likely induced if the frequency of the responding T cell population exceeds a certain threshold. In contrast, activa-


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tion of an infrequent T cell specificity should remain an isolated event and rapidly dissipate without consequences. TCR transgenic mice are the extreme example of a contracted T cell repertoire and could therefore represent an excellent experimental system to analyze the impact of repertoire contraction on the efficacy of peripheral tolerance mechanisms. Autoreactive TCR as transgenes are increasingly used to create murine models of autoimmunity. Even in these transgenic models, clinical disease is often not apparent, while infiltrates in the target organ can be demonstrated [34]. However, disease can be rather easily induced and often only requires nonspecific stimuli. It is likely that quantitative aspects (namely that such an autoreactive receptor is present in overwhelming frequencies with little competition from nontransgenic TCR) are at least as important in these models as qualitative aspects (namely that these mice harbor an autoreactive TCR).

T Cell Population Dynamics – Murine Studies The contraction of the T cell repertoire in patients with RA implies a defect in the mechanisms controlling T cell homeostasis that could precede clinical disease onset (as has been shown for the MRL-lpr or MRL-gld mouse or the different human forms of autoimmune lymphoproliferative syndromes) [35, 36]. Equally important, these repertoire perturbations could follow the initial disease manifestations and be pivotal in sustaining disease activity and progression of the inflammatory process. Accumulation of large numbers of presumably autoreactive T cells has been shown in different strains of SLEprone mice with progression of the disease. These cells develop a senescent phenotype, reminiscent of recent findings on T cell dynamics in RA (see next section) [37, 38]. Mechanisms underlying the abnormalities in RA are unknown. Homeostasis of lymphocyte numbers is an extremely complex process and only recently have concepts been developed that help to interpret these findings. It is generally agreed that the global size of the lymphocyte compartment is regulated [39, 40]. This is not unusual for a physiological organ and appears to be a trivial statement. However, the immune system fulfills its function by, at least temporarily, allowing for clonal expansion. Antigen-specific T cells and B cells respond to antigenic stimulation with clonal proliferation. Therefore, homeostatic mechanisms face the challenge of controlling the size of a system that functions by clonal expansions of its members. At the same time, neither these control mechanisms nor the clonal proliferation should have a major lasting impact on the composition of the compartment. The representation of different lymphocyte subpopulations should be maintained as well as the diversity of

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cell specificities in the naive cell pool. It is obvious that this task is difficult to accomplish. Experiments in different lines of mutant mice have come to the conclusion that the size of different functional lymphocyte subsets are independently controlled, but that there are hierarchies of regulation under extreme conditions. B cell and T cell numbers are independently regulated [41, 42]. The number of T cells is similar in normal mice and mice that lack B cells [41]. Similarly, mice with no TCR, and therefore no T cells, have normal numbers of B cells [42]. The ratio of CD4 and CD8 T cells is generally regulated and preserved. Perturbations, e.g. after adoptive transfer of one subset, are usually only transient and the balance is subsequently restored. However, CD4 and CD8 T cells can compensate for each other. In CD4 or CD8 T celldeficient mice, the remaining subset can compensate for the other and the total number of T cells is maintained at a normal level [43]. Elegant experiments by the groups of Freitas and Rocha [39] have also provided evidence for the concept that naive and memory T cells do not compete with each other for space. In this scenario, extensive stimulation with exogenous or endogenous antigens would not compromise the diversity of the naive T cell compartment. On the other hand, replenishment of naive T cells over time by new thymic emigrants would not conflict with the maintenance of memory responses. In summary, multiple murine studies have led to the general concept that functional lymphocyte subsets have their own ecological niches and do not compete with each other for space [44]. Under physiological conditions, the T cell system is in constant turnover. Experiments in different rodent models have distinguished between short-lived and long-lived lymphocytes. It has been estimated that 20–50% of all T lymphocytes have a life span of several days to a few weeks [45, 46]. Two major mechanisms are involved in lymphocyte replenishment, generation of new thymic emigrants and peripheral self-renewal [44]. The relative quantitative contribution of these two mechanisms depends on a number of variables, foremost the age of the host. Naive T cells are generally considered to be long-lived and are predominantly replenished by new thymic emigrants, a mechanism that would guarantee diversity of the naive T cell population. Thymic output is sufficient to replenish 1% of the peripheral T cell pool daily in young mice and rapidly deteriorates with age, but some thymic function is maintained even in old animals [47]. Whether this reduced thymic function is sufficient to guarantee a naive compartment in older animals or in humans is unclear. It is more likely that self-renewal of circulating cells contributes to the replenishment of the naive T cell pool with increasing age. Longevity and survival of naive T cells depends on the recognition of self-MHC molecules [48]. In contrast, memory cells are short-lived and replenished by self-renewal that appears to be predominantly cytokine-driven [45].


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T Cell Population Dynamics in Humans Data on human T cell dynamics and homeostasis are mostly based on studies involving patients who have undergone profound cancer chemotherapy or have HIV [49, 50]. Following chemotherapy, only young children are able to restore the naive T cell compartment. In contrast, young adults showed persistent CD4 T cell lymphopenia for more than 1 year, an inability to regenerate naive CD4+CD45RO– T cells, and an expansion of atypical CD8 T cells, suggesting that the thymic function is not sufficient to rebuild the peripheral T cell compartment. More recently, the enigma of T cell loss in HIV patients has led to a renewed interest in T cell turnover and generation. These studies have provided a useful framework for the understanding of T cell homeostasis in general and of the results obtained in RA in particular. The lymphopenia in HIV infection was first considered to result from excessive loss that could not be compensated for by increased self-renewal and de novo production (open-drain-open-tap model) [51]. Subsequent studies have shown that a regenerative failure may be more important than increased loss [52]. Several approaches were taken to compare T cell dynamics in healthy individuals and in HIV patients before and during virostatic therapy. Studies of telomere length were difficult to interpret because they only showed telomere shortening of CD8 T cells but not of CD4 T cells, suggesting that at least CD4 T cells do not have an increased replicative history [53]. Enumeration of cycling cells with the cell cycle kinase-specific antibody Ki67 has allowed for more direct estimates of T cell turnover [54]. From these studies it could be extrapolated that circulating CD4 and CD8 T cells had similar half-lives of approximately 150 days in healthy control donors. In vivo studies using deuterium-labeled glucose arrived at a slightly shorter survival time of approximately 80–90 days [52]. In both studies, the life span of CD4 as well as CD8 T cells in HIV patients was shortened. More remarkable, the production of new T cells was significantly decreased in HIV patients, suggesting that a regenerative failure contributed to the peripheral lymphopenia. Virostatic treatment that increased peripheral lymphocyte counts was not able to lengthen T cell survival but reversed the regenerative failure. Studies on the frequency of TCR excision circles (TREC) in the peripheral T cell population have added further support to this interpretation [55]. TREC are formed as episomal DNA during TCR rearrangement, do not replicate, and can therefore be used to estimate the number of thymic emigrants [56]. The number of TREC is decreased in HIV-infected patients. Initiation of virus-specific therapy resulted in an increase of TREC-positive cells, at least in individuals younger than 40 years. In summary, studies involving HIV patients have provided the tools to analyze T cell population dynamics in humans and have allowed for

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assessing the relative contribution of thymic regeneration, peripheral selfrenewal, and peripheral destruction to T cell homeostasis under pathological conditions.

T Cell Population Dynamics in RA Studies in RA have shown a marked contraction in the peripheral repertoire of naive CD45RO– and memory CD45RO+ CD4 T cells [29]. One possible explanation for this observation is that naive and memory T cells are not truly distinct T cell populations and that oligoclonal expansions in the memory compartment may infiltrate the naive T cell population. Indeed, phenotype switches from memory to naive T cells have been observed; however, it is unclear whether these reversions are singular events or of quantitative importance [57]. Alternatively, naive and memory T cells have to be considered as independently regulated cell populations, as nicely demonstrated in murine studies [39]. In support of this view are the data that the human CD4+ CD45RO– T cell populations in RA patients as well as in control individuals have multifold higher TCR diversity than memory CD4+CD45RO+ T cells [29, 58]. If the naive compartment can be considered to be largely independent of events in the memory compartment, the repertoire contraction in naive T cells would either indicate a thymic defect with a reduced generation of new thymic emigrants or a shortened life span of naive T cells [59]. In both cases, self-replication of naive T cells would need to be increased to fill the compartment and to maintain the size of the naive population (fig. 3) [48]. Evidence has been provided that increased peripheral self-replication may be the case in RA patients. Wagner et al. [29] have determined the number of cycling CD4 T cells and have found a significantly increased number of peripheral T cells in the S-G2/M phase in RA patients, indicating increased selfrenewal. This interpretation was supported in subsequent studies comparing the telomere length in peripheral lymphocytes of RA patients and age-matched control donors [38]. Telomere shortening can be taken as evidence of a replicative history. Weng et al. [60] have shown that the telomere lengths in naive as well as memory CD4 T cells decline with age. Surprisingly, the declines in both cell populations were parallel, although the division rates of naive and memory cells are presumed to be different. De Boer and Noest [61] have modeled these data and have come to the conclusion that naive and memory T cells shorten their telomeres at rates set by the division rate of the naive population, irrespective of the division rate of the memory population, suggesting that telomere length studies are most informative in assessing the replicative history of the naive compartment.


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Fig. 3. T cell homeostasis and recognition of self-antigen. Recognition of self-antigen is not only important during positive selection in the thymus, but it is also pivotal in the survival of peripheral naive T cells [46]. In the T-cell-deficient state, T cell tolerance is no longer maintained and recognition of self-antigen results in the activation and clonal expansion of some naive T cells that have higher affinity to self [48].

CD4 T lymphocytes from normal donors lose most of their telomere length when the individuals are between the ages of 40 and 65 years. In contrast, telomere length in CD4 T cells is already disproportionately shortened in RA patients between 20 and 40 years of age and do not show any further decline with advancing age [38]. When naive and memory T cells of agematched controls and patients were separately analyzed, telomere shortening in RA patients was found predominantly in the naive T cell population. This data indicates that the naive T cell compartment in RA patients is under increased replicative stress. Formal studies on T cell turnover and survival, as they have been reported in HIV patients using in vivo labeling with deuteriumlabeled glucose, have not been done in RA. However, the most likely explana-

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tion for these findings is that thymic output is compromised disproportionately for the age of the patient in RA. Indeed, preliminary studies have demonstrated a reduced number of recent thymic emigrants in RA as measured by the concentration of TREC [38].

Emergence of CD28– T Cells in RA The finding that RA patients may have a defect in T cell regeneration provides a novel framework on which to interpret the emergence of large clonal population in RA patients that lack CD28 expression, but recognize autoantigens [25]. As one possibility, RA patients may compensate for the declining generation of new thymic emigrants by increased self-renewal. Selfrenewal is dependent on self-recognition, and T cells with autoreactive specificities would have a competitive advantage [48]. The loss of CD28 expression would then be a result of replicative senescence [62, 63]. There are several lines of evidence that support this view. Studies in RA patients have shown that T cell clones with identical TCR nucleotide sequences can be found that either express or lack CD28 expression, supporting the view that CD28– T cells derive from CD28+CD4+ T cells [25]. In vitro, prolonged culture of T cells with repeated restimulation is associated with the downregulation of CD28 expression [64]. The molecular basis of this downregulation is the gradual loss of two DNA-binding activities that bind to the initiator region of the CD28 promoter. Finally, clonal outgrowth of CD28– T cells can be seen in chronic viral infection, in particular HIV, supporting the view that the clonal proliferation can be interpreted as a response to an antigen, in the case of RA, possibly an endogenous antigen. TCR sequences of clones derived from different, but HLA-DRB1-identical patients show a high degree of homology, adding further support to the hypothesis of an antigen-driven process [65]. Alternatively, clonally expanded CD28– T cells may represent a separate lineage of T cells. These cells not only lack the expression of CD28, but also express an array of molecules that are usually only found on natural killer (NK) cells. Expression of CD57 is a common feature, and MHC class I-recognizing receptors are found on a subset of these cells [66]. In contrast to CD1-restricted NK T cells, CD4+CD28– T cells do not preferentially express an AV24-BV11 TCR. However, they express perforin and have cytotoxic activity [67]. CD28–CD4+ T cells include cells with a low expression of CD8 that represents CD8 aa dimers, a feature characteristic of T cells that have extrathymically matured [68]. This data gives rise to the hypothesis that patients with RA compensate for a thymic deficiency with extrathymic T cell generation. This would not only account for the increased autoreactivity but also for the


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presence and oligoclonal expansion of T cells that functionally resemble NK cells. These two models are not mutually exclusive; increased self-renewal and peripheral selection of autoreactive T cells as well as extrathymic T cell generation could shape the T cell repertoire in RA patients.

Therapeutic Implications of Abnormal T Cell Homeostasis in RA The traditional therapeutic approaches in RA have been based on the model that a limited number of disease-relevant T cells is maintaining disease activity and the suppression or the elimination of these T cells would effectively induce long-term remission. This concept culminated in the T cell depletion treatment studies in the early 1990s with monoclonal antibodies [69] and is about to experience a revival with the efforts to explore autologous bone marrow transplantation to cure RA [70]. Treatments with the antibody Campath-1H, which recognizes the CD52 antigen expressed on all T cells and some other bone-marrow-derived cells, or with several anti-CD4 antibodies were biologically effective and depleted the majority of T cells, including synovium-infiltrating T cells. However, the clinical benefits were transient and the disease relapsed in spite of low peripheral CD4 T cell counts [71, 72]. A defect in thymic T cell regeneration would provide an explanation for this outcome. Patients treated with T-cell-depleting antibodies developed persistent lymphopenia that lasted for years. In particular, these patients were not able to generate any CD4+CD45RO– naive T cells, suggesting that the limited lymphocyte recovery resulted from peripheral self-renewal of surviving T cells [73]. In this self-renewal process, clonally expanded potentially disease-relevant T cells should have a competitive advantage for several reasons. (1) Their clonal size is so large that even the most effective depletion would not sufficiently reduce the clone number below a critical threshold. (2) Expanded clones have been shown to overexpress Bcl-2 and are, therefore, relatively resistant to apoptosis [74]. (3) The specificity profile of increased autoreactivity is likely to favor the self-renewal of these clones. It can, therefore, be predicted that the repertoire in patients after T cell depletion is even more contracted than it is already in untreated RA patients. Indeed, this has been the case [73]. Patients who had been treated with the Campath-1H antibody had a markedly contracted repertoire. T cell oligoclonality in the peripheral blood was so pronounced that it could be easily demonstrated. In spite of peripheral lymphopenia, the synovial tissue was heavily infiltrated and the patients had active disease. Characterization of the TCR sequences of synovial T cells demonstrated that the same T cells that were undergoing increased self-renewal and clonal expansion in the peripheral blood were infiltrating the tissue. Similar

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results can be expected for patients undergoing bone marrow transplantation. Indeed, we have compared the T cell repertoire in an RA patient before and after autologous bone marrow transplantation [Weyand et al., unpubl. observations]. In the pretransplant repertoire, several TCR sequences were identified in the peripheral blood that were clonally expanded and present at frequencies of 1 in 1,000 to 1 in 100,000. All of these sequences were present in the posttransplant repertoire at frequencies that were increased in comparison with the pretreatment repertoire, supporting the interpretation of preferential peripheral self-renewal of disease-relevant T cells in RA. T-cell-directed therapeutic efforts in RA can, therefore, only be successful if they are able to restore the diversity of the peripheral T cell compartment, preferentially by restoring the thymic generation of new T cell emigrants [75]. Pharmacological agents aimed at broadly suppressing T cell function are likely to be of only limited benefit as long as the quantitative imbalance in the T cell repertoire is so profound.

Conclusions The formation of the T cell repertoire is a complex but well characterized process that involves the selection of an immune repertoire in the thymus and subsequent modifications and adaptations in the periphery. Thymic output of new T cells, peripheral recognition of self-antigen, and the peripheral cytokine milieu are important components in this homeostatic process. Studies in murine models of arthritis have demonstrated that the spectrum of T cells selected in the thymus is critical for the development of the disease. In RA, the repertoire has undergone profound quantitative and qualitative changes (fig. 4). RA patients have contracted the naive and memory T cell repertoires with the ultimate emergence of clonal T cell populations that include T cells with specificity to self-antigens. In analogy to the murine models, it is possible that a faulty thymic selection process sets the stage for these aberrations. Indeed, several investigators have tried to link the association of RA with certain HLA-DRB1 polymorphisms to their function in thymic selection. At least equally important, peripheral T cell homeostasis appears to be impaired in RA. These two models are not mutually exclusive; peripheral T cell homeostasis is dependent on the recognition of self-antigen and therefore influenced by the spectrum of T cells initially selected in the thymus. In RA, the peripheral populations of naive and memory T cells have increased replicative histories. Peripheral self-replication of naive and memory T cells is not random but favors certain TCR specificities. The resulting T cell repertoire in RA patients is contracted and contains at most one-tenth of the T cell specificities


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Fig. 4. Schematic diagram of T cell dynamics in RA. The T cell repertoire in RA patients undergoes changes at the level of thymic generation and the turnover of naive and memory T cells. De novo generation of T cells is reduced and self-replication of naive and memory T cells is increased. As a consequence, the diversity of the repertoire is contracted with the clonal outgrowth of autoreactive specificities. T cells express markers of replicative senescence and T cells exhibiting features of NK cells evolve that are important in the extraarticular manifestations of RA.

represented in a normal repertoire. The individual T cells are clonally expanded and can reach large clonal sizes. These large T cell clones acquire features of immunosenescence that compromise some but not all, functional activities. In fact, these cells acquire new properties that are important for the disease process. They lack the expression of the CD28 molecule and are therefore not subject to the normal costimulatory control mechanisms; they gain the expression of regulatory molecules usually expressed on NK cells, the ability to produce large quantities of cytokines, and the ability to kill via a granzyme

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B/perforin mechanism. How these repertoire perturbations impact disease development and disease progression remains to be elucidated. The quantitative changes of repertoire contraction and clonal expansion may render peripheral tolerance mechanisms less effective. The acquisition of novel functions in senescent cells appears to be of particular importance in the extra-articular manifestations of RA where these cells exert critical effector functions [76].

Acknowledgment This study was supported by NIH grants R01 AR41974, R01 AR42527 and R21 GM58604.

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Weyand CM, Brandes JC, Schmidt D, Fulbright JW, Goronzy JJ: Functional properties of CD4+ CD28– T cells in the aging immune system. Mech Ageing Dev 1998;102:131–147. Vallejo AN, Nestel AR, Schirmer M, Weyand CM, Goronzy JJ: Aging-related deficiency of CD28 expression in CD4+ T cells is associated with the loss of gene-specific nuclear factor binding activity. J Biol Chem 1998;273:8119–8129. Vallejo AN, Brandes JC, Weyand CM, Goronzy JJ: Modulation of CD28 expression: Distinct regulatory pathways during activation and replicative senescence. J Immunol 1999;162:6572–6579. Schmidt D, Martens PB, Weyand CM, Goronzy JJ: The repertoire of CD4+ CD28– T cells in rheumatoid arthritis. Mol Med 1996;2:608–618. Namekawa T, Snyder MR, Yen J-H, Leibson PJ, Weyand CM, Goronzy JJ: Killer cell-activating receptors function as costimulatory molecules on CD4+CD28null T cells clonally expanded in rheumatoid arthritis. Submitted. Namekawa T, Wagner UG, Goronzy JJ, Weyand CM: Functional subsets of CD4 T cells in rheumatoid synovitis. Arthritis Rheum 1998;41:2108–2116. Warrington KW, Weyand CM, Goronzy JJ: Expression of MHC class I-recognizing receptors on T cells in rheumatoid arthritis (abstract). Arthritis Rheum 1999;42:1078. Matteson EL: Recent clinical trials in the rheumatic diseases. Curr Opin Rheumatol 1997;9:95–101. Brodsky RA, Smith BD: Bone marrow transplantation for autoimmune diseases. Curr Opin Oncol 1999;11:83–86. Matteson EL, Yocum DE, St Clair EW, Achkar AA, Thakor MS, Jacobs MR, Hays AE, Heitman CK, Johnston JM: Treatment of active refractory rheumatoid arthritis with humanized monoclonal antibody CAMPATH-1H administered by daily subcutaneous injection. Arthritis Rheum 1995;38: 1187–1193. Weinblatt ME, Maddison PJ, Bulpitt KJ, Hazleman BL, Urowitz MB, Sturrock RD, Coblyn JS, Maier AL, Spreen WR, Manna VK: CAMPATH-1H, a humanized monoclonal antibody, in refractory rheumatoid arthritis. An intravenous dose-escalation study. Arthritis Rheum 1995;38:1589– 1594. Jendro MC, Ganten T, Matteson EL, Weyand CM, Goronzy JJ: Emergence of oligoclonal T cell populations following therapeutic T cell depletion in rheumatoid arthritis. Arthritis Rheum 1995; 38:1242–1251. Schirmer M, Vallejo AN, Weyand CM, Goronzy JJ: Resistance to apoptosis and elevated expression of Bcl-2 in clonally expanded CD4+CD28– T cells from rheumatoid arthritis patients. J Immunol 1998;161:1018–1025. Hosaka N, Nose M, Kyogoku M, Nagata N, Miyashima S, Good RA, Ikehara S: Thymus transplantation, a critical factor for correction of autoimmune disease in aging MRL/+mice. Proc Natl Acad Sci USA 1996;93:8558–8562. Martens PB, Goronzy JJ, Schaid D, Weyand CM: Expansion of unusual CD4+ T cells in severe rheumatoid arthritis. Arthritis Rheum 1997;40:1106–1114.

Jo¨rg J. Goronzy, MD, Mayo Clinic, 401 Guggenheim Building, 200 First Street, SW, Rochester, MN 55905 (USA) Tel. +1 507 284 1650, Fax +1 507 284 5045, E-Mail [email protected]

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Leukocyte Homing to Synovium Dhavalkumar D. Patel, Barton F. Haynes Departments of Medicine and Immunology and the Duke University Arthritis Center, Duke University Medical Center, Durham, N.C., USA

A key event in the pathogenesis of rheumatoid arthritis (RA) is the recruitment or homing of activated T cells, monocytes and neutrophils to synovium. The migration of these leukocytes to inflamed synovium involves a complex series of adhesive and activation events including leukocyte rolling on activated endothelial cells (EC), chemokine/chemoattractant receptor activation, increased cell adhesiveness to EC, and eventually transendothelial migration across a chemokine gradient. While the role of adhesion molecules in these events is well established, recent work has recognized chemokines as important mediators of this process. In this chapter, we will describe the events involved in leukocyte migration to synovium, and show how modifying leukocyte migration may affect the pathogenesis of arthritis. First, we will discuss the basic mechanisms and molecules that mediate leukocyte migration. With this background, we will discuss what is currently known about leukocyte migration to synovium and the effects of therapies aimed at modification of leukocyte migration in animal models of arthritis and in clinical trials in humans.

Pathways of Leukocyte Migration Multistep Model Leukocyte Capture and Rolling The vast majority of leukocyte trafficking to sites of inflammation requires multiple regulated steps as outlined in the multistep models proposed by the Butcher [1] and Springer [2] groups. The initial step in this model involves capture of free-flowing leukocytes by vascular endothelium and their rolling

Fig. 1. Schematic of the classical, multistep model of leukocyte migration. Abbreviations are explained in footnotes to tables 1 and 2.

on the endothelium (fig. 1). This process is mediated predominantly by the selectin (CD62) family of proteins including L (leukocyte)-, P (platelet)- and E (endothelial)-selectins that bind carbohydrate ligands [3–6]. CD44 on activated T lymphocytes can also mediate rolling of T cells on activated EC or on plates coated with the CD44 ligand hyaluronan (HA) [7, 8]. This is not entierely surprising because CD44 is a member of the hyalderin family of HA-binding proteins including cartilage link and proteoglycan core proteins [9, 10] whose structure is similar to the selectins [11]. Thus, the first stage of T cell migration to sites of inflammation is mediated by receptor-carbohydrate ligand interactions. Selectins. L-, E- and P-selectin (CD62L, CD62E and CD62P, respectively) are closely related in structure. All three molecules share a unique extracellular region composed of an amino-terminal calcium-dependent lectin domain, an epidermal growth factor (EGF)-like domain, and short consensus repeat (SCR) units homologous to domains found in complement regulatory proteins. L-selectin contains two SCR domains, while E- and P-selectin contain six and nine SCR domains, respectively. A minority of P-selectin proteins contain eight SCR, and a third form of CD62P lacks a transmembrane domain [12]. Human selectins are closely related in amino acid sequence, ranging from D40% identity in the SCR domains to D65% identity in the lectin and EGF domains. This high degree of conservation among the lectin domains results in the recognition of similar, if not identical, carbohydrate epitopes decorating various protein scaffolds.

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While the ligand-binding ability of the selectins is predominantly conferred by the lectin domain, some binding function is also attributed to the EGF domain. For example, the most effective blocking monoclonal antibodies (mAbs) to L-selectin bind epitopes in the lectin domain [13–16] although a few have been mapped to regions in the EGF domain [13, 14, 17]. Functional studies using chimeric selectins have shown that both the lectin and EGF domains are directly involved in cell adhesion [16, 18]. Despite the lack of ligand-binding activity of the selectin SCR domains, these domains are critical for optimal selectin function. The SCR domains serve to extend the lectin and EGF domains of E- and P-selectin from the cell surface, and this appears to be important in promoting interactions which would be spatially unfavorable for a more membrane proximal receptor such as L-selectin [19, 20]. Thus, cooperative interactions among the individual extracellular domains of the selectins promote ligand binding. Selectin function may be regulated by soluble forms of the receptor, formed either by receptor shedding or alternative splicing. The membrane proximal regions of L-selectin, and likely P-selectin, are susceptible to endoproteolytic cleavage by an endogenous membrane-bound protease following cell activation [21]. The cleavage products are functionally active and present at high levels (1.6×0.8 lg/ml for L-selectin and ~40 to ~200 ng/ml for P-selectin) in normal serum where they may downregulate inflammatory responses by blocking selectin function [22–24]. Activated endothelial cells release a truncated form of E-selectin (~94,000 Mr) which lacks an intact cytoplasmic domain [25, 26]. Normal plasma levels of soluble E-selectin have been reported in the range of 0.1 to ~3 ng/ml [25]. Although it has been proposed that L-selectin cleavage from the cell surface is a prerequisite for diapedesis to occur, recent findings do not support this idea [27]. Although closely related in structure, the three members of the selectin family have very different patterns of expression [28]. L-selectin is expressed on all circulating leukocytes except a subset of memory T cells [29, 30]. Among lymphocytes, the majority of circulating virgin/naive T cells express L-selectin and enter peripheral lymphoid tissues, although there are distinct subpopulations of both CD4+ and CD8+ memory cells that lack L-selectin [30, 31]. Recently activated helper T cells with a memory phenotype generally lack L-selectin expression but reacquire surface receptor expression during further maturation into fully competent helper cells [30, 32]. In addition, intrinsic differences in expression levels of L-selectin among lymphocyte subsets regulate, in part, their differential migration patterns [31]. Thus, both naive and mature memory lymphocytes utilize L-selectin to enter lymphoid tissues where effective helper function is provided.

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P-selectin is constitutively found in Weibel-Palade bodies within endothelial cells and in the a-granules of platelets, but not on the cell surface [33]. Within minutes following activation by thrombogenic or inflammatory mediators including thrombin, histamine, complement fragments, oxygen-derived free radicals, lipopolysaccharide (LPS) and cytokines, granules containing P-selectin fuse with the plasma membrane thereby expressing P-selectin on the cell surface. Cell surface P-selectin expression is generally short-lived (minutes), likely due to endoproteolytic cleavage near the cell surface. This is consistent with its role in mediating early leukocyte-EC interactions [3]. However, in vivo studies of P-selectin function suggest that it may also be important at later time points as a cytokine-induced adhesion molecule. E-selectin is expressed by activated but not resting endothelial cells [34], thus providing a means of inducing leukocyte rolling at inflamed sites. E-selectin protein production is strongly induced by a variety of inflammatory mediators, including IL-1b, TNF-a, interferon-c, substance P and LPS. E-selectin expression on human umbilical vein endothelial cells (HUVEC) peaks at 4–6 h following activation and declines to basal levels by 24–48 h [34, 35]. However, E-selectin expression in HUVEC may not be generalized to reflect its expression in other tissues in vivo since sustained expression has been observed in endothelial cells isolated from different vascular beds [36–38]. Therefore, the kinetics of E-selectin expression are variable, likely reflecting differences in both the microvascular bed and the type of inflammatory insult. E-selectin is highly expressed on EC in inflamed synovium, but not in noninflamed synovium [39–42]. Thus, E-selectin may be important for leukocyte homing to RA synovium. Selectin Ligands. The molecular basis of selectin adhesion involves recognition of carbohydrates by the lectin domain [43]. In vitro, selectins recognize a variety of complex carbohydrates, but it is likely that each selectin binds only to a restricted number of high affinity carbohydrate ligands in vivo. All three selectins bind to the tetrasaccharide sialyl Lewisx (sLex, CD15s) and it has, therefore, been identified as a prototype selectin ligand. A number of L-selectin ligands have been identified in vitro, but it is unclear at present which, if any, of those molecules functions as the dominant physiologic ligand for L-selectin. Murine L-selectin has been shown to recognize, in vitro, at least four different heavily glycosylated mucin-like proteins constitutively expressed by high endothelial venules (HEV): GlyCAM-1, CD34, MAdCAM-1 and a 200,000-Mr HEV ligand (sgp200) [44]. Each of these molecules is decorated with sulfated, sialylated and fucosylated O-linked carbohydrate side chains which appear to be essential for L-selectin recognition [45]. Ligands for L-selectin expressed on HEV have previously been identified by the MECA-79 mAb [46–48]. MECA-79 reactivity is also found on venules

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at sites of chronic inflammation [49, 50]. However, HEV from fucosyltransferase VII-deficient mice that lack selectin ligands express normal levels of the MECA-79 epitope [51], raising doubt about the validity of MECA-79 as a marker of L-selectin ligands. Other studies have shown that L-selectin also binds to P-selectin glycoprotein ligand-1 (PSGL-1, see below) expressed by leukocytes [16, 52], although much less efficiently than the binding of P-selectin to PSGL-1 [16]. An unidentified cytokine-inducible ligand for L-selectin has been described for cultured HUVEC and microvascular endothelial cells [14, 53, 54]. Both HEV and endothelial cells of skin venules express a carbohydrate sLex-like epitope defined by the 2H5 mAb [55–57]. The 2H5 epitope is induced on vascular endothelial cells at sites of inflammation and, importantly, this mAb blocks L-selectin-dependent leukocyte binding and migration. Thus, it is likely that the 2H5 mAb recognizes a physiologically relevant L-selectin ligand. PSGL-1 is the dominant physiologic ligand for P-selectin (table 1) [58, 59]. In fact, all P-selectin-dependent leukocyte rolling in vivo appears to be mediated by PSGL-1 [59]. PSGL-1 was originally identified as a Pand E-selectin ligand and is decorated by N-linked glycans and numerous sialylated O-linked glycans, including O-linked polylactosamine determinants that carry sLex. O-linked glycans are required for P-selectin recognition, whereas the N-linked glycans are not. P-selectin binding through the aminoterminal region of PSGL-1 is blocked by a specific anti-PSGL-1 mAb (PL1) and by O-sialoglycoprotein endopeptidase-mediated cleavage of PSGL-1 from the cell surface. Although E- and P-selectin can bind identical carbohydrate moieties, glycoprotein ligands unique to P- and E-selectin have been identified (table 1). E-selectin binds to appropriately glycosylated PSGL-1 on myeloid cells [60]. Eselectin also binds to the cutaneous lymphocyte antigen (CLA), a carbohydrate determinant that is recognized by the HECA-452 mAb and is associated with tissue-selective homing of T cells to sites of chronic cutaneous inflammation [61]. Lymphocyte CLA has been recently identified as a modified form of PSGL-1 [62]. E-selectin also binds a 260,000-Mr glycoprotein on bovine cd T cells [63] and a predominant 150,000-Mr glycoprotein and minor 250,000-Mr protein on mouse neutrophils [64]. The 150,000-Mr glycoprotein, termed the E-selectin ligand (ESL-1), is a variant of a chicken fibroblast growth factor receptor [65]. The above observations have led to the suggestion that E- and P-selectin recognize two types of glycoprotein ligands, one being monospecific and the second common for both endothelial selectins [66]. Recently, it has been recognized that the mucins need to be fucosylated to function as selectin ligands [51] and sulfated tyrosine residues are also critical for function in leukocyte capture [67–71].


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Table 1. Adhesion molecules potentially involved in leukocyte migration Family, molecule, alternate names Ig family CD31 PECAM-1, GPIIa, EndoCAM CD50 ICAM-3

Distribution

Ligand(s)

Function in leukocyte homing

M, G, naive T, NK, P, EC

CD31, aVb3 integrin

Potentially involved in transendothelial migration

Leukocytes, EC

CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1)

Firm adhesion and may be involved in tumor development Firm adhesion at sites of inflammation

CD54 ICAM-1

M, activated T and B, CD11a/CD18, activated EC, activated CD11b/CD18 fibroblasts, subset epithelia CD102 EC, subset PBL, M, P CD11a/CD18, ICAM-2 CD11b/CD18 CD106 M, DC, stromal cells, VLA-4 (CD49d/CD29, VCAM-1, INCAM-110 activated EC a4b1), a4b7

Integrin a family CD11a LFA-1a, aL integrin

All leukocytes

CD54, CD102, CD50, ICAM-4, ICAM-5

G, M, NK, subset of B and T

iC3b, fibrinogen, CD23, CD54, CD102, CD50

G, M, NK, subset of B and T

iC3b, fibrinogen, CD23, CD54? Laminin, collagen I, collagen IV

CD49b VLA-2, a2 integrin, GPIa CD49c VLA-3, a3 integrin

M, activated T, NK, subset EC, fibroblasts, neuronal cells T and B, P, EC, fibroblasts, subset epithelia

Collagen I, II, III, IV; laminin

B, subset epithelia, fibroblasts

Fibronectin, collagen, laminin

CD49d VLA-4, a4 integrin

B, T, M, NK, DC, subset cancers

CD106, MAdCAM-1, fibronectin, invasin, thrombospondin

CD11b aM integrin, Mac-1, CR3 CD11c aX integrin, CR4 CD49a VLA-1, a1 integrin

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Firm adhesion in normal lymphocyte recirculation Firm adhesion at sites of inflammation Firm adhesion, associates with integrin b2 (CD18) to form LFA-1 Firm adhesion, associates with integrin b2 (CD18) to form Mac-1, CD3 Firm adhesion, associates with integrin b2 (CD18) to form CR4 Cell adhesion to ECM, associates with integrin b1 (CD29) to form VLA-1 Cell adhesion to ECM, associates with integrin b1 (CD29) to form VLA-2 Cell adhesion to ECM, associates with integrin b1 (CD29) to form VLA-3 Capture, firm adhesion, cell adhesion to ECM, associates with integrin b1 (CD29) to form VLA-4, associates with b7 to form a4b7 (a ligand for MAdCAM-1)

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Table 1 (continued) Family, molecule, alternate names

Distribution

Ligand(s)

CD49e VLA-5, a5 integrin, FNR a CD49f VLA-6, a6 integrin

T, M, P, EC, fibroblasts, L1, fibronectin muscle cells, subset epithelia, progenitor cells T, M, eosinophils, P, Laminin epithelia

CD51 aV integrin, VNR-a

M, P, subset B, activated T, Vitronectin, fibrinogen, EC, osteoclasts vWF, thrombospondin, fibronectin, collagen

CD103 aE integrin

IEL, subset PBT, subset E-cadherin T cell leukemia/lymphoma

Integrin b family CD18 b2 integrin

All leukocytes

See CD11a, CD11b, CD11c

CD29 Broad See CD49a–f b1 integrin CD61 M, P, subset B, activated T, Vitronectin, fibrinogen, b3 integrin, GPIIb/IIIa EC, osteoclasts vWF, thrombospondin, fibronectin, collagen CD104 b4 integrin b7 integrin

Selectin family CD62E E-selectin, ELAM-1, LECAM-2 CD62L L-selectin, LAM-1, LECAM-1, Leu-8 CD62P P-selectin, PADGEM, GMP-140

M, epithelia, CD4Ö CD8Ö precursor T IEL, subset PBT, subset T and B cell leukemias/lymphomas

Activated EC

laminin 1, 4, 5

Function in leukocyte homing Cell adhesion to ECM, associates with integrin b1 (CD29) to form VLA-5 Cell adhesion to ECM, spreading and migration, associates with integrin b1 (CD29) to form VLA-6 Platelet aggregation, cell adhesion to ECM, associates with CD61 to form VNR (aVb3) Migration to mucosal sites, associates with integrin b7 to form aEb7 Associates with CD11a, CD11b and CD11c; defect in CD18 causes LAD-1 Associates with CD49a–f to function in cell adhesion Platelet aggregation, cell adhesion to ECM, associates with CD51 to form VNR (aVb3) Cell adhesion, migration

MAdCAM-1, E-cadherin

Migration to mucosa, associates with integrin a4 to form a4b7 and with integrin aE to form aEb7

CD15s, sialyl Lewis a, CLA, CD162

Leukocyte rolling on activated EC

PBB, PBT, G, subset NK, GlyCAM-1, MAdCAM-1, Lymphocyte homing, rolling subset hematopoietic CD34 on activated EC malignancies Activated P, activated EC CD162 Leukocyte rolling on activated EC


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Table 1 (continued) Family, molecule, alternate names Selectin ligands CD15 Lewis X, lacto-Nfucopentaose III CD15s Sialyl-Lewis X CD34 CD162 PSGL-1 CLA

Distribution

Ligand(s)

Function in leukocyte homing

M, G, DC

Selectins

Leukocyte rolling

M, G, NK, subset of T, CD62E activated T and B, HEV Hematopoietic progenitor CD62L (controversial) cells, endothelium T, M, G, subset B CD62P, CD62E, CD62L Subset T

CD62E

MAdCAM

Mucosal EC

CD62L, a4b7

PNAd

HEV

CD62L

Others CD44 Broad H-CAM, Pgp-1, Hermes, In(Lu)-related CD44R Epithelia, M, activated CD44v leukocytes

HA, MIP-1b, bFGF, osteopontin, ankyrin

CXCR1

M, T, NK

FKN

FKN

Activated EC

CX3CR1

Leukocyte rolling Leukocyte rolling Rolling of leukocytes on EC, P and other leukocytes A modified form of PSGL-1 that directs T cell homing to sites of chronic cutaneous inflammation Lymphocyte homing to mucosa Leukocyte rolling and migration to lymph nodes

HA, MIP-1b, osteopontin, Lymphocyte rolling, cell ankyrin adhesion to ECM Unclear, but possibly mediates cell movement through ECM and the metastasis of certain epithelial malignancies Integrin-independent leukocyte capture and firm adhesion Integrin-independent leukocyte capture and firm adhesion

CLA>Cutaneous lymphocyte antigen; DC>dendritic cells; EC>endothelial cells; ECM>extracellular matrix; ELAM> endothelial leukocyte adhesion molecule; FNR>fibronectin receptor; G>granulocytes; GlyCAM> glycosylation-dependent cell adhesion molecule; H-CAM>homing-associated cell adhesion molecule; HEV>high endothelial venules; ICAM>intercellular adhesion molecule; IEL>intraepithelial lymphocytes; INCAM> inducible cell adhesion molecule; LAD>leukocyte adhesion deficiency; LECAM>lectin cell adhesion molecule; LFA>lymphocyte function antigen; M>monocytes; MAdCAM>mucosal addressin cell adhesion molecule; NK>natural killer cells; P>platelets; PADGEM>platelet activation-dependent granule-external membrane protein; PBB>peripheral blood B cells; PBL>peripheral blood lymphocytes; PBT>peripheral blood T cells; PECAM>platelet/endothelial cell adhesion molecule; PNAd>peripheral node addressin; PSGL>P-selectin glycoprotein ligand; RBC>red blood cells; VCAM>vascular cell adhesion molecule; VLA>very late activation antigen; VNR>vitronectin receptor; vWF>von Willebrand’s factor.

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CD44. CD44 is an 85-kD glycoprotein that is broadly expressed on most cell types and tissues. It is a member of the hyaldherin family of HA-binding proteins with structural similarity to selectins [10, 11, 72]. The standard, predominant form of CD44 (CD44S) is a highly glycosylated cell surface protein of 80–90 kD. Multiple isoforms of CD44 have been described, and arise by alternative splicing of 9 exons in humans (10 in mouse and rat) [73, 74]. These variant CD44 isoforms are collectively referred to as CD44R (restricted) [75]. The extracellular domain of the mature protein consists of a single globular domain [73, 76] with multiple potential sites for N- and O-linked glycosylation and for chrondroitin sulfate modification [77]. The ability of CD44 molecules to bind its primary ligand, HA [78], is modified by posttranslational glycosylation [79]. CD44 is involved in leukocyte attachment to and rolling on EC, homing to peripheral lymphoid organs and to sites of inflammation, and leukocyte aggregation [7, 8, 73, 77]. Signaling through CD44 can induce cytokine release and T cell activation. This may be associated with activation of protein kinase C which can potentially phosphorylate four of six serine residues in the cytoplasmic domain of CD44: S271, S303, S305 and S317 [72, 73]. Recently, the structure of the HA-binding region of the hyaladherins has been determined, and has structural similarity to the selectins [11]. These observations may explain the involvement of CD44 in leukocyte rolling on endothelial cells and homing to peripheral lymphoid organs and to sites of inflammation. Upregulation of CD44 expression and induction of CD44 ability to bind HA appears to be functionally important in mediation of a variety of inflammatory disease states, including inflammatory synovitis (see below). Firm Adhesion In the second stage of arrested rolling and firm adhesion of leukocytes to EC, leukocytes become activated by a subset of chemokines via signaling through pertussis toxin (PTX)-sensitive G protein-coupled receptors (GPCR), leading to upregulation of integrin adhesiveness and activation-dependent stable arrest [80–82]. The leukocyte integrins active at this stage include the b1 (VLA-4, a4b1, CD49d/CD29), b2 (LFA-1, aLb2, CD11a/CD18; Mac-1, aMb2, CD11b/CD18) and b7 (LPAM-1, a4b7) integrins binding to VCAM-1 (CD106), the ICAMs (CD54, CD102 and CD50) and MAdCAM-1, respectively, on EC. The chemokines that can induce rapid adhesion of rolling leukocytes to ICAM-1 under flow include ELC, SLC, LARC and SDF-1 [82]. Signal transduction through certain chemokine receptors induces a conformational change in integrins to express an activation epitope that is able to bind to its ligand with high affinity and avidity [1]. The increased cell adhesiveness mediated by this mechanism, a process that has been termed ‘inside-out sig-


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naling’, is regulated by the cytoskeletal association of the integrins [83–86] in a poorly defined process involving the small G protein RhoA [87, 88]. Thus, in the multistep model, the role of chemokines is to induce a rolling leukocyte to firmly adhere to endothelium by upregulating the function of the integrin family of adhesion molecules. Chemokines and Chemokine Receptors. Chemokines are structurally related peptides of 8–10 kD that act through cell surface GPCR to mediate diverse functions including cell migration and induction of integrin adhesiveness [89]. They can be grouped based on the structure of the N-terminal cysteine residues into 4 families: the C, CC, CXC and CX3C families. For chemokines, structure does correlate with function. For example, the ELR motif that precedes the first cysteine in all CXC chemokines that act on neutrophils is essential for binding and activation of CXCR1 and CXCR2 [90]. While most chemokines and their receptors have redundant specificity, CC chemokines interact with only CC receptors and vice versa (table 2). The standard nomenclature for chemokine receptors established at the 1996 Gordon Research Conference on ‘Chemotactic Cytokines’ is used in table 2 to list currently known chemokines, chemokine receptors, and their predominant expression patterns [91–99]. A standard nomenclature for chemokines is highly desirable and will hopefully be established soon. Chemokines bind to GPCRs with seven transmembrane spanning domains. Signal transduction through the chemokine receptors is mediated for the most part through PTX-sensitive Gai proteins, but this is controversial since many chemoattractant receptors in transfected cells also couple to Gaq and Ga16-like G proteins that are PTX-insensitive [100–102]. Since leukocytes naturally express predominantly Gai proteins, we will limit our brief discussion here to signaling through PTX-sensitive Gai proteins. Receptor engagement of GPCR induces a conformational change, GTP hydrolysis, and release of active Ga and Gbc subunits which can then activate phospholipase C (PLC), PI3 kinase (PI3K) and receptor tyrosine kinases [89]. PLC activation leads to generation of diacylglycerol and inositol triphosphate which activate protein kinase C and mobilize Ca2+, respectively [89]. Activation of PI3K leads by unknown mechanisms to chemotaxis [103, 104], and activation of the small G protein RhoA leads to increased integrin adhesiveness [87, 88]. Recent studies are showing that signaling through chemoattractant receptors can also lead to activation of transcription factors [105] that regulate proinflammatory cytokine production [106]. Whereas chemokine receptors can activate multiple pathways, engagement of different receptors has different downstream effects. For example, while IL-8 activation of both CXCR1 and CXCR2 can induce chemotaxis and Ca2+ mobilization, only CXCR1 engagement can lead to activation of phospholipase D [107]. The type of signal transduced depends

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Table 2. Chemokine receptors, their expression on leukocytes and their ligands Family, molecule

Old names

XCR family XCR1 CCR family CCR1

CC CKR1

CCR2a, b CCR3

MCP-1Ra, b CKR-3

CCR4 CCR5 CCR6 CCR7

CC CKR5 BLR-2/EBI1

CCR8 CCR9

Distribution on leukocytes

Ligand(s)

Lymphocytes

Lymphotactin/SCM-1

M, iDC, T, Baso

MIP-1a, RANTES, MCP-3, HCC-1/MCIF, MPIF-1/ Ckb-8/MIP-3, MIP-1d M, iDC, T, Th2, Baso MCP-1, 2, 3, 4 Eos, Th2 Eotaxin, RANTES, MCP-2, 3, 4, MIP-1d Baso, Th2, mDC TARC, MDC, RANTES M, iDC, T, Th1 RANTES, MIP-1a, MIP-1b iDC, Act T, B LARC/MIP-3a/Exodus-1 mDC, naive T, B ELC/MIP-3b, SLC/Exodus2/TCA-4 Act T I-309, MIP-1b APC TECK

CXCR family CXCR1 CXCR2

IL-8 RA, IL-8 R1 IL-8 RB, IL-8 R2

CXCR3 CXCR4 CXCR5

Act T, Th1 Fusin/humstr/Lestr Widely expressed BLR-1 B, memory T

IL-8, GCP-2 IL-8, GROa, BROb, GROc, NAP-2, ENA-78, GCP-2 IP-10, Mig, I-TAC SDF-1/PBSF BLC

CX3CR family CX3CR1

V28

FKN

N, iDC N

M, T, NK

Act T>Activated T cells; APC>antigen-presenting cells; B>B cells; Baso>basophils; BLC>B lymphocyte chemoattractant; ELC>Epstein-Barr virus-induced molecule 1 (EBI-1) ligand chemokine; ENA>epithelial cell-derived neutrophil activating peptide; Eos>eosinophils; FKN>fractalkine; GCP>granulocyte chemotactic protein; GRO>growth-related gene product; HCC>hemofiltrate C-C chemokine; iDC>immature dendritic cells; IL>interleukin; IP-10>IFN-c-inducible protein-10; I-TAC>IFN-inducible T-cell a chemoattractant; LARC>liver and activation-regulated chemokine; mDC>mature dendritic cells; M>monocytes; MCIF>melanocyte contact inhibitory factor; MCP>monocyte chemotactic protein; MDC>macrophage-derived chemokine; Mig>monokine-induced by IFN-c; MIP>macrophage inflammatory protein; MPIF>myeloid progenitor inhibitory factor; N>neutrophils; NAP>neutrophil-activating peptide; PBSF>pre-B-cell growth-stimulating factor; RANTES>regulated on activation, normal T cell-expressed and secreted; SCM>single C motif; SDF>stromal cell-derived factor; SLC>secondary lymphoid organ chemokine; TARC>thymus and activation-regulated chemokine; TCA>T cell activation gene; TECK> thymus-expressed chemokine.


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on the chemokine, the receptor, and the types of coupling proteins present within a given cell type. Chemokines and chemokine receptors also regulate the activity of adhesion molecules in coordinating leukocyte migration, but the biochemical mechanisms are not well understood. A specific subset of chemokines namely SLC/ 6-C-kine, SDF-1, LARC/MIP-3a and ELC/MIP-3b can induce the integrinmediated firm adhesion of leukocytes under flow [82]. Further, a study in murine pre-B cells transfected with CXCR1 showed that the small G protein RhoA is likely involved in this upregulation of integrin adhesiveness [87]. In RBL-2H3 cells transfected with CXCR1 and other chemoattractant receptors (fMLPR and C5aR) along with L-selectin, activation of chemokine receptors led to rapid phosphorylation of L-selectin on cytoplasmic serine residues, an event associated with increased L-selectin-mediated adhesion [108]. Recently, a novel role for chemokines in leukocyte migration has been defined. Interactions of fractalkine (FKN), a transmembrane chemokine/mucin hybrid molecule expressed on TNF-activated endothelium, with its receptor CX3CR1 have been shown to mediate the capture, firm adhesion and activation of leukocytes under physiologic flow conditions in a PTX-insensitive and integrinindependent manner (see below) [109]. Thus, chemokines induce the activation and adhesion of circulating leukocytes to endothelium by regulating adhesion molecule function, and in some instances can themselves act as cell adhesion molecules. Integrins and Integrin Ligands. In the classical pathway of leukocyte migration, integrins and their ligands are the primary cell adhesion molecules that mediate firm adhesion of leukocytes to the endothelium. This process in most tissues is mediated primarily by the leukocyte-specific b2 integrins, CD11a/ CD18 (LFA-1) and CD11b/CD18 (Mac-1). The importance of these molecules in leukocyte migration is perhaps best shown by the fact that humans lacking functional b2 integrins due to defects in CD18 develop a syndrome called leukocyte adhesion deficiency syndrome type 1 (LAD-1) characterized by repeated infections and delayed would healing [110]. LFA-1 is expressed to some extent by all leukocytes while Mac-1 is primarily expressed by myeloid cells. While b2 integrins do not provide specificity for leukocyte homing, their function is critically important for leukocyte migration to most sites of inflammation. a4b7 integrins may play a role in targeting leukocytes to mucosal sites (see below). Integrins are heterodimers formed by a combination of at least 15 a and 8 b subunits to form more than 21 different heterodimers (fig. 2), many of which have different functions. Ligand-binding sites reside in the N-terminal regions of both a and b chains [111–114]. The ligand-binding site in the a chain resides within the I (inserted) domain and both chains contain divalent

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Fig. 2. Known associations of integrin a and b chains.

cation chelation sites [115, 116]. Divalent cations are necessary for integrin function [86]. An important point to note is that leukocyte integrins are not constitutively active, and need to be activated to become functional in cell adhesion. This enhanced adhesion may be due either to changes in integrin affinity [117], mobility [118], cytoskeletal association [84, 119], clustering or other postbinding events. Activation of integrin function involves a structural change in the integrin mediated by modification of the cytoplasmic region of the b chain and binding to cytoskeletal components [85, 120, 121], a process that has been termed inside-out signaling [86, 117]. While the b2 and b7 integrins are known to undergo affinity modulation, it is not clear if VLA-4 affinity is regulated in the same way [86]. This activation can be induced by multiple triggers, but signaling through chemoattractant receptors in a rhoAdependent mechanism appears to be the important pathway in leukocyte arrest and firm adhesion [87, 88]. Integrin ligands on endothelium are generally members of the immunoglobulin family of proteins [122]. With the exception of the interaction of Mac-1 with domain 3 of CD54 [123], the most critical domain for integrin binding is usually the N-terminal Ig domain (domain 1) that extends the


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farthest from the cell surface [124]. Many reviews on the structure, function and regulation of integrins and their receptors exist, including an excellent review by Shimizu et al. [86]. Thus, we will not in this chapter review this subject extensively. The patterns of expression of integrin ligands on vascular endothelium provide some clues to their functions in normal leukocyte trafficking and in leukocyte migration to sites of inflammation. CD102 (ICAM-2) is constitutively expressed in vascular endothelium, and its expression is not modulated by proinflammatory cytokines such as TNF-a and IL-1 [125], suggesting that it may function in normal leukocyte trafficking. CD54 (ICAM-1) and CD106 (VCAM-1) are markedly upregulated on vascular endothelium at sites of inflammation and by proinflammatory cytokines [126–128] and these molecules are important for leukocyte migration to sites of inflammation. CD50 (ICAM-3) is highly expressed on vascular endothelium in tumors [129, 130] and may be involved in tumorigenesis or trafficking of leukocytes to tumors. MAdCAM-1 is constitutively expressed in lamina propria venules and on HEV in mesenteric lymph nodes and Peyer’s patches, and is upregulated in the gut during inflammatory responses [49, 131, 132] suggesting a role for MAdCAM-1 in the mucosal immune response. Transendothelial Migration Less is known about the third stage of leukocyte trafficking whereby the leukocyte migrates through the endothelial cell monolayer, the basement membrane and into the tissue. It has been hypothesized that the adhesion molecule CD31 (PECAM) plays an important role in this step, but this is not entirely clear. CD31 is a member of the immunoglobulin family of proteins, functions as a homophilic adhesion molecule and may also act as a heterophilic adhesion molecule, binding to avb3 [133–135]. There is some evidence supporting a role for CD31 in transendothelial migration. Blocking CD31 on the endothelium inhibits the transmigration of leukocytes across endothelial cell monolayers in vitro, as does blocking CD31 on monocytes or neutrophils [136]. Blocking CD31 function in vivo in animal models reduces leukocyte emigration into the peritoneum and lung alveolae [137, 138]. After firm adhesion to the endothelium, the leukocyte must receive new signals and/or become desensitized to the old signals for directed migration toward the tissue and release of leukocyte-EC contacts. FKN Pathway Recently, a new pathway by which leukocytes can be induced to firmly adhere to endothelium and traffic into inflamed tissues has been described. This process is mediated by the chemokine FKN, expressed on endothelial

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cells, and its receptor, CX3CR1, expressed on PBMC. FKN and CX3CR1 function as cell adhesion molecules under both static and dynamic conditions [109, 139]. Unlike other chemokine/GPCR interactions that require signal transduction and integrin activation for cell adhesion to occur, the adhesive interaction between FKN and CX3CR1 is independent of signal transduction or integrin function [109, 140]. Therefore, FKN and CX3CR1 provide an integrin-independent mechanism for leukocyte migration. Signaling through CX3CR1 can, however, also activate integrins [141]. Fractalkine FKN is unique amongst the chemokines in that it is a transmembrane, chemokine/mucin hybrid molecule [142–144]. The chemokine domain of FKN shares high homology with the CC family of chemokines, but has an insert of three amino acids between the two N-terminal cysteine residues, conferring a CX3C structural motif [142, 143]. The mucin domain of FKN has 26 potential O-linked glycosylation sites, and is heavily glycosylated [142, 143, 145]. Consistent with other mucins (selectin ligands), the FKN mucin domain has a long stalk-like ultrastructure, but unlike other mucins involved in leukocyte migration, it does not appear to have a binding function [145]. In this respect, the FKN mucin domain shares similarity with the SCR of L-, E- and P-selectin that function to extend the ligand-binding lectin+EGF domains while having no intrinsic binding activity [13–20]. In addition to a membrane form, FKN-transfected cells also produce a soluble, secreted 95-kD form that is chemotactic for monocytes, NK cells and T lymphocytes [139, 142]. The mouse homologue of FKN, neurotactin, may also be chemotactic for neutrophils [143], but this has not been confirmed. In tissues, FKN mRNA is most highly expressed in brain, heart, kidney and adrenal gland with low level expression in lung. FKN is constitutively expressed at low levels in neurons and astrocytes in brain, and is markedly upregulated with TNF treatment and in inflamed brain [142, 146, 147]. It is also expressed on epithelial cells and dendritic cells [148–150]. Resting endothelium contains low to undetectable levels of FKN, and its expression is greatly enhanced by IL-1 and TNF [109, 142, 151, 152]. Recently, it was shown that FKN could act as a cell adhesion molecule in static binding assays [139] and that FKN also mediates the capture and firm adhesion of monocytes, CD8+ T cells and NK cells under physiologic flow conditions [109]. CX3CR1 Chemokines act through seven-transmembrane GPCRs to exert their biologic effects. Based on this and the structural similarity of FKN to CC chemokines, Imai and colleagues [139] identified the orphan receptor V28


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(also called CMK-BRL1) as the receptor for FKN. CX3CR1 shares the most homology with CC chemokine receptors, and is expressed in human T lymphocytes, NK cells and monocytes [139], and its rat homologue (RBS11) is expressed in leukocytes and microglia [146]. Ca2+ flux through CX3CR1 in many cell types is completely PTX-sensitive [139, 147] indicating that it couples exclusively to Gai, and not other Ga proteins. Recently, it has been shown that signaling through CX3CR1 induces MAPK and PKB activation in microglia, but the functional significance of this is not known [147]. Evidence is now being gathered indicating that CX3CR1 is expressed primarily on effector lymphocytes and monocytes and may be important for their effector function [153] or the migration of these cell types into target tissues. The roles of FKN and CX3CR1 in normal and diseased states are now being elucidated. FKN may participate in autoimmune kidney inflammation [154, 155]. CX3CR1 can act as a coreceptor for some strains of HIV [156, 157] and CMV [158], indicating a potential role in viral pathogenesis. Indeed, a naturally occurring polymorphism in CX3CR1 (I249M280) is associated with rapid progression to AIDS in a European cohort of HIV-infected individuals [159]. FKN is expressed highly in neurons of the brain and interacts with CX3CR1 expressed in the microglia [147, 160], suggesting a means by which these neural cell populations may communicate. Exogenously added FKN can cause the migration of primary rat microglial cells in vitro [147]. A role for FKN and CX3CR1 during nerve regeneration has been postulated [146]. Roles for the FKN pathway have been postulated in the pathogenesis of Crohn’s disease, solid organ transplant rejection (specifically, kidney and heart) and in the early stages of atherosclerosis. The role of this pathway in the pathogenesis of autoimmune synovial inflammation has yet to be determined.

Regulation of Homing to Inflamed Synovium Homing of specific leukocyte subsets to tissues in an organ-specific manner is highly regulated. There are several steps at which this regulation can occur. At the rolling step, regulation can be achieved by increasing selectin expression on the EC surface, selectin ligand expression on the EC or leukocyte or activating leukocyte CD44 to bind HA. At the stage of firm adhesion, there are several levels of regulation including tissue-specific expression of either adhesion molecules (CLA on skin-homing leukocytes and MAdCAM in mucosal tissues) or chemokines (BLC and SLC among others) as well as leukocytespecific expression of adhesion molecules (VLA-4) and chemokine receptors (virtually all chemokine receptors are differentially expressed on leukocyte

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subsets). Leukocyte entry into the tissue after firm adhesion could be regulated by the presence of a lack of appropriate chemotactic factors to induce directional migration, and the presence or lack of leukocyte adhesion molecules to bind with those in the extracellular matrix. It has also been recently recognized that another important stage of regulation is the ability of cells to be retained within tissues. The mechanisms involved in leukocyte retention in tissues are presently being evaluated. Regardless of the mechanism used, there must be regulation of at least one molecule (adhesion molecule or activation molecule) on either the endothelium or leukocyte side of the equation for appropriate leukocyte homing to occur. Adhesion Molecules in RA Much evidence exists that the infiltration of leukocytes into inflamed synovium and the development of arthritis is adhesion molecule-dependent. Rheumatoid synovium contains HEV-like vessels, similar to those found in peripheral lymph nodes, which support leukocyte binding and migration [153, 161, 162]. Increased E-selectin expression has been consistently demonstrated on RA synovial endothelium, both in vitro and in situ [39–42]. Antiinflammatory therapies, such as anti-TNF-a antibody, pulse intravenous methylprednisolone, and intramuscular gold, decrease vascular endothelial Eselectin expression in synovial biopsy specimens [41, 163, 164]. In addition, intense, diffuse synovial P-selectin expression and/or P-selectin-mediated monocyte adhesion has been described in some [162], but not other [40] patients with RA. Although sserum levels of L- and P-selectin are elevated in RA patients, only P-selectin levels correlate with disease activity [165, 166]. Serum E-selectin levels are increased in patients with active disease, but fall in response to anti-inflammatory therapy [167]. Thus, in RA, each of the selectins appears to play a role in recruiting leukocytes into the inflamed synovium. Furthermore, both soluble P- and E-selectin may serve as markers of disease activity and may potentially be used to gauge response to therapy. Both P-selectin and E-selectin, but not L-selectin appear to be important for the recruitment of neutrophils and monocytes to the joint in animal models of arthritis. Antibodies to P-selectin alone reduced the accumulation of neutrophils and monocytes in rat adjuvant-induced arthritis (AIA) [168, 169]. AntiCD62E enhanced the inhibition of neutrophil and monocyte accumulation with anti-CD62P, but had no effect when used alone; anti-CD62L had no effect either alone or in combination with anti-CD62P [168, 169]. However, CD62P-deficient mice developed collagen-induced arthritis (CIA) more rapidly and more severely than wild-type mice indicating that P-selectin is not necessary for inflammatory arthritis to occur [170]. Further studies will be needed to clarify the roles of selectins in animal models of arthritis.


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CD44 may be important for the capture of lymphocytes by synovial vascular endothelium. Early studies indicated that the adhesion molecules used by lymphocytes that migrate to synovium may be different than those used by lymphocytes that migrate to lymph nodes and mucosal sites. In one study, lymphoblastoid cell lines that bound to either lymph node HEV or mucosal HEV in a modified Stamper-Woodruff assay [171] did not bind to synovial HEV, and antibodies that blocked the binding of peripheral blood lymphocytes to lymph node HEV had no effect on lymphocyte binding to synovial HEV [172]. Although it has been known that CD44 is dramatically upregulated in RA synovium [173], it has only been recently that CD44 has been identified as an important adhesion molecule for migration to synovium. The basis for this has been severalfold. First, more recent Stamper-Woodrufftype studies using a broad panel of antibodies identified that the binding of lymphoblasts to synovial HEV could be partially blocked by anti-CD44 antibodies, but that blocking the a4 integrin, b1 integrin, b2 integrin, b7 integrin, and L-selectin pathways had no effect on binding to synovial HEV [174]. Second was the recognition that CD44 mAbs that inhibit CD44-HA interactions were able to inhibit the production of edema and leukocyte migration into the synovium in mouse models of arthritis [175, 176]. CD44-HA interaction also appear to play a major role in other types of migration such as the homing of superantigen-stimulated Vb8+ T cells to the peritoneum [177]. Third, inflammatory arthritis disease activity in humans has correlated with the ability of pathogenic T cells to roll on HA [178–180]. In addition, TNF-a, upregulates HA binding to CD44 in monocytes, and the anti-inflammatory cytokines IL-4 and IL-13 downregulate monocyte CD44-HA binding [181, 182]. All of these findings together indicate a possible role for CD44 in the pathogenesis of inflammatory arthritis. Presumably, integrins and their ligands must be involved in the homing of leukocytes to the synovium since they are instrumental in the firm adhesion of leukocytes to the vascular endothelium. However, the data regarding the roles of specific integrins are contradictory. In vitro, blocking either a4 integrins, b1 integrins, b2 integrins or b7 integrins has no effect on the ability of lymphocytes to bind to RA synovial vascular endothelium [174]. In vivo, in SCID mice engrafted with human RA synovium (SCID/hu), antibodies to CD11a, aEb7 and CD54 but not CD103 inhibited mononuclear cell migration to the synovium [183, 184]. However, anti-LFA-1 (CD11a/CD18) and antiMac-1 (CD11b/CD18) had no effect on T lymphocyte migration to inflamed joints in AIA [185]. Anti-CD18 and anti-CD49d in combination inhibit a majority of neutrophil accumulation in AIA joints [186, 187]. For monocytes, anti-LFA-1 and anti-VLA-4 combine to effectively block their migration to synovium in AIA [188]. CD54-deficient mice had markedly reduced incidence

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and severity of CIA [189]. Thus, most of the evidence indicates a role for b2 integrins, CD54 and possibly CD49d in the migration of neutrophils and monocytes to inflamed joints. Based on the above data and the important role of b2 integrins and CD54 in leukocyte migration to sites of inflammation, the first attempts to specifically block leukocyte entry into the synovium in man have been aimed at blocking CD54 with monoclonal antibodies [190, 191]. While there was limited clinical efficacy in patients with refractory RA [190], anti-CD54 mAbs did induce an alteration in T cell recruitment with increased numbers of circulating lymphocytes (but not neutrophils or monocytes) and persistent T cell hyporesponsiveness leading to a second, phase I/II open label study in patients with early RA [191, 192]. In this second open label trial, 5/10 patients had a ?50% reduction and 7/10 patients had a ?20% reduction in tender/swollen joint counts [191]. The mechanism by which anti-CD54 mAbs may function in RA are unclear since increases in peripheral blood lymphocytes did not correlate with improvement in disease activity [190, 191]. Chemokines in RA It is rapidly becoming clear that chemokines and their receptors are intimately involved in regulating organ-specific leukocyte trafficking and inflammation. Since the groundbreaking finding that a defect in BLR-1 (now called CXCR5), a GPCR on B cells, led to severe defects in the migration of B cells to spleen and Peyer’s patches but normal migration to mesenteric lymph nodes [193], many groups have confirmed that alterations of chemokines or their receptors lead to defects in organ-specific trafficking. With the recent explosion in the discovery of new chemokines and their receptors (table 2), investigators have identified that many chemokines like TARC, MDC, SLC/6Ckine, LARC/MIP-3a and BLC are expressed in an organ-specific manner [91, 97, 194–196] and that they regulate leukocyte migration to those organs [98, 193, 197]. Similarly, chemokine receptor expression is also tightly regulated and defines leukocyte subsets by cell type as well as function. Chemokines, like adhesion molecules, can be divided into two general functional categories: constitutive and inflammatory. The constitutively produced chemokines such as BLC, ELC and SLC function in normal leukocyte migration such as that of B cell and naı¨ve T lymphocyte homing to lymph node. The receptors for these constitutively produced chemokines (CXCR5 for BLC and CCR7 for ELC and SLC) are expressed predominantly on B cells and naı¨ve T cells, respectively. The inflammatory chemokines such as MIP-1b, RANTES, eotaxin, Mig and IP-10 are predominantly expressed in inflamed tissues and direct the migration of specific leukocyte subsets. Certain inflammatory chemokines can be subdivided into those that act primarily on


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neutrophils, eosinophils/basophils, Th1 cells or Th2 cells. For example, IP-10 and Mig that act through CXCR3, a receptor primarily expressed on Th1 cells [93], are thought to be involved in the pathogenesis of multiple sclerosis [198, 199], a Th1-type disease. CCR5 is a second chemokine receptor that is predominantly expressed on Th1 cells as well as monocyte lineage cells [93]. CCR3 and CCR4 appear to be differentially expressed on Th2 cells compared to Th1 cells [93]. Those chemokines that act through CXCR1 and CXCR2 (IL-8, GRO, ENA78, NAP-2) act primarily on neutrophils. Thus, leukocyte chemokine receptor expression provides a means of regulating migration in a tissue-specific, leukocyte subset-specific, Th1- versus Th2-specific manner. Altering chemokine receptor surface expression can also lead to a dynamic regulation of leukocyte trafficking. For example, immature dendritic cells (DC) in the circulation can be recruited to a site of inflammation based on their expression of CCR5 and CCR6 [200–204]. When in the inflamed tissue, they can take up antigen and be induced to mature into mature DC that are better capable of antigen presentation. In this process, the chemokine receptor expression is switched from CCR5/CCR6 to CCR7 whereby the mature DC is now capable of responding to lymph node-produced SLC but not the inflammatory chemokines and thus moves away from the inflamed tissue into the lymph node where it can present antigen to naı¨ve T cells [201, 203–208]. Thus, chemokines and their receptors are key regulators of the inflammatory process. Our current understanding of the role of chemokines in the pathogenesis of human RA is based primarily on extensive studies evaluating the pattern of chemokine expression in synovial tissues and fluids, and on limitied studies of chemokine and chemokine receptor antagonists in animal models of inflammatory arthritis. With rare exceptions, the synovial fluid levels of all the chemokines that have been tested thus far are elevated in RA compared to osteoarthritic controls. This is not unexpected since the inflammatory infiltrate in RA is composed of nearly all leukocytes including monocytes, lymphocytes and mast cells in synovial tissue and predominantly neutrophils in the synovial fluid. Whether at the mRNA or protein level, expression of IL-8, ENA-78, MIP-1a, RANTES, MCP-1 and IP-10 is increased in RA compared to OA [89, 209]. Only MIP-1b has been shown in a single study to be elevated in OA compared to RA synovial fluid [210]. IL-8 and ENA-78, which are primarily chemoattractants for neutrophils and monocytes, are highly increased in the synovial fluid of patients with RA compared to OA and are expressed by synovial macrophages, synovial fluid mononuclear cells and IL-1- or TNF-a-activated synovial fibroblasts [211–213]. MCP-1, primarily a monocyte chemoattractant, is elevated in the synovial fluid of RA patients and is produced by synovial macrophages, activated (IL-1, TNF-a or IFN-c) synovial fibroblasts, and acti-

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vated (IL-1, TNF-a, LPS or TGF-b) chondrocytes [214]. The published expression patterns of the macrophage-lymphocyte chemokines, MIP-1a, MIP-1b and RANTES (which all act through CCR5 among other receptors) in RA is confusing. MIP-1a which is expressed by synovial macrophages, TNF-aactivated synovial fibroblasts and synvoial fluid mononuclear cells is elevated in RA synovial fluid [215]. MIP-1b has been shown to be elevated in OA compared to RA synovial fluid [210]. RANTES mRNA is inducible by IL-1 and TNF-a in RA synovial fibroblasts [213, 216, 217], but the levels of RANTES protein are about the same in RA as in OA [216]. Very little is known, in RA, about the lymphocyte-specific chemokines such as IP-10, Mig, I-TAC, Ltn, LARC, SLC, ELC or BLC. IP-10 mRNA can be induced by IL-1 and TNF-a [218]. Based on the hypothesis that RA is a Th1type disease, we have evaluated the expression of chemokines that attract Th1 cells in RA. IP-10, Mig, MIP-1a and MIP-1b are highly elevated in RA synovial fluid and tissue compared to OA, and there is a gradient of the chemokine from the blood into inflamed synovial tissue [Patel and Whichard, unpubl. data]. Further, virtually all cells in the synovial fluid and tissue express the Th1 chemokine receptors CCR5 and CXCR3 [219]. The role of CCR5 in the pathogenesis of RA is unclear since some studies indicate that there is no decreased prevalence of RA in individuals with the CCR5D32 mutation [220, 221] that encodes a defective receptor, some studies indicate that there is a decreased prevalence of RA in these individuals [222], and some studies indicate that the lack of CCR5 affects some RA variable such as IgM rheumatoid factor production [221]. In figure 3, we have summarized the known information on chemokines in RA, and have also added our unpublished observations. One of the central themes to be appreciated is that TNF-a and IL-1 activate cells of the synovial microenvironment including monocyte lineage type A synovial lining cells, fibroblasts, EC and chondrocytes to produce chemokines. Therapies such as steroids, anti-TNF and other biologic agents that are known to inhibit joint inflammation also inhibit synovial chemokine production [223; Smeets et al., unpubl. data]. With the recent identification of SLC as a chemokine involved in T cell trafficking to secondary lymphoid organs and BLC as a chemokine involved in B cell trafficking to secondary lymphoid organs [97, 98, 193], it will be of great interest to evaluate SLC and BLC in the context of the subset of RA with lymphoid follicles and germinal centers. Additionally, it may be that the pathogenesis of the granulomatous subset of RA which has high levels of IFN and TNF is mediated in part by the Th1 chemokines IP-10, I-TAC and Mig. Recently, there has been great interest in evaluating the role of chemokine and chemokine receptor antagonists in either the treatment or prevention of


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Fig. 3. Chemokines in the rheumatoid synovial microenvironment. Abbreviations are explained in footnotes to tables 1 and 2.

RA. Clearly, chemokines are an important component of the inflammatory response. In certain circumstances such as the myocarditis associated with Coxsackie virus infection, the lack of a chemokine (MIP-1a) may prevent the inflammatory response [224]. However, with the great redundancy of chemokines and their receptors, inhibiting single chemokines or their receptors may not be effective as monotherapy in RA. They may have a role as adjuvants combined with other antirheumatic drugs. Biologicals aimed at inhibiting chemokine-mediated inflammatory responses have had some efficacy in animal models of RA. Antibodies to MIP-1a and MIP-2 ameliorated disease in CIA in mice, but was unable to prevent the disease [225]. MetRANTES, a CCR antagonist that interacts with

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CCR1, CCR3, CCR4 and CCR5, ameliorated disease activity in CIA in a dose-dependent manner suggesting that chemokine antagonists with a broad effect may have potential therapeutic utility in RA [226]. However, this approach may also have significantly more side effects than agents aimed at single receptors. MCP-1 (9–76), a truncated form of MCP-1 that is a potent antagonist of CCR2, prevents the onset of AIA in the autoimmunity-prone MRL-lpr mice [227]. Full length MCP-1, on the other hand, enhances AIA [227]. Antibodies to MCP-1 also reduced joint disease in rat AIA [228]. The platelet factor-4 octapeptide CT-112 (TTSQVRPR) partially inhibited the progression of CIA [229]. Thus, in the limited number of studies that have been performed, chemokine receptor antagonists have had some efficacy in ameliorating disease severity in animal models of RA. Except under special circumstances, therapies aimed at preventing a single chemokine or chemokine receptor interaction are unlikely to have great efficacy as monotherapy in the treatment of RA. Certainly those therapies that target a broad spectrum of chemokines or receptors are more likely to be efficacious but may have more side effects. Since a great many small molecule inhibitors of GPCRs (in particular a- and b-adrenergic and opioid receptors) are being successfully used in the clinic, there has been a great interest in producing small molecule inhibitors of chemokine receptors. Currently several compounds have been reported that inhibit CCR1, CCR5, CXCR2 and CXCR4 [230–234] and many more are on the way. Studies on the utility of these agents in RA remain to be performed.

Conclusion In the last 5 years, there has been a great explosion in our knowledge about the mechanisms by which leukocytes migrate into tissues, and the molecules that are involved in regulating this migration. While we have known that adhesion molecules are essential components of the trafficking process, we had not realized the great complexity, redundancy and specificity afforded by chemokines and their receptors. The challenge is going to be to identify pathways or molecules that are specific for migration to synovium. Although some adhesion molecules, chemokines and their receptors (for example, CLA, MAdCAM-1, BLC and CXCR5) target leukocytes to specific tissues, no synovium-specific molecules have yet been identified. It may be that such synoviumspecific pathways do not exist. However, based on an increasingly large body of work accumulating on the mechanisms of leukocyte homing to synovium, several new targets for RA therapies including CD44, CCR5 and CXCR3 amongst others have recently been identified.


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Acknowledgments This work was supported by National Institutes of Health grant AR39162.

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Dhavalkumar D. Patel, MD, PhD, Box 3258, 223 MSRB, Duke University Medical Center, Durham, NC 27710 (USA) Tel. +1 919 684 4234, Fax +1 919 681 9399, E-Mail [email protected]


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Lymphoid Microstructures in Rheumatoid Synovitis Cornelia M. Weyand, Andrea Braun, Seisuke Takemura, Jo¨rg J. Goronzy Departments of Medicine/Rheumatology and Immunology, Mayo Clinic and Foundation, Rochester, Minn., USA

Introduction The lesion of rheumatoid arthritis (RA) is a tissue infiltrate of inflammatory cells accumulating in the synovial membrane. Tissue-infiltrating cells are lymphocytes and macrophages; neutrophils are rarely seen in rheumatoid synovitis. Professional antigen-presenting cells are also represented in the infiltrates, providing the opportunity for adaptive immune responses to occur at this unusual tissue site. While the identification and characterization of antigens presented and recognized in the synovial layer is of obvious interest, it is equally important to understand the factors in the synovial microenvironment that facilitate immune recognition. Optimal immune responses require not only the interactive effort of several different and highly sophisticated cell populations, but also depend upon the spatial relationship of cells involved in the generation of immune responses. The immune system has accommodated for this need by the formation of primary, secondary, and tertiary lymphoid structures. A unique feature of the immune system is that antigens are transported to lymphoid tissues where appropriate cell densities and the proper architecture are available for priming of naive T cells and B cells. In contrast to many other chronic inflammatory diseases, the lymphoid infiltrates in rheumatoid synovium establish a complex arrangement and actually form microstructures of tertiary lymphoid tissues. The formation of extranodal lymphoid tissues in synovial membrane cannot remain without consequences and raises several important questions that will be addressed in this review. What is unique about the layer of synovial lining that allows for the recruitment of lymphocytes and macrophages and also for the generation

of lymphoid microstructures? Understanding this target tissue susceptibility may be critical in getting insights into the immunopathogenesis of RA. Susceptibility of the synovium to participate in the establishment of lymph nodelike structures could introduce a bias for immune responses to preferentially manifest in the joint. Careful examination of rheumatoid synovitis has indicated that multiple patterns exist. In a subset of patients, classical granulomata are encountered in the inflamed synovial membrane. Yet other patients form lymphoid follicles that can represent typical germinal centers. The existence of these two quite distinct lymphoid microstructures already suggests that more than one type of immune response can be hosted in the joint. It also emphasizes that synovial tissue, as well as other components of the joint, may have an impact on the direction an immune response takes. It is almost predictable that the contribution of different regulatory and effector cells will not be the same, dependent upon which lymphoid architecture has been established. Finally, it would be worthwhile to explore how the formation of tertiary lymphoid tissue affects the immune system as a whole, as it is likely that the pathogenic immune responses in the joint occur in the context of more complex dynamics imposed upon the global immune system. Besides the potential benefit of understanding the rules of immune recognition events in the rheumatoid joints, a more detailed investigation of the joint lesion may also be helpful in dissecting the heterogeneity of RA, a clinical syndrome almost certainly encompassing more than one disease. Assigning a pathway of immune stimulation to a patient could have prognostic value and could provide a rationale for targeted immunointervention [1].

CD4 T Cells Are the Ultimate Regulators of Synovial Inflammation Findings emphasizing the T cell dependence of RA synovitis include the dominance of T cells among tissue-infiltrating cells in the inflamed synovial membrane and the genetic association of the disease with HLA class II genes [2–5]. T cell receptor repertoire studies have documented that the synovial infiltrate contains multiple clonally expanded T cell populations, supporting the view that the disease involves activation and clonal proliferation of CD4 T cells in the tissue [6–17]. T cells with identical T cell receptor sequences have been detected in different joints of the same patient, suggesting that they respond to an arthritogenic antigen [18, 19]. The opposing view that synoviocytes become autonomous and progress with tissue destruction independent of the regulatory control of T cells has been fueled by at least three lines of evidence. First, Firestein et al. [20, 21] have shown that the tumor

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suppressor gene p53 is overexpressed in rheumatoid synovium and frequently contains somatic mutations. Second, despite the large number of tissue-infiltrating T cells that are clonally expanded and express activation markers, T-cell-derived cytokines are not very abundant [22]. Last and maybe most importantly, RA has been proven to be rather resistant to T-cell-directed therapies. Compared to other chronic inflammatory diseases such as psoriasis, treating of RA with cyclosporine is only moderately effective [23]. Administration of T-cell-specific monoclonal antibodies induced profound and long-term peripheral T cell lymphopenia; however, the clinical effects were only shortlived and patients developed progressive arthritis in spite of low numbers of peripheral T cells [24]. These findings are less conclusive, for several reasons, than they appear to be. T-cell-derived cytokines are not produced abundantly in any type of chronic inflammatory response. Antigen-specific T cells comprise only a minority of T cells in a chronic lesion. However, mediators released by T cells are explicitly potent, and small numbers of activated T cells are sufficient to stimulate the innate immune system that then acts as an amplification loop. Also, cytokine production by T cells is transient after T cell activation and T cell stimulation is not synchronized, predicting that cytokine-producing T cells have to be represented in low frequencies. Finally, the T cell depletion studies were not effective in depleting clonally expanded and putatively diseaserelevant T cells. On the contrary, such T cells had a competitive advantage in restoring the lymphocyte pool after T cell depletion. Analysis of tissue-infiltrating cells in the synovium of antibody-treated patients with pronounced peripheral lymphopenia demonstrated that the T cell infiltrate was essentially unaffected, while the repertoire of circulating T cells was markedly contracted, and the clonal T cell populations present in the synovial infiltrate became dominant among circulating T cells [25]. Therefore, the failure of T-cell-depleting therapies does not indicate T cell independence of the synovial inflammation. Rather, T cell depletion in patients has not been efficacious, and the repopulation dynamics of T lymphocytes in RA appears to favor the proliferation of surviving T cells and not the thymic generation of new T cells. To perform more conclusive studies on the role of T cells in the synovial inflammation, we and other investigators have developed a human synoviumSCID mouse chimera model [26–28]. Synovial tissue implanted into SCID mice is engrafted within 1 week, the tissue is vascularized with human capillaries that apparently connect to the murine vasculature, and the inflammatory response including activation of T cells, macrophages, and synovial fibroblasts persists for several months. Klimiuk et al. [29] have depleted human T cells in such synovium-SCID mouse chimeras by administering anti-CD2 monoclonal antibody. Dependent on the dose of anti-CD2 antibody used, synovial interferon (IFN)-c production declined. This decline was strictly paralleled by a sharp

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reduction in in situ transcription of the monokines interleukin (IL)-1b and tumor necrosis factor (TNF)-a (fig. 1). In addition, T cell depletion disrupted the transcription of IL-15. IL-15 has recently been shown to represent an important amplification mechanism, resulting in the enhanced production of TNF-a [30, 31]. Independent of the recognition of antigen, IL-15 in the synovial tissue activates T cells to provide a cell contact-dependent signal to macrophages for the production of TNF-a. Data from the synovium-SCID mouse chimeras suggest that this amplification loop is ultimately dependent on signals derived from antigen-specific T cells [28]. Declining cytokine production was followed by a decrease in the production of metalloproteinases 1 and 3. Conversely, adoptive transfer of autologous synovial CD4 T cells boosted the in situ production of IFN-c as well as TNF-a. Taken together, synovial CD4 T cells are the ultimate regulators of synovial inflammation. IFN-c has emerged as the critical cytokine that not only induces synoviocyte activation but that also appears to be necessary for macrophage survival in the engrafted synovial tissue. Reconstitution with exogenous IFN-c after T cell depletion completely restored the integrity of the synovial graft with increased macrophage survival and also sustained monokine and metalloproteinase production.

T Cell Responses in the Synovium: More than One Pathway The T cell depletion experiments in the human tissue-SCID mouse chimeras drew a seemingly uniform picture of rheumatoid synovitis in that the synovial inflammation was T-cell dependent and IFN-c was the key regulator. This uniformity contrasts with the histological variability of the synovial inflammation [32]. In a case series of synovial tissue specimens obtained either at the time of joint replacement or by arthroscopic synovectomy, three histological patterns have been distinguished. Onethird of all samples contained follicular lymphoid aggregates. In a slightly higher percentage of patients, the infiltrate of T cells, B cells and macrophages lacked a clear topographical arrangement, but formed a diffuse infiltrate. In the minority of tissue samples, a granulomatous pattern characterized by necrotic centers lined with a collar of epitheloid histocytes was found. These different patterns appeared to be rather specific for each patient. In particular, synovial granuloma formation and follicular aggregates did not coexist in the same tissue. Also, there was a high correlation in tissues samples obtained from different joints or at different points in time from the same patient. To explore whether these different topographical arrangements correlated with cytokine profiles, IFN-c and IL-4 were semiquantified as marker cytokines for Th1 and Th2 cells, respectively (fig. 2). CD4 T cells can be grouped into


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Fig. 1. Effects of T cell depletion on synovial cytokine and metalloproteinase production. Human rheumatoid synovium was implanted into SCID mice, and the human tissue-mouse chimeras were treated with anti-CD2 monoclonal antibody. Grafts were harvested 6 days after treatment, and cytokine and metalloproteinase mRNA production were semiquantified by PCR-ELISA. Antibody treatment resulted in the depletion of up to 90% of all tissueinfiltrating T cells. IFN-c production in the synovial grafts declined with increasing doses of antibodies. In parallel, mRNA tissue concentrations of the macrophage/synoviocytederived cytokines IL-1b, TNF-a and IL-15, as well as metalloproteinases (MMP) 1 and 3, decreased, demonstrating that the production of these mediators is T-cell dependent [reproduced with permission from 29].


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Fig. 2. Heterogeneity of the synovial inflammation in RA. Synovial tissues from RA patients were classified by using the topographical arrangement of the mononuclear infiltrate as a histopathological criterion. The number of IFN-c and IL-4 transcripts in tissue samples from the different categories was semiquantified. Results of the cytokine measurements are shown as box plots with medians, 25th and 75th percentiles, and whiskers representing the 10th and 90th percentiles. Diffuse synovitis was characterized by low production of IFN-c and IL-4. In follicular synovitis, the production of IFN-c dominated, while in granulomatous synovitis, IFN-c and IL-4 were both abundantly transcripted [reproduced with permission from 32].

functional subsets that differ in their homing pattern and in the pattern of cytokines they produce. TH1 cells secrete IFN-c, IL-2 and TNF-b, whereas TH2 cells produce IL-4, IL-5 and IL-13 [33, 34]. IL-10 is also considered a TH2 cytokine, although it can be produced by both subsets. These two sets of cytokines are, at least in part, mutually antagonistic, and it has been argued that an imbalance in cytokine expression is a crucial event in the development of autoimmune diseases [35]. In tissue with diffuse synovitis, both marker cytokines IFN-c and IL-4 were detected in low quantities. In contrast, granulomatous tissue was associated with high concentrations of both IFN-c and IL-4. Follicular synovitis had a unique pattern in that IFN-c was present at intermediate concentrations; however, IL-4 was completely absent. Surprisingly, IL-10 was found in high concentrations in the tissue samples with lymphoid aggregates. The source of


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the IL-10 is not clear, but it possibly derives from B cells. Thus, none of the tissues displayed a typical TH1 pattern. The production of the macrophage products, IL-1b and TNF-a, correlated closely with the amount of IFN-c mRNA, emphasizing the causal relationship between IFN-c and monokine production demonstrated in the synovium-SCID mouse T cell depletion model [29]. These data provide a new framework for the interpretation of synovial cytokine studies. In earlier reports, RA could be distinguished from other inflammatory arthritides by a TH1 response, whereas TH2 cytokines are found in reactive arthritis [36]. It has, therefore, been concluded that RA is a disease of immune deviation and that commitment to the TH1 cytokine pathway is relevant in the pathogenesis of the disease. Consequently, IL-4, IL-10 and IL-13 have been explored as therapeutic interventions in animal models as well as in early human treatment studies [37–39]. Additional studies have confirmed that IFN-c is the dominant T cell cytokine in RA, although other TH2 cytokines have been detected in varying degrees [12, 40, 41]. In particular, IL-6 and IL-10, both of them TH2 cytokines but also produced by non-T cells, have been found to be present in high concentrations [42, 43]. The emerging model proposes that RA can be associated with more than one cytokine pattern with individual patterns possibly being important in shaping the progression of the disease. Leprosy is a classical example of such a pathogenic mode. In leprosy, tissue destructive inflammation develops in the setting of multiple patterns of cytokines [44]. The clinical variant of lepromatous disease is characterized by TH2 cytokines and can be clearly distinguished from tuberculoid disease that is dominated by TH1 cytokines. Taken together, the cytokine patterns in RA cannot be easily explained along the lines of TH1 and TH2 pathways. With the caveat that all data are based on established disease and do not allow conclusion on early events, there is no evidence that RA is characterized by a single cytokine pattern that is critical in the development of the disease. Evidence suggests that the patterns of cytokines secreted contribute to the organization of the inflammatory infiltrate and thus influence the outcome of the disease. The different patterns are maintained in individual patients and may be in part genetically determined.

Diversity of Lymphoid Microstructures Generated in Rheumatoid Synovium Granuloma Formation in Synovial Tissue Rheumatoid synovitis presents as a diffuse mononuclear infiltrate in about one half of the patients; in the other patients the infiltrate develops a characteristic microarchitecture, either in forms of lymphoid aggregates or granulomata.


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Granuloma formation is characteristic for RA not only in the synovium but also in extra-articular tissues. The mechanisms leading to granuloma formation have not been elucidated, and it is unclear why this type of granulomatous inflammation is rather specific for a subset of RA patients. Rheumatoid nodules form in a radiating pattern around an area of tissue necrosis. The central region of necrosis is rimmed by palisading fibroblasts surrounded by an area of neovascularization and infiltrating mononuclear cells, including activated macrophages and T cells. The central necrosis may be the result of vascular injury that either occurs spontaneously or is precipitated by a microtrauma. The formation of the anatomical structure around the central area of necrosis is regulated by cytokines. IFN-c activates macrophages and allows for their transformation into histiocytes [45]. The role of IL-4 in granulomata is less clear; however, formation of hypersensitivity granuloma can be a hallmark of TH2 responses, as seen in schistosoma egg-induced granuloma formation [46]. The release of high amounts of transforming growth factor-b in these structures may actually contribute to the increased matrix production that is characteristic for rheumatoid nodules. Cytokine production alone does not explain the uniqueness of granulomatas and does not allow conclusions on why some, but not all, patients develop this lesion. Correlative association studies have identified several risk factors that are associated with rheumatoid nodule formation. These may be, at least in part, genetically determined and may help in understanding the pathogenesis of this histological variant. Patients with nodule formation generally produce very high titers of rheumatoid factor. Genetically, they are characterized by the presence of two disease-associated HLA-DRB1*04 alleles, assigning a crucial component in nodule formation to CD4+ T cells. Finally, patients with nodular disease also have an expanded frequency of CD4 natural killer (NK) T cells [47]. These T cells combine the expression of T cell receptor ab heterodimers with cell surface molecules that are typically found on NK cells [48]. Specifically, these cells express MHC class I-recognizing receptors of the KIR/KAR immunoglobulin superfamily as well as the C-type lectin molecule CD161. CD4 NK T cells are functionally characterized by their ability to exhibit cytotoxicity via granzyme/perforin mechanisms and to produce high amounts of IFN-c [49, 50]. The correlation between increased frequency of these cells and susceptibility to nodule formation may suggest a direct involvement of cytotoxic cells in the initial vascular injury, as well as in the subsequent cytokine amplification loop. Formation of Lymphoid Aggregates in Synovial Tissue Lymphoid aggregate formation in the synovial tissue has long been considered one of the histological hallmarks of RA. However, it only occurs in about


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30% of all patients. Also, it is not specific for rheumatoid inflammation but can be found in other arthritides. It was first described in RA in the early 1980s [51, 52] and has subsequently been found by Steere et al. [53] in Lyme arthritis. Synovial lymphoid aggregates resemble many features of ectopic lymphoid tissue and, in fact, can assume microscopic characteristics reminiscent of germinal centers. Germinal centers are specialized microanatomical structures that are required for the generation of high-affinity antibodies and the selection of memory B cells in response to antigens [54, 55]. Germinal centers arise from B cells that accumulate among the processes of follicular dendritic cells and undergo proliferation, apoptosis and immunoglobulin gene hypermutation. Indeed, Schroder et al. [56] have demonstrated B cell hypermutation in the synovial membrane of RA, clearly demonstrating germinal center activity. Ectopic lymphoid organ formation in inflammatory diseases is unusual and appears to occur preferentially in certain target tissues such as the synovium and mucosa-associated tissue. Understanding how ectopic lymphoid tissues are formed may be accelerated by recent progress in defining the signals that control the development of normal secondary lymphoid tissue. In these studies, lymphotoxin (LT) has emerged as a critical signaling molecule with dual function [57]. Studies in LT-deficient mice have demonstrated that LT provides crucial signals that are required for the formation of secondary lymphoid tissues. The major function has been assigned to the LT a1b2 heterotrimer expressed as a cell surface molecule and not to the soluble LT a3 homotrimeric peptide. Some of the actions of the membrane-bound LT are directed to lymphoid tissue during organogenesis; the target cell for this action is unknown. However, the effect can only be achieved during a short window of time during development and is then fixed. In addition, membrane-bound LT action is required for the integrity of the lymphoid tissue. This is particularly true for the formation of follicular dendritic cell clusters, which are strictly dependent on the expression of LT on B cells. The ability of lymphoid follicles to fully develop germinal centers is dependent on T cell help. Molecular mechanisms include the CD40-CD40 ligand (CD40L) and CD28-CD80/CD86 interactions [58, 59]. Patients with the primary immunodeficiency hyper-IgM syndrome have lymph nodes with primary follicles lacking germinal center reactions and are not able to generate memory B cells. The molecular defect underlying this syndrome has been localized to a mutation of the CD40L gene [60]. Lymphoid aggregates are not a uniform structure in the rheumatoid synovium. Germinal centers develop in about one half of all patients who form lymphoid aggregates [61]. The remainder do not have evidence of B cell proliferation and loss of IgD expression on B cells. Characteristically, these aggregates also lack a network of follicular dendritic cells. In contrast, functional


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germinal centers have follicular dendritic cell networks, B cell proliferation, and downregulation of IgD expression. The reasons why lymphoid aggregates form in some, but not other, tissues are not understood; however, progress has been made in defining the molecular mechanisms that distinguish a nondeveloped lymphoid aggregate from a fully functional germinal center. One of the central players appears to be the membrane form of LT that is exclusively expressed in synovial tissue with fully developed germinal centers but not in tissue samples lacking germinal center reactions (Braun et al., unpubl. observations). LT-b is mainly produced by a small subset of immature B cells in the follicular mantle zone and also by some B cells in the germinal center. The target cell regulating these LT-expressing B cells is unclear. However, based on murine experiments, LT may be involved in the formation and the maintenance of the network of follicular dendritic cells. In this context, it is of particular interest that recent studies have shown that synovial fibroblasts can acquire markers of follicular dendritic cells after exposure to TNF-a [62]. The second characteristic marker that distinguishes lymphoid aggregates from fully developed germinal centers is the presence of a subset of CD8 T cells in the lymphoid follicles [61]. This is surprising because CD8 T cells have not been considered important contributors to germinal center formation. In fact, in murine experiments, virtually all of the T cells in the germinal centers express the CD4 marker. Follicular CD4 T cells are thought to influence germinal center formation by recognizing antigen on B cells and engaging CD40 on the B cells via CD40L. We, therefore, explored the question whether follicular CD8 T cells also express CD40L. Indeed, CD8+CD40L+ cells were a hallmark of classical germinal centers while CD4+CD40L+ cells were found in both germinal centers as well as in lymphoid aggregates without follicular dendritic cells. CD4+CD40L+ cells were functionally different from normal CD8 T cells in that they expressed IFN-c but not perforin mRNA. These studies suggest that a subset of CD8 T cells is crucial in the development of fully functional germinal centers in the synovial tissue [60]. To address the question whether CD8 T cells truly accumulate in follicular structures and recognize antigen, a microdissection technique was used to isolate T cells from distinct germinal centers and to analyze the follicular T cell receptor repertoire. These studies showed that identical T cell receptor b-chains were found in distinct germinal centers from the same patient, suggesting that clonally expanded T cells are seeded into different germinal centers where they recognize the same antigen [63]. More than one half of the shared T cell receptor sequences were derived from CD8+ T cells and not from CD4+ T cells, supporting the notion that antigen recognition by CD8 T cells is of functional importance in the transformation of a lymphoid aggregate into a functional germinal center.


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What are the implications of the formation of tertiary lymphoid microstructures in the rheumatoid synovium? Synovial tertiary lymphoid structures resemble secondary lymphoid tissues with the notable difference that they are formed outside of lymphoid organs such as lymph nodes, Peyer’s patches and spleen. It is, therefore, likely that similar functional considerations apply. Secondary lymphoid tissues are thought to enhance the sensitivity of antigen recognition due to the topographical organization of their cellular constituents. Zinkernagel et al. [64] have demonstrated that induction of an immune response requires only a minute amount of antigen if the antigen is directly administered into a secondary lymphoid organ. In comparison, a significantly higher amount of antigen is needed if it is administered into the skin. It may be for this reason that secondary lymphoid tissues are located at strategic sites where foreign antigen is likely to be encountered. In addition, they control the maturation and differentiation of the immune response, in particular the generation of high affinity memory B cell responses [55]. In analogue, it could be postulated that the lymphoid organization in the synovium enhances antigen presentation and antigen recognition and allows to maintain T cell responses to low-abundant antigens. To explore the contribution of follicle formation to T cell responses in the synovium, we have used human synovium-SCID mouse chimeras and have depleted B cells by treating the animals with anti-CD20 monoclonal antibody. Depletion of B cells resulted not only in the disbanding of the lymphoid aggregates but also in a loss of tissue-infiltrating T cells [65]. Overall, the in situ activation of T cells rapidly declined. Transcription of IFN-c in the synovial tissues harvested from treated animals was significantly reduced compared with untreated animals. In parallel, the transcription of IL-1b declined. These studies demonstrated that lymphoid follicles and, in particular, follicular B cells are pivotal for antigen-dependent T cell stimulation. Thomas et al. [66] have shown an abundance of interdigitating dendritic cells in the synovial tissue. The B cell depletion studies suggest that dendritic cells are not sufficient to guarantee optimal T cell activation. This conclusion was further substantiated in adoptive transfer studies [65]. CD4 T cell clones were derived from synovial tissue, and clones were selected that had T cell receptor sequences demonstrated to be represented in the synovial germinal centers. Adoptive transfer of such T cell clones into mice engrafted with autologous tissue from the same patient homed to the synovial graft where they were activated and started to produce IFN-c. Similar results were obtained when these clones were transferred into mice engrafted with HLA-DRB1*04-matched tissue but not into mice with HLA-DRB1-mismatched tissue, supporting the notion that activation of the adoptively transferred T cells was dependent on HLA-DRrestricted recognition of antigen. However, matching for HLA-DR genotypes


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was not the only factor controlling T cell function in the graft. Synovial tissues with diffuse mononuclear cell infiltration and without any topographical organization of the infiltrate did not support the activation of adoptively transferred T cells regardless of the HLA-DRB1 type, again emphasizing that B cells organized into lymphoid aggregates are critical in the activation of synovial T cells. Synovial T Cells with Anti-Inflammatory Action The complexity of the lymphoid microstructures in rheumatoid synovium predicts close regulation of immune processes in this inflammatory tissue. Factors determining the particular pathway of immune activation are obviously different among patients, and the presence of specialized T cell subsets may be critical in guiding the inflammatory reaction in a certain direction (e.g. CD4+CD28– T cells in granulomatous RA and CD8+CD40L+ T cells in ectopic germinal centers). Patients with diffuse synovitis are characterized by a lack of sophisticated microstructures, which could either result from the absence of such T cell subsets or, alternatively, could indicate active inhibition. Suppressive mechanisms would also provide an explanation for the subdued T cell response in diffuse synovitis. The studies described so far have focused on identifying mechanisms that are responsible for the topographical organization of the infiltrate and the formation of lymphoid microstructures. In this model, the lack of a critical factor or cell would explain the absence of a lymphoid structure in some patients, such as the absence of certain T cell subsets (CD4+CD28– T cells in granulomatous RA, CD8+CD40L+ T cells in patients with ectopic germinal centers). Alternatively, downregulatory mechanisms may be important in suppressing the expression of inflammatory cytokines in the tissue or interfering with a topographical organization of the infiltrate, and the subdued T cell response in the patients with a diffuse synovitis may be the result of active inhibition. Several downregulatory pathways have been described to be functional in synovial T cells. TNF-a, a dominant cytokine in rheumatoid inflammation, has also been shown to inhibit T cell responses [67, 68]. Although this may partially explain the functional impairment of tissue-infiltrating T cells, it does not explain the heterogeneity of inflammatory lesions. TNF-a and IFN-c show a direct and not an inverse correlation, consistent with the model that IFN-c induces TNF-a production [29]. A second hypothesis, promoted to explain the subdued T cell response, postulates that the rheumatoid synovium, although highly vascularized, is a tissue with high oxidative stress. Oxidative stress can lead to a downregulation of the CD3 f-chain. This mechanism has been widely demonstrated in tumor-infiltrating lymphocytes and can be directly attributed to reactive oxygen species produced by activated


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macrophages [69]. Impaired T cell receptor signaling has also been seen in T cells harvested from rheumatoid synovium, providing indirect evidence for possible effects of oxidative stress [70]. Release of toxic oxygen metabolites is a rather nonspecific mechanism and may function as a negative feedback loop in inflammation. More specific regulatory pathways have not been detected. The two cytokines that are generally considered to suppress macrophage activation, IL-4 and IL-10, do not inversely correlate with the production of proinflammatory cytokines in the synovium. On the contrary, the highest production of IL-4 is found in granulomatous tissue, and production of IL-10 is most abundant in tissues with active germinal centers [32]. To identify novel regulatory pathways, we have used the human synoviumSCID mouse model of RA and have adoptively transferred synovial T cell subsets into mice engrafted with synovium from the same donor [28]. Surprisingly, transfer of CD8 T cells inhibited production of IFN-c and the proinflammatory monokines IL-1b and TNF-a, whereas CD4 T cells boosted the production of these cytokines. CD8 T-cell-mediated inhibition of cytokines correlated with increased in situ production of TGF-b and IL-16. Immunohistochemistry demonstrated that IL-16 was derived mainly from tissue-infiltrating CD8 T cells. Further support implicating IL-16 came from blocking experiments in which the suppressive action of adoptively transferred CD8 T cells could be partially reversed by anti-IL-16 antibodies. Also, administration of recombinant IL-16 inhibited IFN-c production in the graft, mimicking the action of CD8 T cells. IL-16 is thought to be a natural ligand of the CD4 molecule that can modulate the function of CD4-expressing cells [71, 72]. It is generally considered to be a chemotactic molecule for CD4+ cells, although it does not have any resemblance to chemokines, and it is unclear how CD4 triggering would influence chemotaxis. More recent studies have proposed that CD4 triggering may deliver a tolerogenic signal, possibly via the cytoplasmic sequestration of lck recruited to CD4 [73, 74]. Experimental data suggest that activation of CD8 T cells in synovial tissue induces the production and cleavage of pro-IL-16 and the subsequent secretion of active IL-16 that then suppresses the activation of CD4-expressing cells. Heterogeneity of the Synovial Inflammation and the Nature of the Antigen It is conceivable that the heterogeneity in synovial inflammation is not only determined by host factors but also reflects the spectrum of antigens recognized in the synovial tissue. In particular, granuloma formation has been associated with certain antigens, but it is also possible that the induction of extralymphoid follicular structures is dependent on the nature of the antigen. While the evidence that CD4 T cells recognize antigen in the synovial inflammation is unequivocal, the nature of the antigen remains an enigma. Several autoantibody systems have


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been identified in subsets of RA patients. Not surprisingly, CD4 T cell response can be demonstrated to the antigens recognized by autoreactive B cells [75, 76]; however, it is unclear whether these antigens drive the synovial inflammation. The adoptive transfer experiments described above suggest that the antigen recognized is shared between different patients with lymphoid aggregates but may be different in patients with diffuse synovitis (Braun et al., unpubl. observations). Preliminary evidence suggests that patients with diffuse, follicular and granulomatous inflammation cannot be distinguished based on their HLA-DR4 haplotypes, questioning a role of antigen selection and presentation in determining the type of lymphoid microstructure. Obviously, immunoregulatory actions of CD8 T cells could also depend on the antigen recognized in the context of the MHC class I molecules. A series of recent studies has identified Epstein-Barr virus (EBV)-derived proteins as the antigen driving clonally expanded synovial CD8 T cells and has renewed the interest in the role of EBV in RA. EBV was implicated in RA in the 1980s when Alspaugh et al. [77] described elevated levels of antibodies to viral antigens in RA patients. Subsequent studies have shown several abnormalities in the EBV response of RA patients; however, they could not confirm the hypothesis that EBV infection is associated with RA [78]. In 1996, David-Ameline et al. [79] described that clonally expanded CD8 T cells from RA patients are frequently specific for antigens expressed on lymphoblastoid B cells, and Scotet et al. [80] identified the antigens as two EBV transactivators, BZLF1 and BMLF1. These EBV antigens are certainly not the primary antigens in RA. DNA of EBV as well as RNA of several latent and lytic EBV genes can be detected in RA synovium; however, they are also present in control tissues including synovium from osteoarthritis patients [81]. Furthermore, CD8 T cell responses to EBV antigens are not RA-specific but appear to be a major component in any type of chronic inflammation regardless of whether the inflammation was induced by an exogenous antigen or whether it is considered to be autoimmune [82]. Nevertheless, EBV-specific responses by CD8 T cells appear to have an important immunoregulatory role in the synovial inflammation. Whether they represent an amplification mechanism and recognition of EBV antigens on B cells by CD8+CD40L+ T cells is important in germinal center formation or whether EBV antigens activate anti-inflammatory CD8 T cells producing IL-16 remains to be determined.

Conclusions T cells, activated by antigen, are the ultimate regulatory cell in rheumatoid synovitis and control the production of inflammatory mediators, as well as the production of tissue-injurious metalloproteinases. However, the T cell


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Fig. 3. Schematic diagram of T cell regulatory pathways in the rheumatoid synovium. T cell responses in rheumatoid synovitis can take one of several pathways. Different types of lymphoid microstructures are created, possibly reflecting differences in the lymphocyte recruitment or diversity in the antigen-presenting process. There is a strong correlation between the patterns and amounts of cytokines produced in the tissue environment and the variant of synovitis that emerges.

response in RA synovium has proven to be more complex than originally imagined. There is no universal response pattern in RA, and patients differ widely in the recruitment of different cellular constituents, the production of cytokines and the formation of tertiary lymphoid structures. Besides the classical CD4 T cells, CD4+CD28– T cells, CD8 T cells and B cells have emerged as important players (fig. 3). B cells are present in follicular structures in about one third of the patients and are critical antigen-providing and/or presenting cells. Production of LT-b by a subset of B cells is critical in the formation of follicular dendritic cell networks that allow for the formation of germinal


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centers and the local maturation of antibody responses. CD8 T cells have a dual function by supporting the generation of germinal centers and by downregulating the activity of CD4+ T cells and macrophages. It is likely that these two functions are not exhibited by the same cell but can be assigned to different subsets of CD8 T cells. Finally, CD4+CD28– T cells have NK cell features and possibly inflict vascular injury as well as macrophage activation. Depending on the cellular constituents, different cytokine patterns predominate, and tertiary lymphoid structures such as germinal centers and granulomata evolve. These different response patterns may be, in part, determined by different susceptibility genes but could also reflect differences in antigens recognized by different subsets of lymphocytes. In particular, EBV antigens appear to be important in triggering CD8 T cells. Future studies have to explore how these variants of synovial inflammation correlate with disease progression and outcome of the synovial inflammation. From a clinical point of view, the dissection of different inflammatory phenotypes is becoming more and more important as increasingly selective treatment modes are developed. It is likely that cytokines and cytokine inhibitors have a role in some disease variants but not in others. Targeting the appropriate patient subset would greatly improve clinical study design and optimize patient management.

Acknowledgment This study was supported by NIH grants R01 AR41974, R01 AR42527 and R01 AI44142.


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Cornelia M. Weyand, MD, Mayo Clinic, 401 Guggenheim Building, 200 First Street, SW, Rochester, MN 55905 (USA) Tel. +1 507 284 1650, Fax +1 507 284 5045, E-Mail [email protected]


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The Role of TNFa and IL-1 in Rheumatoid Arthritis M. Feldmann, F.M. Brennan, B.M.J. Foxwell, R.N. Maini The Kennedy Institute of Rheumatology, Hammersmith, London, UK

Cytokine expression has been studied for over a decade in rheumatoid patients’ serum synovium and synovial fluid. IL-1 was the first cytokine detected in synovial fluid [1], and with its already described capacity to augment joint destruction in experimental models [2, 3], it became widely believed that IL-1 was of major importance in rheumatoid arthritis (RA). However soon after TNFa was described [4], with properties D90% identical, also expressed in RA tissues [5–7], and capable of inducing joint damage in experimental models [8]. This redundancy of cytokine properties, with multiple cytokines apparently capable of doing the same thing present in diseased tissues, suggested to some workers in the field that cytokines may not be good therapeutic targets, on the basis that blocking one would leave the other(s) to drive the biological properties. Our own view of this data compelled us to study cytokine regulation in the rheumatoid synovium to understand whether the apparent redundancy was of importance, and to attempt to define which, if any, cytokines may be effective therapeutic targets. The approach taken was to use the diseased tissue. We had found, first in mRNA analysis, and later of proteins, that dissociated RA synovium placed in culture without any extrinsic stimulus nevertheless produced appreciable quantities of the multiple pro-inflammatory cytokines [9] assessable at the time, e.g. IL-1, TNFa, IL-6, IL-8, GM-CSF for the 5–7 days when the cell composition remained similar to the starting material. This simple culture system provided a model system to study what signals regulated the spontaneous cytokine production, which mimicked that occurring in vivo. The first cytokine chosen for analysis of its regulation in synovial culture was IL-1. Many signals were present in synovium known to be capable of

Fig. 1. Cytokine cascade in RA. Pro-inflammatory cytokines in RA synovium interact in a network or cascade [modified and reproduced with permission from 40].

regulating it, e.g. GM-CSF, TNFa, LTa (TNFb), immune complexes or IFNc. To our surprise, it turned out that blocking TNFa (but not TNFb or IFNc) downregulated IL-1 bioactivity almost completely [10]. It is noteworthy that a bioassay was used: this is more difficult and time-consuming than an ELISA (not available at appropriate sensitivity in 1988) but automatically integrates the activity of the cytokine and its inhibitors, and so gives the most functionally relevant data. The blockade of TNFa yielding marked diminution of IL-1 as a consequence led us to re-evaluate the relationship of a large panel of pro-inflammatory cytokines expressed in RA synovium. We found that blocking TNFa also downregulated GM-CSF [11], IL-6, IL-8 [12], whereas blocking IL-1 with IL-1Ra blocked GM-CSF, IL-6, IL-8 [12] but had no effect on TNF. This led us to propose that there is present in a chronic inflammatory site a proinflammatory ‘cascade’ or hierarchy, with TNFa the pivotal cytokine (fig. 1). This concept was of importance in the development of anti-TNFa therapy, as it predicted that in vivo also, the blockade of TNFa would have profound effects on other pro-inflammatory mediators. Confirmation of the potential utility of anti-TNF therapy was obtained in animal models of arthritis [13–15], and this led to clinical trials of antiTNFa therapy. The biology of IL-1 suggests that it needs to be regulated precisely. Thus there are two endogenous inhibitors, the type II IL-1 receptor [16], in either its cell surface or shed form, and the IL-1 receptor antagonist [17], in IL-1a/ b-related molecule which binds to the type I IL-1 receptor (the signalling receptor) but does not transmit any signals. The capacity of the latter to block the action of both IL-1a and IL-1b prompted an evaluation of its role as a

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Fig. 2. Histological profile of collagen-induced arthritis joints treated with anti-TNF antibody, isotype control or untreated [reproduced with permission from 15].

potential therapeutic, first in sepsis and then in RA. The results in sepsis, while encouraging initially were unsuccessful, probably due to the large amounts of IL-1Ra needed to block IL-1 signalling, and the poor pharmacokinetics of this inhibitor [18].

Studies in Collagen-Induced Arthritis Demonstrate that Both TNFa and IL-1 Are Involved in Arthritis after Onset A number of groups have each independently demonstrated that antibodies to TNFa [13–15] or IL-1 [19] could each ameliorate collagen-induced arthritis. As the results have used a wide variety of reagents, at widely differing concentrations and injection frequency it is not possible to compare efficacy of blocking TNF or IL-1, despite the attempts of some authors to do so on inadequate data – single doses of an antibody, for example. Two key points are worth considering, first that TNF and IL-1 are synergistic in many assays, so that blocking either one would be expected to be beneficial, and second that if IL-1 production is driven by TNFa, much of the benefit of anti-TNFa may in fact be due to reduced IL-1. This was first shown in synovial cultures, and is dramatically illustrated in the TNFa transgenic mice of Kollias and colleagues [20], whose arthritis can be inhibited by anti-TNFa or by blocking the IL-1 receptor. The second is that blocking TNFa in mice protects joint architecture as determined by histological analysis (fig. 2).

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However, while these studies are consistent with knowledge of the biology of TNFa and IL-1, they do not necessarily reveal what really happens in humans with RA.

Anti-TNF Therapy of RA Ameliorates Virtually All Aspects of the Disease The only way to really understand the role of a molecule in the pathogenesis of human disease is to evaluate response to blocking its action. The trials of anti-TNFa antibody initiated with the chimaeric antibody cA2, now termed infliximab or RemicadeTM began in 1992, and through phases I [21] and II [22–24] provided ample opportunity to study its efficacy and mechanisms of action. Phase III has provided an opportunity to study joint destruction by serial X-ray studies. The results have been remarkably consistent with all the three antibodies [25, 26] and three receptor constructs tried in RA proving efficacious [27–29]. This multitude of trials has amply documented the importance of TNFa in RA, and the mechanism of action studies, only performed (to our knowledge) with Remicade, has deepened our understanding of how TNFa orchestrates the events in chronic inflammation. Figures 3–5 summarize the clinical data in RA with Remicade, which is published. Figure 6 illustrates that the TNF-Rp75 fusion protein, etanercept (EnbrelTM), is also very effective [27] comparable to Remicade on symptoms and signs.

The Multiple Mechanisms of Action of Anti-TNFa Explain Its Marked Efficacy The studies with Remicade have demonstrated five major mechanisms of action, summarized in table 1. Downregulation of the Cytokine Cascade This was the rationale for therapy, and it was confirmed in vivo. Serum IL-6, IL-1, VEGF, MCP-1 and IL-8 are some of the cytokines downregulated which can be accurately quantitated in serum [reviewed in 30, 31]. Reduction in Leucocyte Trafficking to Joints This was suggested by the reductions in adhesion molecules (E selectin, ICAM-1, VCAM-1) detectable both in serum by ELISA [32] as well as by


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Fig. 3. Open-label single-centre phase I trial (short) of cA2 (Remicade) in RA patients. Results illustrated as swollen joint count and C-reactive protein (CRP) [reproduced with permission from 21]. a p=0.02; b p=0.01; c p=0.002; d p=0.001.

immunohistology [33] in joints by reductions in serum and joint chemokine expression, and also by rapid alterations in leucocyte counts. Formal proof of reduced leucocyte trafficking has come from labelled granulocyte studies, before or 2 weeks after Remicade therapy which revealed a 40% reduction in granulocytes in joints [34]. While ethical restrictions do not permit similar studies with lymphocytes, rapid augmentation of lymphocyte counts on the day of therapy is consistent (since these are long-lived cells) with diminished trafficking to joints. We believe that reduced trafficking is a very important aspect in the mechanism, and contributes to the reduced cellularity of joints. Increases in apoptosis have been noted after anti-TNFa, but are not yet fully understood, and require further studies. Reduction in VEGF and Angiogenesis There are very rapid changes in joint swelling, which suggest reductions in fluid in the joint, as well as cellular changes. VEGF was also cloned as ‘vascular permeability factor’, and it seems likely that reductions in VEGFreducing vascular permeability factor are important in reducting joint swelling.


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Fig. 4. Clinical response of 73 patients treated with placebo, 1 or 10 mg/kg of cA2 in a randomized, double-blind placebo-controlled multicentre trial (phase II, short). Swollen joint count, and reduction in disease activity measurement (C-reactive protein, CRP) are shown [produced previously and reproduced with permission, 22]. b p=0.01; d p=0.001. The shaded bar represents the normal range.

However, reductions in VEGF are also important in reducing the generation of the new vessels which feed the augmented cell mass in the synovium [35]. Less vessels probably eventually means less inflammatory mass, with the consequent benefit. Restoration of Blood Profiles RA patients have a reduced haemoglobin level which probably contributes to their lethargy and tiredness [36]. They also have a high granulocyte count, high platelet count and high fibrinogen levels. The latter two are of importance, as they are independent risk factors for atherosclerosis, which is the leading cause of death in RA patients. In theory anti-TNFa therapy might reduce vascular disease, an assumption that has not been tested.


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Fig. 5. The clinical response of RA patients treated long term with cA2 (Remicade). Placebo and Remicade treatment every 8 and 4 weeks are shown. Serial measurements according to American College of Rheumatology criteria [reproduced with permission from 24].

Fig. 6. Treatment of RA with recombinant human p75 TNF-R-Fc fusion protein (etanercept). Patients received subcutaneous injections of placebo or etanercept at 0.25, 2 or 16 mg/m2 of body surface area, twice weekly for 3 months. Mean swollen joint counts and tender joint counts are shown over a 5-month period. The shaded bar represents the treatment period [reproduced by kind permission from 27].

Remicade Therapy and Enbrel Therapy Offer Joint Protection Announcements have been made in recent months that clinical data to be presented at the American College of Rheumatology National Meeting, November 1999, showed that there were marked reductions in X-ray changes after TNF blockade: with Remicade the median level of X-ray progression


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Table 1. How is cytokine washout with Remicade translated into clinical benefit? (1)

Downregulation of many pro-inflammatory cytokines in vivo: Il-1, GM-CSF, IL-6, IL-8 and other chemokines Reduction in leucocyte trafficking: reduction in adhesion molecules and chemokines Reduction in angiogenesis: reduction in VEGF Reduction in joint destruction: reduction in IL-1, MMPs K X-ray changes Haematological normalization: haemoglobin C, platelets B, fibrinogen B : cardiovascular risk B ?

(2) (3) (4) (5) Conclusion

TNFa close to core of disease

was halted, and was maintained at 1 year at the initial level in contrast to the MTX-alone group which progressed. This data increasingly suggests that anti-TNFa therapy affects nearly all aspects of the disease, although in the late, therapy-resistant patients it is not curative. Perhaps in the future it will be possible to treat early patients, with possibly different results. Anti-IL-1 Therapy with IL-1Ra Has a Moderate Anti-Inflammatory Effect and Moderate Joint-Protective Effects As already discused IL-1Ra has a short half-life, and as it needs 10–100 times more IL-1Ra than IL-1a or b to block signalling, IL-1Ra is a poor drug. Nevertheless, trials of IL-1Ra given subcutaneously daily were performed, and had moderate results [37]. The overall conclusion was the effects on joint symptoms and signs were clearly inferior to that of anti-TNFa at the time this trial was reported, the data on joint protection was ahead of that with anti-TNFa (fig. 7, 8). Thus there were press reports that attempts were made by Amgen to establish whether in the light of joint protection, the drug might be licensed without a phase III trial, but apparently that will not be the case.

Conclusions TNFa and IL-1 are the best-studied pro-inflammatory cytokines, and are expressed in rheumatoid synovium. However, their relative importance in the pathology of RA has been debated. Here the rationale for therapy with antiTNFa and IL-1ra is reviewed, along with the clinical results and the mechanism of action studies. These are abundant for anti-TNFa, along with the extensive mechanism of action studies. Regrettably the available human data does not


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Fig. 7. Serial swollen joint counts in patients receiving placebo or IL-1ra [reproduced by kind permission from 37].

Fig. 8. Larsen and erosive joint scores (+SEM) in patients receiving placebo or IL-1ra [reproduced by kind permission from 37]. a p=0.02; b p=0.004 vs. placebo.

yet permit any conclusions concerning the relative merits of TNF blockade over IL-1 or vice versa, because of the poor pharmacokinetics of IL-1ra. It does not perform like an antibody or a dimeric receptor. A real comparison of TNFa and IL-1 blockade will require the use of antibodies to IL-1b or to the type I IL-1 receptor. At the moment the hypothesis favoured by some that


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TNFa controls joint inflammation but IL-1 controls joint destruction is not established, but not excluded either. The marked joint protection with antiTNFa antibody (Remicade) indicates that TNFa blockade alone is enough to protect joints. However, at this point in time it is still possible that the mechanism of anti-TNFa-induced joint protection involves reduction of endogenous IL-1. What can be excluded is that it will be essential to target IL-1 directly to reveal substantial joint protection. Our current work is aimed at understanding what regulates the excess TNFa production in RA synovium. This work is following a number of paths. One is based on observations that TNFa production from macrophages in RA synovium is T cell-dependent. We have thus been studying the action of T cells. Cell contact appears essential, as does an atypical form of T cell activation, involving cytokines acting in concert. At the molecular level, we have been studying intracellular signals of importance in regulating RA TNFa production. NFjB is important, as judged by 70% reduction in synovial TNFa if an adenovirus overexpressing IjBa is used to infect synovium [38, 39]. These studies indicate that there will be intracellular targets, which will be able to mimic the benefit of anti-TNFa.

Acknowledgment Prof. M. Feldmann is a member of the Scientific Advisory Board of Centocor, which makes Remicade, previously known as cA2 or infliximab.

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Saklatvala J: Tumour necrosis factor a stimulates resorption and inhibits synthesis of proteoglycan in cartilage. Nature 1986;322:547–549. Brennan FM, Chantry D, Jackson AM, Maini RN, Feldmann M: Cytokine production in culture by cells isolated from the synovial membrane. J Autoimmun 1989;2(suppl):177–186. Brennan FM, Chantry D, Jackson A, Maini R, Feldmann M: Inhibitory effect of TNF a antibodies on synovial cell interleukin-1 production in rheumatoid arthritis. Lancet 1989;ii:244–247. Haworth C, Brennan FM, Chantry D, Turner M, Maini RN, Feldmann M: Expression of granulocyte-macrophage colony-stimulating factor in rheumatoid arthritis: Regulation by tumor necrosis factor-a. Eur J Immunol 1991;21:2575–2579. Butler DM, Maini RN, Feldmann M, Brennan FM: Modulation of proinflammatory cytokine release in rheumatoid synovial membrane cell cultures. Comparison of monoclonal anti-TNFa antibody with the IL-1 receptor antagonist. Eur Cytokine Netw 1995;6:225–230. Thorbecke GJ, Shah R, Leu CH, Kuruvilla AP, Hardison AM, Palladino MA: Involvement of endogenous tumour necrosis factor a and transforming growth factor b during induction of collagen type II arthritis in mice. Proc Natl Acad Sci USA 1992;89:7375–7379. Piguet PF, Grau GE, Vesin C, Loetscher H, Gentz R, Lesslauer W: Evolution of collagen arthritis in mice is arrested by treatment with anti-tumour necrosis factor (TNF) antibody or a recombinant soluble TNF receptor. Immunology 1992;77:510–514. Williams RO, Feldmann M, Maini RN: Anti-tumor necrosis factor ameliorates joint disease in murine collagen-induced arthritis. Proc Natl Acad Sci USA 1992;89:9784–9788. Symons JA, Eastgate JA, Duff GW: Purification and characterization of a novel soluble receptor for interleukin-1. J Exp Med 1991;174:1251-1254. Arend WP, Welgus HG, Thompson RC, Eisenberg SP: Biological properties of recombinant human monocyte-derived interleukin 1 receptor antagonist. J Clin Invest 1990;85:1694–1697. Fisher CJJ, Slotman GJ, Opal SM, Pribble JP, Bone RC, Emmanuel G, Ng D, Bloedow DC, Catalano MA: Initial evaluation of human recombinant interleukin-1 receptor antagonist in the treatment of sepsis syndrome: A randomized, open-label, placebo-controlled multicenter trial. Crit Care Med 1994;22:12–21. Van den Berg WB, Joosten LAB, Helsen M, Van De Loo FAJ: Amelioration of established murine collagen-induced arthritis with anti-IL-1 treatment. Clin Exp Immunol 1994;95:237–243. Probert L, Plows D, Kontogeorgos G, Kollias G: The type 1 interleukin-1 receptor acts in series with tumor necrosis factor (TNF) to induce arthritis in TNF-transgenic mice. Eur J Immunol 1995; 25:1794–1797. Elliott MJ, Maini RN, Feldmann M, Long-Fox A, Charles P, Katsikis P, Brennan FM, Walker J, Bijl H, Ghrayeb J, et al: Treatment of rheumatoid arthritis with chimeric monoclonal antibodies to TNFa. Arthritis Rheum 1993;36:1681–1690. Elliott MJ, Maini RN, Feldmann M, Kalden JR, Antoni C, Smolen JS, Leeb B, Breedveld FC, Macfarlane JD, Bijl H, et al: Randomised double blind comparison of a chimaeric monoclonal antibody to tumour necrosis factor a (cA2) versus placebo in rheumatoid arthritis. Lancet 1994; 344:1105–1110. Maini RN, Breedveld FC, Kalden JR, Smolen JS, Davis D, Macfarlane JD, Antoni C, Leeb B, Elliott MJ, Woody JN, Schaible TF, Feldmann M: Randomized placebo-controlled trial of multiple intravenous infusions of anti-TNFa monoclonal antibody with or without weekly methotrexate in rheumatoid arthritis. Arthritis Rheum 1998;41:1552–1563. Maini R, St Clair EW, Breedveld F, Furst D, Kalden J, Weisman M, Smolen J, Emery P, Harriman G, Feldmann M, Lipsky P: Infliximab (chimeric anti-tumour necrosis factor alpha monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: A randomised phase III trial. ATTRACT Study Group. Lancet 1999;354:1932–1939. Rankin EC, Choy EHS, Kassimos D, Soowith M, Kingsley G, Isenberg DA, Panayi GS: A doubleblind, placebo-controlled, ascending dose trial of the recombinant humanised anti-TNFa antibody CDP571 in patients with rheumatoid arthritis (RA): A preliminary report. Arthritis Rheum 1994; 37:S295. Salfield J, Kaymakcalan J, Tracey D, Roberts A, Kamen R: Generation of fully human anti-TNF antibody D2E7 (abstract). Arthritis Rheum 1998;41/9(suppl):S57.


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Moreland LW, Baumgartner SW, Schiff MH, Tindall EA, Fleischmann RM, Weaver AL, Ettlinger RE, Cohen S, Koopman WJ, Mohler K, Widmer MB, Blosch CM: Treatment of rheumatoid arthritis with a recombinant human tumor necrosis factor receptor (p75)-Fc fusion protein. N Engl J Med 1997;337:141–147. Hasler F, van de Putte L, Baudin M, Lodin E, Durrwell L, McAuliffe T, van der Auwera P: Chronic TNF neutralization (up to 1 year) by lenercept (TNFR 55 IgG1, Ro45-2081) in patients with rheumatoid arthritis. Results for open label extension of a double-blind single-dose phase I study. Arthritis Rheum 1996;39:S243. Rau R, Sander O, den Broeder A, van Riel PLCM, van der Putte L, Kruger K, Schattenkreher M, Fenner H, Lassman A, Kupper H, Rempoi J: Long term efficacy and tolerability of multiple i.v. dose of the fully human anti-TNF-antibody D2E7 in patients with rheumatoid arthritis. Arthritis Rheum 1998;41:S55. Charles P, Elliott MJ, Davis D, Potter A, Kalden JR, Antoni C, Breedveld FC, Smolen JS, Eberl G, deWoody K, Feldmann M, Maini RN: Regulation of cytokines, cytokine inhibitors, and acutephase proteins following anti-TNF-alpha therapy in rheumatoid arthritis. J Immunol 1999;163: 1521–1528. Feldmann M, Elliott MJ, Woody JN, Maini RN: Anti-tumour necrosis factor-a therapy of rheumatoid arthritis. Adv Immunol 1997;64:283–350. Paleolog EM, Hunt M, Elliott MJ, Feldmann M, Maini RN, Woody JN: Deactivation of vascular endothelium by monoclonal anti-tumor necrosis factor a antibody in rheumatoid arthritis. Arthritis Rheum 1996;39:1082–1091. Tak PP, Taylor PC, Breedveld FC, Smeets TJM, Daha MR, Kluin PM, Meinders AE, Maini RN: Decrease in cellularity and expression of adhesion molecules by anti-tumor necrosis factor a monoclonal antibody treatment in patients with rheumatoid arthritis. Arthritis Rheum 1996;39: 1077–1081. Taylor PC, Peters AM, Paleolog E, Chapman PT, Elliott MJ, McCloskey R, Feldmann M, Maini RN: Reduction of chemokine levels and leukocyte traffic to joints by tumor necrosis factor a blockade in patients with rheumatoid arthritis. Arthritis Rheum 2000;43:38–47. Paleolog EM, Young S, Stark AC, McCloskey RV, Feldmann M, Maini RN: Modulation of angiogenic vascular endothelial growth factor (VEGF) by TNFa and IL-1 in rheumatoid arthritis. Arthritis Rheum 1998;41:1258–1265. Davis D, Charles PJ, Potter A, Feldmann M, Maini RN, Elliott MJ: Anaemia of chronic disease in rheumatoid arthritis: In vivo effects of tumour necrosis factor a blockade. Br J Rheumatol 1997; 36:950–956. Bresnihan B, Alvaro-Gracia JM, Cobby M, Doherty M, Domljan Z, Emery P, Nuki G, Pavelka K, Rau R, Rozman B, Watt I, Williams B, Aitchison R, McCabe D, Musikic P: Treatment of rheumatoid arthritis with recombinant human interleukin-1 receptor antagonist. Arthritis Rheum 1998;41:2196–2204. Foxwell BMJ, Browne K, Bondeson J, Clarke C, de Martin R, Brennan FM, Feldmann M: Efficient adenoviral infection with IjBa reveals that TNFa production in rheumatoid arthritis is NF-jB dependent. Proc Natl Acad Sci USA 1998;95:8211–8215. Bondeson J, Foxwell BMJ, Brennan FM, Feldmann M: Defining therapeutic targets by using adenovirus: Blocking NF-jB inhibits both inflammatory and destructive mechanisms in rheumatoid synovium but spares anti-inflammatory mediators. Proc Natl Acad Sci USA 1999;96:5668–5673. Feldmann M, Brennan FM, Maini R: Rheumatoid arthritis. Cell 1996;85:307–310.

Prof. Marc Feldmann, The Kennedy Institute of Rheumatology, 1, Aspenlea Road, Hammersmith, London, W6 8LH (UK) Tel. +44 181 383 4406, Fax +44 181 563 0399, E-Mail [email protected]


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Innate Response Cytokines in Inflammatory Synovitis: A Role for Interleukin-15 Iain B. McInnes, Bernard P. Leung Centre for Rheumatic Diseases and Department of Immunology, University of Glasgow, UK

T lymphocytes of activated, memory phenotype are widely distributed within the inflamed synovial compartment in rheumatoid arthritis (RA). Most data suggest that their presence is pro-inflammatory, although the precise nature of their contribution to either initiation or perpetuation of synovitis remains unclear [1, 2]. In particular, factors that sustain their activation status, in the relative absence of IL-2, are poorly understood. Most cytokines present within synovium are of macrophage and synovial fibroblast derivation [3, 4]. Interleukin-15 (IL-15) is a cytokine with quaternary structural similarities to IL-2 [5, 6] that is produced primarily by macrophages. This article will review data which indicate that IL-15, together with other cytokines of the innate immune response, represents a potential link between T cell and macrophage activation in RA synovial membrane.

Interleukin-15 Biology IL-15 is a 14- to 15-kD cytokine expressed at the mRNA level in numerous normal human tissues in a broad range of cell types, including activated monocytes, dendritic cells and fibroblasts [5, 7]. IL-15 mediates activity through a heterotrimeric receptor (IL-15R) that consists of the IL-2 receptor (IL-2R) b-chain and c-chain, together with a unique a-chain (IL-15a), which is alternatively spliced to yield three active forms, each capable of high-affinity binding to IL-15 [5–10]. Whereas IL-2Ra is primarily expressed on activated

T cells, IL-15Ra mRNA has been identified in numerous human tissues and cells, including activated T cells [8, 11]. Whether IL-15Ra in the absence of bc-chain expression transduces signal is unclear. Recent data suggest that its cytoplasmic tail contains regions homologous to TRAF2 binding domains, analogous to CD40, and may compete with TNFRI for TRAF2 binding [12]. IL-15Ra-deficient mice have recently been generated that exhibit lymphopenia due to reduced proliferation and homing of mature lymphocytes, particularly of the CD8+ subset. A key role in the development of several cell lineages of the innate immune response such as natural killer (NK) cells was also confirmed [13]. A 60- to 65-kD receptor for IL-15 has also been identified on mast cells (IL-15RX), which requires neither IL-2Rb nor IL-2Rc for signalling [7, 14]. This receptor recruits distinct signalling pathways. Whereas the IL15Rabc complex signals through JAK1/3 and STAT3/5, IL-15RX utilizes JAK2 and STAT5 [7, 10, 14]. Regulation of IL-15 Production An early and puzzling observation was the abundant expression of IL-15 mRNA at levels which were disproportionate to the limited detection of protein in tissues. That this reflects tight regulatory control of IL-15 translation and secretion has recently been elucidated [15]. Several transcription sites have been identified in 5 regulatory regions, including NF-jB, NF-IL-6, GCF, IRF-E and c-IRE [7, 16, 17]. Whereas many cytokines are regulated by modification of transcription and message stabilization, IL-15 appears subject to significant posttranscriptional regulation. IL-15 mRNA 5-untranslated region (UTR) contains twelve AUG triplets that significantly reduce the efficiency of translation. Fusion of the IL-15 mRNA with a human T-cell leukaemia virus 1 R region in the HuT-102 cell line deleted this AUG-rich 5UTR sequence, leading to high levels of constitutive IL-15 secretion [18]. Replacement of the IL-15 48-aa signal peptide with that of IL-2 or CD33 induces significantly higher levels of IL-15 production in transfection systems, indicating that this region is also normally involved in downregulating IL-15 protein release [15, 19]. Several studies also suggest a third regulatory element in the C-terminus region since FLAG fusion proteins exhibit higher levels of secretion than native construct [7, 15]. Thus IL-15 secretion is tightly regulated by at least three regulatory mechanisms. Two isoforms of IL-15 have thus far been identified. Secreted IL-15 is derived from a long signalling peptide containing a 48-aa leader sequence, whereas a second isoform which contains a short signalling peptide of 21-aa is retained within the cell and has been localized to non-endoplasmic regions in both cytoplasmic and nuclear compartments [19–23]. The biologic significance of the latter is at present unclear. Altered glycosylation of the IL-15-

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48-aa isoform may further regulate intracellular trafficking [7]. These multiple levels of regulation presumably provide an available pool of mRNA, a suggestion that is compatible with the perceived early functional role of IL-15, but under normal circumstances prevent undesirable IL-15 expression in tissues. Factors demonstrated thus far to induce IL-15 secretion by human cells are diverse and include human herpesvirus 6 and 7, Mycobacterium leprae, Mycobacterium tuberculosis, Staphylococcus aureus, lipopolysaccharide and ultraviolet irradiation [24–29]. However, to establish consistent IL-15 secretion in vitro has proven difficult in most systems thus far investigated. Bioactivities Commensurate with the broad expression of IL-15R, diverse pro-inflammatory activities have been attributed to IL-15. Effects on NK and T cells are most prominent. IL-15 induces proliferation of mitogen-activated CD4+ and CD8+ T cells, T-cell clones and cd T cells, with release of soluble IL-2Ra, and enhances cytotoxicity both in CD8+ T cells and lymphokineactivated killer cells [5, 6, 30–32]. CD69 expression is upregulated on CD45RO+ but not CD45RA+ T cell subsets, consistent with the distribution of IL-2Rb expression [33]. IL-15 also induces NK cell activation, measured either by direct cytotoxicity, antibody-dependent cellular cytotoxicity or production of cytokines [27, 34–36]. It has recently been directly implicated in promoting NK cell-mediated shock in mice [37]. Moreover, several reports demonstrate a role for IL-15 in thymic development of T cell and, particularly, NK cell lineages. IL-15 likely also functions as an NK cell survival factor in vivo by maintaining Bcl-2 expression [38–41]. IL-15 exhibits T cell chemokinetic activity [42, 43] and induces adhesion molecule (e.g. intercellular adhesion molecule-3) redistribution [44]. It further induces chemokine (CC-, CXC- and C-type) and chemokine receptor (CC but not CXC) expression on T cells [45]. Thus, IL-15 can recruit T cells and, thereafter, modify homo- or heterotypic cell-cell interactions within inflammatory sites. Whether IL-15 prejudices T helper 1 (Th1) or Th2 differentiation in addition to recruitment is controversial. IL-15 primes naive CD4+ T cells from TCR-transgenic mice for subsequent IFNc expression, but not IL-4 production [46]. Antigen-specific responses in T cells from human immunodeficiency virus-infected patients in the presence of high-dose IL-15 exhibit increased IFNc production, particularly if IL-12 is relatively deficient [47]. Similarly IL-15 induces IFNc/IL-4 ratios which favour Th1 dominance in mitogen-stimulated human T cells [48]. However, IL-15 induces IL-5 production from allergen-specific human T-cell clones, implying a positive role in Th2-mediated allergic responses [49]. Moreover, administration of soluble IL-15-IgG2b fusion protein in murine hypersensitivity models

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clearly implicates IL-15 in Th2 lesion development [50]. Thus, through its function as a T cell growth factor, IL-15 can probably sustain either Th1 or Th2 polarization. IL-15 mediates potent effects beyond T/NK cell biology. It promotes B-cell proliferation and immunoglobulin synthesis in vitro, in combination either with CD40 ligand, or immobilized anti-IgM [51]. IL-15 has also been proposed as an autocrine regulator of macrophage activation, such that low levels of IL-15 suppress whereas higher levels enhance pro-inflammatory monokine production [52]. Moreover, since human macrophages constitutively express bio-active membrane-bound IL-15, such autocrine effects are likely of early importance during macrophage activation [53]. Similarly, neutrophils express IL-15Ra and IL-15 can induce neutrophil activation, cytoskeletal rearrangement and protection from apoptosis [54, 55]. Finally, addition of IL15 to rat bone marrow cultures induces osteoclast development and upregulates calcitonin receptor expression [56]. IL-15 apparently represents a mechanism whereby host tissues can contribute to the early phase of immune responses, providing enhancement of polymorphonuclear and NK cell responses, and subsequently T cell responses, prior to optimal IL-2 production. The corollary to such pleiotropic activity may be a propensity to chronic, rather than selflimiting, inflammation should IL-15 synthesis be aberrantly regulated.

IL-15 Expression in Synovial Membrane Several data indicate that IL-15 mRNA and protein are present in RA synovial membrane. Using RT-PCR, elevated IL-15 mRNA levels have been detected in RA compared with reactive arthritis synovial biopsies [57]. Moreover, levels were higher in biopsies obtained from patients prior to commencement of immunosuppressive therapy [57]. IL-15 can be detected by ELISA in around 60% of RA, but not osteo-arthritis synovial fluids (SF), with a median concentration of 198 pg/ml when present, similar to levels of TNFa detected in parallel assays [58]. Low levels of IL-15 are also detected in sera of 40% of RA patients. In both serum and SF, levels correlate directly with TNFa and remain after prior removal of the rheumatoid factor, which likely interfered with early IL-15 assays, leading to overestimation of the concentrations present. These observations have recently been confirmed [59a], although lower or absent expression of IL-15 has also been reported [59b, 60]. This discrepancy may reflect the assay systems employed or the presence of inhibitory factors within SF that can interfere with IL-15 detection. IL-15 has also been measured in RA SF using soluble IL-15Ra chain in a novel receptor capture assay [58], in which IL-15 levels in RA SF correlate closely with those detected by ELISA.


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IL-15 has also been detected in rheumatoid pleural effusions [61]. Immunohistochemical localization of IL-15 in synovium indicates that lining layer macrophages represent the predominant cellular source in situ, although expression in synovial fibroblasts and in endothelial cells [62–64] is also evident. Whether synovial T cells also express IL-15 remains controversial [63]. In contrast, IL-15 expression, although detectable, is reduced in psoriatic and reactive arthritis synovial membranes as compared to RA [63, 65]. Of interest, both psoriatic and reactive arthritis synovium contain IL-2, with which IL-15 may exhibit counterregulatory activities. Spontaneous production IL-15 by primary RA synovial membrane cultures and by isolated synovial fibroblasts has recently been reported [59]. In similar studies, we have demonstrated upregulation of IL-15 mRNA and intracellular protein expression by TNFa and IL-1b in purified synovial fibroblasts, although we have been unable to consistently detect IL-15 secretion thus far. Studies characterizing the regulation of intracellular processing events leading to LSP-IL-15 production in synovial membrane are awaited, as is formal comparison of LSP and SSP isoform expression [7]. Nevertheless, the foregoing data together suggest that IL-15 is consistently expressed in RA synovial membrane.

Potential Biologic Effects of IL-15 in RA The potential activities of IL-15 in RA have been elucidated from neutralization studies in which bio-activity within RA SF is blocked using antiIL-15 antibodies, from in vitro data in which recombinant IL-15 is added to synovial culture systems, and from arthritis models in which IL-15 has been specifically targeted. In vitro studies addressing lymphocyte migration in RA indicate that IL-15 accounts in part for the chemokinetic activity of RA SF, in combination with at least IL-8, MCP-1 and MIP-1a [43, 62]. Responding T cells are predominantly of the CD3+, CD45RO+ subset. IL-15 simultaneously upregulates CD69 and leucocyte function-associated molecule 1 (LFA-1) [66, 67] and induces redistribution of adhesion molecules, including ICAM-1–3, CD43 and CD44, to uropods to further facilitate migration [44]. Injection of IL-15 into the footpad of Corynebacterium parvum or type II collagen-primed mice induces a sustained local inflammatory infiltrate, consisting primarily of CD3+ lymphocytes, associated with local lymphadenopathy, indicating that such in vitro observations are likely of in vivo relevance [62]. Subsequent studies have demonstrated IL-15 expression on synovial endothelial cells, where it mediates activated, memory T cell migration into synovial membrane tissues transplanted into SCID mice [64]. Moreover, IL-15 upregu-

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lates endothelial cell hyaluronan expression and promotes intraperitoneal migration of T cells through a CD44-dependent pathway [68]. Thus IL-15 likely promotes T cell recruitment to synovial membrane through effects on both lymphocytes and endothelial cells. The critical role of TNFa in RA pathogenesis has been clearly demonstrated in studies investigating the clinical utility of anti-TNFa antibodies and soluble TNF receptors [69, 70]. Purified SF, but not PB T lymphocytes produce cytokines in vitro in response to IL-15, including TNFa together with IFNc, IL-17 and IL-10, suggesting that direct effects of IL-15 on T cells may contribute to synovial cytokine production. Histologic studies, however, suggest that TNFa in synovial membrane is predominantly macrophage-derived [71, 72]. Moreover, the addition of IL-15 to RA synovial membrane cultures induces TNFa secretion, which is localized by intracellular FACS analysis primarily in macrophages [Gracie and McInnes, unpubl. data]. Whereas IL-15 can mediate direct effects on macrophages [52], we have proposed that IL-15 might drive cognate interactions between T cells and macrophages, which clearly lie in juxtaposition within RA synovial membrane. Following stimulation with nonphysiological mitogens, paraformaldehyde-fixed T cells and T cell clones induce pro-inflammatory cytokine production by macrophages and fibroblasts through cell contact [66, 67, 73]. Freshly isolated synovial T cells similarly induce TNFa synthesis by blood- or synovial-derived macrophages through cell-membrane contact, with no requirement for secretory factor synthesis [66]. This activity is maintained in vitro by addition of IL-15. Moreover, IL-15 confers similar properties upon CD45RO+ PB T cells, such that IL-15-activated PB cells from RA patients induce TNFa synthesis in synovial macrophage/synoviocyte cocultures. Neutralization studies implicate at least CD69, LFA-1 and ICAM-1 [66, 74]. Complex interactions between secreted and cognate pathways are predicted. Thus, IFNc production in T cell/monocyte cocultures is suppressed by IL-15 neutralization, indicating cooperative activity of IL-12 and IL-15 through effects on IL-12Rb1 expression, enhanced CD40 expression and direct effects on CD4+ T cell cytokine production [75]. Of interest, secreted IL-15 was not detected in such cultures. Feedback loops such as these could, therefore, perpetuate synovial inflammation through T cell/ macrophage interactions. Data consistent with this central role for T cells are provided in vivo by elegant experiments demonstrating that anti-CD2mediated T cell depletion in synovial grafts in NOD-SCID mice leads to suppression of TNFa and IL-15 levels and to MMP expression [76]. Furthermore, in coculture experiments we have recently established that synovial T cell/ monocyte interactions induce IL-15 secretion in vitro (unpubl. observations), providing a mechanism for autocrine and paracrine amplification of IL-15 and TNFa synthesis (fig. 1).


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Fig. 1. IL-15 in synergy with other cytokines of the innate response may contribute to RA synovial inflammation through T cell recruitment and activation. Thereafter T cell interactions with macrophages–/–synovial fibroblasts, through cell contact and soluble factor release can promote further T cell activation through induction of at least IL-15 and IL-18 production. These events likely enhance downstream production of metalloproteinases (MMP), prostaglandins (PG), reactive oxygen (ROI) and reactive nitrogen intermediates (RNI) as indicated. Further effects on other cells of the innate response are implicated, represented in the figure as neutrophil activation.

A further potent effect of IL-15 lies in protection from apoptosis [7, 77, 78]. Thus, IL-15 retards apoptosis in vivo and in vitro in T and B lymphocytes induced either by cytokine withdrawal, by anti-Fas or anti-antigen receptor antibody or by dexamethasone [77]. Despite these properties, the predominant pathways that account for rescue from apoptosis in RA SF seem independent of IL-15 signalling [78], but rather reflect the activities of type I interferons. The reason for this discrepancy is unclear, but may reflect the complexity of pro- and anti-inflammatory cytokine networks in SF, or that IL-15 present in SF (low picogram range [58]) is quantitatively less important than interferon (low nanogram range [78]). IL-15 constitutes an important cytokine of the innate immune response. Several data suggest that neutrophils can be activated by IL-15 [54, 55]. More-

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over, in studies investigating chemokine, cytokine and CD11b expression and f-MLP-induced chemiluminescence, enhanced responsiveness to IL-15 is detected in RA compared with normal control-derived neutrophils [79]. Neutrophil activation by RA SF may be partially ameliorated by IL-15 neutralization, providing further evidence that bio-active IL-15 is present in SF. Whereas the role of NK cells in RA synovitis remains poorly understood, the potent effect of IL-15 on NK cell biology indicates that this is also a plausible synovial target for IL-15. Studies investigating this possibility are awaited. Thus, several data obtained in vitro indicate that IL-15 in bio-active form is present in the RA synovial membrane.

Modulation of IL-15 in vivo Considerable information about the implication of cytokine expression in synovitis has been obtained using inflammatory arthritis models. Administration of IL-15 during priming with type II collagen in incomplete Freund’s adjuvant induces development of an erosive inflammatory arthritis in DBA/1 mice, which closely resembles that obtained on immunization with collagen in complete Freund’s adjuvant. This effect is synergistically enhanced on co-administration of IL-12 or IL-18, suggesting that combinations of ‘innate’ cytokines can promote onset of erosive inflammatory arthritis [80]. Soluble IL-15Ra (sIL-15Ra) administration provides a mechanism to manipulate IL-15 bio-activities in vivo. When sIL-15Ra was injected daily following antigen challenge the development of collagen-induced arthritis was suppressed, associated with delayed development of anti-collagen-specific antibodies (IgG2a) and with reduced antigen-specific IFNc and TNFa production in vitro [81]. On discontinuation of sIL-15Ra administration collagen-induced arthritis developed to levels comparable with controls, suggesting that antiinflammatory effects were transient. Similarly, an IL-15/Fcc2a fusion protein, which can antagonize the activities of IL-15 in vitro, suppresses the onset of DTH responses in vivo, associated with reduction in CD4+ T cell infiltration [82]. Together these data clearly indicate that IL-15/IL-15R interactions are important in the development of immune responses in vivo.

A Biologic Role for Cytokines of the Innate Immune Response in RA Synovitis? Antigen-independent proinflammatory processes that implicate elements of both innate and specific immune responses are of likely importance within


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the RA synovial membrane. We previously proposed that IL-15 could contribute to perpetuation of synovial inflammation in RA via promotion of such mechanisms [83]. Thus, IL-15 can recruit and expand CD45RO+ memory T cell subsets in the synovial membrane, in which, under the continuing influence of IL-15, newly recruited T cells can promote TNFa production either directly or via cognate interaction with macrophages. In positive-feedback loops thus generated, IL-15 synthesis by activated synovial macrophages or fibroblasts can induce continued T cell recruitment, with consequent maintenance of macrophage activation and TNFa production through further cell contact (fig. 1). Inflammation need not, therefore, be perpetuated primarily by local recognition of antigen. Equally, such non-antigen-driven inflammatory processes could complement ‘arthritogen’-specific responses. Whether IL-15 is present in sufficient quantity in RA synovium to sustain this scenario remains unclear. However, it is now clear that IL-15 mediates many biologic functions intracellularly or in paracrine loops through membrane-bound expression [7, 21, 23, 53]. In this context, absolute levels detected in biologic solutions including SF and culture supernatants may underestimate its net contribution. Consistent with this, IL-15 blockade in vitro and in vivo by specific moieties modulates inflammatory models in which detection of significant IL-15 levels has proven elusive. It is likely that other cytokines produced during innate immune responses might synergize with IL-15. IL-18, a recently discovered cytokine with structural homology with IL-1b, is expressed in RA synovial membrane [84]. In synergistic combination with IL-15, IL-18 mediates both synovial T cell activation, and ‘IL-1-like’ activation of macrophage function, thereby promoting TNFa production. IL-6 and TNFa may similarly contribute directly to cytokine-mediated ‘bystander’ T cell activation in combination with IL-15 [74]. Moreover, T-cell-contact-mediated activation of fibroblasts [73] leads to cytokine and metalloproteinase secretion suggesting that diverse target cells may exhibit co-ordinate pro-inflammatory activities through cell contact. We have, therefore, modified our original hypothesis to provide for amplification of synovial inflammation by T cells, driven synergistically by combinations of cytokines of the innate response. Cytokine-mediated non-specific activation of T cells has been reported. Polyclonal T cell activation follows injection of type I IFN or IL-15 in vivo in mice and resting human CD4+ T cells can be activated to produce cytokines and provide B cell help in vitro by a combination of IL-2, IL-1b and TNFa [85, 86]. Moreover, soluble cytokines may further modify the effects of cognate interactions with target cells. For example, GM-CSF enhances cytokine-activated T cell-induced TNFa synthesis by blood-derived monocytes, whereas IL-10 is inhibitory [74]. By this means, the co-operative action of soluble or cell-bound cytokine

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products of cognate interactions may feedback to ‘fine-tune’ macrophage activation.

IL-15 Expression in Other Chronic Inflammatory Diseases Elevated IL-15 expression has now been observed in several human chronic inflammatory diseases. IL-15 mRNA and protein levels are elevated in peripheral blood mononuclear cells and in inflamed colon from ulcerative colitis and to a lesser extent Crohn’s disease patients [87]. Within colonic lesions, IL-15 is expressed not only in macrophages but also in intestinal epithelial cells, upon which IL-15 exerts autrocrine effects [88]. Moreover, intraepithelial lymphocytes exhibit enhanced proliferative, cytotoxic and cytokine secretory responses to IL-15, compared with IL-2. We recently observed elevated secretion of IL-15 from explant cultures of colonic tissues from ulcerative colitis but not Crohn’s disease patients and found that such IL-15 production correlated with spontaneous TNFa release [unpubl. data]. Elevated serum IL-15 expression is described also in chronic hepatic diseases, in which it correlates with disease activity [89] and in cerebrospinal fluid and serum of multiple sclerosis patients [90, 91]. Finally, IL-15 has been detected at high levels in alveolar macrophages from pulmonary sarcoid patients [92]. IL-15 promotes IFNc production and CD28 expression on CD4+ T cells isolated from sarcoid T cell alveolitis lesions and further enhances co-stimulatory molecule expression on alveolar macrophages [93]. Thus, the significance of the biologic activities elucidated for IL-15 in RA synovitis can likely be extended to include several important human inflammatory diseases.

Conclusions That dysregulation of innate immune responses might contribute to the perpetuation of autoimmune diseases provides exciting therapeutic potential. In this context, bystander cytokine-mediated T cell contact might represent a general mechanism whereby polyclonal T cell recruitment can contribute to ongoing inflammation. Cytokines such as IL-15 and IL-18 are apparently of early importance during immune responses. Failure to downregulate such cytokine expression appropriately, or continued upregulation by unknown factors, could therefore lead to chronicity rather than resolution of inflammation. The efficacy of TNFa and IL-1c blockade in RA has elegantly demonstrated the therapeutic utility, albeit transient, of cytokine-directed therapy in the treatment of chronic inflammatory diseases. The identification of IL-15-


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mediated T cell and monocyte activation in the synovial membrane in synergy with other cytokines of the innate response, apparently operating upstream from the effects of TNFa, provides a novel target for such biologic therapeutic approaches. Studies conducted thus far in animal models indicate that specific targeting of IL-15 can mediate immunomodulation, although convincing suppression of established inflammation has not yet been achieved. Future studies investigating the potential for IL-15 blockade in combination with other cytokine targeting agents are now required.

Acknowledgments The support of the Nuffield Foundation (Oliver Bird Fund), the Wellcome Trust and the Arthritis Research Campaign (UK) is acknowledged. Foo Y. Liew, J. Alastair Gracie, Holger Ruchatz, Xiao Qing Wei, Max Field, Peter Wilkinson and Roger D. Sturrock provided invaluable contributions to many experiments and ideas expressed above. Clinical support from Hilary Capell, John Hunter, Duncan Porter and Rajan Madhok is acknowledged.


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Iain B. McInnes, Centre for Rheumatic Diseases, University Department of Medicine, Level 3, Queen Elizabeth Building, Glasgow Royal Infirmary, 10, Alexandra Parade, Glasgow G31 2ER (UK) Tel. +44 141 211 4687, Fax +44 141 211 4878, E-Mail [email protected]


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Regulation of Apoptosis of Synovial Fibroblasts John D. Mountz a, b, Huang-Ge Zhang a a

b

Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham and Birmingham Veterans Administration Medical Center, Birmingham, Ala., USA

Apoptosis Signaling by Molecules with Death Domains Apoptosis is a physiologic process that mediates the death of selected cells [1–12]. In contrast to necrosis, which results from a strong nonspecific or toxic cell injury, apoptosis is initiated by ligand-receptor interactions that are highly regulated and tightly coupled to the phagocytosis of cells undergoing apoptosis [13–18]. Several molecules that mediate or inhibit apoptosis of immune cells have now been identified including Fas, tumor necrosis factor-receptor type 1 (TNFR1), and death domain-related (DR) DR3, DR4, DR5 and DR6 [19–25] (fig. 1). TNF-related apoptosis-inducing ligand (TRAIL) was identified as a new member of the TNF family which mediates cell death in a wide variety of malignant cell lines and primary tumor cells. There are currently four receptors identified that interact with TRAIL including DR3, DR4, DR5, DR6 and a decoy receptor DcR1/trid [20–25]. TRAIL induces two different signals, cell death mediated by caspases and gene induction mediated by nuclear factorkB (NF-jB). Inhibition of TRAIL-induced activation of NF-jB augments apoptosis induction by TRAIL and attenuates apoptosis resistance. Currently, TRAIL and its receptors are of major interest due to their potential roles and application in cancer therapy. Fas/APO-1 (CD95) and either death domain family members trigger apoptosis through an approximately 90-amino acid death domain (amino acids 201–292 of Fas) which is required to signal apoptosis [11, 12]. Upon trimerization by Fas ligand or TNFR1 by tumor necrosis factor-a (TNFa),

Fig. 1. Death domain family of receptors. The death domain family receptors include Fas, TNFR1, cytoplasmic avian leukosis-sarcoma virus receptor (CAR)-1, death receptor 3 (DR3), DR4, DR5 and DR6. The decoy receptor (DcR)1/TRAIL receptor without an intracellular domain (TRID) does not have a death domain but binds to TRAIL. The extracellular region of these receptors carries 2–6 repeats of cystine-rich subdomains that have approximately 25% homology. Fas has 3 cystine-rich subdomains and TNFR1 has 4 such subdomains. The numbers of amino acids between the cystine-rich domain and the membrane are shown. The intracytoplasmic death domain is shown by the rectangular box.

the cytoplasmic death domain forms a death-inducing signaling complex (DISC) [26]. This DISC acts to dock adapter and signaling molecules that signal apoptosis, including Fas-associated protein with death domain (FADD or MORT1) [27–31] which then recruits FADD-like interleukin-converting enzyme (FLICE) now referred to as caspase 8 [32–36] (fig. 2). For TNFR1 signaling, TNFR-associated death domain (TRADD) acts as an adaptor molecule.

Rheumatoid Arthritis and Apoptosis Initial investigations of apoptosis in rheumatoid synovium indicated that apoptotic cells were confined to the synovial lining layer and that infiltrating T cells express high levels of Bcl-2 and were resistant to Fas apoptosis [37–39]. Other studies reported decreased expression of Bcl-2 in synovial fluid T cells or no significant difference in Bcl-2 expression in synovial tissue T cells in

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Fig. 2. Apoptosis signaling pathway. Apoptosis signaling by a DISC was mediated by binding of FADD and Fas-like interleukin 1-converting enzyme (FLICE), otherwise known as caspase 8, to Fas. The formating of the DISC depends upon cross-linking of the Fas molecule. This leads to production of active caspase 8. In the case of TNF1, the first molecule to bind to DISC is the TNFR apoptosis death domain (TRADD), followed by assembly of the same molecules as described for Fas.

rheumatoid arthritis (RA) compared to osteoarthritis and reactive arthritis [40, 41]. Soluble Fas, capable of binding Fas ligand and inhibiting Fas apoptosis, is increased in synovial fluid of patients with RA [42, 43]. Other investigators reported increased expression of Fas ligand on activated T cells in RA synovium [44–48], increased expression of Fas, and also high sensitivity to apoptosis [49–54]. Therefore, most studies support the notion that T cells in the synovium of patients with RA are activated and correspondingly express increased levels of Fas, Fas ligand and a trend to undergo Fas-mediated apoptosis. However, the question remains if this apoptosis is sufficient for the elimination of T cells that promote inflammatory disease. The synovial fibroblast which undergoes hyperplasia in patients with RA has been reported to have several defects in Fas, Fas ligand, apoptosis and expression of other apoptosis molecules such as p53. Apoptotic synovial lining cells are largely type A (macrophage-like) with little apoptosis of type B (fibroblast-like) synovial cells that is evident. These synovial fibroblasts have been demonstrated to be sensitive to apoptosis in an HTLV-1 tax transgenic mouse model when high levels of anti-Fas antibody were injected intra-articularly [55]. These experiments were carried out using a novel anti-Fas mono-

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clonal antibody (RK-8) which did not cause significant liver toxicity which can cross-link Fas and induce apoptosis in some strains of mice. Also, transfection of human Fas ligand into RA synovial fibroblasts which have been transplanted into SCID mice can also induce apoptosis [56, 57]. Similar results were obtained using anti-Fas monoclonal antibody to induce apoptosis of human RA tissue engrafted into SCID mice [58]. TNFa has been shown to inhibit [59] or facilitate [60] Fas signaling in human RA synovial fibroblasts. Together these results indicate that Fas apoptosis signaling may be defective in human synovial fibroblasts and that this signaling can be modulated by other cytokines such as TNFa and transforming growth factor-b that are present in abundance in the joint tissue [61]. An important pathway of apoptosis resistance for some synovial cells appears to be the expression of mutant p53 [62]. It was hypothesized that free radical production associated with the highly oxidative metabolism present in the inflammatory and invasive areas of synovium may lead to mutations in the p53 tumor suppressor gene [63]. Therefore, p53 is proposed to be a critical regulator of fibroblast-like synovial cell proliferation, apoptosis and invasiveness. Abnormalities of p53 function might contribute to synovial lining expansion and joint destruction in RA.

Signaling Pathways of Apoptosis in RA Synovial Cells The signaling pathway for Fas in synovial fibroblasts has not been extensively studied but several observations indicate that Fas signaling is downregulated. Fas apoptosis has been shown by one investigator to involve the June kinase and the AP1 pathway [64] as well as ceramide signaling [65]. Other investigators have found defects of the mitogen-activated protein map kinase/ kinase to be defective in RA synovial fibroblasts (RASF) [66]. Interactions with TNFR1 produce a proapoptotic signal by recruitment of TNFR1 receptor death domain protein (TRADD) to the DISC of the TNFR1 trimer [67, 68]. TRADD recruits the Fas-associated death domain (FADD) which in turn recruits caspase 8 and signals apoptosis [69]. Simultaneously, an antiapoptosis pathway involves recruitment of RIP and TRAF2, which leads to activation of NF-jb inducing kinase (NIK) [70] (fig. 3). This results in phosphorylation of IjBa and IjBb and translocation of NF-jB to the nucleus. This second signal predominates in RASF, and NF-jB translocation to the nucleus plays a role in transcription of several genes including TNFa, IL-1b, IL-6 as well as collagenase, stromylysin and adhesion molecules [71, 72]. At the same time, NF-jB translocation inhibits apoptosis in many cell lines including tumor cell lines [73], but the mechanism of induction of antiapoptosis genes in RASF is


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Fig. 3. Regulation of NF-jB nuclear translocation. TNFR signaling by TNFa acts through TRAF2 and Rip to activate NIK. This phosphorylates IjBa and IjBb chains. The phosphorylated IjBa and IjBb are rapidly degraded in the proteosome. The NF-jB 50-kD and the NF-jB 65-kD dimer undergo nuclear translocation which is guided by the nuclear localization (NLS). Nuclear translocation of NF-jB leads to upregulation of a number of genes including antiapoptosis genes.

not known. RASF, like tumor cell lines, also do not undergo apoptosis in response to TNFa, and, therefore, we propose that RASF may be selected in vivo to use TNFa as a growth factor as well as to induce production of cytokines and invasive enzymes. Other pathways of pro- and antiapoptosis in RASF include TNFR signaling. TNFR signaling can activate a potent anti-inflammatory pathway via the NF-jB nuclear translocation [74]. Analysis of synovium of rats in the streptococcal cell wall (streptococcal cell wall model) of arthritis indicated that a mutant form of inhibitor-jBa (IjBa) which prevents nuclear translocation of NF-jB results in enhanced apoptosis of rats in this model. Phosphorylation of IjBa results in release of NF-jB and can be inhibited by mutant forms of IjBa lacking the serine phosphorylation site [75, 76] (fig. 4). Serine phosphorylation has been shown to be necessary for release of IjBa from NF-jB, resulting in ubiquitinization of IjBa and degradation by proteosomes. A mutant form of IjBa lacking 36 amino acids which includes this serine residue has previously been shown to bind tightly to NF-jB, but cannot be phosphorylated by NIK (fig. 4). Inhibition of NF-jB nuclear translocation results in apoptosis in a variety of cell types originally resistant to TNFa-induced apoptosis [77]. Furthermore, fibroblasts and macrophages from NF-jB subunit p65-deficient mice are more sensitive to TNF-induced apoptosis [78]. Under

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Fig. 4. Truncated AdIjBa inhibits NF-jB nuclear translocation. Activation of IjBkinase normally leads to proteosome degradation of IjBa and IjBb and associated nuclear translocation of NF-jB. An adenovirus that expresses high levels of a truncated IjBa chain which lacks the phosphorylation site was constructed. Transfection of RASF with this construct results in high levels of expression of a truncated Ijba chain referred to as DIjBa or IjB-dominant negative (IjB-DN). This dominant negative form of Ijba competes with the normal Ijba chain. The absence of the phosphorylation site on the mutant Ijba chain prevents phosphorylation and association of Ijba and Ijbb from NF-jB. This in turn prevents nuclear translocation of NF-jB.

normal conditions, NF-jB is present in the heterotrimer consisting of p50 and p65 subunits bound to dephosphorylated IjBa [78–80]. When IjBa undergoes phosphorylation by IjB kinase, IjB undergoes unbiquination and degradation. The p50-p65 NF-jB complex undergoes translocation to the nucleus resulting in gene activation.

AdCMVIjB-DN and TNFa Induces Apoptosis of RASF We constructed an adenovirus that expresses high levels of a truncated form of IjBa, denoted as AdCMVIjB-DN. Transfection of RASF with this recombinant adenovirus leads to overexpression of DIjBa which cannot be phosphorylated and prevents nuclear translocation of NF-jB. This leads to an unopposed apoptosis signal by RASF upon culture with TNFa. Overexpression of this mutated IjBa by an AdCMVIjBa-DN construct results in inhibi-


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d Fig. 5. Increased apoptosis after treatment with AdCMVIjBa-DN plus TNFa. Primary RASF cell lines (2¶104) were transfected with either control AdCMVGFP (a, c) or with AdCMVIjB-DN (b, d ). Twenty-four hours later, cells were treated with TNFa (10 ng/ml), and analyzed after culture for an additional 12 h. Viable, nonapoptotic cells are indicated by green fluorescence. Early and intermediate apoptotic cells are indicated by decreased cytoplasmic green fluorescence and increased blue nuclear fluorescence and condensation. Late state apoptosis and cell fragmentation are indicated by nuclear condensation and extensive blue nuclear staining. Magnification was 10¶ (a, c ) and 40¶ (b, d ).

tion of nuclear translocation of NF-jB after TNFa stimulation of RASF. Primary RASF cell lines (2¶104) were transfected with either control AdCMVGFP (fig. 5a, c) or with AdCMVIjB-DN (fig. 5b, d). Apoptosis was analyzed 12 h after stimulation with TNFa (10 ng/ml). There was no significant apoptosis in synovial cells that were transfected with AdCMVGFP and treated

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d Fig. 6. Treatment with AdCMVIjBa-DN plus TNFa in vivo. Approximately 1¶106 of primary RA synovial cells were injected intra-articularly (i.a.) into both knee joints of 20 male SCID mice at 6–8 weeks of age. Four weeks later, both knee joints of 10 mice were i.a. injected with 1¶109 pfu of AdCMVIjB-DN. As a control, both knee joints of 10 mice were i.a. injected with 1¶109 pfu of AdCMVLacZ. Two days later, 5 mice in each group were treated with human TNFa (10 lg/kg of body weight) and 5 mice were treated with PBS, followed by sacrifice 24 h later. Multiple sections of the joints were stained with either H&E (a, b) or stained for apoptosis by TUNEL (c, d ). a–d The RASF adjacent to bone is shown.

with TNFa. In contrast, incubation of primary RA synovial cell lines with AdCMVIjBa-DN plus TNFa induced apoptosis of 80–90% of synovial cells at 12 h. These results indicate that in the presence of AdCMVIjBa-DN, TNFa exerts a potent apoptosis signal on primary RASF.

AdCMVIjBa-DN plus TNFa in vivo Induces Apoptosis of RASF in a SCID Mouse Model Four weeks after intra-articular of RASF into SCID mice, there was significant local hyperplasia of primary and SV40-transformed RASF in all mice. Mice were then intra-articularly injected with 109 plaque-forming units (pfu) of either


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e f Fig. 7. Increased TNFa-induced apoptosis of RA cell lines after treatment with AdCMV XIAP-AS. Primary RASF (2¶105) were transfected with either AdCMVGFP (50 pfu/cell) (a, d), AdCMV XIAP-AS (10 pfu/cell) (b, e ) or AdCMV XIAP-AS (50 pfu/cell) (c, f ). After 1 h the cells were washed and cultured for an additional 18 h. TNFa was added and the cells were analyzed for apoptosis 12 h later by fluorescent microscopy. Viable, nonapoptotic cells are indicated by intact cells (a, d ). Early and intermediate apoptotic cells are indicated by decreased cell size and increased nuclear condensation. Late state apoptosis and cell fragmentation are indicated by nuclear condensation. Magnification was 10¶ (a, b, c) and 40¶ (d, e, f ).

control AdCMVLacZ or AdCMVIjBa-DN. After 2 days, the mice were treated with TNFa (10 lg/kg of body weight, i.p.). Twenty-four hours later, all mice were sacrificed and the knee joints were processed for histology analysis. There was no effect on the RASF cells of SCID mice after treatment with AdCMVLacZ and TNFa (fig. 6a). In contrast, SCID mice treated with AdCMVIjBa-DN plus TNFa exhibited extensive pycnotic nuclei and cell destruction 24 h after administration of TNFa. To determine if TNFa induced apoptosis in vivo, knee joints were sectioned and analyzed by in situ TUNEL staining. There was extensive apoptosis of synovial fibroblasts transfected with AdCMVIjBa-DN plus TNFa but not in RASF treated with control AdCMVLacZ plus TNFa (fig. 6c, d).

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TNa Induces XIAP Expression in RASF Inhibitor of apoptosis (IAP) gene products play an evolutionarily conserved role in regulating programmed cell death in diverse species ranging from insects to humans. Human XIAP, cIAP1 and cIAP2 directly inhibit caspase-3, -6 and -7 [81–84]. The IAPs also can block cytochrome c-induced activation of pro-caspase-9 and inhibit Fas-mediated apoptosis. The murine homologue of the human X-linked IAP, called miap, has been mapped to the X chromosome [85]. Stimulation of cells with TNFa has been shown to generate two signals: one that initiates programmed cell death, and another that leads to activation of the transcription factor NF-jB that induces IAPs and also promotes production of proinflammatory factors [86–88].

AdCMV XIAP-AS Converts TNFa Apoptosis-Resistant RA Cell Lines to TNFa Apoptosis-Sensitive Cell Lines NF-jB induction of XIAP may inhibit TNFa-induced apoptosis in primary RA synovial cell lines. Antisense XIAP complementary to the 5 portion of the coding region (nucleotide position from –28 to +180) XIAP gene was driven by the cytomegalovirus early promoter, and the GFP marker gene was also coexpressed in the recombinant adenoviral vector (AdGFPXIAPas). To determine if XIAP might play a role in the inhibition of apoptosis of RA cell lines, RA cell lines were transfected with different pfu/cell of either AdCMV XIAP-AS antisense (AdCMV XIAP-AS) or a control AdCMVGFP vector. After 18 h, cells were either untreated, or treated with TNFa (10 ng/ml) and analyzed for apoptosis 12 h later. There was no apoptosis in RASF transfected with the AdCMVGFP followed by treatment with TNFa (fig. 7a, d). In contrast, there was apoptosis of approximately 50% of cells treated with 10 pfu/ cell of AdCMV XIAP-AS (fig. 7b, e) and 80% cells treated with 50 pfu/cell of AdCMV XIAP-AS (fig. 7c, f ). These results indicate that inhibition of the TNFa-induced antiapoptosis gene XIAP can facilitate TNFa apoptosis of RA synovial cell lines.

Regulation of Fas Apoptosis in RASF Most investigators have reported that RASF are resistant to anti-Fas apoptosis [56–60]. The susceptibility of five different RASF cell lines to apoptosis was investigated by treating the cell lines with different concentrations of anti-Fas (CH-11) monoclonal antibody (fig. 8). There was low anti-


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Fig. 8. RASF are resistant to Fas apoptosis. The Fas-sensitive T cell line, CEM6 and RASF primary cell line were incubated with different concentrations of the human anti-Fas antibody CH-11. The cells were incubated for 24 h and apoptosis was determined by Hoechst staining. The results represent the mean percent of cells undergoing apoptosis×standard error of the mean. Four different RASF cell lines were analyzed and the average apoptosis indicated.

Fas-induced apoptosis of RASF after 48 h of incubation with CH-11 concentration up to 10 lg/ml. In contrast, there was high apoptosis at 48 h (and 24 h, data not shown) of CEM-6 cells at a 100-fold decreased concentration of CH-11 of 0.1 lg/ml. These results indicate that RASF are highly resistant to signaling through Fas. The mechanism of Fas apoptosis resistance in RASF may be proximal, at the level of caspase 8 activation, or more distal, involving defects in the mitochondrial amplification loop or in activation of terminal caspases (fig. 9). In T cells, Bcl-2 family members play a major role in the regulation of Fas apoptosis signaling, and have been reported to be upregulated in RASF [89]. The family of Bcl-2-related proteins share homology at four conserved Bcl-2 homology (BH1-4) domain regions, which control the ability of these proteins to dimerize and function as regulators of apoptosis [37]. Conserved domains

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Fig. 9. Members of the Bcl-2 family. Protein-protein interactions between Bcl-2 family members regulate apoptosis function. Bax can form homodimers or heterodimers with either Bcl-2 or Bcl-XL blocks cell death. Bad, a proapoptotic Bcl-2 family member, heterodimerizes with Bcl-2 and Bcl-XL and promotes cell death. Bid can be directly activated by Fas and promote cell death. Members of the Bcl-2 family have been suggested to play a role in mitochondrial ion channel formation and promote the release of cytochrome c for mitochondria.

BH1, BH2 and BH3 participate in the formation of various dimer pairs as well as the regulation of cell death. The Bcl-2 family includes the death antagonists Bcl-2, Bcl-XL, Mcl-1 and A1 as well as the proapoptotic molecules Bas, Bcl-XS, Bak, Bik, Bid, Bim and Bad. The overall ratio of the death agonists to antagonists determines the susceptibility to a death stimulus. Bcl-XL, Bcl-2 and Bax can also form ion-conductive pores in artificial membranes. Bid and Bad possess the minimal death domain BH3, and the phosphorylation of Bad connects proximal survival signals to the Bcl-2 family. Bcl-2 and Bcl-XS display a reciprocal pattern of expression during lymphocyte development. Fas apoptosis is associated with sequential caspase (cleave after Asp residues) activation beginning with caspase 8 (fig. 9). Caspases are expressed constitutively in most cells, residing in the cytosol as a single chain proenzyme. These are activated to fully functional proteases by a first proteolytic cleavage to divide the chain into large and small caspase subunits and a second cleavage to remove the N-terminal domain (prodomain). Inefficient activation of caspase-8 results in direct activation of Bid, a proapoptotic member of the Bcl-2 family, and the C-terminal fragment acts on mitochondria triggering


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b Fig. 10. Decreased mitochondrial permeability transition in RASF after induction of apoptosis. a RASF were induced to undergo apoptosis by incubation with anti-Fas (1 lg/ml) for 48 h. The mitochondrial permeability was analyzed by DiOC6 (0.5 lM ) and the nucleus was visualized by Hoechst staining. So far the mitochondrial permeability was analyzed by DiOC6 5 lM and the nucleus was visualized by Hoechst staining. b As a control, CEM-6 T cells were incubated with anti-Fas (1 lg/ml) for either 6 (early) or 24 h (late) and stained with DiOC6 and Hoechst staining as described above. Before treatment with CH-11 the CEM-6 T cells have prominent mitochondrial organelles visible in the cytoplasm and the

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cytochrome c release [90–92]. The released cytochrome c binds to apoptotic protease activating factor-1, which self-associates and binds to caspase 9 [93]. This is associated with a drop in inner mitochondria membrane potential corresponding to the opening of the inner membrane permeability transition pore complex, and loss of the ability to take up certain dyes [94]. In cells in which this mitochondria amplification loop is important, antiapoptotic Bcl-2 family members can suppress Fas-induced apoptosis [95]. Bcl-2 and Bcl-X act to prevent cytochrome c release and thus interfere with this pathway. Caspase 9 and 8 then act on terminal caspases 3, 6 and 7 which are terminal caspases that are activated prior to apoptosis [96]. Alternatively, strong signaling through Fas leads to high levels of caspase 8 activity and direct activation of terminal caspases 3, 6 and 7 and apoptosis [97–100].

Defective Mitochondrial Permeability Transition in RASF after Treatment with Anti-Fas Effective signaling through Fas with resultant apoptosis can utilize the mitochondrial amplification loop pathway as well as a direct pathway for activation of caspase 3 in apoptosis. The mitochondrial amplification loop pathway is associated with release of cytochrome c and decrease in the mitochondrial permeability transition. The mitochondrial permeability transition can be visualized with the voltage-sensitive dye DiOC6. DiOC6 is taken up by mitochondria of T cells that are not undergoing apoptosis (fig. 10a). Apoptosis can be visualized by coincubation of T cells with Hoechst stain, which stains chromatin of cells undergoing apoptosis. In early apoptosis, there is a decreased number of cells with DiOC6 staining of mitochondria associated with an increased number of cells demonstrating nuclear staining by Hoechst stain. The apoptotic nucleus can be easily distinguished by their multilobular morphology and nuclear fragmentation. During the late stages of anti-Fasinduced apoptosis of T cells, there are few mitochondria visible by DiOC6 staining and a high percentage of cells indicate a nuclear fragmentation in Hoechst staining (fig. 10a). Treatment of primary RASF with anti-Fas induced minimal apoptotic changes after 48 h. However, there is no change in the mitochondrial permeability as determined by the voltage-sensitive dye DiOC6. Therefore, anti-Fas apoptosis of RASF occurs without MP transition. This nucleus is not stained by Hoechst stain. Six hours after treatment with anti-Fas, there is decreased uptake in clustering of mitochondria DiOC6 staining and also nuclear condensation as revealed by Hoechst staining. Late changes show very low mitochondrial staining with intense nucleus changing consistent with apoptosis.


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result indicates that anti-Fas apoptosis either does not use the mitochondrial amplification loop, or that a novel mechanism of mitochondrial signaling occurs that does not result in decrease of the mitochondrial permeability transition (fig. 10b). Defective mitochondrial permeability transition in RASF after treatment with anti-Fas suggests that there is a negative regulator of Fas apoptosis between caspase 8 activation and Bid/Bad activation upstream from the mitochondrial amplification loop.

Akt Regulation of Fas Apoptosis of RASF Akt is a proto-oncogene that causes leukemia in mice and is overexpressed in human tumors [101–104]. Akt is a PKB serine/threonine protein kinase and has a pleckstrin homology domain that aids membrane interactions. Akt is in the same family as the XID PKB that is mutant in XID mice. Akt inhibits apoptosis in cytochrome c release and also inhibits mitochondrial membrane permeability transition that can be induced by Bax and Bid. Akt promotes cell survival by inhibiting the apoptosis cascade before cytochrome c release. Addition of exogenous cytochrome c induced apoptosis in the presence of active Akt. Akt activity can be inhibited by the PI3-kinase inhibitor, wortmannin (fig. 11). Akt may be a regulator of RASF apoptosis. This was analyzed by culture of RASF in the presence of anti-Fas, wortmannin, or wortmannin plus antiFas (fig. 12). There was low apoptosis of primary RASF in the presence of anti-Fas (1 lg/ml). There was no effect of wortmannin on apoptosis of RASF at 50 nM, but wortmannin was toxic to RASF at 200 nM. The addition of wortmannin (50 nM ) plus anti-Fas (1 lg/ml) induced apoptosis of 70×5% of RASF compared to control anti-Fas at 48 h. The apoptosis process proceeded slowly, and at 24 h there was minimal apoptosis of anti-Fas-treated RASF with and without wortmannin. Pretreatment of RASF by wortmannin (50 nM for 24 h) resulted in increased susceptibility to apoptosis after treatment with anti-Fas plus GAM. These results indicate that the PI3-kinase inhibitor wortmannin likely inhibits Akt, which results in increased apoptosis sensitivity by anti-Fas on primary RASF (fig. 12).

Current Therapy for RA and Apoptosis Most current therapies used for RA have been shown to induce apoptosis [105]. Chloroquine inhibits growth of human umbilical vein endothelial cells by induction of apoptosis [106]. This is associated with upregulation of

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Fig. 11. PI3-kinase signaling of AKT. Signaling through many receptors including IL-3, angiotensin II and TNFR activates the p85 and p110 domains of PI3-kinase. This leads to conversion of phosphatidylinositol-4,5 (P2) to phosphatidylinositol-3,4,5 phosphate (P2). This phosphorylated phosphatidylinositol binds the pleckstrin homology domain of Akt and sequesters this to the membrane. This results in phosphorylation of Akt to the active form. The active form of Akt has several functions including regulation of protein synthesis, glycogen metabolism and also regulation of proliferation and apoptosis. One mechanism for regulation of apoptosis is phosphorylation of the Bcl-2 proinflammatory family member Bad resulting in inhibition of apoptosis.

Bcl-X without any change in Bcl-2. The folate antagonist methotrexate (MTX) is used extensively to treat RA as well as other chronic inflammatory diseases. MTX exerts an antiproliferative property by inhibition of dehydrofolate reductase and other folate-dependent enzymes. MTX efficiently induced apoptosis of T cells in a concentration of 0.1–10 lM. This apoptosis did not depend on Fas or Fas ligand but required cell cycle. In vitro activation of PBMCs from arthritis patients after MTX injection resulted in increased propensity towards apoptosis [107]. Phototherapy, useful in the treatment of rheumatic disease, can operate by direct induction of the Fas, Fas ligand system [108] and also by Fas-independent apoptosis [109]. Soluble TNFR2 (etanercept) and soluble TNFR1 can inhibit later stages of inflammatory disease [110–114].


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Fig. 12. Increased Fas signaling and RASF apoptosis after treatment with the protein kinase c inhibitor, wortmannin (WM). RASF was treated with anti-Fas, wortmannin (50 nM), wortmannin (50 nM) + anti-Fas (1 lg/ml) or preincubated with wortmannin (50 nM) for 24 h followed by treatment with anti-Fas (1 lg/ml) for an additional 24 h. Apoptosis was determined after 48 h by staining with DiOC6 and Hoechst stain.

In contrast, we and others have previously shown that TNFa can inhibit development of autoimmune disease, and that TNFR1 knockout mice crossed with lpr/lpr mice develop an accelerated autoimmune disease [115, 116]. These different results of TNF are due to the dual signaling pathway of TNFR. One pathway involves a proapoptotic mechanism and acts through TRADD and then FADD and caspase 8 as shown above for Fas. However, this pathway appears to be blocked in RA synovial cells. The second pathway, a proinflammatory pathway, is mediated primarily by a nuclear translocation of NF-jB, which induces transcription of proinflammatory cytokines as well as transcription of antiapoptosis molecules such as IAP. Sulfasalazine, which is widely used for RA as well as inflammatory bowel disease, can induce apoptosis of neutrophils in vitro [117]. Neutrophil apoptosis can be blocked by specific inhibitors including a tyrosine kinase inhibitor, protein kinase A inhibitors and antioxidants. These results indicate that phosporylation of tyrosine kinase and protein kinase A as well as generation of reactive oxygen species are involved in sulfasalazine-mediated neutrophil apoptosis. Together, these results indicate that a better understanding of apoptosis pathways, and

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better mechanisms to trigger these apoptosis pathways will be beneficial in the treatment of rheumatic diseases.

Summary and Conclusions In 1992, the first gene to cause systemic autoimmune disease in mice was identified as the Fas gene that is mutated in lymphoproliferative (lpr mice). These mice exhibited a defect in activation-inducted cell death of T cells and B cells in vivo. This leads to the failure of proper clearance and removal of immune cells and defective downmodulation of an immune response. This then leads to the speculation that apoptosis defects, including defects in Fas, Fas ligand and Fas apoptosis signaling, may play a role in defective downmodulation of the hyperimmune response observed in human autoimmune diseases. Over the past 7 years, many scientists have analyzed different proapoptotic genes such as Fas, Fas ligand, Bcl-X, caspases as well as antiapoptosis pathways including defects in Fas and Fas ligand, Bcl-2 and caspase inhibitors. Potential genetic defects have been analyzed at the RNA, protein and functional level in humans with autoimmune disease. Somewhat surprisingly, most studies indicate that there is excessive apoptosis of PBMCs in autoimmune disease and human autoimmune disease suggesting that human autoimmune disease is not due to defective apoptosis of immune cells. Some studies indicate that there is decreased apoptosis of parenchymal cells such as RASF that undergo hyperplasia. Gene therapy and other modulators of apoptosis, such as wortmannin, can be used to faciliate apoptosis of RASF.

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John D. Mountz, MD, PhD, University of Alabama at Birmingham, 701 South 19th Street, LHRB 473, Birmingham, AL 35294-0007 (USA) Tel. +1 205 934 8909, Fax +1 205 975 6648, E-Mail [email protected]


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Biologics in the Treatment of Rheumatoid Arthritis: Mechanisms of Action Arthur Kavanaugh, Peter Lipsky Department of Internal Medicine, The University of Texas Southwestern Medical Center at Dallas, Tex., USA

Recent years have witnessed remarkable advances in the development of biologic agents for the treatment of rheumatoid arthritis (RA). In the 1980s, a number of approaches to biologic therapy were considered to be potentially viable. Using various types of agents that were directed against several distinct targets, clinical trials were undertaken to test these approaches. Many agents would eventually be proven not to be feasible therapies, and many others would be abandoned for other considerations. However, at the end of the 1990s two biologic agents were introduced into the clinic for the treatment of RA. The success of these agents, both of which are inhibitors of tumor necrosis factor (TNF), provides substantial impetus for further development of other biologic agents. There have been several factors underlying the interest in the development of biologic agents for treating RA. Until the late 1970s, RA was considered a relatively benign disease [1–3]. Based upon observational population studies, it was considered that the majority of RA patients would experience a remission of disease within a few years. Although it was recognized that certain patients with RA could develop deformities of their joints, it was thought that overall most patients could expect to have a more benign disease course. Therefore, the treatment paradigms for RA such as the ‘therapeutic pyramid’ were cautious and rather passive. Typically, the socalled disease-modifying antirheumatic drugs (DMARDs) would only be used after rheumatoid inflammation had been persistent for an extended period of time [1–3]. Beginning in the 1980s, it became appreciated that RA

was actually a much more serious disease. Some of this relates to a change in the classification of RA; previously, the term RA encompassed a variety of arthritides, many of which were transient or had a generally favorable outcome. As currently classified, the vast majority of RA patients have a chronic disease course; spontaneous remission is distinctly unusual. Moreover, it became appreciated that many patients have a poor outcome, with quite substantial morbidity and even accelerated mortality. In addition, it was shown that the disease progresses more rapidly than was once considered. Many patients who ultimately experience significant morbidity and potentially decreased life expectancy can be found to have evidence of aggressive disease, for example periarticular bony erosions, within months of disease onset [1–3]. This growing understanding of the serious nature of RA led to a change in treatment paradigms. More aggressive therapy, for example earlier and more extensive use of DMARDs, became standard. In addition, combinations of DMARDs were used. Despite this more aggressive approach to therapy, it became clear that for a number of patients, the disease could not be sufficiently controlled. In part, this related to the fact that most patients do not remain on a given DMARD for long periods of time, because of either inefficacy or toxicity [4]. Dissatisfaction with presently available DMARDs provided impetus for the development of novel therapies. Interestingly, most DMARDs were discovered to be of benefit in RA anecdotally, or they were introduced based on presumed mechanisms that were subsequently disproven. Although DMARDs have been associated with various in vitro activities, their precise mechanisms of action in RA patients remain incompletely delineated. As more has become known of the pathogenesis of RA, it has been expected that future therapies might target pathogenic abnormalities relevant to the disease process in a more specific manner. Progress in defining the immunologic abnormalities of RA has been a central factor enabling the development of biologic agents (fig. 1) [5, 6]. The initiation of RA probably relates to the exposure of a genetically susceptible host to some unidentified environmental stimulus. At present, the relevant etiopathogenic agent or agents have not been conclusively identified. The most important implication of this from a therapeutic standpoint is that treatments directed at such an agent are not presently available. Nevertheless, substantial progress has been made in unravelling the pathogenesis of this systemic inflammatory disorder. The propagation of inflammation reflects an active, ongoing, immunologically driven process. There is considerable evidence that several components of the immune response are dysregulated in patients with RA. Alterations in the synovial vasculature occur early in the disease course and may play a vital role facilitating its propagation. Such changes include angiogenesis, and alteration of the endothelium into an

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Fig. 1. The immunopathogenesis of rheumatoid arthritis. Interactions between immunocompetent cells, including those resident within the synovium as well as those that recirculate to that inflammatory site, result in the elaboration of proinflammatory mediators. The various components of the immune system that are dysregulated in this systemic inflammatory disorder are potential targets for immunomodulatory biologic therapies.

activated, ‘high endothelial venule’ phenotype. This high endothelial venule phenotype allows enhanced accumulation of inflammatory and immunocompetent cells within the synovium, and may contribute to their activation [7, 8]. Recirculating CD4+ T lymphocytes, particularly those with a ‘memory’ phenotype, accrue in large numbers in the rheumatoid synovium, utilizing specific adhesion molecules [8–10]. These T cells, many of which appear to be activated and have a pattern of cytokine secretion skewed towards a ‘Th1’ phenotype, play a central role in the initiation and orchestration of the autoimmune response [9–12]. Resident synovial cells such as fibroblasts, macrophages, dendritic cells, mast cells, endothelial cells as well as bloodderived B cells and neutrophils are critical to various facets of rheumatoid inflammation and the active ongoing immunologic activity [13, 14]. Interactions among these various cells result in the liberation of various mediators, including cytokines, chemokines, prostaglandins, degradative enzymes, and

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numerous other mediators. These soluble mediators produce local tissue damage and cause many of the signs and symptoms of disease. In addition, cytokines and chemokines also exert prominent effects on immunocompetent cells [15–18]. Understanding the role of the various components of the immune response in RA has been an intrinsic part of the development of biologic agents because it has provided specific targets for novel immunomodulatory therapy. Another critical development central to the development of biologic therapies has been advances in biopharmaceutical discovery and manufacture. Beginning with the elucidation of monoclonal antibodies (mAb), through the synthesis of soluble receptor fusion proteins and other elegant constructs, progress in the creation of novel biologic agents has allowed specific components of the immune response to be targeted in autoimmune diseases such as RA [19]. These powerful tools have allowed specificity to an extent heretofore unachievable. The use of these agents has provided proof of concept; that is, validation that inhibition of a particular target may affect the course of disease. Further refinements in the development of biologic agents, both ongoing and in the foreseeable future, will facilitate the creation of additional therapeutic agents that have optimal pharmacokinetic, pharmacodynamic and other characteristics.

Trials of Biologic Agents Numerous trials have been conducted using biologic agents in patients with RA [19–23]. Initially, many trials focused on T cells. Several relevant T cell surface molecules have been targeted using mAb, soluble cytokines linked to toxins, and other agents. In general, results of the trials inhibiting T cells have followed a disappointing path; early unblinded studies seemed to indicate potential efficacy, whereas subsequent more rigorous blinded trials produced clinical results similar to placebo. However, relevant information can be gleaned from these trials that can be expected to impact future therapeutic trials. In addition to targeting T cells, a number of trials have targeted inflammatory cytokines, such as interleukin-1 (IL-1) and TNF-a. These trials, particularly those utilizing TNF-a inhibitors, have shown considerable promise. Significant results from studies of these agents have allowed the introduction of biologic agents to the clinic. These trials have also laid the groundwork for what will surely be a number of other studies analyzing methods of optimizing this approach to treating RA. In this chapter, we will briefly discuss the implications of results from trials targeting CD4 T cells, and then focus on the results of trials inhibiting TNF-a.


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Trials Targeting CD4 Rationale for Inhibition of CD4/Animal Studies There is ample evidence supporting a role for CD4+ T cells in the etiopathogenesis of RA [9, 10, 24]. Such evidence includes the immunohistochemical demonstration of abundant activated CD4 T cells in the rheumatoid synovium, clinical efficacy of T cell-directed therapeutic interventions (e.g. lymphocytapheresis, cyclosporine, etc), and perhaps most importantly, the association of RA with certain MHC class II alleles. Among the population of CD4+ T cells, it appears that memory cells with a bias towards a Th1 type of immune response may be most relevant to human RA [11, 12]. The CD4 molecule, a member of the immunoglobulin superfamily, binds non-polymorphic regions of MHC class II molecules. CD4 associates with CD3 on the surface of T cells, and functions to decrease the threshold of activation of the MHC class II/antigen/T cell receptor complex [25]. This relates in part to the synergistic activation of protein tyrosine kinases associated with the CD4 molecule (p56lck) and those associated with CD3/TCR (p59fyn). While its role in specific T cell activation provides rationale for inhibition of CD4 in RA, it was the exciting results from animal studies that laid the groundwork for interest in this approach to therapy in human disease. Antibodies to CD4 have been used in a variety of animal models of human disease [26–28]. In arthritis models in rodents, pretreatment with anti-CD4 prevents the establishment of disease. In addition, treatment of animals with ongoing arthritis has also proven successful. Perhaps most importantly, treatment with anti-CD4 successfully achieved antigen-specific unreponsiveness, or tolerance, to antigens introduced during the time of therapy. This was considered to be of great relevance to human RA; because the etiologic antigen(s) in RA are not known, specific targeting is not possible. However, by extrapolation from other models of chronic immunologic reactivity, intermittent exposure to relevant antigen is required to maintain the immunologic memory that presumably underlies the long-term propagation of immunedriven inflammation in RA [29]. Therefore, it was hoped that treatment with anti-CD4 might result in tolerance to relevant but unidentified etiologic antigens in RA and thereby allow the reestablishment of normal immunologic homeostasis. Moreover, in animal models, therapy with anti-CD4 antibodies modulated the immune response to the extent that the treating antibodies themselves, even though they were of foreign origin, were not immunogenic. Such characteristics would be very desirable in a therapeutic agent for human use.

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Human Studies Human studies of anti-CD4 therapy began with murine mAb. The reasons for using mAb of mouse origin is that they are the easiest to create and develop. Although potentially immunogenic, it was hoped that the immunomodulatory effects of inhibiting CD4 might attenuate the immunogenicity of the foreign antibody. Initial open trials, using various anti-CD4 antibodies, seemed to indicate potential clinical efficacy for a number of the treated patients [30, 31]. Interestingly, although clinical efficacy was achieved for some patients in virtually all trials, effects on acute phase reactants (ESR, CRP) seemed variable with different agents. By contrast, rapid and sustained loss of CD4+ cells from the peripheral blood was achieved uniformly with all antibodies. Circulating CD4+ counts decreased to less than half of pretreatment values, or even lower. Depletion of CD4+ cells was noted within an hour of treatment, and persisted for months in some patients. Importantly, there was no association between the extent of decrease in numbers of CD4+ cells and clinical efficacy in these trials, suggesting that depletion of T cells was neither required nor sufficient for clinical efficacy. Indeed, despite notable decreases in peripheral CD4 counts, the patients did not appear to exhibit clinical immunodeficiency (e.g. opportunistic infections were not common). Dissimilar to results in animal studies, the murine anti-CD4 antibodies were immunogenic. Human antimouse antibodies were demonstrated in most, but not all, patients, and human anti-mouse antibodies increased with subsequent readministration. Nevertheless, the treating antibodies were generally well tolerated. In an effort to avoid some of the problems with murine antibodies, chimeric antibodies to CD4 were next utilized in trials of patients with RA. In open trials, the antibody cM-T412 induced a substantial biologic effect, with pronounced decreases in CD4+ cell counts. At the higher doses studied, this antibody also resulted in clinical efficacy, with improvements persistent through several months of follow-up for some patients [32]. Encouraging results from this open trial provided the rationale for controlled trials with this antibody. In these trials, substantial depletion of CD4+ T cells was again observed. However, in contrast to the results from the open trials, no clinical efficacy was observed at any of the treatment doses [33]. Although the T cell depletion was long-lived, persisting for months in many patients, patients did not demonstrate significant clinical immunodeficiency. Nevertheless, concern about the persistent T cell depletion coupled with the lack of apparent efficacy led to the abandonment of further study of this agent. There are several considerations and implications resulting from the negative clinical results in these studies. It was observed that there was a correlation between coating of cells in the peripheral blood with anti-CD4 and clinical response [34]. Therefore, the relatively low doses of antibody utilized in the


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study may not have resulted in sufficient concentrations to achieve relatively long-lived coating of CD4 cells in all relevant sites. Of course from a practical standpoint, the profound decrease in CD4 cell counts probably precludes further study of higher doses of these antibodies. In a study of cM-T412 in patients with MS, it was noted that unprimed or ‘naı¨ve’ CD45RA+ cells were much more sensitive to the depleting effects of this antibody than activated memory cells (CD45RO+). Moreover, cells with a Th1 profile were relatively spared, whereas those with a Th2 profile were affected [35]. Because memory populations of T cells with a Th1 profile are presumed to be etiologically relevant to RA, this suggests that the antibody preferentially targeted the wrong subset of T cells. Thus, fine analysis of the effects of anti-CD4 therapies is required. Perhaps most importantly, the results solidified an appreciation of the concept that depletion of CD4+ T cells should not be the ultimate goal of CD4-directed therapies. Rather, modulation of the function of CD4 cells might be a more therapeutically relevant outcome. This conclusion is supported by a number of animal studies wherein non-depleting antibodies to CD4 are able to achieve the desired immunomodulatory effect [24, 36]. Indeed, in some of these models, nondepleting antibodies have been more effective than depleting antibodies. Interestingly, it has been shown in an allograft transplant model that the maintenance of tolerance was mediated by a population of CD4+ cells [37]. This highlights the idea that indiscriminate depletion of CD4 cells is probably not the optimal goal. There have been several studies assessing other, presumed nondepleting anti-CD4 antibodies in RA. In one series of studies, a chimeric ‘primatized’ (V regions from cynomolgous monkey, human IgG1 constant regions) antibody IDEC-CE9.1 was used [38]. There were encouraging preliminary results in an open trial, with clinical efficacy and a very transient (mostly =7 days) depletion in circulating CD4 T cells. In a larger controlled trial, however, there was depletion of CD4 cells that persisted for weeks to months. In addition, a number of patients who received the highest treatment dose developed leukocytoclastic vasculitic skin lesions. This study highlights that results seen in small preliminary studies may not always be confirmed in larger studies that involve more patients and larger amounts of antibody. Another study assessed a humanized anti-CD4 antibody (Orthoclone OKTcdr4a; murine complementarity determining regions grafted onto a human IgG4). In a small (11 patients) placebo-controlled trial, there was no depletion of CD4+ cells. Clinical response when patients received active treatment exceeded those for placebo-treated patients [39]. Importantly, treatment with OKTcdr4a led to a rapid decrease in levels of CRP, suggesting that modulation of CD4+ T cell function decreased the production of those proinflammatory cytokines that increase hepatic production of acute phase reac-

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tants. In vitro analysis showed that treatment with anti-CD4 resulted in reduced IL-2 and IFN-c by peripheral blood T cells. This suggests that clinical improvement with anti-CD4 treatment may relate to modulation of function and cytokine secretion by Th1 CD4+ T cells. Of note, modulation of Th1/Th2 balance has been noted with anti-CD4 therapy in animal models. Future Directions Although there has been limited clinical success to date utilizing CD4directed therapies, this may still be a viable future approach for biologic treatment. Certainly, there is a rationale for inhibition of CD4 as a means to truly modify immune responses. This has been very clear from numerous animal models and studies. The lack of success with many approaches used in the clinic so far has generated important lessons to be learned for future development of anti-CD4 therapies. Certainly, modulation of CD4+ T cells, rather than depletion, should be the ultimate goal. Not only is the goal important, but characteristics of the treating agent are quite relevant to the ultimate success of anti-CD4 agents. In addition to not depleting CD4 cells, the agent must achieve a high enough serum and synovial concentration to adequately modify relevant populations of CD4 cells. The ultimate mechanisms involved in anti-CD4 therapy remain to be elucidated. Whereas depletion of cells is presumably not desirable, alteration of Th1/Th2 phenotype, generation of specific populations of suppressor cells, negative signaling of CD4 cells, or even effects on other cells expressing CD4, such as monocytes or dendritic cells, might be operative. As more is learned of the mechanisms of action, synergy with other biologic agents might become possible.

Trials of TNF-a Inhibitors In RA, there is substantial evidence that cytokines, particularly proinflammatory cytokines such as TNF-a and IL-1, subserve a crucial role in disease propagation and expression [40–42]. Thus, these cytokines were considered particularly appropriate therapeutic targets in RA. Of note, cytokines tend to exhibit pleiotropy (i.e. one cytokine can mediate diverse functions) and redundancy (i.e. several cytokines may mediate the same activity) in their functions. Moreover, in vivo they function in complex networks or cascades, rather than individually. Therefore, around the time of the initial trials addressing this concept, there had been a concern that inhibiting only a single proinflammatory cytokine would not be expected to have a substantial effect, as the effects of other factors could nullify the inhibition of a single target. However, to date a number of recent trials have successfully utilized varied


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methods to modulate the availability or function of proinflammatory cytokines, effectively alleviating this concern. Tumor Necrosis Factor-a Background. TNF-a, the prototype of a family of molecules involved in immune regulation and inflammation, was named for its ability to induce necrosis of tumors when injected into certain tumor-bearing animals. Although the initial observation of this effect was made in the 1890s, most knowledge of the role of TNF-a has been ascertained over the past 2 decades [43]. Soon after it was cloned in 1984, TNF-a was found to be identical to another molecule that had been called cachectin; it was also found to be approximately 30% homologous to the molecule known as lymphotoxin (LT) [44]. Genes encoding both TNF-a (cachectin) and the two forms of LT (formerly called TNF-b; now LT-a and LT-b) are closely linked on chromosome 6, between loci for class I and class II molecules of the major histocompatibility complex [43]. Although it can be synthesized and secreted by various types of cells, at inflammatory sites such as the rheumatoid synovium, TNF-a is produced to a large extent by cells of the monocyte/macrophages lineage. It is synthesized and secreted in very large quantities by macrophages in response to various proinflammatory stimuli such as bacterial lipopolysaccharide. Production of TNF-a is closely regulated at both transcriptional and posttranscriptional levels; that is, at the level of both mRNA stability and translational efficiency. TNF-a is synthesized and expressed as a transmembrane protein and can be functional on the cell surface. However, as a result of the actions of a specific metalloproteinase (referred to as TNF-a-converting enzyme), it is cleaved and released from the cell surface. Secreted TNF-a functions as a soluble homotrimer of 17-kD subunits. LT exists in two forms; LT-a and LT-b. LT-a, which also functions as a soluble homotrimer, is produced and secreted almost exclusively by lymphocytes after antigenic or mitogenic stimulation. Both TNF-a and LT-a bind to 2 specific cell surface receptors; the 55-kD (also known as CD120a or TNF-RI) and the 75-kD TNF receptors (also known as CD120b or TNF-RII) [43]. These receptors, which are type I transmembrane proteins, are present on numerous cell types. It is thought that CD120a and CD120b can mediate largely overlapping activities in most tissues, although important differences in the signaling properties of the two receptors have been noted [43, 45]. Differences between the receptors have also been suggested in affinity for ligand and in some aspects of function [46, 47]. Soluble forms of both CD120a and CD120b, which bind TNF-a or LT-a with high affinity and compete with cell surface receptors for binding, can be detected in blood. LT-b, which functions as a heterotrimer of 1 LT-a and 2 LT-b

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Table 1. Activities of TNF-a Effects on the vasculature Upregulates adhesion molecules (ICAM-1, VCAM-1, E-selectin) via activation of NF-jB Modulates adhesion molecules (CD44), altering their function Stimulates angiogenesis Alters the normal anticoagulant function of the endothelium towards procoagulant activities (e.g. stimulates tissue factor production, downmodulates thrombomodulin) Effects on cells Lymphocytes (costimulates lymphocytes, plays a role in lymphoid tissue development) Dendritic cells (promotes maturation of dendritic cells and their migration from nonlymphoid tissue into secondary lymphoid organs) Activates neutrophils and platelets Induces proliferation of fibroblasts/synoviocytes Effects on mediators Induces synthesis of proinflammatory cytokines (e.g. IL-1, IL-6, GM-CSF) Induces synthesis of proinflammatory chemokines (RANTES, IL-8, MIP-1a, MCP-1) Induces other inflammatory mediators: prostaglandins (e.g. PGE2 via COX-2 upregulation), leukotrienes, platelet activating factor, nitric oxide (via inducible nitric oxide systhase activation), reactive oxygen species Induces synthesis of metalloproteinases (e.g. collagenases, gelatinases, stromolysins) that mediate bone and cartilage damage Other effects Mediates pain Mediates cachexia Mediates fever Mobilizes calcium from bone (‘osteoclast activating factor’ activity) Modulates apoptosis

subunits, is a transmembrane protein present on the surface of T cells and some other cells. LT-b does not bind to the 55- or 74-kD TNF-R; rather, it binds to a distinct cell surface receptor (LT-bR). TNF and the TNF-R are members of a family of related pairs of coreceptor molecules that include: Fas-ligand/Fas (CD95), CD40L (CD154)/ CD40, CD27L(CD70)/CD27, CD30L/CD30 and others. Molecules in this family play a critical role not only in the activation of cells, but also in programmed cell death or apoptosis. It is not known at this point if modulation of apoptosis plays a role in the efficacy of TNF inhibitors that has been noted in the clinic. Activities of TNF-a. TNF-a mediates numerous inflammatory and immunoregulatory activities. Indeed, it can be involved to some extent in virtually all facets of the inflammatory cascade (table 1). These diverse effects


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provide the basis for the important role TNF-a plays in the pathogenesis of RA. In addition, modulation of these various activities presumably provides some mechanistic explanation for the clinical efficacy of TNF-a inhibitors noted in trials with RA patients. Interestingly, little is known about the hierarchy of these disparate activities as they relate to the role of TNF-a in the immunopathogenesis of RA. Likewise, it might be hypothesized that inhibition of TNF-a clinically might effect these activities, but perhaps the level to which given activities are inhibited might vary with characteristics of the inhibitor, such as concentration achieved in vivo, specificity for target(s), affinity and avidity and others. Deficiencies of TNF-a in animal models highlight its relevance to normal immunocompetence and host defense. Thus, animals that have been made genetically deficient (knockouts) in TNF/TNF-R molecules lack features of normal development in their secondary lymphoid tissues, and are susceptible to certain infections upon challenge [48]. This has implications for potential adverse effects that might be expected from the use of TNF-a inhibitors in RA. There are several activities of TNF-a that may be of particular relevance to its role in RA and other systemic inflammatory diseases. For example, TNF-a exerts prominent effects on the vasculature. Indeed, the originally described ability of TNF-a to induce necrosis of tumors relates to the vasoocclusive changes it causes. Exposure of endothelial cells to TNF-a stimulates the production of tissue factor and downmodulates thrombomodulin. This changes the normal anticoagulant function of the endothelium towards procoagulant activities, which can play a role in inflammatory responses. Perhaps of greater relevance to RA is the ability of TNF-a to upregulate the expression of several endothelial adhesion receptors, including E-selectin (CD62E), ICAM-1 (CD54) and VCAM-1 (CD106); effects mediated via activation of the gene transcription factor NF-jB. TNF-a also alters the sulfation of the adhesion receptor CD44, increasing its binding ability [49]. The effects of TNF-a on adhesion receptors facilitate the adhesion and subsequent transendothelial migration of circulating leukocytes into the inflamed synovium [8, 50]. Adhesion molecule interactions are central to the pathogenesis of RA, and may be among the most important effects of TNF-a inhibition in this disease. Finally, TNF-a plays a role in the stimulation of new blood vessel growth (angiogenesis), which is critical to the growth and propagation of the rheumatoid synovium. Many of the notable effects of TNF-a in RA relate to its ability to cause signs and symptoms of inflammation. For example, TNF-a can directly mediate pain, fever and cachexia [51, 52]. Moreover, TNF-a exerts substantial effects on other inflammatory mediators. Thus, TNF-a induces the synthesis of prostaglandins (by inducing expression of cyclooxygenase-2), leukotrienes,

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platelet activating factor, nitric oxide (TNF-a stimulates the inducible form of nitric oxide synthetase), and reactive oxygen species. The effects of these mediators not only confer many of the signs and symptoms of inflammation, they also potentiate the inflammatory response. The rapid improvement in certain symptoms of RA that has been seen in patients treated with TNF-a inhibitors may be predicated on downmodulation of inflammatory mediators. Although it affects many inflammatory mediators, the effects of TNF-a on other cytokines may be most critical. Mediated by NF-jB activation, TNF-a induces the synthesis of several key proinflammatory cytokines, including IL-1, IL-6 and GM-CSF. It also stimulates production of various chemokines such as RANTES, IL-8, MCP-1, and MIP-1a [52, 53]. The combined effect of these mediators on cell recruitment, cell activation and other inflammatory activities may be fundamental to the pathogenesis of RA. Because TNF-a induces the synthesis of so many other mediators, it serves a central role in this process; therefore, it is a particularly attractive target for therapy. Inhibition of these activities presumably contributes to the mechanism of action of TNF-a inhibitors in the clinic; however, the extent to which inhibition of these various activities contributes to the overall efficacy seen in RA patients has not been clearly delineated. TNF-a exerts other effects that may be relevant to tissue damage in RA. It induces synthesis of matrix metalloproteinase enzymes (e.g. collagenases, gelatinases, stromolysins) that mediate bone and cartilage damage. Stimulation of fibroblast proliferation contributes to the formation of invasive pannus tissue. Damage to bone and surrounding structures is accentuated because TNF-a also decreases the de novo of matrix constituents and facilitates mobilization of calcium from bone, by contributing to the activation of osteoclasts. Whereas the induction of inflammation mediated by TNF-a is important to the signs and symptoms of RA, it may also contribute to the immunologic process that drives this chronic disease. It has been suggested that productive immune responses are facilitated by cognate immune interactions that take place in the setting of an active inflammatory milieu [54, 55]. Thus, inflammatory cytokines released in the course of tissue injury and in responses to various noxious stimuli may activate antigen-presenting dendritic cells and subsequently promote specific immunoreactivity. A correlate of this hypothesis would be that inhibition of TNF could be truly immunomodulatory. In addition, TNF-a exerts effects on various immunocompetent cells, particularly lymphocytes [18]. TNF/TNF-R interactions can induce or regulate apoptosis. Therefore, the inhibition of TNF-a may well be expected to modulate the immune response. The extent to which these immunomodulatory effects may be involved in the clinical efficacy noted in patients treated with TNF-a inhibitors remains to be determined.


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Evidence for a Role of TNF-a in RA. While consideration of the above proinflammatory activities strongly suggests a potential role for TNF-a in RA, more direct evidence comes from two sources: analysis of RA patients and animal models. Paralleling the progress that has been made in immunologic methods over the past decades, investigators have employed various techniques to detect cytokines in the rheumatoid synovium. These studies show that fairly consistently, there is an abundance of immunoreactive TNF-a in the synovial fluid and synovial tissues of RA patients [56, 57]. On the other hand, there are some individual patients who appear to have a paucity of stainable TNFa in synovial samples. This is interesting when considering the fact that although responses to TNF-a inhibitors by RA patients in clinical trials have been quite extensive, clinical responses are not universal. It might be possible that for subsets of RA patients, perhaps those with insignificant clinical response to TNF-a inhibition, this proinflammatory cytokine might be less abundant and perhaps of lesser importance in the overall immunopathogenesis of RA. In addition to TNF-a itself, the levels of soluble forms of the naturally occurring TNF-R are generally increased in the serum and synovial fluid of patients with RA [58]. The complete in vivo functions of these soluble receptors are not known [59]. However, it is assumed that they serve primarily an inhibitory function, binding to TNF-a and LT-a and interfering with their ability to bind to cell surface receptors. While their concentrations are increased in RA, it has been hypothesized that their concentration is insufficient to counterbalance the abundance of TNF-a in RA. Of note, a similar situation has been noted regarding the balance between IL-1 and IL-1Ra in Lyme arthritis [60]. Additional evidence for a potential role of inhibiting TNF-a in arthritis comes from animal studies. Using susceptible strains of animals, various models such as collagen-induced arthritis, adjuvant arthritis, and streptococcal cell wall arthritis, bear semblance to some features of human RA. Anti-TNF-a therapy has proven efficacious in each of these models [61, 62]. Such therapy has been shown to attenuate inflammation and to reduce joint destruction partially. Perhaps of greatest relevance to human RA, anti-TNF therapy in animal models was effective not only when treatment was begun at the onset of disease, but also later in the disease course, when the arthritis had become established. It should be noted that blockade of IL-1 or IL-6 is also effective in preventing or treating many of these animal models. Moreover, it has been claimed that TNF-a may play a more critical role in mediating inflammation, whereas IL-1 may be more important in the causation of cartilage damage [42]. With the recently noted success of the TNF-a inhibitor, infliximab, in

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arresting the progression of joint damage in RA, this concept may need to be revisited. It may be possible that in human RA, TNF-a can have a predominant effect on bony destruction. Alternatively, inhibition of TNF-a may result in very substantial downmodulation of IL-1, thereby explaining the beneficial effect on bone damage. Additional evidence of the role of TNF-a in arthritis comes from studies of transgenic mice. Mice that overexpress human TNF-a spontaneously develop an erosive, inflammatory arthritis [63]. The arthritis can be effectively abrogated by blocking TNF-a. Of note, in this model the inflammation can also be attenuated by blocking IL-1. This highlights the potential role of TNF-a in the induction of the inflammatory cytokine cascade [64]. In summary, these data indicated that TNF-a should be an important therapeutic target in RA. The data laid the groundwork for the clinical trials of TNF-a inhibitors, and provides important considerations relevant to the potential mechanisms of action of these agents used in RA patients. Inhibitors of TNF-a in Patients with RA Agents/Trial Designs A number of studies have evaluated inhibitors of TNF-a in RA (table 2) [65–79]. Almost all of the studies published to date have utilized one of the following agents: (1) a chimeric monoclonal anti-TNF-a antibody (infliximab, RemicadeÔ; previously designated cA2); (2) a recombinant p75TNF-R (CD120b)-Fc fusion protein (etanercept, Enbrel Ô); (3) a humanized monoclonal anti-TNF-a antibody (CDP571). A number of additional TNF inhibitors are in early stages of clinical development. Infliximab is a chimeric mAb that consists of the variable regions of a murine anti-TNF-a mAb engrafted onto a human IgG1 molecule. The resulting construct is approximately 70% human in origin. Infliximab has a high affinity for trimeric TNF-a (KdD100 pM ) and has been shown to inhibit both secreted and cell-associated TNF-a effectively in numerous in vitro systems [42]. Etanercept was developed by linking DNA encoding the extracellular portion of the CD120b TNF-R (p75TNFR) with DNA encoding the Fc portion of human IgG1 (CH2, CH3, and hinge domains) [73, 74]. The resulting dimeric construct is expressed in a mammalian cell line, and binds soluble trimers of TNF-a or LT-a with higher affinity than the naturally occurring monomers of soluble TNFR. CDP571 is a humanized anti-TNF-a mAb, consisting of the complementarity-determining regions (CDR) of a murine anti-TNF-a mAb engrafted onto a human IgG4 molecule. The resulting construct is approximately 95% human. It binds trimeric TNF-a with high affinity, comparable to that of infliximab.


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Table 2. Trials of TNF-a inhibitors in RA Ref. Trial design No.

Agent

65

open

66

DBPCRCT

Agent/dosage

Concurrent DMARD

Patients

Results/comments

infliximab infliximab: 20 mg/kg divided over 2 weeks

none

n>20; 85% RF+; mean disease duration: 10 years, mean DMARDS failed: 4

substantial improvement in all variables; at week 6 all patients were responders by the composite Paulus criteria

infliximab infliximab: single dose of 0, 1 or 10 mg/kg

none

n>73; 81% RF+; mean disease duration: 8 years, mean DMARDS failed: 3.2

significant improvement in antiTNF groups; extent and duration of response increase with higher dose [Paulus 20% improvement at week 4: 79% (10 mg), 44% (1 mg), 8% (placebo)]

67

open, repeated infliximab infliximab: 1 dose none treatment of 20 mg/kg; up to [of patients in 3 further doses of Ref. 37] 10 mg/kg

n>8 [follow-up of patients from Ref. 31 who were treated when arthritis flared]

all treated patients responded; trend towards shorter duration of response with repeated treatments; 4/8 patients did not receive all doses due to adverse effect

68

DBCRCT, followed by open label treatment

infliximab infliximab: single dose of 0, 5, 10 or 20 mg/kg; open label: 3 doses of 10 mg/kg at 8-week intervals

MIX 10 mg/week

n>28; 82% RF+; mean disease duration: 6.2 years, mean DMARDS failed: 2.8

81% anti-TNF-treated patients, 14% placebo responded by ACR composite criteria; extent and duration of response dose-dependent; many patients responded to repeat treatments; some responses sustained through 40-week followup period

69

DBPCRCT

infliximab infliximab: 0, 1, 3 or 10 mg/kg at weeks 0, 2, 6, 10 and 14

MTX 7.5 mg/week or placebo

n>101; mean disease duration 10 years, mean DMARDS failed: 2.4

concurrent MTX enhanced and prolonged clinical response to cA2; D80% of patients receiving MTX+ 3 or 10 mg/kg cA2 sustained response through the 26 weeks of the trial

70

DBCRCT study

infliximab infliximab: 0, 3 or 10 mg/kg every 4 or 8 weeks

MTXq12.5 n>428; 81% RF+; mg/week mean disease duration 8.4 years; 37% had prior joint surgery

71

DBPCRCT

CDP571

single dose of 0, none 0.1, 1 or 10 mg/kg

n>36; mean disease dose-dependent improvement in duration 6 years, mean several clinical parameters DMARDS failed: 3.5

72

repeated treatment [of patients in Ref. 43]

CDP571

1.0 or 10 mg/kg (up to 4 doses)

n>14 [follow-up of patients from Ref. 36]

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ACR 20% response at week 30: placebo 20%, infliximab 52% (3 and 10 mg/kg given every 4 or 8 weeks had comparable efficacy); ACR 50%: placebo 5%, infliximab 28%; ACR 70%: placebo 0%, infliximab 12%; efficacy sustained through 54 weeks for most patients; X-ray progression halted in infliximab groups

dose-dependent response to repeat treatments

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Table 2 (continued) Ref. Trial design No.

Agent

75

DBPCRCT

76

Agent/dosage

Concurrent DMARD

Patients

Results/comments

etanercept 0, 2, 4, 8 or 16 none mg/m2 twice weekly ¶4 weeks (after loading dose)

n>22 (6 assessed for safety only, 16 for safety and efficacy)

joint and pain scores decreased in 45% of treated patients compared with 22% of placebo

DBPCRCT

etanercept 0, 0.25, 2 or none 16 mg/m2 twice weekly ¶12 weeks

n>180; 77% had RA ?5 years

ACR 20%: placebo 14%, 0.25 mg/m2 33%, 2 mg/m2 46%, 16 mg/m2 75%. ACR 50%: placebo 7%, 0.25 mg/m2 9%, 2 mg/m2 22%, 16 mg/m2 57%

77

DBPCRCT

etanercept 0, 10 or 25 mg twice weekly ¶6 months

none

n>234

ACR 20%: placebo 11%, 10 mg 51%, 25 mg 59%; ACR 50%: placebo 5%, 10 mg 24%, 25 mg 40%

78

open follow-up

etanercept 25 mg twice weekly

none

n>105

persistent efficacy through 2 years of follow-up

79

DBPCRCT

etanercept 0 or 25 mg twice weekly ¶6 months

MTX 15–25 n>89; 86% RF+, mg/week mean disease duration 13 years, mean DMARDs failed: 2.7

ACR 20%: 27% placebo, 71% etanercept; ACR 50%: 3% placebo, 39% etanercept; ACR 70%: 0% placebo, 15% etanercept

DBPCRCT>Double-blind, placebo-controlled, randomized clnical trial; RF>rheumatoid factor; MTX>methotrexate.

There are important similarities in the designs of most of the trials assessing TNF-a inhibitors in RA. All trials have enrolled patients with an established diagnosis of RA who also had active disease. Activity was defined as having some combination of numbers of swollen joints, numbers of tender joints, elevated concentrations of acute phase reactants, and prolonged early morning stiffness. In addition, most trials have enrolled groups of RA patients that might be considered somewhat refractory, by virtue of long disease duration, and failure of several DMARDs (table 2). Trials are currently underway evaluating the efficacy of TNF inhibitors in other types of RA patients, for example, patients with relatively early disease. During the trials conducted to date, patients were allowed to continue stable doses of NSAIDs and prednisone (usually p7.5 or 10 mg/day). Efficacy was often assessed utilizing composite criteria, such as the Paulus criteria or the ACR criteria [80, 81]. Such criteria, which require improvement in multiple variables, are more stringent than analysis of improvement in one or only selected clinical variables. For example, in order to be classified as a responder according to ACR criteria, patients must demonstrate: (1) q20% improvement in swollen joint count, (2) q20% improvement in tender joint count and


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(3) 20% improvement in 3 of 5 other measures [patient global assessment of disease activity, physician global assessment of disease activity, patient assessment of pain, an acute phase reactant (e.g. ESR), and a measure of disability (e.g. the Health Assessment Questionnaire, HAQ)]. The Paulus criteria and EULAR criteria are similar. As would be expected, trials progressed in phases from early, open trials through the more rigorous double-blind, placebo-controlled, randomized clinical trials. Finally, after initial studies of TNF-a inhibitors as monotherapy, additional trials have used these agents in combination with a traditional DMARD, methotrexate. Clinical Efficacy (see also table 2) Infliximab. The initial study of infliximab, an open-label trial, demonstrated significant efficacy for this anti-TNF-a mAb [65]. All patients treated with this mAb, which is administered intravenously, demonstrated a clinical response utilizing the composite Paulus criteria. The extent of improvement was substantial, with individual parameters such as swollen joint counts, pain score, ESR and CRP all improving by more than 50% for the whole group. The duration of response after this single treatment course ranged from 8 to 25 weeks. Treatment was well tolerated. Based upon these promising results, a more rigorous double-blind placebo-controlled trial was performed. In the subsequent double-blind trial of 4 weeks’ duration, efficacy was significantly superior in the two groups receiving a single dose of infliximab compared to the placebo group [66]. Utilizing the Paulus criteria to define response, only 2/24 (8%) placebo-treated patients experienced a response, compared to 11/25 (44%) patients treated with 1 mg/kg infliximab and 19/24 (79%) patients treated with 10 mg/kg infliximab. In addition to dose dependency of response, the duration of clinical response was also greater for patients receiving the higher dose of infliximab. As in the open trial, the extent of improvement among responding patients in the double-blind trial was quite substantial. For the infliximab-treated groups, more than 50% improvement was noted in many individual parameters. Indeed, analysis using a modification of the Paulus criteria to require more than 50% improvement (instead of the typical 20%) in the individual parameters resulted in 58% responders in the high dose group and 28% of the lower dose group at 4 weeks after therapy. The effects observed in this study appeared to be of a more profound nature than that seen in other clinical studies of other agents in RA. In an open label follow-up study, 8 patients from the double-blind trial received up to 4 additional treatments with infliximab [67]. Although all treated patients experienced a response, there was a trend towards shorter duration of response with repeated treatments. In addition, adverse effects appeared to be more frequent; 4/8 patients did not receive all planned doses.

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While the utility of infliximab in RA had thus been clearly established, clinical benefit was transient when the anti-TNF-a mAb was used as monotherapy. Using results from several studies, the median duration of response after a single dose of infliximab has been calculated to be 3 weeks with 1 mg/kg, 6 weeks with 3 mg/kg and 8 weeks with 10 mg/kg [42]. In an attempt to sustain the clinical improvements seen in trials of infliximab as monotherapy, additional studies were carried out assessing the use of this anti-TNF-a mAb in RA patients who were using a DMARD concurrently. These studies enrolled patients who had active disease despite using methotrexate. The hypothesis was that the clinical response achieved with the anti-TNF-a mAb might be sustained by the concurrent use of methotrexate. Moreover, in clinical practice many RA patients are treated initially with methotrexate. Thus, patients with active RA despite concurrent therapy might represent the type of patient most likely to receive anti-TNF-a therapy in the clinic. A randomized, double-blind, placebo-controlled study enrolled patients with active RA despite receiving concurrent methotrexate (treatment with methotrexate q3 months; stable dose of 10 mg/week for q4 weeks) [68]. 28 patients received a single dose of either 0, 5, 10 or 20 mg/kg anti-TNF-a mAb and were followed for 12 weeks. Patients continued treatment with methotrexate (10 mg/week) throughout the trial. As assessed by the ACR composite criteria, clinical response was achieved much more frequently among patients receiving anti-TNF-a mAb (17/21, 81%) as compared to placebo (1/7, 14%). As in the other infliximab trials, the magnitude of clinical response was notable. The mean number of tender joints among all infliximab-treated patients decreased from 30.1 at baseline to 13.3 at week 12, and mean CRP decreased from 3.0 at baseline to 1.1 at week 12. The duration of clinical response appeared to be dose-dependent; 2/6 (33%) of the responding patients treated with 5 mg/kg cA2 sustained a clinical response through 12 weeks of followup, compared to 7/11 (64%) of the responding patients who received 10 or 20 mg/kg. In addition, patients were eligible to receive 3 further treatments with 10 mg/kg infliximab at 8-week intervals. Approximately two thirds of patients receiving infliximab in the open trial were able to maintain a clinical response throughout the 40 weeks of this study. Another double-blind placebo-controlled trial assessed the effects of 3 doses of anti-TNF-a mAb (1, 3 or 10 mg/kg) or placebo in 101 patients with active RA. In addition to treatment with infliximab at weeks 0, 2, 6, 10 and 14, patients also received concurrent therapy with either methotrexate (7.5 mg/ week) or placebo [69]. The primary outcome of the study was clinical response, defined according to the 20% Paulus criteria. Overall, approximately 60% of patients receiving infliximab achieved a response. In the infliximab treatment groups, concurrent therapy with methotrexate seemed to prolong and accentu-


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ate the clinical response. This effect was most prominent at the lowest dose; median total time of response at 1 mg/kg infliximab was greater than 16 weeks with methotrexate, but less than 4 weeks without methotrexate. In the groups receiving the higher doses of anti-TNF-a mAb (3 or 10 mg/kg) plus methotrexate, approximately 80% of patients responded, and approximately 60% sustained a clinical response through the 26 weeks of the trial. While the effect of methotrexate was less pronounced at the higher doses, synergy was observed when the Paulus 50% criteria was analyzed. Median total time of response (Paulus 50%) at 10 mg/kg infliximab was greater than 13 weeks with methotrexate, but less than 6 weeks without methotrexate. Interestingly, the addition of methotrexate appeared to increase the blood levels of infliximab, especially those resulting from the lower dose. The addition of methotrexate may have altered the metabolism and perhaps the immunogenicity of the chimeric antiTNF-a mAb. Interim data have recently become available from a trial (the ATTRACT trial) of 428 patients who had active RA despite receiving concurrent therapy with methotrexate at median doses of q15 mg/week [70]. Patients received 3 or 10 mg/kg of infliximab, or placebo, at intervals of 4–8 weeks. The primary clinical endpoint was efficacy, analyzed according to ACR criteria, at 30 weeks. Whereas only 20% of the patients receiving placebo responded according to ACR 20% criteria, 52% of the infliximab groups responded. Interestingly, for the infliximab-treated patients, efficacy was comparable at both doses and both dosing frequencies. When more rigorous response criteria were assessed, the infliximab groups again had significant improvements as compared to placebo. Thus, using ACR 50% criteria, 28% of infliximab patients responded, compared to 5% of placebo. In addition, none of the placebo patients achieved a 70% ACR response, whereas 12% of the infliximab patients experienced this level of response. Additional data from 1 year follow-up on patients in this trial have recently become available. It appears that for the majority of patients, the clinical responses achieved at the earlier time point were sustained through the 1-year evaluation time point. Of note, whereas the response rate for the patients receiving the lower dose and less frequent interval (3 mg/kg every 8 weeks) seemed to be relatively flat, rates of responses in the other groups continued to increase over time. From a mechanistic standpoint, this may raise the consideration of a slower immunomodulatory effect achieved by TNF-a inhibition that becomes evident at a later time point than the very rapid response rates seen in the first weeks of therapy. Alternatively, the improved response rates over time could be explained in part by discontinuation by patients with lesser responses before the 1-year time point (although the data do not support such an explanation) or intrinsic variation in disease severity.

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Serial X-rays were obtained during the ATTRACT trial to determine whether treatment might have a beneficial effect on the progression of structural damage in the patients. In patients receiving methotrexate (and, for many patients, low dose steroids and NSAIDs) alone, there was progression in joint damage as measured by an increase in the modified Sharp scores at weeks 30 and 54. In contrast, patients who received any doses of infliximab overall had an arrest of progression of joint damage. Of note, this was achieved at all doses/dose schedules of infliximab. Interestingly, progression of joint damage depended more on treatment group than clinical response. Thus, patients in the methotrexate-alone groups who achieved some clinical efficacy still had progression of X-ray damage whereas infliximab-treated patients had an arrest of joint damage irrespective of their clinical responses. This raises the possibility that there may be some dissociation among the various process measures used for assessment of RA that may be differentially achieved with various immunomodulatory agents. CDP571 mAb. A double-blind, placebo-controlled study assessed the efficacy of the humanized anti-TNF-a mAb CDP571 in patients with active RA [71]. Single intravenous doses of 0.1, 1.0 and 10 mg/kg were assessed. Responses were dose-dependent. Significant improvements in tender joint count and pain score were noted for patients receiving 10 mg/kg at weeks 1 and 2 after therapy; decreases in CRP were also marked for this group at these time points. Although small patient numbers and individual trials preclude specific comparison, it appears that cA2 may achieve a somewhat greater effect than CDP571 on certain clinical variables (e.g. swollen joint count) [42]. After the single treatment in the blinded trial, some patients received additional open label treatments with CDP571 at 8-week intervals [71, 72]. Clinical response was again dose-dependent, with patients receiving 10 mg/kg demonstrating greater improvements in all variables than those receiving 1 mg/kg. Mild adverse effects were noted with repeat administration. Etanercept. The initial trial of etanercept demonstrated substantial efficacy for this soluble TNF-R fusion protein in a small, dose-escalating, double-blind study [75]. Twenty-two patients were randomized to receive etanercept or placebo subcutaneously twice weekly for 4 weeks after a single intravenous loading dose. Doses of etanercept used were: 4 mg/m2 (load)/ 2 mg/m2 (maintenance), 8 mg/m2 (load)/4 mg/m2 (maintenance), 16 mg/m2 (load)/8 mg/m2 (maintenance), and 32 mg/m2 (load)/16 mg/m2 (maintenance). Of the 16 patients analyzed for efficacy (6 patients were analyzed for safety only), there was a 45% mean improvement in joint and pain scores among patients receiving etanercept compared with 22% improvement in patients receiving placebo. The main adverse effect in the etanercept groups was injection site reaction that did not require study discontinuation. Based upon


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these promising results, a more rigorous double-blind placebo-controlled trial was performed [76]. In the subsequent trial, 180 patients with refractory RA were randomized to receive one of three doses of etanercept (0.25 mg/m2, 2 mg/m2, 16 mg/m2) or placebo, administered subcutaneously on a twice-weekly schedule for 3 months. Substantial dose-dependent efficacy was noted [76]. Utilizing the ACR 20% measure of improvement, response rates at 3 months were: placebo: 14%, 0.25 mg/m2: 33%, 2 mg/m2: 46%, 16 mg/m2: 75%. Notably using the more stringent ACR 50% measure of improvement, response rates at 3 months were: placebo: 7%, 0.25 mg/m2: 9%, 2 mg/m2: 22%, 16 mg/m2: 57%. Treatment was generally well tolerated, with injection site reactions being the most common event. In all groups, efficacy achieved was transient, and measures of disease activity began to rise soon after treatment was discontinued. A subsequent study used a similar design. Two hundred and thirty-four patients with active RA were randomized to receive placebo or one of 2 doses of etanercept (10 or 25 mg) subcutaneously twice weekly for 6 months. As in the earlier study, significant and dose-dependent efficacy was noted [77]. At 6 months, response rates using the ACR 20% measure were: placebo: 11%, 10 mg: 51%, 25 mg: 59%. Using the ACR 50% response criteria, response rates were: placebo: 5%, 10 mg: 24%, 25 mg: 40%. Because the efficacy dissipated after discontinuation of etanercept, a longer, open trial was conducted in order to assess the feasibility of long-term treatment. In this study, 105 patients who had participated in earlier trials were eligible to receive longterm therapy with etanercept at a twice weekly dose of 25 mg subcutaneously [78]. Of note, dropout rates were minimal, and efficacy has been maintained for a number of patients through 2 years of treatment. The efficacy of etanercept has also been evaluated in patients with active RA despite concurrent methotrexate at doses of 15–25 mg/week [79]. In a 24week study, 89 patients were randomized (1:2) to receive placebo or etanercept (25 mg) twice weekly. At 24 weeks, response rates using the ACR 20% measure were 27% in the patients receiving methotrexate plus placebo, compared to 71 in patients receiving etanercept plus placebo. Using the ACR 50% response criteria, response rates were 3% in the placebo group and 39% in the etanercept group. The most common adverse effect was mild injection site reactions that were seen in 42% of patients receiving etanercept, compared to 7% receiving placebo; of note, none of these reactions required exit from the study protocol. Etanercept has also been evaluated and shown promising results in the treatment of juvenile chronic arthritis [82]. Use of TNF-a Inhibitors in Diseases other than RA. In addition to RA, anti-TNF-a mAb have been utilized in a variety of other diseases, including Crohn’s disease, ulcerative colitis, sepsis and HIV infection. The greatest experi-

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ence has been in Crohn’s disease, an immunologically driven inflammatory bowel disease with many pathologic similarities to RA. Both infliximab and CDP571 have been shown to be efficacious in double-blind, placebo-controlled trials in this condition [83, 84]. Adverse Effects Important adverse effects that might potentially be observed with any TNF-a inhibitor include risk of infection, risk of malignancy, and the development of autoimmune manifestations. In addition, there may be untoward reactions that are specific to the particular agent utilized, such as immunogenicity (with its attendant risk of adverse effects) and local reactions. Because it mediates myriad activities relevant to normal inflammation and immunity (see table 1), TNF-a serves a central role in host defense. Therefore, increased susceptibility to specific infections (e.g. Listeria monocytogenes, Mycobacteriae, Histoplasma, etc) is a potential concern with any agent that inhibits TNF-a. This is perhaps illustrated most clearly by animal experiments. For example, ‘knockout’ animals that have been genetically engineered to be deficient in the TNF-I or II receptors are highly susceptible to infection with L. monocytogenes and Mycobacterium [43, 85, 86]. Also, inhibition of TNF-a abrogates the protective effect of mast cell reconstitution in mast cell-deficient mice challenged in peritonitis and pneumonitis models [87, 88]. Complicating the analysis of any increased risk of infection that could be caused by TNF-a inhibitors in diseases such as RA and Crohn’s disease is a high baseline prevalence of infection in these patients. In RA, infections occur more frequently and are an important contributor to the accelerated morbidity of this condition [1, 89]. How much of this susceptibility relates to the disease itself and how much is caused instead by the effects of immunomodulatory therapies (e.g. steroids, cytotoxic drugs) is difficult to dissect. Of note, the subset of RA patients with the greatest susceptibility to infection (i.e. those with severe, active disease) has also been the type of patients most commonly enrolled in trials of TNF-a inhibitors. In RA trials, a number of infections have occurred among patients receiving TNF-a inhibitors (infliximab, etanercept and CDP571), especially upper and lower respiratory infections. In some studies (e.g. infliximab, etanercept), a slightly greater propensity to develop upper respiratory infections has been seen in patients receiving higher doses of the agents. However, the incidence of severe infections has been comparable to that seen in the placebo groups, and significant sequelae occurring as a consequence of infection also appears similar to that in placebo groups. While several opportunistic infections have been noted during studies of TNF inhibitors (e.g. mycobacterial, fungal), the increased predilection to infection of RA patients, particularly those with


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severe, refractory disease, must be taken into account. In summary, at present the data do not substantiate that treatment with anti-TNF-a mAb results in a significantly increased susceptibility to infection in patients with RA. However, because of the relatively small numbers of patients treated to date as well as the relatively short mean duration of therapy, this remains an area of concern and deserves further study. Immunosurveillance is also critical to the host defense against malignancy. Therefore, there is also a theoretical risk of increasing the risk of malignancy with immunomodulatory therapies such as TNF-a inhibitors. As is the case for infections, patients with RA also appear to have an increased susceptibility to certain malignancies, particularly lymphoproliferative cancers [89–91]. This risk must be kept in mind when evaluating results and implying causality in therapeutic trials. In trials of the various TNF-a inhibitors in RA patients, several cases of hematologic malignancies and other malignancies have been reported. At present, it does not appear that the number of cancers observed significantly exceeds the rate that would be expected in this population, although the relatively short patient exposure to these agents does not permit a definitive conclusion. This is another area that will require further study in the future. An interesting observation from the trials of TNF-a inhibitors has been that approximately 10% of treated patients developed antibodies to doublestranded DNA (anti-DNA). As for predisposition to infection and malignancy, the development of autoantibodies seems to be a common adverse effect for all agents capable of inhibiting TNF-a. The significance of the development of autoantibodies is uncertain, as there has been a paucity of cases in which patients clearly developed symptoms suggestive of SLE [42]. The mechanisms underlying the development of these autoantibodies have also not been defined. Interestingly, in some animal models of lupus, TNF-a is protective of disease progression whereas inhibition of TNF-a exacerbates it [92, 93]. In addition, it has been hypothesized that TNF-a, driven perhaps by malarial or other parasitic infection, is protective against SLE in West African populations [94]. Because molecules in the TNF/TNF-R family serve a critical role mediating normal apoptosis, it is possible that inhibition of TNF-a modulates this in some manner, facilitating the development of autoantibodies. The significance of the development of anti-DNA antibodies with the use of TNF-a inhibitors requires further study. Considerations of antigenicity are germane to any biologic therapy. The development of antibodies to a therapeutic agent could diminish its serum half-life and, thereby, decrease its efficacy. In addition, these antibodies could produce adverse effects by means of immune complex formation or the development of immediate hypersensitivity. However, determination and quantifica-

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tion of antibodies to the biologic agents used in clinical studies is a complex issue. By definition these agents are novel, and specific assays to test for antibodies to the agents have not been widely standardized. Other factors, for example the presence of rheumatoid factor, can potentially interfere with the assays. These factors make direct comparisons about the relative antigenicity of different biologic agents difficult and potentially tenuous. Various factors affect the immunogenicity of foreign antigens. The degree of antigenic variation or ‘foreignness’ is an important factor. Thus, murine mAb are more immunogenic than chimeric mAb, which are more immunogenic than humanized mAb. However, even completely human products (e.g. insulin, factor VIII) can be immunogenic and elicit antibody responses in certain circumstances. Other factors affecting immunogenicity include: (1) dose (both high and low doses of an antigen are capable of inducing tolerance), (2) route of administration (cutaneous administration is more likely to induce antibody responses than intravenous or oral administration), (3) frequency of treatment, and (4) immunomodulatory effects of the agent or of concurrent treatment. The impact of concurrent therapy may be particularly relevant to RA. For example, concurrent use of immunosuppressive medications is known to suppress the immunogenicity of foreign antigens. In addition, some agents modulate the immune system such that they induce tolerance to themselves. These factors may be relevant in the treatment of RA. In studies performed to date, single doses of the chimeric anti-TNF-a antibody infliximab have been associated with the formation of human antichimeric antibodies (HACA) in 0–5% up to 25% of patients [42]. The frequency of HACA may vary with dose, with higher doses inducing less of a response [69]. In some studies, the persistence of the treating antibody at the time of HACA measurement may have affected the assays. With repeated administration, the prevalence of HACA may increase to 50% or more [42]. Although the data available to date are not extensive, the development of antibodies may have clinical relevance; in one small study, the duration of clinical response decreased with repeated administration [67]. Importantly, concurrent therapy may affect immunogenicity. In one study, concurrent therapy with methotrexate decreased the prevalence of HACA [69]. Thus, among patients receiving cA2 as monotherapy, HACA were seen in 53% (1 mg/kg cA2 dose), 21% (3 mg/kg) and 7% (10 mg/kg); in patients taking concurrent methotrexate, the corresponding frequencies of HACA were 17, 7 and 0%. These data show that the development of antibodies to the treating agent varied with dose, and was affected by methotrexate therapy. This suggests that adjusting the treatment paradigm in terms of dosages and concurrent therapies may be a potentially useful method of decreasing immunogenicity of anti-TNF-a mAb.


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For patients treated with etanercept, the most common unique adverse effect has been the development of local reactions at the site of injection. In general, these have been mild and resolved spontaneously, and only rarely required withdrawal from the studies. Interestingly, reactions at sites of previous injections have been noted to develop after injections at other sites. Mechanism of Action of TNF-a Inhibitors Whereas TNF-a inhibitors have clearly been shown to be efficacious, there is still some speculation as to the precise mechanisms by which they exert their beneficial clinical effect. As described above, TNF-a can exert myriad proinflammatory and immunomodulatory actions, several of which are presumably relevant to the clinical efficacy seen in trials of RA patients. Perhaps the most straightforward mechanism of action would relate to binding and inhibition of inflammatory mediators. In support of this, substantial decreases in IL-6 [65, 95, 96] and IL-1 [95] have been demonstrated after anti-TNF-a mAb therapy. Decreases in IL-6 provides an explanation for the pronounced improvements in acute phase reactants seen during treatment. Because it too produces numerous inflammatory effects, decreases in IL-1 would be associated with improvement in many of the signs and symptoms of inflammation. Other mediators that are increased in vivo by TNF-a are also decreased after therapy, including IL-1Ra, sCD14, IL-8, MCP-1, nitric oxide, collagenase and stromolysin [42, 95, 97]. The effects on matrix metalloproteinases (collagenase, stromolysin) may be important in attenuating joint damage in RA. In summary, these data highlight the central role of TNF-a in triggering the inflammatory cytokine cascade in RA, and confirm its utility as a therapeutic target. Inhibition of various inflammatory mediators may also provide some explanation for the improvements noted in patients treated with TNF inhibitors. As noted, TNF-a exerts diverse effects on the vasculature, such as activating the endothelium and upregulating adhesion molecule expression. This may provide an explanation for one of the most important mechanisms of action of TNF-a inhibitors. In one of the placebo-controlled trials of cA2, soluble adhesion molecules were measured in the serum [98]. Because they are expressed and then shed from the cell surface upon activation, soluble forms of adhesion molecules provide an indirect measure of endothelial activation. Treatment with anti-TNF-a mAb resulted in a dose-dependent decrease in soluble forms of ICAM-1 and E-selectin [98]. In addition, an increase in the numbers of circulating lymphocytes and a decrease in circulating neutrophils were noted. These changes in circulating lymphocytes are reminiscent of those achieved in another study of RA patients in which the adhesion molecule ICAM-1 was directly targeted [99]. Interestingly, in the anti-TNF-a study, changes in soluble E-selectin, soluble ICAM-1, and circulating lymphocytes

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correlated with clinical outcome, implying mechanistic relevance. Confirmation of these effects of anti-TNF-a mAb on endothelial activation and inflammatory cell recirculation has come from additional studies, in that the decrease in serum sICAM-1 has been reproduced by another group [95]. More directly, histopathologic analysis of a small number of synovial biopsy specimens has shown reduction in the expression of E-selectin and VCAM-1 consequent to anti-TNF-a therapy [100]. Of note, this decrease in adhesion molecule expression was accompanied by a reduction in the infiltration of T lymphocytes into the synovium [100]. Labeling studies have shown a decreased accumulation of neutrophils into joints with anti-TNF-a therapy [97]. In summary, these data indicate that a crucial mechanism of action of anti-TNF-a mAb in RA patients may be the inhibition of TNF-a-dependent endothelial cell activation. This, in turn, could alter cellular recirculation and inhibit the accrual of cells into the rheumatoid synovium. There are other potential mechanisms of TNF-a inhibitors. TNF/TNF-R interactions can regulate apoptosis, including that mediated by Fas/Fas-ligand interactions. Therefore, inhibition of TNF-a may modulate apoptosis in the rheumatoid synovium and, for example, downregulate synovial hyperplasia [101]. In diseases that are characterized by a predominant Th1 phenotype, such as RA and Crohn’s disease, it is also possible that TNF-a inhibitors may alter the predominant milieu within the inflammatory site, possibly via an increase in IL-10 [84, 102]. Finally, because some of the agents used to inhibit TNF-a can fix complement, it is possible that lysis of cells bearing TNF-a on their surface may occur, although there is a paucity of evidence supporting this potential mechanism of action. Additional mechanisms may still be delineated as experience with TNF-a inhibitors as therapeutic agents expands. Comparisons of TNF-a Inhibitors There are several considerations relevant to the comparison of novel TNF-a inhibitors (table 3). Important characteristics that vary among the agents include: (1) avidity for ligand (including off-rate of binding), (2) halflife and (3) specificity (e.g. does the agent bind TNF-a, LT-a or both?). The optimal characteristics for these variables remain a matter of speculation. For example, an agent with higher avidity may achieve better results, but may also increase the susceptibility to infection. Likewise, an agent that has a very long half-life could be more convenient to administer, but such prolonged inhibition of TNF-a may have untoward effects. Agents that block both TNF-a and LT-a may exert different effects and be associated with different adverse effects than agents specific for TNF-a. Similarly, agents that interact with both soluble as well as cell-bound forms of the cytokines may exert immunomodulatory effects not seen with agents that might preferentially affect the function of soluble


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Table 3. Considerations relevant to the comparison of TNF-a Inhibitors Characteristics Specificity Avidity Half-life Frequency and route of administration Pharmacokinetics, pharmacodynamics Complications Risk of infection/malignancy/autoimmune disease Agent-specific adverse effects Immunogenicity Development of adverse effects Loss of efficacy Development of local reactions Cost Cost efficacy/cost utility

forms of TNF-a [103]. The optimal characteristics will need to be defined by long-term comparison studies. Another area of comparison between TNF-a inhibitors relates to complications. Some complications (e.g. risk of infection, malignancy, autoimmune disease) would be expected to be common to all TNF-a inhibitors, although factors such as avidity and half-life may affect the incidence or severity. Other complications may depend on the particular agent, and may be best delineated by comparison studies. Finally, because biologic agents would be expected to be more expensive than traditional drugs, cost analyses will be germane to their utility. However, TNF-a inhibitors have proven to have quite impressive efficacy for some treated patients. Therefore, optimal cost analyses in RA require balancing the costs of the agents themselves against those costs that can be alleviated (e.g. lost wages) by controlling this chronic, pernicious disease [104].

Future Directions in Biologic Therapy Expectations generated by the successes demonstrated for TNF-a inhibitors, and relevant information gleaned from studies of anti-CD4 therapies are part of the impetus for what can be expected to be further exciting development in the use of biologic agents in RA and other autoimmune diseases.

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Much interest is indeed focused on inhibition of TNF-a. A variety of other approaches to inhibition of TNF-a are under development, including inhibitors of TNF-a-converting enzyme, inhibitors of phosphodiesterase IV (which regulates TNF-a production), and gene transfer to overexpress the TNF-R [105–107]. Some currently available agents may also mediate their effects at least in part by inhibition of TNF. This includes thalidomide (which enhances the degradation of TNF-a mRNA), and methotrexate (via induction of adenosine, which downregulates TNF-a synthesis) [108, 109]. There are many avenues that should allow refinement and optimization in the use of anti-TNF-a mAbs in RA. As experience is gained with biologic agents targeting other parts of the immune response, such considerations will also apply to them. An extremely relevant and important area will be the definition of subsets of patients for whom particular therapies may be most efficacious and least toxic. In animal studies, it has been shown that different therapies may be more or less effective at different stages of arthritis [61]. In RA, it has also been suggested that patients may be heterogeneous in their clinical presentations as well as their response to certain therapeutics. Although TNF-a inhibitors have been effective for the majority of treated patients, it is possible that certain patients may be particularly responsive to this type of therapy, and resistant to adverse effects. The same may be true for antiCD4 and other immunomodulatory therapies. The identification of relevant therapeutic subsets will require additional research. Interestingly, genetic polymorphisms in TNF-a, IL-1b, IL-10 and other molecules have been noted. Such genetic polymorphisms, either alone or in combination with other genetic factors such as MHC haplotype, may correlate with outcomes in RA [110]. Research into this and other factors may allow the definition of subsets of RA for whom TNF-a inhibitors and other biologic agents would be particularly useful. Another area in which refinement in the use of biologic agents can be expected is the treatment paradigms. Early studies used biologic agents as monotherapy. More recent studies have begun to assess the effect of concurrent use of methotrexate as an adjunct to TNF-a inhibitors. This combination, which bears semblance to the ‘induction-remission’ approach to cancer chemotherapy, has proven effective. In addition to prolonging and possibly potentiating the clinical response, such combination therapy also appeared to decrease the metabolism and perhaps the immunogenicity of agents such as mAb. Further study of this approach to therapy is anticipated. Definition of effective combinations of agents may allow lower doses of each therapy to be utilized, thereby obviating toxicity. In animal models, the combinations of anti-TNF-a and anti-CD4 therapies have been synergistic in the therapy of arthritis [111]. Preliminary studies in humans have also suggested success of this combined approach [112]. Other


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potential approaches include combinations of agents blocking inflammatory cytokines such as TNF-a and IL-1. Further study in this area promises exciting progress in the use of biologic agents in the treatment of RA.

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69

70

71

72 73 74

75

76

77

78

79

80

81

82

83 84 85

86 87 88

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89 90 91 92 93 94 95

96

97

98

99

100

101

102

103

104 105 106

107

108

Wolfe F, Mitchell D, Sibley J, et al: The mortality of rheumatoid arthritis. Arthritis Rheum 1994; 37:481–494. Cibere J, Sibley J, Haga M: Rheumatoid arthritis and the risk of malignancy. Arthritis Rheum 1997;40:1580–1586. Gridley G, McLaughlin J, Ekbom A, et al: Incidence of cancer among patients with rheumatoid arthritis. J Natl Cancer Inst 1993;85:307–311. Jacob C: Tumor necrosis factor a in autoimmunity: Pretty girl or old witch? Immunol Today 1992; 13:122–125. Jacob C, McDevitt H: Tumour necrosis factor-a in murine autoimmune ‘lupus’ nephritis. Nature 1988;331:356–358. Adebajo A: Does tumor necrosis factor protect against lupus in West Africans? Arthritis Rheum 1992;35:839. Lorenz H-M, Antoni C, Valerius T, Rrepp R, Gru¨nke M, Schwerdtner N, Nu¨sslein H, Woody J, Kalden JR, Manger B: In vivo blockade of TNF-a by intravenous infusion of a chimeric monoclonal TNF-a antibody in patients with rheumatoid arthritis: Short term cellular and molecular effects. J Immunol 1996;156:1646–1653. Choy E, Kassimos D, Kingsley G, et al: The effect of an engineered human anti-tumour necrosis factor antibody on interleukin-6 and bone markers in rheumatoid arthritis patients. Arthritis Rheum 1995;38(suppl):S185. Taylor P, Chapman P, Elliot M, Schaible T, Peters A, Feldmann M, Maini R: Reduced granulocyte traffic and chemotactic gradients in rheumatoid joints following anti-TNF-a therapy. Arthritis Rheum 1997;40(suppl):S80. Paleolog E, Hunt M, Elliott M, Feldmann M, Maini R, Woody J: Deactivation of vascular endothelium by monoclonal anti-tumor necrosis factor a antibody in rheumatoid arthritis. Arthritis Rheum 1996;39:1082–1091. Kavanaugh A, Davis L, Nichols L, Norris S, Rothlein R, Scharschmidt L, Lipsky P: Treatment of refractory rheumatoid arthritis with a monoclonal antibody to intercellular adhesion molecule-1. Arthritis Rheum 1994;37:992–999. Tak PP, Taylor PC, Breedveld FC, Smeets TJM, Daha M, Kluin PM, Meinders AE, Maini RN: Decrease in cellularity and expression of adhesion molecules by anti-tumor necrosis factor a monoclonal antibody treatment in patients with rheumatoid arthritis. Arthritis Rheum 1996;39:1077– 1081. Ohshima S, Saeki Y, Mima T, et al: Tumor necrosis factor a interferes with FAS mediated apoptotic cell death on RA synovial cells: Possible mechanism for synovial hyperplasia and clinical benefit of anti-TNF-a therapy in rheumatoid arthritis. Arthritis Rheum 1997;40(suppl):S79. Ohshima S, Saeki Y, Mima T, Suemera M, Shida M, Shimuzu M, McCluskey R, Kishimoto T: Possible mechanism for the long-term efficacy of anti-TNF-a antibody (cA2) therapy in RA. Arthritis Rheum 1996;39(suppl):S242. Scallon B, Moore M, Trinh H, Knight D, Ghrayeb J: Chimeric anti-TNF-alpha monoclonal antibody cA2 binds recombinant transmembrane TNF-alpha and activates immune effector functions. Cytokine 1995;7:251–259. Kavanaugh A, Heudebert G, Cush J, Jain R: Cost evaluation of novel therapeutics in rheumatoid arthritis (CENTRA): A decision analysis model. Semin Arthritis Rheum 1996;25:297–307. Black R, Rauch C, Kozlosky C, et al: A metalloproteinase disintegrin that releases tumour necrosis factor-a from cells. Nature 1997;385:729–736. Semmler J, Wachtel H, Endres S: The specific type IV phosphodiesterase inhibitor rolipram suppresses tumor necrosis factor-a production by human mononuclear cells. Int J Immunopharmacol 1993;15:409–413. Le C, Nicolson A, Morales A, Sewell K: Suppression of collagen-induced arthritis through adenovirus-mediated transfer of a modified tumor necrosis factor a receptor gene. Arthritis Rheum 1997;40:1662–1669. Moreira A, Sampaio E, Zmuidzinas A, Frindt P, Smith K, Kaplan G: Thalidomide exerts its inhibitory action on tumor necrosis factor a by enhancing mRNA degradation. J Exp Med 1993; 177:1675–1680.

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272

109 110

111

112

Sajjadi F, Takabayashi K, Foster A, Domingo R, Firestein G: Inhibition of TNF-a expression by adenosine: Role of A3 adenosine receptors. J Immunol 1996;156:3435–3442. Hajeer A, Worthington J, Silman A, Ollier W: Association of tumor necrosis factor microsatellite polymorphisms with HLA-DRB1*04-bearing haplotypes in rheumatoid arthritis patients. Arthritis Rheum 1996;39:1109–1114. Williams R, Mason L, Feldmann M, Maini R: Synergy between anti-CD4 and anti-tumour necrosis factor in the amelioration of established collagen-induced arthritis. Proc Natl Acad Sci USA 1994; 91:2762–2766. Morgan A, Hale G, Rebello P, Richards S, Waldmann H, Emery P, Isaacs J: Combination therapy with a TNF antagonist and a CD4 monoclonal antibody in rheumatoid arthritis. A pilot study. Arthritis Rheum 1997;40(suppl):S81.

Peter E. Lipsky, MD, National Institute of Health, 9000 Rockville Pike, Rm. 9N228, Bethesda, MD 20892 (USA) Tel. +1 301 496 2412, Fax +1 301 402 0012, E-Mail [email protected] Arthur F. Kavanaugh, MD, Thornton Hospital, 9310 Campus Point Drive, #A111, La Jolla, CA 92037-0943 (USA) Tel. +1 858 657 7044, Fax +1 858 657 7045, E-Mail [email protected]


273

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Author Index

Albani, S. 51 Bockermann, R. 17 Braun, A. 168 Brennan, F.M. 188 Carson, D.A. 51 Cope, A.P. 64 Corne´lis, F. 1 Feldmann, M. 188 Foxwell, B.M.J. 188

Haynes, B.F. 133 Holmdahl, R. 17 Huber, B.T. 94

Nagy, Z.A. 36

Jirholt, J. 17 ˚ 17 Johansson, A

Patel, D.D. 133 Prakken, B.J. 51

Kavanaugh, A. 240

Sinigaglia, F. 36 Steere, A.C. 94

Leung, B.P. 200 Lipsky, P. 240 Lu, S. 17

Olofsson, P. 17

Takemura, S. 168 Weyand, C.M. 112, 168

Goronzy, J.J. 112, 168 Gross, D. 94

Maini, R.N. 188 McInnes, I.B. 200 Mountz, J.D. 216

Zhang, H.-G. 216

274

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Subject Index

Affected sib-pair analysis, rheumatoid arthritis 5, 7, 8 Akt, rheumatoid arthritis synovial fibroblast regulation 230 American College of Rheumatology, criteria for rheumatoid arthritis 17 Angiogenesis, tumor necrosis factor-a inhibition by inhibitors 192, 193 role 250 Animal models, see Transgenic mice Apoptosis death domain protein signaling 216, 217 induction by rheumatoid arthritis therapies chloroquine 230, 231 methotrexate 231 sulfasalazine 232 tumor necrosis factor-a blockade 231, 232, 265 interleukin-15, prevention 206 synovial fibroblasts, rheumatoid arthritis Akt regulation 230 anti-Fas effects on mitochondrial permeability transition 229, 230 caspases 226, 227, 229 expression of modulators 217, 218 Fas apoptosis regulation 225–227, 229, 230, 233 induction by adenovirus-producing IjBa 221–224 induction by adenovirus-producing XIAP 225

prospects for facilitation 233 signaling defects 218, 219 pathways 219–221 TRADD 219 tumor necrosis factor receptor 201, 220 XIAP induction by tumor necrosis factor-a 225 TRAIL mediation 216 Avridine-induced arthritis features 23 regulators 24 Candidate gene approach 4, 5 Cartilage protein-induced arthritis cytokine regulation 190, 191 features 27 genetic control mice 28–30 rats 27, 28 immunization with CII 27 Caspases, apoptosis in rheumatoid arthritis synovial fibroblasts 226, 227, 229 CD4, see T cell CD28-deficient T cells in rheumatoid arthritis 125, 126, 128, 182, 183 CD44, leukocyte migration, role 141, 150 CDP571 adverse effects 261–263 clinical efficacy in rheumatoid arthritis 259

275

CDP571 (continued) Crohn’s disease treatment 260, 261 description 253 mechanisms of action 264, 265 Chemokines antagonism in therapy 153–155 classification 142 constitutive versus inflammatory 151, 152 leukocyte migration adhesion, roles 142, 144 fractalkine pathway of transendothelial migration 146, 147 regulation of leukocyte homing to inflamed synovium 151–155 levels in arthritis 152, 153 receptors CX3CR1 147, 148 leukocyte distribution 143, 152 ligands 143, 152 signal transduction 142 Chloroquine, apoptosis induction 230, 231 Course, rheumatoid arthritis 240, 241 Cytokine response, rheumatoid synovium 171–174, 188, 242 Diabetes gene elucidation candidate gene approach 4, 5 combination of approaches 6 genome scan 5 linkage disequilibrium mapping 6 rheumatoid arthritis association 4 Disease-modifying antirheumatic drugs (DMARDs), use 240, 241 DNA, antibody development with tumor necrosis factor-a inhibitors 262 dnaJP1 expansion of rheumatoid arthritis T cells 58 S1 cross-reactive T cells 59, 60 Enbrel adverse effects 261–264 clinical efficacy in rheumatoid arthritis 259, 260 description 253 mechanisms of action 264, 265

Subject Index

Epstein-Barr virus (EBV), rheumatoid arthritis implications 181, 183 Etanercept, see Enbrel Fas anti-Fas effects on mitochondrial permeability transition 229, 230 apoptosis regulation 225–227, 229, 230, 233 Fibroblast, see Rheumatoid arthritis synovial fibroblasts, apoptosis Fractalkine (FKN) pathway of leukocyte transendothelial migration 146, 147 receptor 147, 148 structure 147 tissue distribution 147, 148 Gene elucidation approaches candidate gene approach 4, 5 combination of approaches 6 genome scan 5 linkage disequilibrium mapping 6 rheumatoid arthritis affected sib-pair analysis 5, 7, 8 animal models 19, 20, 30 genome scanning 7, 8 GENOPOLE, resource for association studies 10 linkage disequilibrium mapping 8–10 prospects 10, 11, 19, 30 transmission disequilibrium testing 4, 7 Genome scan diabetes 5 rheumatoid arthritis 7, 8 GENOPOLE, resource for association studies 10 Glucose-6-phosphate isomerase (GPI), self-reactivity 76, 77 Granuloma, formation in synovial tissue 174, 175 HCgp-39 self-antigen responses in transgenic mice 69–72

276

sources 66 T cell responses in transgenic mice 66–69 Heat shock proteins, antigenicity in molecular mimicry 55, 56, 115 Heredity, rheumatoid arthritis 2 High endothelial venule phenotype 242 HLA-DQ arthritogenic peptide presentation 45, 46 mouse homolog 64 HLA-DRB1 alleles in rheumatoid arthritis 2, 3, 36, 56, 115 class II binding motifs 38, 39 collagen type II peptide binding 42, 44 DRb-chain as peptide donor 45, 46 ethnic preferences in disease 36 immunointervention prospects 46, 47 Lyme disease alleles 94, 97, 100 peptide interactions 37, 38 phenotype in lymphoid aggregate T cells 178, 179 protective alleles 37, 41, 47, 85 quantitative matrices and computational prediction of peptide binding 39 selective autoantigen presentation 41–45 severity of disease 36 shared epitope effects on peptide-binding specificity 40, 41 overview 37, 56, 57 sustained response to negatively charged peptides 43, 44 transgenic mice, see Transgenic mice Human anti-chimeric antibodies (HACA), development with tumor necrosis factor-a inhibitors 263 IjBa, rheumatoid arthritis synovial fibroblast apoptosis induction with tumor necrosis factor-a 221–224 Incidence, rheumatoid arthritis 17 Inflammation, systemic response in rheumatoid arthritis 113

Subject Index

Infliximab, see Remicade Integrins leukocyte adhesion, role 141, 142, 144–146 regulation of leukocyte homing to inflamed synovium 150, 151 structures 144, 145 Interleukin-1 (IL-1) collagen-induced arthritis, role 190, 191 inhibitors 189, 190 receptor antagonist therapy in rheumatoid arthritis 195, 196 tumor necrosis factor-a relationship 188, 189 Interleukin-15 (IL-15) activity in rheumatoid arthritis 204–207 apoptosis prevention 206 blockade in therapy 210 cellular bioactivity 202, 203 expression chronic inflammatory disease 209 synovial membrane 203, 204 innate immune response, role 206–209 isoforms 201, 202 modulation in vivo 207 neutrophil activation 206, 207 proinflammatory activity 202 receptor 200, 201, 207 regulation of production 201, 202 T cell effects 202, 203, 209, 210 Interleukin-16 (IL-16), anti-inflammatory actions 180 Leukocyte migration adhesion molecules, table 138–140 regulation of homing to inflamed synovium adhesion molecules 149–151 chemokines 151–155 overview 148, 149 Remicade effects 191, 192 rheumatoid infiltrate composition 168 stages capture and rolling 133–137, 141 firm adhesion 141, 142, 144–146 transendothelial migration 146–148

277

LFA-1, Lyme disease candidate autoantigen 100 pathogenic model 103–105 Linkage disequilibrium mapping diabetes 6 rheumatoid arthritis 8–10 LT, see Lymphotoxin Lyme disease animal models 105, 106 antibiotic-resistant disease 96, 97 arthritis course 94, 96 autoimmune disease comparison 106, 107 causative agent 94, 95 clinical features 96 HLA-DRB1 alleles 94, 97, 100 LFA-1 candidate autoantigen 100 pathogenic model 103–105 molecular mimicry 94, 95 OspA pathogenic model 103–105 sequence variations 102, 103 T cell responses 98, 100 tick vector 95, 96 Lymphoid aggregate, formation in synovial tissue 175–179 Lymphotoxin (LT) forms 248 receptors 248, 249 secondary lymphoid tissue formation, role 176, 177 Mac-1, leukocyte adhesion, role 144, 145 Major histocompatibility complex class II, see HLA-DRB1 Methotrexate (MTX) apoptosis induction 231 combination therapy 267 Mineral oil induced arthritis features 23 regulators 24 Molecular mimicry dnaJP1 expansion of rheumatoid arthritis T cells 58 S1 cross-reactive T cells 59, 60

Subject Index

heat shock proteins as antigens 55, 56 HLA-DRB1 shared epitope effects on peptide-binding specificity 40, 41 overview 37, 56, 57 Ia52-Hil5 cross-reactive T cells 54, 55 Lyme disease 94, 95 microbial peptides in rheumatoid arthritis 57–59 multistep mimicry hypothesis for rheumatoid arthritis 57 thymic selection 53, 54 Monocyte migration, see Leukocyte migration MTX, see Methotrexate Mycobacterium cell wall induced arthritis genetic analysis 23 mouse specificity 20 T cell, role 20, 21 Neutrophil interleukin-15 activation 206, 207 migration, see Leukocyte migration Nuclear factor-jB (NF-jB), rheumatoid arthritis synovial fibroblast apoptosis, role 220, 221 OKTcdr4a, trials 246, 247 OspA Lyme disease pathogenic model 103–105 sequence variations 102, 103 T cell responses 98, 100 T cell responses in transgenic mice 72–74 Pristane-induced arthritis features 23, 24 genetic predisposition 25, 26 markers 24, 25 regulators 24 Remicade adverse effects 261–263 clinical efficacy in rheumatoid arthritis 256–259 Crohn’s disease treatment 260, 261

278

description 253 mechanisms of action 264, 265 angiogenesis inhibition 192, 193 blood profile restoration 193 cytokine cascade downregulation 191 joint protection 194, 195, 197 leukocyte trafficking reduction 191, 192 Rheumatoid arthritis synovial fibroblasts (RASF), apoptosis Akt regulation 230 anti-Fas effects on mitochondrial permeability transition 229, 230 caspases 226, 227, 229 expression of modulators 217, 218 Fas apoptosis regulation 225–227, 229, 230, 233 induction by adenovirus-producing IjBa with tumor necrosis factor-a 221–224 induction by adenovirus-producing XIAP 225 prospects for facilitation 233 signaling defects 218, 219 pathways 219–221 TRADD 219 tumor necrosis factor receptor 201, 220 XIAP induction by tumor necrosis factor-a 225 Selectins E-selectin 134–136 leukocyte migration, role 134–136 ligands 136, 137, 141 L-selectin 134, 135 P-selectin 134–136 regulation of leukocyte homing to inflamed synovium 149 structures 134, 135 Sex, rheumatoid arthritis distribution 3, 17 Sulfasalazine, apoptosis induction 232 Synovium-SCID mouse chimera 170, 171, 178, 180 T cell see also Molecular mimicry, Thymic selection, Tolerance

Subject Index

anti-inflammatory synovial cells 179, 180 CD4 cell regulation of synovial inflammation 169–171 CD4 cell targeting in therapy animal studies 244 combination therapy 267, 268 human trials chimeric antibodies 245, 246 dosing 245, 246 murine antibodies 245 OKTcdr4a 246, 247 overview 243 prospects 247 rationale 244 CD8 cells in lymphoid aggregates 177, 181, 183 CD28–cells in rheumatoid arthritis 125, 126, 128, 182, 183 clonal expansion of autoreactive cells in rheumatoid arthritis 116, 117 cytokine response in rheumatoid synovium 171–174 developmental overview 51 migration, see leukocyte migration population dynamics studies humans 122, 123 mice 120, 121 rheumatoid arthritis 123–125 repertoire in rheumatoid arthritis contraction 117–120, 127 formation 115, 116, 127, 182 self-antigen cross-reactivity with foreign antigen, see Molecular mimicry T helper cytokine profiles 171, 173 therapeutic implications of abnormal homeostasis in rheumatoid arthritis 126, 127 transgenic mouse studies, see Transgenic mice T cell capture (TCC), antigen-specific T cell identification 54, 59, 60 T cell receptor (TCR) candidate genes in rheumatoid arthritis 3, 4

279

T cell receptor (TCR) (continued) KRN transgenic mouse model 75–77, 88 tumor necrosis factor-a uncoupling of signal transduction 82, 83 TDT, see Transmission disequilibrium testing Thymic selection adaptation 53, 54 dynamics 53, 54 self-antigen recognition as driving force 52, 53 transgenic mouse manipulation 114 TNF-a, see Tumor necrosis factor-a Tolerance, central tolerance defects in mouse rheumatoid arthritis models 113, 114 TRADD, rheumatoid arthritis synovial fibroblasts, role 219 TRAIL, apoptosis mediation 216 Transgenic mice CD4 transgenic mouse 66–69 disease checkpoint progression in nonsusceptible host 84–88 HCgp-39 self-antigen responses 69–72 T cell responses 66–68 HLA-DRB1*0402 transgenic mouse cytokine effector response 87 epitope presentation 86 immunization and self-reactive T cells 86, 87 immunoregulation of T cell response 87, 88 rationale 85 I-Ab knockout mouse 66 immunodominance and autoreactivity linkage 74, 75 OspA, T cell responses 72–74 T cell receptor KRN model 75–77, 88 thymic selection manipulation 114 tumor necrosis factor-a ARE element deletion 78, 79 chronic exposure effects on T cells effector response driving in chronic inflammation 83, 84 reversible nondeletional T cell hyporesponsiveness 80–82

Subject Index

uncoupling of signal transduction 82, 83 human cytokine expression 78 Transmission disequilibrium testing (TDT), rheumatoid arthritis 4, 7 Tumor necrosis factor-a (TNF-a) angiogenesis, role 250 biological activities 249–251 blockade in rheumatoid arthritis, see also CDP571, Enbrel, Remicade adverse effects 261–264 agents 191 apoptosis induction 231, 232, 265 approaches 267 combination therapy 267, 268 considerations for inhibitor comparison 265, 266 effects on other cytokines 188, 189 efficacy 191 immunogenicity of agents 263 mechanisms 191–195, 197, 264, 265 rationale in therapy 247, 248 trials in rheumatoid arthritis, see also CDP571, Enbrel, Remicade design 255 responder criteria 255, 256 table 254, 255 collagen-induced arthritis, role 190, 191 cytokine interactions 251 expression in rheumatoid arthritis 252 inflammatory mediator regulation 250, 251 interleukin-15 effects in induction 205 polymorphisms in rheumatoid arthritis 3, 267 receptors 248, 249 rheumatoid arthritis synovial fibroblast apoptosis, receptor signaling 201, 220 sources 248 tissue damage, role 251–253 transcriptional control 78, 197, 248 transgenic mice ARE element deletion 78, 79

280

chronic exposure effects on T cells effector response driving in chronic inflammation 83, 84 reversible nondeletional T cell hyporesponsiveness 80–82 uncoupling of signal transduction 82, 83 human cytokine expression 78 translational repression 78, 79, 248

Subject Index

Twin studies, rheumatoid arthritis 2, 18 Vascular endothelial growth factor (VEGF), Remicade effects 192, 193 XIAP apoptosis induction in rheumatoid arthritis synovial fibroblasts 225 induction by tumor necrosis factor-a 225

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Rheumatoid Arthritis (Current Directions in Autoimmunity)

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