New insights into the pathogenesis of rheumatoid arthritis

Rheumatology 2000;39(suppl. 1):3–8

New insights into the pathogenesis of rheumatoid arthritis C. M. Weyand Department of Medicine, Division of Rheumatology, Mayo Clinic, Rochester, MN, USA Abstract T lymphocytes play a critical role in the inflammatory process of rheumatoid arthritis (RA). Studies in a new animal model of RA, created by implanting human inflamed synovium into SCID mice, have confirmed that the production of matrix-degrading enzymes and pro-inflammatory cytokines is ultimately under T-cell control. T-cell dysfunction in RA patients also alters T-cell dynamics, resulting in profound abnormalities in T-cell pool composition. The cause and consequences of altered T-cell dynamics in RA are not yet understood, but factors determining T-cell homeostasis include the generation of new T cells, loss of T cells during immune responses and self-renewal of T cells within the system. Understanding the mechanisms that govern the formation of the T-cell pool in RA emphasizes the dynamic and quantitative aspects of lymphocyte behaviour in RA and has profound therapeutic implications when devising strategies to counteract T-cell dysfunction. KEY WORDS: Rheumatoid arthritis, Pathogenesis, T cells, Animal models, T-cell clonal expansion, T-cell homeostasis.

of macrophage-like cells, which modulate innate and adaptive immunity, will be mentioned briefly. It is noteworthy that other cells not discussed here in detail may also contribute to RA. B cells produce autoantibodies that may be pathogenic; fibroblasts secrete pro-inflammatory cytokines and metalloproteinases that have harmful effects on tissue; and endothelial cells control the influx of inflammatory cells into the synovium.

Rheumatoid arthritis (RA) remains a major medical challenge because the exact cause of the disease is still undefined. The diagnostic and prognostic tools, as well as the therapeutic approaches currently available, require further optimization, despite novel advances that have facilitated the management of RA. RA is a chronic, inflammatory, systemic disease characterized by joint pain and swelling, joint destruction and pannus formation. The RA pannus consists of a hypertrophic synovial membrane composed of hyperplastic synoviocytes and inflammatory cells that infiltrate the synovial membrane. T cells, B cells, CD68+ macrophage-like cells, mast cells and endothelial cells are all present in the RA synovium and contribute to the inflammatory processes. This review will focus essentially on the role of T cells, the key element of the adaptive immune response in RA. T cells recognize antigen on the surface of antigenpresenting cells. Consequently, antigen-presenting cells are intimately involved in regulating T-cell function. The antigen-presenting cells of relevance in the disease process are macrophages and macrophage-like cells. They form the interface between the innate and adaptive immune systems and, in addition to presenting antigen, they are responsible for amplifying inflammatory responses and mediating tissue injury. The involvement

T cells are critical players in the pathogenesis of RA The aetiology of early events in RA remains undefined. The search for a bacterial or viral agent initiating the disease has been unrewarding, despite the variety of molecular and cellular approaches used. It is, therefore, possible that there is not a single cause but, rather, a combination of genetic and environmental factors that contributes to the initiation of RA. In contrast, important progress has been made in defining the molecular, cellular and genetic mechanisms underlying the progress and perpetuation of the disease. In this respect, a number of hypotheses that are not mutually exclusive have been postulated. One of these hypotheses suggests that RA is an antigen-driven, T-celldependent disease and that the inflammatory events are initiated by CD4+ T cells recognizing antigens in the synovial tissue. It is now clear that the synovial T-cell infiltrate is diverse, but it contains clonally expanded CD4+ populations, suggesting antigen-specific recog-

Correspondence to: C. M. Weyand, Department of Medicine, Division of Rheumatology, Mayo Clinic, 200 1st Street, SW, Rochester, MN 55905, USA.

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© 2000 British Society for Rheumatology

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nition. However, to date, no specific, common antigen has been identified in the synovium of different RA patients. In recent years, an alternative hypothesis has been proposed that implies an aberration of peripheral tolerance mechanisms, an anomaly of lymphoproliferation and a deviation in T-cell homeostasis in the disease process [1]. Specifically, the composition of the global T-cell repertoire has been found to be altered in RA cohorts with the emergence of functionally unusual CD4 T cells. Based on accumulating data, it has become apparent that dysregulation of T-cell immunity in RA may not be limited to the inflammatory lesion. Rather, the establishment of chronic destructive lymphoid tissue in the synovial membrane occurs in the context of a fundamental defect in T-cell tolerance. Instead of having acquired tolerance to self-antigens, the immune system of RA patients appears to harbour high frequencies of functionally active T cells with self-reactivity. The model implies that the expansion of these autoreactive T cells derives from an underlying abnormality in controlling lymphoproliferation and T-cell homeostasis. T-cell-dependent functions in the synovial membrane Initial studies suggested that one of the early events in RA may be the recognition of an arthritogenic antigen in the joint, resulting in a chronic and detrimental immune response. This concept was supported by the dominance of T cells in the infiltrates, by the genetic association of RA with human lymphocyte antigen (HLA) molecules, which mediate antigen presentation to T cells, and by the production of autoantibodies in the synovial membrane. However, it is important to mention that, to date, convincing evidence of a joint-specific endogenous antigen is still lacking [2]. It is possible that a variety of antigens are involved in the T-cell response and that the antigens or epitopes presented to T cells vary during the course of the disease. The spectrum of antigens driving T- and B-cell activation in the rheumatoid lesions may indeed be novel in each affected patient. T cells also appear to play a pivotal role in the conversion of the synovium into a lymphoid tissue. The infiltrates in the synovial tissue have been pathologically described as follicular aggregates consisting mainly of CD4+ T cells and B cells, with CD8+ T cells occupying interfollicular tissue. It is interesting that in some patients true lymphoid follicles with germinal centres appear, demonstrating that lymph node-like structures are created in the joint. The formation of germinal centres (complex lymphoid microstructures mediating antigentrapping and adaptive immune responses) is T-cell dependent, and elimination of T cells completely prevents their generation. Classical germinal centres consist of CD20+ B cells and CD4+ T cells. The centre of the follicular structures is occupied by CD23+CD21+ cellular networks representing follicular dendritic cells, which can trap an immunocomplex containing antigen and present it to B cells. In the follicular centres, B cells undergo affinity maturation and selection by hypermutating the immunoglobulin gene and then probing with the result-

ing antibodies for optimal fit to an antigen. Detailed analysis of synovial membranes from RA patients has demonstrated that T-cell populations not typically associated with germinal centre function, such as CD68+ macrophages and CD8+ T cells, are also actively involved. Quantification of cell subsets by image analysis of immunostained synovial tissue sections and by flow cytometry of tissue-derived lymphocytes indicated that the proportions of CD4+, CD20+ and CD68+ cells were invariant, and therefore that precise processes govern the organization of the synovial lesions. CD8+ T cells were found in the perifollicular region, with a fraction of these cells expressing the CD40 ligand, a molecule of the tumour necrosis factor (TNF) receptor superfamily. These findings suggested a previously unsuspected role for CD8+ T cells in the formation of follicular centres and in the aggravation of pathological immune responses in the synovium [3]. The CD4+ T cells in the synovium have several important features. First, they express the CD45RO isoform, characterizing them as memory cells and indicating that the cells have been exposed to antigens. In contrast, the number and trafficking of naive T cells in the synovial membrane are very low. In this respect, the synovium is different from secondary lymphoid organs such as the lymph nodes. Second, synovial CD4+ T cells express CD69, an early marker of activation. Because T cells in the blood of RA patients rarely express CD69, cellular activation in the synovium must occur, possibly as a consequence of antigen recognition, passage through endothelial cells or exposure to cytokines present in the tissue microenvironment. A correlation between CD69 expression on synovial T cells and disease severity has been reported, directly implicating these activated CD4 T cells in the disease process [4]. Third, synovial T cells produce a number of molecules that support inflammation, such as CD40 ligand, a member of the TNF receptor superfamily, and in particular interferon (IFN)-γ, which may account for many of the inflammatory manifestations of the disease. Interaction of CD40 ligand with CD40 on B cells induces B-cell proliferation and the production of immunoglobulins. CD40 ligand also induces monocyte activation and dendritic cell differentiation, processes with potentially critical influence on the RA disease process [5]. An important aspect of memory T cells accumulated in the synovium is that they are deficient in some of their expected functions. Synovial T cells express phenotypes that suggest vulnerability to apoptosis (low Bcl-2, high Bax and Fas ligand) [6]; however, the rate of T-cell death in the synovium appears to be low. In contrast, when isolated from the tissue, lesional T cells do not survive well. Therefore, it has been suggested that the synovium inhibits T-cell apoptosis and promotes T-cell survival potentially through special functions provided by fibroblasts [6]. In addition, tissue-derived T cells proliferate poorly, and their signalling mechanisms appear to be altered. In contrast to the high production of IFN-γ, other cytokines, such as the immunoregulatory mediators

T cells in RA pathogenesis

interleukin (IL)-2 and IL-4, are produced in very small amounts [7]. Direct evidence for the pivotal role of T cells in RA is provided by experiments performed in immunocompromised NOD–SCID mice engrafted with rheumatoid synovial tissue isolated from patients with active disease. Human synovium–SCID mouse chimeras were treated with anti-CD2 antibody, resulting in the depletion of 80–90% of the tissue-resident T cells. The loss of T cells led to a marked decline in the production of IL-β (70%), TNF-α (86%) and IL-15 (84%), mediators released by macrophages and synoviocytes. In addition to pro-inflammatory cytokines, the transcription of metalloproteinases 1 and 3, proteases involved in tissue destruction, was also diminished by 72%. Immunocytochemical studies suggested that the decrease in cytokine production was potentially due to the disappearance of CD68+ synovial cells, which are dependent on T cells for their survival. Accordingly, adoptive transfer of autologous tissue-derived T-cell lines and T-cell clones into synovium–SCID mouse chimeras was able to increase tissue production of IFN-γ, but also of TNF-α. The action of adoptively transferred T cells could be mimicked by injecting recombinant human IFN-γ. This observation suggested that one of the essential agents mediating synovial inflammation is IFN-γ [8]. Recent treatment trials in RA patients have provided important information on molecules that play critical roles in the inflammatory process. Most appealing have been studies demonstrating the therapeutic effect of TNF-α blockade. Patients treated with either antiTNF-α antibody or soluble TNF-α receptor constructs have shown marked improvement during the treatment. However, the beneficial effects disappeared upon cessation of antibody administration, indicating that the immune reactions leading to the inflammatory manifestation of the disease continue and are only transiently suppressed, but are not eliminated. Finally, reduction of TH1 activity following treatment of patients with monoclonal antibodies to CD4, which interferes with the function of T cells without depleting them, diminished inflammation [9] and resulted in transient clinical improvement [10]. These beneficial effects could be attributed to a decrease in IL-2 and IFN-γ mRNA levels following 7 weeks of treatment. In summary, activated T cells in the synovium provide mediators that regulate the functional ability of macrophages, fibroblasts and B cells, and occupy a critical position in promoting rheumatoid synovitis. Clonal expansion of T cells in the synovium Activation of T cells is followed by proliferation and clonal expansion. Therefore, efforts have been directed toward determining the nature and composition of T-cell populations in the synovium and analysing T-cell receptors (TCRs) that are preferentially found in the tissue. This could be helpful in the identification of antigen-specific clones that are important in the progression of the disease. Various approaches have

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been used to tackle this issue and have led to conflicting results. Although the synovial T-cell infiltrates in RA are diverse, clonally expanded CD4+ T cells are consistently present. A number of reports have indicated that RA patients are prone to developing oligoclonality that is also found in the blood, a concept that will be elaborated on in more detail later in this paper. Therefore, clonal dominance in the synovium may result from local antigen-specific stimulation or may be the consequence of the infiltration of expanded clonotypes present throughout the lymphoid system. To distinguish between these two possibilities, CD4+ T cells were isolated from blood and synovial fluid or tissue of RA patients with active disease. Clonal populations were detected by reverse transcriptase–polymerase chain reaction (RT– PCR) using specific primers for the TCR-BV and -BJ regions. This was followed by size fractionation and direct sequencing of dominant size classes of TCR transcripts. The results indicated that all patients had synovial clonotypes that were undetectable in the blood. These clonotypes were small in size and persisted over time, were found in independent joints, and represented activated T cells expressing the IL-2 receptor (IL-2R). However, there were also additional clonotypes that were present both in affected joints and in the blood, thus exhibiting an unrestricted tissue distribution. These T-cell clones were equally frequent among activated (IL-2R+) and non-activated (IL-2R–) T-cell subpopulations. Therefore, it was concluded that at least two different mechanisms could explain the T-cell clonal outgrowth in the synovium: T-cell clones restricted to the joints appeared to react to local antigens, whereas the clones with unrestricted distribution patterns indicated a more generalized defect in the maintenance of T-cell diversity [11].

RA may be a systemic disease involving aberrant T-cell function Assuming that T lymphocytes are key regulatory cells in rheumatoid synovitis, it follows that therapeutic T-cell depletion could have beneficial effects in RA. Based on this rationale, several experimental therapies have been developed. In one trial, RA patients were treated with Campath-1H antibody, which depleted T cells by binding the CDw52 molecule. About half of the patients responded to this treatment and exhibited an initial improvement. However, the beneficial effects were not long-lasting and disappeared shortly after treatment. Importantly, the patients developed persistent lymphopenia, very similar to that observed in patients infected with HIV, suggesting abnormalities in the mechanisms regulating T-cell homeostasis and T-cell regeneration. The process of T-cell replenishment in Campath-1Htreated patients was impressively slow; CD4 T-cell counts in the blood reached only 105 cells/µl 34 weeks after the antibody injection. To explore whether these patients had fundamental deficiencies in regenerating a diverse and functional T-cell compartment, the molecular diversity

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of repopulating T cells was assessed in the peripheral blood and synovial tissue of Campath-1H-treated patients. TCR β-chains in random samples of T-cell clones were sequenced, and frequencies of T cells in the blood and synovial tissue were estimated by semiquantitative hybridization with oligonucleotide probes specific for the TCR α-chain V–D–J region of selected clones. The results indicated that the diversity of repopulating T cells was significantly restricted with the emergence of dominant clonotypes. Importantly, the clonotypes found in the blood compartment were also the dominant constituents of T cells in the synovial tissue [12]. It is noteworthy that despite the depletion of T cells in the periphery, T-cell infiltrates in the synovium were maintained at the same level as in RA patients not treated with Campath-1H. One potential explanation is that the microenvironment in the synovium functioned as a lymphoid organ. In fact, the synovial T cells appeared to repopulate the periphery following Campath-1H treatment because the repopulating cells were predominantly memory T cells. The most likely explanation is that patients were unable to generate new T cells and that the T-cell pool in the periphery was replenished by the clonal expansion of cells that were already available [12]. Although dominant clonotypes in the periphery were first observed in patients treated with T-cell-depleting agents, subsequent studies indicated that RA patients who were not subjected to this treatment also had clonally expanded CD4+ T-cell populations [13]. Two different models were suggested. The first model postulated that the clonal expansion of selected T-cell clones was the consequence of chronic antigenic stimulation. The second model predicted a defect in T-cell homeostasis. Model 1 would imply that only a minor component of the T-cell pool was abnormal and disease relevant, whereas Model 2 proposes that the entire T-cell compartment was altered, resulting in the emergence of T-cell clones of varying sizes, some of them so greatly expanded that they would become detectable as large clonotypes. To distinguish between these two possibilities, the diversity of the peripheral CD4 T-cell repertoire was examined by determining the frequencies of arbitrarily selected TCR β-chain sequences. Whereas in healthy individuals the median frequency of individual TCR β-chain sequences was 1 in 2.4 × 107 CD4+ T cells, in RA patients this median was increased 10-fold, indicating that T cells had replicated. Instead of having an enormous spectrum of diverse T cells fill the pool in RA patients, the repertoire was contracted and diversity was compromised. Moreover, the skewing of repertoire diversity involved not only memory but also naive T cells, indicating that the changes observed were not due to antigen recognition, but resulted instead from aberrant T-cell repertoire formation. Contraction of T-cell diversity was not the consequence of chronic infection because the frequencies of T cells in patients with chronic hepatitis C infection were indistinguishable from those in normal controls [3]. To determine whether the aberrations in TCR diversity predisposed an individual to RA and to examine the role

of disease-associated HLA-DR alleles in the formation of the T-cell pool, TCR repertoires were compared in naive (CD4+CD45ROnull) T cells obtained from patients with RA and HLA-matched or HLA-mismatched normal individuals. Comparison of the frequencies of TCR BV–BJ combinations in three different BV gene segment families indicated that the repertoires of naive T cells in RA patients and in HLA-DR matched or unmatched donors were clearly distinct. Moreover, the BV–BJ gene combinations in clonally expanded synovium-specific T-cell clones were biased for combinations that exhibited higher frequencies in naive T cells of RA patients. Taken together, these findings suggested that in addition to HLA-DR, other factors common in RA patients contribute to the formation of a unique TCR repertoire. The naive T-cell repertoire appears to influence the repertoire of the clonally expanded synovial T cells, raising the possibility that defective thymic education in RA patients results in the selection of self-reactive T cells [14]. Recent observations also indicate additional features of CD4+ T-cell clones found in RA that may have important consequences for the disease process. Phenotypic characterization of autoreactive T-cell clones following the isolation and in vitro propagation of CD4+ T cells demonstrated the lack of the most important costimulatory molecule, CD28 [15], which is a receptor for the B7 molecule. Although CD4+CD28null cells are also found in normal individuals, expansion of their numbers is characteristic of RA patients. Assessment of clonal dominance by BV–BC-specific RT–PCR, size fractionation and sequencing indicated that CD4+CD28null cells are oligoclonal and represent the T-cell clonotypes that populate the blood as well as the synovium of RA patients [16]. Functional aspects of clonally expanded CD4+CD28null T cells have been examined. These unusual cells are present in the synovial infiltrates. They express intracellular perforin and can kill Fc-receptor-bearing targets via anti-CD3-redirected lysis. These functional properties identify CD4+CD28null T cells as potentially tissue-damaging effector cells [17]. In addition, the CD4+CD28null cells do not express CD40 ligand, indicating that their primary role is not to promote B-cell differentiation [18]. One of the interesting aspects of CD4+CD28null T cells is their association with disease patterns. Specifically, the highest proportions are found in patients with nodular RA and those with rheumatoid organ disease [19]. In addition, a possible direct involvement of CD4+CD28null T cells in vascular injury is suggested by their emergence in patients with rupture of atherosclerotic plaque and acute coronary syndrome [20]. In summary, these results raise the possibility that the assembly of the T-cell pool and the maintenance of T-cell homeostasis follow different rules in RA patients than in normal controls. The repertoire of CD4+ T cells is contracted due to the replacement of normally infrequent T-cell specificities by largely expanded T-cell clones. The large T-cell clones of RA patients are of a specific and

T cells in RA pathogenesis

unusual phenotype (CD4+CD28null) and produce high levels of pro-inflammatory cytokines [21]. Considering the systemic nature of these abnormalities, therapeutic approaches should be sought that can recreate a highly diverse and functionally competent T-cell compartment in RA patients [22].

RA is a genetic disease Multiple genetic determinants appear to contribute to the risk of developing RA. Investigations are now focusing on determining the spectrum, identity and properties of genes associated with RA. The relative risk of developing RA in monozygotic twins is 12- to 62-fold higher than in unrelated individuals, whereas in dizygotic twins or siblings sharing only 50% of their genes the risk is only 2- to 17-fold higher. This significant difference indicates a genetic basis for the development of RA. To date, the best identified genes as a risk factor for RA are the HLA genes. However, it is not clear at what stage of the disease these genes become relevant in the disease process. HLA genes participate in immune function by presenting antigens to T cells and inducing their activation. They also regulate thymic selection of immature T cells. Initial studies have indicated an enrichment of HLA-DRB1*04 in patients with RA. The HLADRB1*04 family contains multiple allelic variants with amino acid polymorphisms in the HLA-DRB1 gene. It has been postulated that a sequence spanning amino acid positions 70–74 in the third hypervariable region of the HLA-DRB1*04 gene contributes to RA, as indicated by multiple studies in different ethnic groups (the shared epitope hypothesis) [23]. Investigations aiming to associate different HLA-DR4 subtypes with disease severity have indicated a correlation between certain subtypes and the clinical manifestation or pattern of the disease [24–26]. In terms of erosions and extra-articular features, patients with the HLA-DRB1*0401 subtype exhibited greater disease severity, whereas patients with the HLA-DRB1*01 subtype manifested a milder form. Thus, accumulated evidence suggests that the contributions of the different alleles, all sharing a common epitope, are not equal. Another aspect of the HLA alleles in RA is that there may be additive effects of haplotypes. HLA-DRB1*0401/0404 individuals have a higher risk of developing the most severe form of RA, Felty’s syndrome. In general, patients with two copies of RA-associated HLA-DRB1 genes exhibited more severe symptoms [26]. Also, a correlation between HLA-DR sequence polymorphisms and rheumatoid factor production has been reported [27]. Comparison of HLA-DRB1 sequences in rheumatoid-factor-positive and rheumatoid-factor-negative patients has indicated that the most prominent feature of seropositive individuals is a lysine substitution in position 71, whereas in seronegative patients this position is occupied by arginine. In summary, although the HLA association of RA was initially thought to indicate selection and presentation

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of arthritogenic antigen, recent findings are more in line with a contribution of HLA polymorphisms to the progression of the disease, possibly reflecting more generalized effects of immunoregulation. More recently, emphasis has shifted to non-HLA genes, which undoubtedly have a role in RA. Their number is not known and no consensus exists on their identity. The search for non-HLA genes associated with RA may be approached in two different ways. First, the genome of two affected siblings may be searched for common genetic similarities. Genetic regions that are shared by the two siblings at a frequency higher than expected can have potential disease relevance. The second approach focuses on examining the representation of some candidate genes in RA versus normal individuals. Candidate genes can be defined by implicating certain molecular pathways in RA pathogenesis. One example would be to examine whether polymorphisms in cytokines implicated in RA demonstrate enrichment among patients. In conclusion, it has been estimated that genetic factors account for ~15% and non-genetic factors for ~85% of RA. HLA genes appear to have relevance in the progression and patterning of the disease. Their precise interference with immune responses involved in RA remains a matter of investigation. It is foreseeable that genome-wide searches could pinpoint genes so far considered irrelevant to RA. With increasing information on the human genome, thorough exploration of the distribution of gene polymorphisms in affected patients and control donors will likely become the most effective means of identifying novel disease-risk genes.

Conclusions The events that initiate RA are still undetermined. A number of factors, including infections, non-specific inflammation and injury, may contribute to the onset of the disease. Yet, different determinants may affect the progression and pattern of the disease. It is clear that distinct allelic variants of HLA-DRB1*04 molecules influence the course of RA and the severity of the clinical symptoms. Compelling evidence indicates that T cells play a pivotal role in RA. They exhibit a key role at different checkpoints of the disease. The formation of sophisticated lymphoid structures in the synovial membrane, which determines the destructive nature of synovitis, is intimately dependent on T-cell helper function. However, abnormal T-cell immunity in RA is not limited to the joint. RA is characterized by massive abnormalities in the composition of the T-cell pool, suggesting a fundamental defect in T-cell homeostasis. Whereas a normal T-cell compartment is highly diverse, filled by an enormous spectrum of different T cells, the rheumatoid T-cell compartment has a contracted diversity with the emergence of large clonal T-cell populations. Expanded T-cell clonotypes in RA patients have functional characteristics that identify them as being potentially tissue-injurious effector cells. Therefore, novel therapeutic approaches in RA should

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have two goals: (1) to abrogate T-cell activation in the synovial membrane and (2) to attempt to restore the diversity of the T-cell compartment by inhibiting the outgrowth of T-cell clonotypes and by fostering new T-cell generation.

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