Cartilage-specific molecules such as type II collagen (CII) have long been suspected to be targets of pathogenic autoimmune responses in the induction or perpetuation of joint inflammation in rheumatoid arthritis (RA). The hypothesis of CII-directed autoimmunity as a relevant pathogenic mechanism in RA is an appealing explanation for the chronicity of the arthritic process as well as for its fatal consequences for the integrity of joint cartilage. Several lines of evidence further support the concept of a CII-driven autoimmune process in RA. Direct evidence for the arthritogenic potential of CII autoimmunity is derived from experimental disease models of collagen-induced arthritis (CIA) in rodents as well as in nonhuman primates (1). In these models, the arthritis is initiated by complement-fixing autoantibodies that bind to CII in the cartilage matrix, and the formation of these antibodies upon immunization is major histocompatibility complex–restricted and T cell–dependent (2). In humans, anti–CII IgG in rheumatoid cartilage and synovium and circulating autoantibodies to native and denatured CII have been detected in the sera of RA patients (3–6). Moreover, anti–CII IgG–producing B cells have been detected in rheumatoid synovium and synovial fluid (7, 8), suggesting an intraarticular antigen-driven immune process.
In the present study, we focused on the question of whether the epitope specificity of anti-CII autoantibody formation in RA differs from that in other rheumatic diseases, such as osteoarthritis (OA), relapsing polychondritis (RP), and systemic lupus erythematosus (SLE). Recently, a systematic analysis of conformation-dependent autoantibody binding in the murine CIA model in DBA/1 mice revealed a discrete recognition of sterically privileged sites on collagen fibrils, suggesting the possible importance of epitope accessibility in the cartilage matrix for the formation of arthritogenic immune complexes (9). Since recognition of the identified immunodominant domains is associated with arthritis development in CIA and since the CII domains are conserved in their amino acid sequences in mouse, rat, and human collagen, we have selected these epitopes for an analysis of the fine specificity of circulating human anti–CII IgG autoantibodies.
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- PATIENTS AND METHODS
In addition to cartilage-specific proteins, ubiquitously expressed proteins have been suggested as candidate autoantigens in the pathogenesis of RA. Thus, strong evidence for a potential role of glucose-6-phosphate isomerase (G6PI) has recently been provided by the arthritogenic potential of anti-G6PI IgG in a murine arthritis model and by the detection of elevated anti-G6PI autoantibodies in RA sera and synovial fluids in up to 64% of the samples tested (18). However, RA, as defined by the commonly accepted classification criteria, is a rather heterogenous disease, and several different pathways may result in similar clinical manifestations, as reflected by the animal models that can be induced by distinct provocations. Several lines of evidence suggest a critical role of genetically linked autoimmunity to cartilage-specific CII in arthritic joint destruction. CII-specific B cells in the inflamed synovium and in synovial fluid have been shown to produce IgG autoantibodies that can bind to native collagen structures, indicating an intraarticular antigen-driven immune process in RA (7, 8, 19). The locally produced autoantibodies may be largely consumed in the joint by complex formation with the cartilage matrix (20), thereby giving rise to activation of the complement cascade. Consistent with antibody escape into the circulation, CII-specific IgG is also detectable in the sera of RA patients, although at a lower frequency compared with the joint compartment (21). Therefore, the serum titers of CII-specific autoantibodies might reflect, at least to a certain extent, the vigor of the local immune response.
Consequently, several studies have been performed to detect correlations between circulating anti-CII antibodies and clinical parameters in RA patients (3–6). However, formation of autoantibody to CII also occurs in other rheumatic diseases, e.g., in RP (22), SLE (5), and degenerative joint disease. Although immunoreactivity to denatured collagen seems to prevail in OA sera, autoantibodies to triple-helical CII are also detectable (23). It therefore appears rather difficult to decide whether the occurrence of anti-CII autoantibodies plays a role in the pathogenesis of RA or is merely a result of the exposure of destroyed cartilage. More direct experimental evidence of a pathogenic potential has been demonstrated by the ability of human and murine CII-specific autoantibodies to transfer arthritis to a normal mouse (21, 24). However, the fine specificity of the arthritogenic anti-CII autoantibodies has remained obscure. In this respect, the arthritis transfer experiments and the ELISA analysis of CII epitope-specific human IgG in the present investigation provide the first experimental evidence for the pathogenicity of autoantibody formation directed to an evolutionary conserved conformational determinant that is located between aa 359–369 of the CII triple helix.
Our results revealed that the CII region that is an immunodominant target of arthritogenic murine B cell responses in CIA is also recognized by human IgG autoantibodies in RA sera. Moreover, the prevalence of IgG autoantibodies binding to this particular conformational epitope in RA clearly varies from other rheumatic conditions, suggesting disease-related differences in fine specificity of CII-directed autoimmunity. The intriguing hypothesis that differences in B cell epitope specificities might be linked to distinct functional aspects of humoral autoimmune responses is further supported by the predominant IgG binding to the CII region aa 926–936 (F4 epitope) measured in OA compared with RA sera. Thus, a mouse mAb (CII-F4) with specificity for this region exhibited a remarkable disease-ameliorating effect in arthritis transfer experiments in combination with 2 arthritogenic CII-specific mouse mAb. Although the underlying mechanism of the protective effect of mAb CII-F4 is presently unknown, at least 2 hypothetical modes of action can be envisioned. One possibility is the induction of antiidiotypic immunoregulatory responses directed to cross-reactive idiotopes on anti-CII antibodies (25). Alternatively, a steric hindrance of proteolytic degradation of cartilage collagen in the N-telopeptide region close to the crosslinks in the fibril by stromelysin could account for the arthritis-ameliorating effect of the F4 mAb, because the F4 epitope colocalizes in the quaternary collagen structure with the stromelysin cleavage sites on adjacent molecules (9, 26).
For elucidation of disease-associated qualitative differences in fine specificities of CII-directed humoral autoimmunity, a novel technology was introduced in the present investigation. In this approach, recombinant chimeric collagens were replaced by small synthetic collagen-mimetic peptides that were synthesized according to a protocol described by Grab et al (17) for the assessment of conformation-dependent epitope recognition in ELISA. The assay conditions have the advantage that they do not require additional control for the exclusion of false-positive results that may arise from antibody recognition of the type X collagen frame surrounding the CII epitope inserts in the recombinant chimeric collagens, and it was also found to mimic the antibody recognition of native CII. The application of the new ELISA technology confirmed the role of the C1III epitope as a preferential target of CII-directed IgG autoantibody responses in RA sera, whereas J1-specific IgG remained at the limit of detectability. Remarkably, the levels of C1III-directed autoantibody binding in RA not only greatly exceeded the values determined in OA and SLE sera, but also the ELISA results obtained with the RP samples, although inflammatory cartilage destruction and CII autoimmunity are common features in both disease conditions. However, the primary target tissue of the destructive autoimmune process in RP is the extraarticular cartilage, e.g., in the ear, nose, and respiratory tract, whereas the joint is only occasionally affected by nonerosive inflammation.
In RP, the involvement of extraarticular cartilage is accompanied by autoimmune responses to matrilin 1, a cartilage matrix component that is not expressed in joint cartilage, suggesting its potential importance as the major autoantigen (27). Accordingly, CII autoantibody formation in this condition might arise as a secondary phenomenon during epitope spreading, thus lacking the epitope specificity that is required for the induction of erosive joint inflammation. In rodents and, as shown by the results of our study, in humans as well, autoantibody responses with C1III fine specificity are associated with an inflammatory disease leading to the destruction of hyaline cartilage in the joints. The autoantibodies are directed to a collagen structure that is conserved in evolution; it is localized on the cyanogen bromide fragment 11 of CII that has previously been shown to possess all the structural requirements of an arthritogenic immunogen for the induction of CIA in DBA/1 mice (28, 29).
The systematic assessment of sera from patients with different rheumatic diseases for immunoreactivity to CII epitopes that were originally characterized in an experimental arthritis model in rodents revealed striking differences in fine specificities of the immune response that usually remain invisible with detection systems that use the entire antigen. Two of the epitopes under investigation (J1 and E/F10II) were not recognized by human autoantibodies despite the conserved amino acid sequence in human CII and were detected as major epitopes in the mouse CIA model. A possible explanation is that sera obtained from CIA mice are obtained during the priming response and during the early acute phase of arthritis, phases of disease that are not possible to address in humans.
The focus of this pilot study on well-defined collagen structures relevant to the pathogenesis of murine experimental arthritis implies that the recognition of additional epitopes in human anti-CII autoimmunity during early events of the disease or the spreading to other CII determinants in the course of the disease cannot be excluded. However, the new technical approaches using recombinant collagen chimeras and synthetic triple-helical collagen mimetics also offer promising strategies for future prospective investigations of larger patient populations to answer these yet-unresolved questions of CII-directed autoimmunity in the pathogenesis of inflammatory joint disease. Therefore, this first comparative study on epitope specificity of circulating anti-CII autoantibodies in different rheumatic diseases demonstrates the potential of the approach to uncover hidden associations between clinical conditions and immunorecognition that may relate to the underlying pathogenic mechanisms and should encourage further work on the fine specificity of joint-specific autoimmunity.