In contrast to the usual functions of fibroblasts, fibroblast-like synoviocytes (FLS) have been hypothesized to be important in joint inflammation and destruction (1–4). In rheumatoid arthritis (RA), FLS adopt an inflammatory phenotype and secrete cytokines, proteases, and other mediators. FLS secrete chemokines that enhance leukocyte recruitment through synovial endothelium (5). The most abundant infiltrating leukocyte is the T lymphocyte, which may come into close proximity to FLS, and interaction between these cells could contribute to RA pathology. When FLS and T cells are cocultured in vitro, activation of both cell types is observed. FLS produce inflammatory mediators such as interleukin-6 (IL-6), IL-8, and prostaglandin E2 (6), and T cells up-regulate the activation markers CD69 and CD25 (7). The functional consequences of these cocultures mirror the molecular characteristics of RA synovium, which suggests that similar interactions could be occurring within the inflamed RA joint. These experiments were performed without addition of antigen; thus, the bidirectional activation observed was by mechanisms independent of exogenous antigen presentation.
The etiology of RA is still unknown, although several hypotheses have been proposed; however, a strong genetic contribution to RA is well established. Within the Caucasian population, the most important genetic association is with the major histocompatibility complex (MHC) locus—specifically with HLA–DR4 and other closely related DR alleles (8). One of the RA-associated DR4 alleles, HLA–DRB1*0401, may also be a predictor of disease severity, with homozygous individuals showing worse outcomes (9–12). The RA-associated DR4 alleles all share a common motif near the peptide binding groove at residues 70–74 of the β-chain (13–15). When human HLA–DRB1*0401 is expressed as a transgene in mice, it confers susceptibility to induction of arthritis by immunization with certain autoantigens, even on otherwise genetically resistant backgrounds (16).
Prior work has yielded mixed results regarding the capacity of fibroblasts to function as true antigen-presenting cells (APCs) for MHC-restricted responses to exogenous antigens from pathogens (17–20). The present study sought to evaluate the ability of FLS to function as APCs for specific autoantigens present within the joint and relevant to RA. Mouse T cell hybridomas were employed that are specifically responsive to arthritogenic peptides of human cartilage gp-39 (HC gp-39, YKL-40) or human type II collagen (CII) presented by the human class II MHC allele HLA–DRB1*0401, as measured by production of murine IL-2. These hybridomas were developed from a mouse transgenic for the human class II allele HLA–DR4*0401. Since these T cell hybridomas have been shown to recognize their respective antigens when presented by human dendritic cells or human monocytes that express *0401 (21), they were used to evaluate the APC potential of FLS for arthritogenic autoantigens.
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- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
Presentation of autoantigens to T cells in synovial tissue could be very important in the initiation and perpetuation of inflammatory arthritis. The RA-associated MHC alleles possess a common sequence motif referred to as the “shared epitope” (13). Structural analysis of the shared epitope region shows that it is positioned near the MHC peptide binding groove (15); thus, it influences bound peptides or affects interactions with T cell receptors (TCRs) (14). This suggests that antigen presentation may be an important pathogenic mechanism in RA.
Prior work has documented T cell–FLS interaction leading to activation of both cell types (6, 7, 23), but previous data regarding fibroblasts as APCs have been mixed. Investigators in early studies using dermal fibroblasts observed that they were poor generators of allogeneic responses (17). This defect was not due to inadequate expression of class II MHC, but rather to the lack of an accessory molecule that could be provided by conventional APCs. However, it was noted that dermal fibroblasts could stimulate previously activated alloreactive T cells. Expanding on this work, the capacity of dermal fibroblasts to function in antigen presentation was evaluated. Dermal fibroblasts were able to process exogenous antigen, but did not function well as APCs without accessory cell help (18, 24). In both of those studies, IFNγ was used to induce class II MHC, but human autoantigens were not evaluated. These previous studies do document fibroblast expression of functional class II MHC.
FLS of RA synovium express high levels of class II MHC in vivo and ex vivo (25), suggesting that the potential exists for antigen presentation by FLS in RA. Previous work indicated that FLS can process and present bacterial antigens to T cell clones via a class II MHC–restricted mechanism (19). FLS can also present bacterial superantigens to polyclonal peripheral blood lymphocytes, activating a significant subset of T cells. However, these reports focus on exogenous antigens from pathogens. Our current study has documented the ability of FLS to function as APCs for human autoantigenic peptides and endogenous human proteins present in SF, indicating that these interactions could be occurring in vivo within an inflamed joint, in the absence of infection.
There is also evidence that under some conditions FLS might not activate T cells, but instead induce anergy, in experiments that assessed the APC and allostimulatory functions of FLS (26). In these studies, FLS were able to load antigen onto class II MHC, but allogeneic responses depended upon the addition of accessory cells expressing CD80, and blockade of CD80 abolished the response (i.e., FLS are poor allogeneic stimulators, similar to dermal fibroblasts ). When FLS without accessory cells were cultured with T cells, the T cells adopted a phenotype resembling anergy, characterized by up-regulation of CD25, reduced proliferation, and reconstitution of proliferation by exogenous IL-2. This result implies that FLS cause anergy due to a lack of costimulatory molecules, but that bystander cells expressing costimulatory molecules could overcome this. The potential for accessory costimulation exists abundantly within RA synovium due to the close proximity of FLS to B cells, macrophages, and dendritic cells, as well as other T cells.
The evidence that FLS can express functional class II MHC is strong, but the function of FLS MHC expression has not been thoroughly explored. Work in transgenic mice suggests that the specific class II MHC allele subtype has a striking effect on T cell polarization. Evaluation of the T cells from DR4-transgenic mice reveals functional differences between RA-associated and non–RA-associated alleles. T cells from *0401-transgenic mice, possessing the RA-associated allele, differ from T cells from *0402-transgenic mice, which carry a non–RA-associated allele, by their cytokine profile after antigen stimulation. Mice transgenic for *0401 show a skew toward a Th1-mediated immune response and make greater levels of IFNγ and tumor necrosis factor α (TNFα) after stimulation with antigen compared with *0402-transgenic mice (20).
A possible reason for differing T cell responses to antigens presented by different DR4 alleles is that the peptide binding repertoire of RA-associated DR4 alleles is distinct from that of other DR4 alleles. HLA–DRB1*0401 presents immunodominant peptides from the autoantigens HC gp-39 and human CII, which are peptides distinct from those presented by non-RA DR4 alleles (20). Perhaps the presentation of a unique panel of peptides by RA-associated MHC activates a distinct set of T cells in the periphery and educates a corresponding set of T cells in the thymus, thus producing the predisposition to autoimmunity (27, 28).
Even though a definitive autoantigen (or autoantigens) has yet to be consistently identified in this process of T cell development and activation in RA, HC gp-39 and human CII are plausible candidates. These autoantigens are found within cartilage and synovial tissues and are arthritogenic, able to induce inflammatory arthritis in susceptible strains of rodents (29). HC gp-39 is made by chondrocytes and macrophages, 2 cell types that are found in RA joints. Elevated serum levels of HC gp-39 appear to correlate with increased RA disease activity (30–32). Peripheral blood mononuclear cells from RA patients show an increased proliferative response to HC gp-39 antigen compared with those from healthy controls (33). The prevalence of HC gp-39–responsive T cells in peripheral blood of RA patients was similar to that seen in controls in 1 study (34), but RA T cells produced increased IFNγ in response to HC gp-39, whereas controls showed an IL-10 response, indicating that there is a skew toward inflammation in RA patients, while controls show a regulatory response (35). Histologic studies have even identified HC gp-39 complexed with HLA–DRB1*0401 on APCs within human RA synovial tissue sections (36). These observations support the idea that HC gp-39–responsive T cell clones might contribute to RA pathogenesis or joint inflammation.
Like HC gp-39, human CII is a human autoantigen, also used to induce arthritis in mice as a model of RA (37). It is only found within cartilage and synovial fluid or tissue. Although autoantibodies to human CII and human CII–responsive T cell clones are not specific for RA (38), T cell clones in RA patients have been identified that have a TCR repertoire similar to that of human CII–expanded T cells (39). There is also evidence that T cell responses to altered forms of CII are important in RA (40). Furthermore, cocultures of human CII–reactive T cells with FLS increased the production of IFNγ, IL-17, TNFα, IL-15, and IL-18 (23). These studies suggest that T cell responses are altered in RA patients such that responses to antigens might be skewed toward a proinflammatory response centered around HC gp-39 and/or human CII antigens, even when measurable T cell proliferative responses are not remarkably elevated.
In the current study, HLA–DRB1*0401–positive fibroblasts from different tissue sources were able to present immunodominant peptides from the arthritogenic proteins HC gp-39 and human CII to antigen-specific T cell hybridomas. The key requirement appears to be the expression of the correct class II MHC allele. Since T cell hybridomas represent previously activated T cells, minimal costimulation may be needed for reactivation. Nonetheless, any activation of the T cell hybridoma indicates that a functional peptide–class II MHC complex is present on the FLS. If irrelevant peptide or a class II MHC mismatch is present, no T cell stimulation occurs. The significance of other structural interactions outside of MHC–TCR is not yet clear. The CD54–CD11a and CD58–CD2 interactions are often important for professional APC interaction with T cells. Inhibiting these interactions did not substantially disrupt FLS APC function. Perhaps cross-species limitations of some costimulatory receptor–ligand interactions and the relative lack of dependence of hybridomas on costimulatory signals minimize the roles of these molecules in the system that we used.
FLS are also able to extract antigens from SF and present the antigens to both HC gp-39 and human CII T cell hybridomas. The exact nature of the antigenic material in SF is still unknown. It may include predigested peptide fragments and/or intact proteins, such as HC gp-39. Whether these antigens are passively loaded onto FLS or are ingested and processed by FLS remains to be discovered, and it is possible that both processes can occur. In the current experiments, it was important to exclude possible mechanisms of T cell–FLS interaction that did not involve antigen presentation, and to prove that FLS induction of the IL-2 by T cell hybridomas was through antigen presentation. Thus, activation of T cell hybridomas by FLS, as measured by IL-2, showed MHC restriction, was antigen specific, required IFNγ, and was dependent on functional class II MHC. Moreover, SF from patients with RA or OA almost never induced secretion of IL-2 by hybridomas in the absence of FLS.
In the present study, we did not find a correlation between the concentration of HC gp-39 in SF and the ability of FLS to present antigens to the HC gp-39 T cell hybridoma. The specificity of the HC gp-39 ELISA regarding which region of the protein is recognized by antibodies is unknown; the recognized portion could be inconsequential to antigen presentation. Moreover, it is possible that there are HC gp-39 degradation products present in SF that were not recognized by the HC gp-39 ELISA. These fragmented portions of HC gp-39 could result in a “functionally” high concentration of HC gp-39, while leaving the measurable HC gp-39 low. Conversely, the measurable HC gp-39 could be in relative abundance, while the portions responsible for antigen presentation are reduced, leading to high measured concentrations of HC gp-39 with little stimulatory effects on the hybridoma.
In addition to uncertain protein concentration, SF also contains many factors that could affect antigen presentation, enhancing or inhibiting it. Inflammatory and inhibitory cytokines (e.g., IL-1 and IL-10) are present in SF and may affect immune cell function. Failure of FLS to present recombinant peptides in the presence of certain SF samples supports the concept of inhibitory factors within those SF samples (data not shown). It is also notable that while professional APCs can effectively present HC gp-39 and human CII antigens with overnight incubation with antigen and 20-hour coculture times with T cells, the kinetics are different for FLS as APCs. FLS require prolonged incubation with antigen and longer coculture times with T cells. This suggests that FLS might not process and/or present antigen as efficiently as their professional counterparts, or that they might use entirely different mechanisms to achieve the same ends. Given the chronicity of RA, the prolonged time needed for FLS–T cell interactions would not be an obstacle to the occurrence of such interactions in vivo. These issues will require further analysis in order to identify additional factors that govern FLS APC function in vitro and in vivo.
FLS exist under unique conditions that provide mechanisms for stimulation of T cells specific to arthritogenic autoantigens. FLS express high levels of class II MHC in RA synovium and are chronically exposed to autoantigens present within SF. Our studies indicate that FLS can express a functional autoantigen–class II MHC complex. In the context of an inflamed RA joint, FLS could take up antigen, display antigenic peptides on MHC, and activate T cells in an antigen-dependent mechanism as suggested by findings of our in vitro studies. However, the data in this report do not establish that FLS can take up and process antigens with the same efficiency as professional APCs. Nevertheless, the role of nonclassic APCs (such as fibroblasts) as participants in autoimmunity deserves further consideration.
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
Dr. Fox had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Tran, Davis, Lundy, Kovats, Fox.
Acquisition of data. Tran, Davis, Tesmer, Endres, Motyl, Smuda, Somers, Chung, Urquhart, Lundy, Fox.
Analysis and interpretation of data. Tran, Davis, Motyl, Lundy, Fox.
Manuscript preparation. Tran, Endres, Lundy, Kovats, Fox.
Statistical analysis. Tran.
Manuscript review. Somers, Chung, Urquhart.