In this study, we show that citrullinated fibrinogen, an antigen that is frequently targeted by autoantibodies in RA patients, can induce arthritis in mice that carry the RA-associated MHC class II SE allele DRB1*0401. This disease was restricted by both the posttranslational modification of the antigen and the HLA transgene, as arthritis was not seen in mice administered unmodified fibrinogen or in wild-type (B6) mice administered either form of fibrinogen. Citrulline-specific T and B cell responses were prominent in DR4-IE tg mice, but they were not absolutely restricted by the SE, as antibody reactivity to some citrullinated antigens could be detected by ELISA and antibody microarray profiling in B6 mice. Further, although the arthritis in DR4-IE tg mice led to joint damage, and like RA, was accompanied by synovial cell hyperplasia, we did not find a robust and persistent polymorphonuclear cell infiltrate within joint tissues, suggesting that in this model, HLA-restricted responses to citrullinated fibrinogen can recapitulate some, but not all, aspects of the rheumatoid pathology.
Evidence that, in large part, the SE regulates the production of anti-citrulline antibodies in RA patients implies a causal relationship with disease pathogenesis mediated through CD4 T helper cell activation (26
). We have shown previously that the increased affinity of peptide-bound citrulline versus arginine for RA-associated MHC class II molecules correlates with CD4 T cell activation, but whether these peptides can be processed from citrullinated fibrinogen was not known. In this study, we addressed this question by assessing T cell recall responses in DR4-IE tg and B6 mice after immunization with CithFib. The presence of the HLA SE transgene afforded citrulline-specific T cell proliferation accompanied by IFN-γ production after in vitro challenge with CithFib and CitmFib. We also assessed peptide-specific recall responses to predicted DR4-binding epitopes and confirmed that a strong HLA SE-restricted T cell response was generated against both heterologous regions of hFib, and to a region of the α chain that was identical between species. The later peptide (Fibα R84Cit) contained citrulline at the critical P4 SE position and T cell responses in DR4-IE tg mice were restricted by this posttranslational modification. Notably, this was the only peptide within fibrinogen that we identified with this stimulatory property after conversion from arginine to citrulline at P4. The significance of T cell reactivity to this epitope is not known, but sequencing data from in vitro citrullination shows that modification of arginine at position 84 of the α chain is variable for hFib, whereas the same amino acid is consistently converted to citrulline after in vitro modification of mFib (unpublished data) (25
). Site-specific citrullination within antigens highlights another level of variability that could impact the development of autoimmune responses. Moreover, whereas epitope specificity of the PAD enzymes have been assessed in vitro, naturally occurring citrullination sites within fibrinogen (or other antigens) have not been determined in vivo, or in other disease conditions besides RA.
In regard to HLA-restricted antibody responses, we found that characteristics of RA serum reactivity toward citrullinated antigens were paralleled in these mice. First, as in human patients (28
), anti-citrulline antibodies were not completely restricted by the MHC class II SE, but instead levels were significantly higher in DR4-IE tg mice. This was evident by ELISA and antibody microarray profiling when assessing reactivity to CitmFib. Second, antibody reactivity to citrullinated antigens was broad, targeting sequences not only within fibrinogen, but also antigens such as vimentin (the target of anti-Sa antibodies in RA patients), fibromodulin, cartilage oligomeric protein (COMP), vitronectin, biglycan, and clusterin. Some of these proteins remain to be confirmed as targets of PAD in vivo, but all have been implicated either in RA pathogenesis or synovial/cartilage homeostasis. An unresolved issue with respect to anti-citrulline antibody specificity and evolution in RA patients is whether this broad response is the result of epitope spreading or merely cross-reactivity, which is a question that is not directly addressed in this study, but one that is currently being pursued. If epitope spreading is the cause, it would suggest that many of the proteins targeted by anti-citrulline antibodies are functionally and/or physically associated. This may be true for some of the citrullinated aforementioned antigens, as fibrinogen, vimentin, and vitronectin have been shown to associate during platelet activation, whereas fibromodulin, COMP, and biglycan are integral components of cartilage (29
). The polyreactive nature of this antibody response also confounds the interpretation of the potential arthritogenicity of autoantibodies solely directed against citrullinated fibrinogen in this model and in RA.
It was interesting that several DR4-IE tg mice immunized with CithFib responded uniquely to citrullinated peptides from vimentin. The reactivity to Cit vimentin peptides derived from the C-terminal region of vimentin downstream of a caspase 6/8 cleavage site of the protein (e.g., vim 256-275cit) was noteworthy. Autoantibodies to citrullinated vimentin or Sa are strongly associated with disease severity and SE carriage in RA patients (8
), and a role in disease pathogenesis has been speculated for years. Vimentin's role as an intracellular intermediate filament protein has been extensively studied; however, it is now evident that this protein is also present extracellularly, and could therefore interact directly with autoantibodies. Extracellular vimentin has been detected on platelets, macrophages, neutrophils, and T cells (30
). It is not known if extracellular vimentin is citrullinated, but vimentin is seen in situations of cellular activation and apoptosis, two conditions where intracellular calcium fluxes occur, a process necessary to activate peptidylarginine deiminase.
A distinct feature of the arthritis seen in DR4-IE tg mice was pronounced synoviocyte dysregulation, but a relative paucity of polymorphonuclear cells within the joints compared with other mouse models of arthritis. The reason for this is not known, but it is possible that a transient and self-limiting inflammatory infiltrate (not captured at the time points of our histological examination) could have provided a stimulus for synovial hyperplasia. A discordant relationship between anti-citrulline immune responses and synovial inflammatory cell infiltration has, however, been reported in RA patients. Baeten et al. have shown that anti-citrulline antibody titers do not correlate with histological parameters of synovial inflammation, nor does local inflammation correlate with SE carriage (14
). Although not formally addressed in the current study, autoantibodies in this model could perpetuate disease by directly altering synovial fibroblast or chondrocyte cellular homeostasis, as has been shown previously with IgG (36
). Alternatively, a persistent and robust inflammatory cell infiltrate in the joint could be facilitated by additional genetic insults (possibly in PTPN22 or FcγRIII) that are independent of the SE (38
This is the first description of citrulline-dependent arthritis in mice. Previous studies in mice have shown that the state of citrullination can influence disease severity in collagen-induced arthritis and that citrullination can break tolerance, leading to the development of citrulline-specific antibodies (42
). Recent work has also shown that murine monoclonal antibodies specific for citrullinated fibrinogen can augment arthritis through passive transfer with anti–collagen II antibodies (45
). Our studies show that the influence of the HLA transgene in citrulline-specific immune responses influences the magnitude and diversity of reactivity rather than the mere presence or absence of a response. These factors likely influence disease expression, as thresholds for T cell reactivity and antibody titers, as well as IgG epitope specificity, are limiting factors in other murine models of arthritis (46
). Threshold effects may also be relevant to human disease, as prospective analysis of anti–cyclic citrullinated peptide antibodies in healthy individuals who later go on to develop RA show a marked increase in titer before disease onset (50
There are still unresolved issues pertaining to the model of arthritis presented in this study that require further exploration. For instance, it is not known why <40% of the mice develop disease, and we have not identified any 1 parameter of the immune response that significantly differentiates arthritic and nonarthritic DR4-IE tg mice immunized with CithFib (although arthritic mice did show a trend to higher cytokine responses and antibody production to certain antigens). In preliminary studies, we have induced a transient arthritis in DR4-IE tg mice after the transfer of citrulline-specific CD4+
T cells or anti-citrulline antibodies, directly implicating these mediators in the disease process (Fig. S3 and unpublished data) (52
We have also found that CitmFib, an antigen that can evoke a citrulline-specific immune responses in DR4-IE tg mice, is not arthritogenic. Our examination of heterologous regions of fibrinogen that have been found to be citrullinated by mass spectrometry (25
), coupled with IgG antibody microarray and ELISA data, does suggest that reactivity to Fibα121-140Cit may be important to this species-specific response. It is possible that reactivity to this sequence provides a platform for cross-reactivity to other proteins (such as vimentin), but a role in pathogenesis requires further examination.
In conclusion, these results indicate that in genetically susceptible DR4-IE tg mice, citrullinated fibrinogen can drive an autoimmune response that is associated with the development of arthritis. Further exploration of this model will help elucidate the role and contribution of SE-restricted T and B cell responses to citrullinated antigens in the pathogenesis of RA.