Two possible explanations may be considered for the high specificity of autoantibodies directed to citrullinated antigens for RA. One possible explanation is that there is an RA-specific overexpression of citrullinated antigens in the rheumatoid synovium that leads to an immune response. Alternatively, presence of citrullinated proteins may be a common phenomenon in any inflamed (synovial) tissue but RA patients may have an abnormal humoral response to them.
The first possibility is supported by the finding of genetic polymorphisms in the PAD4 gene [27
]. One haplotype of PAD4 was associated with susceptibility for RA. Although it was not shown whether the PAD4 encoded by the susceptible haplotype exhibits an altered enzymatic function, it was shown that the RA-susceptible haplotype increases PAD4 mRNA stability. In theory, this could result in more PAD4 enzyme being produced and subsequently lead to increased citrullination of proteins and a higher chance of developing anti-CCP antibodies [7
]. Unfortunately, the increased presence of citrullinated proteins was not investigated in that study.
Some studies dealing with the second possibility have recently been published. Masson-Bèssiere and coworkers [26
] showed that various citrullinated proteins are present in the synovial tissue of RA patients. They could be detected in the cytoplasm of various mononuclear cells as well as in deposits of extravascular fibrin (as described above). Although no synovial tissue specimens of non-RA patients were initially investigated, a recent follow up of that study [28
] showed that citrullinated fibrin may also be present in synovial tissue of patients with other forms of joint inflammation. By contrast, Baeten and colleagues [29
] found citrulline staining in about half of the RA patients studied but in none of the control individuals. However, the staining pattern they observed was quite different from that in the study conducted by Masson-Bèssiere and coworkers [26
]. The immunohistochemical staining was observed in a few, widely dispersed cells but not in extracellular structures. The explanation for this discrepancy is probably that the antibody used in the study by Baeten and colleagues was developed for the detection of free L
-citrulline and not for the detection of citrullinated proteins [30
]. This is illustrated by the fact that the staining could be completely abolished by competition with free L
]. In addition, staining patterns similar to the ones described by Baeten and coworkers [29
] were observed in a separate study after staining with an irrelevant control antibody (rabbit anti-FITC) [31
]. Based on cellular morphology and colocalization with CD38, it appeared most likely that rheumatoid factor-producing plasma cells are detected by this antibody rather than citrullinated proteins [31
Finally, data from our studies, obtained using several types of anti-citrullinated protein antibodies, suggest that citrullinated proteins can be detected in RA patients but also in control individuals (such as patients with osteoarthritis or reactive arthritis; our unpublished data, and data from Smeets and coworkers [31
]). Presence of citrullinated antigens in synovial tissue was also not associated with the presence of anti-CCP autoantibodies in serum or synovial fluid. These results thus suggest that the presence of citrullinated proteins in the synovium is not specific for RA. This conclusion is supported by our recent observation that, also in mouse models of arthritis, various synovial proteins are citrullinated during inflammation [32
]. This suggests that citrullination (of synovial proteins) is not an RA-specific phenomenon, but rather it is an inflammation-related process.