Protein modification through posttranslational citrullination in the rheumatoid joint is thought to play an important role in perpetuation of local chronic inflammation in the presence of specific anticitrulline immunity. This is the first report showing that synovial citrullination is actively modulated by antirheumatic treatment.
Previous studies characterized synovial expression of citrullination, showing a general increase in the level of citrullination in the presence of active inflammation [2
]. These early reports used one particular method for detection of citrullination, consisting of the chemical modification of the citrullinated tissue proteins to allow their recognition by an antibody raised against similarly modified citrullinated proteins [16
]. However, this technique is no longer available, and in our hands, the only other antibody performing well in immunohistochemistry is F95 [10
]. As with the modified anti-citrulline antibody, F95 is supposed to recognize a large array of citrullinated molecules independent of the amino acid context. We previously demonstrated that this antibody is able to recognize specifically at least one RA-relevant citrullinated antigen, citrullinated fibrinogen [17
]. However, a somewhat more restricted pattern of citrullination, as compared with the modified anti-citrulline antibodies, has been observed both in synovial biopsies [11
] and in other tissues, such as lungs of smoking healthy individuals [18
]. By using this new antibody, De Rycke et al.
] were able to describe an RA-specific intracellular F95 staining. We confirm this finding by the virtually absence of F95-positive cells in synovial biopsies of healthy individuals.
Only few reports of PAD synovial expression are currently available [2
]. The most thorough investigation of synovial PAD expression was performed by Foulquier et al.
], showing correlation between expression of both PAD2 and PAD4 expression with the degree of synovial inflammation. We confirm these findings and identify the same correlation for the CP expression.
The novel finding of the current study is the reduction in citrullination and PAD expression induced by intraarticular GC, which was not observed after MTX treatment. One potential explanation for this difference is the different time points to obtain the follow-up biopsy, 2 weeks after treatment initiation with GCs, as compared with 8 weeks for MTX. However, the two different time points were chosen in accordance with the clinical expectation of maximal effect of the administered drug, which would theoretically increase the chance to observe a change, not only for GCs, but also for MTX. We were able also to demonstrate a direct effect of GCs independent of inflammation in our synovial explants. This is further supported by the results of the in vitro
experiments, in which we observed a dose-dependent effect of GCs despite obvious difficulties in standardization of a quantitative analysis by using immunohistochemistry on cells. Interestingly, although GCs decreased citrullination and PAD4 expression in SFMCs, no such effect was observed in PBMCs. This could be due to the lower baseline levels and, as a consequence, to the lower sensitivity to detect changes of expression of the investigated molecules in PBMCs as compared with SFMCs. Conversely, it could be due to different regulatory mechanisms and cellular activation states of PBMCs, as compared with SFMCs, as previously suggested [20
]. Despite major advances in understanding the central role of citrullination and anti-citrulline immunity in RA pathogenesis, we face a striking lack of knowledge regarding regulatory factors responsible for induction, perpetuation, and/or amelioration of the process of citrullination, both locally in the joint and even more generally in other tissues where citrullination occurs either under physiologic (skin) [21
] or pathologic conditions (lungs of smokers) [18
]. It is generally accepted that citrullination accompanies inflammation, and it has been suggested that induction of citrullination by inflammatory stimuli such as TNF occurs after PAD4 activation and induction of signaling pathways dependent on NF-κB [23
]. Interestingly, the antiinflammatory effects of GCs are at least partially dependent on NF-κB, as demonstrated by their lack of effect in animal models of acute inflammation in NF-κB knockout mice [24
]. In contrast, MTX appears to have limited effects on NF-κB activation, as demonstrated by high levels of activation in PBMCs of MTX-treated RA patients that can be reversed by anti-TNF agents [25
]. These findings suggest that GCs might affect citrullination through PAD4 downregulation through an NF-κB-dependent mechanism.