ANTIBODIES DIRECTED TO DIFFERENT CITRULLINATED TARGETS ARE VALUABLE TOOLS FOR ANALYSING EXPRESSION OF CITRULLINATED PROTEINS IN SYNOVIAL TISSUE OF PATIENTS WITH RHEUMATOID ARTHRITIS

Background: Autoantibodies against citrullinated proteins are highly specific for rheumatoid arthritis (RA), but neither the biochemical nature nor the location of the eliciting antigen is definitely known. Citrullinated proteins characterized in human synovial tissue include fibrinogen and vimentin, but it is unclear how many additional citrullinated proteins are expressed in the joint.Objectives: To investigate expression of citrullinated proteins in human synovial tissue by performing detailed immunohistochemical analyses in synovial specimens from patients with RA or osteoarthritis (OA) using poly- and monoclonal antibodies (Abs) directed to L-citrulline or citrullinated proteins (anti-citrulline Abs).Methods: Synovial membranes were obtained at the time of joint surgery. Frozen sections were stained either with affinity-purified rabbit polyclonal anti-citrulline Abs obtained from Quartett (Berlin, GER) and Chemicon (Lake Placid, USA), respectively, or a polyclonal Ab to a synthetic deiminated polypeptide of mouse keratin (cit-keratin) obtained from Upstate (NY, USA) or a monoclonal Ab (mAb) to citrullinated mouse fibrinogen (cit-fibrin) from ModiQuest (Nijmegen, The Netherlands). To analyse the localisation of citrullinated proteins tissue sections were double-stained with the various anti-citrulline Abs and mAbs against cell lineage-specific antigens or fibrinogen.Results: In specimens both from RA and OA patients, citrullinated proteins were found highly expressed in the lining layer and sublining areas as well as in approximately 20% of B cells, whereas T cells were generally negative. The majority of stained cells were CD68 positive macrophages while staining of fibroblasts was generally weaker. The two anti-L-citrulline Abs produced nearly identical staining patterns which were similar to the pattern obtained with the anti-cit-keratin Ab. However, the pattern obtained with the anti-cit-fibrin mAb was somewhat different since this Ab also stained extracellular areas, particularly at the surface of the lining layer where also intense staining by the anti-fibrinogen Ab was observed. Furthermore, and in contrast to the other Abs, the anti-cit-fibrin mAb stained endothelial cells. Although staining was more intense in specimens from RA patients, no qualitative differences were seen between RA and OA synovial tissue.Conclusion: All four anti-citrulline Abs generated similar staining patterns in RA and OA synovial tissue, irrespectively of the nature of the antigen used for immunisation, but fibrinogen as well as endothelial cells appeared to be exclusively stained by the anti-cit-fibrin mAb. Thus, commercially available anti-citrulline Abs are very useful tools for characterization of citrullinated antigens even when raised to an antigen that is not expressed in the joint (such as cit-keratin). It will be a challenging task to elucidate the nature of the antigens stained by the various anti-citrulline Abs.Citation: Ann Rheum Dis, volume 67, supplement II, year 2008, page 148Session: Humoral aspects – Autoantibodies