Citrullinated proteins have increased immunogenicity and arthritogenicity and their presence in arthritic joints correlates with disease severity.

Autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in patients with rheumatoid arthritis and have been suggested to be involved in the disease pathogenesis. The targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deiminase (PAD), which converts positively charged arginine to polar but uncharged citrulline. The aim of this study was to explore the effects of citrullination on the immunogenicity of autoantigens as well as on potential arthritogenicity. Thus, immune responses to citrullinated rat serum albumin (Cit-RSA) and to unmodified rat serum albumin (RSA) were examined as well as arthritis development induced by immunisation with citrullinated rat collagen type II (Cit-CII) or unmodified CII. In addition, to correlate the presence of citrullinated proteins and the enzyme PAD4 with different stages of arthritis, synovial tissues obtained at different time points from rats with collagen-induced arthritis were examined immunohistochemically. Our results demonstrate that citrullination of the endogenous antigen RSA broke immunological tolerance, as was evident by the generation of antibodies directed against the modified protein and cross-reacting with the native protein. Furthermore we could demonstrate that Cit-CII induced arthritis with higher incidence and earlier onset than did the native counterpart. Finally, this study reveals that clinical signs of arthritis precede the presence of citrullinated proteins and the enzyme PAD4. As disease progressed into a more severe and chronic state, products of citrullination appeared specifically in the joints. Citrullinated proteins were detected mainly in extracellular deposits but could also be found in infiltrating cells and on the cartilage surface. PAD4 was detected in the cytoplasm of infiltrating mononuclear cells, from day 21 after immunisation and onwards. In conclusion, our data reveal the potency of citrullination to break tolerance against the self antigen RSA and to increase the arthritogenic properties of the cartilage antigen CII. We also show that citrullinated proteins and the enzyme PAD4 are not detectable in healthy joints, and that the appearance and amounts in arthritic joints of experimental animals are correlated with the severity of inflammation.

Recombinant human monoclonal autoantibodies specific for citrulline-containing peptides from phage display libraries derived from patients with rheumatoid arthritis.

OBJECTIVE: To isolate and characterize monoclonal autoantibodies (Mab) directed to citrullinated antigens from patients with rheumatoid arthritis (RA). METHODS: Using lymphocytes from bone marrow or peripheral blood from RA patients, we constructed antibody fragment libraries representing the antibody repertoire of these individuals. Antibody fragments recognizing a citrulline-containing peptide were selected from these patient libraries. Individual antibody clones were analyzed for germline gene usage and reactivity toward citrullinated (auto)-antigens. RESULTS: Sequence analysis of the cDNA encoding the 21 distinct antibody fragments that were obtained revealed a restricted germline gene usage. Individual antibody clones were positive in both antiperinuclear factor (APF) and antikeratin antibody (AKA) tests, stained citrullinated filaggrin and fibrinogen on Western blots, and reacted with subsets of citrulline-containing peptides in ELISA, but not with noncitrullinated peptides. CONCLUSION: Our report describes the first recombinant human Mab fragments reactive with citrulline-containing peptides. The restricted germline gene usage of these antibodies, and the fact that the VH alleles used are not present in all individuals, may indicate the existence of a genetic predisposition for the development of anticitrulline antibodies in individuals with these germline alleles. The selected antibody clones may facilitate studies on the role of these autoantibodies and their target antigens in the development of RA.