Background/Purpose: Citrullinated vimentin (cVIM) is one of the major autoantigens in patients with rheumatoid arthritis (RA), recognized by anti-citrullinated peptide antibodies. Autophagy is a self-cannibalism system to adapt to starvation but also regulates various cellular functions. Since autophagy is involved in antigen presentation with major histocompatibility complex (MHC) class II and accelerates peptidylarginine deiminase activity, we hypothesized that activation of autophagy causes citrullination of vimentin and its interaction with MHC class II in synovial fibroblasts (SF), one of the effector cells in RA.

Methods: SF were derived from synovial tissue specimens obtained from RA patients during joint replacement surgery. To evaluate antigen presenting capacity of SF, the cell surface expression of MHC class II and B7 molecules were analyzed by flow cytometry after 72h treatment with IFN-γ. Anti-cVIM antibodies were measured in RA patients’ sera using enzyme-linked immunosorbent assay. Intracellular citrullinated autoantigens in SF were analyzed by western blotting using anti-cVIM antibody positive patient’s serum as a primary antibody. To induce autophagy, SF were incubated in serum-free medium for 2h or treated with 10 μM of the proteasome inhibitor MG132 for 24h. To inhibit autophagy, SF were treated with 5 mM of 3-methyladenin. Intracellular cVIM was evaluated by western blotting and immunocytochemistry using anti-vimentin and -citrulline antibodies. To evaluate the interaction between MHC class II and cVIM, lysates of IFN-γ treated SF were immunoprecipitated by anti-HLA-DR antibody, followed by western blotting for vimentin and citrulline. In addition, proximity ligation assay was performed using anti-HLA-DR, -vimentin, and -citrulline antibodies.

Results: MHC class II, B7-H1, and B7-DC were expressed on SF following treatment with IFN-γ, while B7-H3 was expressed on SF regardless of the presence of IFN-γ. Anti-cVIM positive RA patients’ sera recognized 54 kDa protein in SF. By co-immunoprecipitation using anti-vimentin and -citrulline antibodies, the 54 kDa protein recognized by RA sera was revealed to be citrullinated vimentin. Following induction of autophagy by serum-free starvation or proteasome inhibition, intracellular cVIM was increased in SF but the effect was cancelled by the autophagy inhibitor 3-methyladenin. The interaction between MHC class II and cVIM was demonstrated by co-immunoprecipitation. Furthermore, proximity ligation assay revealed that the MHC class II-cVIM interaction significantly increased following induction of autophagy.

Conclusion: Our current data indicate that SF contribute to the autoimmunity in RA through citrullination of vimentin and its interaction with MHC class II promoted by autophagy.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/autophagy-promotes-citrullination-of-vimentin-and-its-interaction-with-major-histocompatibility-complex-class-ii-in-synovial-fibroblasts/