APOPTOSIS AND THE SUBSEQUENT GENERATION OF AUTO-ANTIGENS AS INITIAL TRIGGERS IN THE PATHOGENESIS OF EXPERIMENTAL AUTOIMMUNE ARTHRITIS

Background: Rheumatoid Arthritis has a definite autoimmune pathogenesis. Rising evidence let suggest that deregulated and/or deficient clearance of apoptotic materials may lead to the release of potential auto-antigens provoking the break of self-tolerance mechanism. Major auto-antigens in RA include immunoglobulin G, the target of rheumatoid factor (RF), citrullinated proteins and the heterogeneous nuclear ribonucleoprotein (hnRNP) A2. Objectives: To gain more insight in the role of apoptotic processes in lymphoid tissues, in the generation of arthritogenic auto-antigens we are studying autoimmune responses in the pristane-induced arthritis (PIA) model which shows some striking similarities with human RA including symmetrical polyarthritis, massive bone erosion and the presence of RF and autoantibodies to hnRNP-A2. Methods: Pristane (2,6,10,14-tetramethylpentadecane) was injected intradermal at the base of the tail in DA.1F rats. Lymph nodes were analyzed by immunohistochemistry and immunoblotting at various time points after pristane application. The fate of potential neoantigens, particularly hnRNP-A2 during apoptosis of lymphoid cells was studied in cultured cells by inducing apoptosis with agents such as Etoposide or anti-Fas antibody, or by cultivating cells in the presence of pristane/cyclodextrine complexes To investigate apoptotic cleavage of hnRNP-A2 in vitro caspase digestion assays were performed. Results: Three days after pristane application the amount of apoptotic cells was strongly increased in draining inguinal lymph nodes of injected rats compared to naïve animals. Immunoblotting analysis of inguinal lymphnodes revealed cleavage of hnRNP-A2. Furthermore, a cleaved product of hnRNP-A2 was also detected in cellular apoptosis assays performed with human peripheral blood cells and rat splenocytes. Cleavage was most pronounced after apoptosis induction via the Fas receptor. Interestingly, the cleaved product was preferentially detected in cell supernatants compared to cell lysates whereas the intact protein was found to similar amounts in lysates and supernatants. Furthermore, both caspase 3 and caspase 6 were able to efficiently cleave recombinant hnRNP-A2 in vitro. The in-vivo relevance of these findings was further strengthened by results obtained with pristane-cyclodextrine complexes which were able to induce apoptosis in both human and rat cells. Conclusion: Pristane induces apoptosis in draining lymphnodes leading to cleavage of hnRNP-A2 by proteolytic enzymes such as caspase 3 or caspase 6. This suggests a mechanism for the generation of neoepitopes which are subsequently targeted by arthritogenic T cells. These data further strengthen the view of hnRNP-A2 being a key player in the pathogenesis of PIA and possibly also human RA. Disclosure of Interest: None declaredCitation: Annals of the Rheumatic Diseases, volume 69, supplement 3, year 2010, page 188Session: Arthritis etiology, pathogenesis and animal models (Poster Presentations )