THU0064 Antibodies against carbamylated proteins from patients with rheumatoid arthritisactivate endothelial cells

Background Inflammation contributes to the excess of cardiovascular morbidity in rheumatoid arthritis (RA), by promoting endothelial activation; this brings toward the production of adhesion molecules and the activation of signalling mediators. Antibodies against carbamylated proteins (anti-CarP) detected in RA patients correlate with subclinical atherosclerosis. Objectives Aims of the present study were: 1) to determine the effect of anti-CarP antibodies purified from the sera of RA patients, on the production of VCAM1 and ICAM1 as well as the activation of IRAK1 and NF-kB by human endothelial cell line EAhy926. 2) To evaluate endothelial cell apoptosis induced by anti-CarP. Methods An indirect ELISA was used to detect the presence of anti-CarP in the sera of RA patients. To purify anti-CarP, carbamylated-FCS used as an antigen was spotted onto a nitrocellulose filter and incubated with patient’s sera that recorded the highest titer. Antibodies were eluted with glycine 100 mM, pH 2.5 and neutralised with Tris-HCl 1M, pH 8. Antibodies concentration was measured by using a colorimetric Bradford assay. The immortalised hybridoma cell line EAhy926 was cultured in Dulbecco’s Modified Medium containing 10% fetal bovine serum, 1 mM l-glutamine, 100 U/ml penicillin and 10 ml HAT. After cell stimulation with purified anti-CarP at different time points (30 min-48 h) and different concentrations (5–20–50 µg/ml), supernatants were gathered to investigate the production of VCAM-1, ICAM-1 using commercial ELISA kits while activation of IRAK1 and NF-kB was detected by Western Blot analysis using cell lysates. Apoptosis was measured using FITC-conjugated annexin V (AV) and a propidium iodide (PI) apoptosis detection kit at different times (30 min-48 h). Results After EAhy926 stimulation with anti-CarP we observed: 1) induction of VCAM-1 but not ICAM production in cell supernatants; 2) activation of IRAK1 and NFkB transcription factor in cell lysates and 3) induction of endotelial cell apoptosis. Conclusions These data suggest a potential involvement of anti-CarP antibodies in endothelial cell activation allowing to further speculate on its possible effect in excess cardiovascular risk in RA patients. References [1] Pecani A, et al. Arthritis Res Ther2016; [2] Spinelli FR, Pecani A, et al., BMC Musculosceletal Disord2017; [3] Gonzales-Gay MA, et al. Clin and Exp Rheumatol2006; [4] Jing Shi, et al. Autoimmun Rev2014. Acknowledgements .EULAR Scientific Bursaries, Programme of 2017 Disclosure of Interest None declared