There were no differences in PQ, QRS, or corrected QT intervals

e the accumulation of inflammatory cells in the synovial tissue of RA patients. Using gene-deficient mice, we recently demonstrated that lack of both CD55 and CD97 indeed ameliorates disease activity in collagen-induced and K/BxN serum transfer models of RA. The high and cell type-specific expression of CD55 raises the question how CD55 expression is triggered in FLS. FLS can be activated by cytokines and molecular patterns, originating from damaged cells, present in the synovial fluid. We therefore tested the ability of a range of pro-inflammatory cytokines and Toll-like receptor ligands to induce CD55 expression in cultured FLS. We show that CD55 was strongly upregulated by “2899909 triggering of TLR3, an endosomal pattern recognition receptor involved in the detection of double-stranded RNA. Stimulation of the cytoplasmic dsRNA sensors melanoma differentiationassociated gene 5 and retinoic acid-inducible gene-I induced CD55 expression as well. Notably, activation of MDA5, but hardly TLR3 or RIG-I triggering, caused cell death in cultured FLS. Finally, we show that TLR3 activation enhanced the binding of CD97-loaded beads in FLS in a CD55-dependent manner, suggesting that dsRNA increases the interaction of FLS with CD97-positive leukocytes. in Dulbecco’s Eagle’s medium supplemented with 10% heat inactivated fetal calf serum, L-glutamine, HEPES, and antibiotics . Non-adherent cells were removed after 24 h, and adhering cells were grown to sub-confluence and subsequently split by trypsinization. Synovial fibroblasts were used for experiments from passage 3 until passage 9; at that “8813645 time cultures were free of macrophages and non-fibroblasts. Primary dermal fibroblasts, obtained from biopsy samples of normal skin, were kindly provided by Dr. Marcel Teunissen. The cells were cultured in Ham’s F-12 medium with 10% FCS and used for experiments between passage 3 and 5. Reagents and Stimulation Assays Synovial and dermal fibroblasts were cultured in 6-well plates and allowed to grow to 7090% confluence. After serum starvation over night in DMEM containing 1% FCS, the cells were stimulated for 48 h with the following agents: tumor necrosis factor a, interferon a, IFNb, interleukin 1b, IL-6 , IFNc, lipoteichoic acid from Staphylococcus aureus, polyinosinic-polycytidylic acid; from 0.01250 mg/ml), lipopolysaccharide from Escherichia coli K-235, imiquimod , and CpG 221244-14-0 oligonucleotides. When indicated, hydroxychloroquine was added to the cultures 2 h prior to stimulation with poly. For intracellular delivery of poly and 59-triphosphate RNA transfection reagent Fugene HD was used according to the manufacturer’s protocol. Materials and Methods Isolation and Culture of FLS and Dermal Fibroblasts Synovial tissue samples were obtained by needle arthroscopy from patients with different forms of arthritis. RA patients fulfilled the American College of Rheumatology criteria, non-psoriatic spondylarthritis patients fulfilled European Spondylarthropathy Study Group criteria, patients with psoriatic arthritis fulfilled the Classification Criteria of Psoriatic Arthritis study group criteria, and patients with inflammatory osteoarthritis fulfilled established criteria and had a joint effusion in the absence of rheumatic disease other than OA. Clinical data on patients and medication are presented in Flow Cytometry For measurement of CD55, CD46, and CD59 surface expression, FLS were detached from 12-well plates with TrypLETM, washed with PBS/0.5% bovine serum albumine, and i