APOPTOSIS AND CYCLOOXYGENASE-2 EXPRESSION IN HUMAN SYNOVIAL FIBROBLASTS IN CULTURE

Background: Hyperplasia and inflammation in synovial membrane are the main pathological features of rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLS) which accumulate in synovium are believed to be critically involved in etiopathology of RA. Mounting evidence indicate that apoptosis of FLS is disturbed during RA. Prostagladin E2 (PGE2) found at elevated levels in synovial tissues has been shown to play important role in the process of rheumatoid inflammation. Cyclooxygenase-2 (COX-2) is overexpressed in rheumatoid joints and is considered to be a main source of PGs.Objectives: To investigate relationship between cyclooxygenase–2 expression, prostaglandin E2 generation and apoptosis in a model of cultured human fibroblast-like synoviocytes.Methods: Synoviocytes were isolated by enzymatic digestion of synovial specimens obtained from 14 patients undergoing surgery (9 with RA and 5 with osteoarthritis). Cells between 3-5 passage, which constituted homogenous population of FLS were pre-treated for 24 hours with a mixture of IL-1beta and TNF-alfa (10ng/ml) and then incubated for 60 minutes with aspirin (ASA, 200 microM). As control conditions incubation only with cytokines, only with ASA or control medium was performed. Apoptosis was measured by Annexin V-FITC and PI flow cytometric assay. COX-2 protein expression was determined by intrastaining method with anti-human COX-2-RPE mAb and FACS analysis. PGE2 generation was measured by ELISA.Results: In basal conditions 2% of FLS showed signs of apoptosis. Cytokines stimulated FLS apoptosis (4%; p<0,01). We observed negligible spontaneous expression of COX-2 (2% of FLS were positive for COX-2), also PGE2 generation was low (0,24ng/ml). Cytokines dramatically stimulated COX-2 protein expression (55%; p<0,005) and PGE2 generation as well (6,3ng/ml; p<0,005). ASA used in such treatment conditions inhibited PGE2 generation, but had no effect on FLS apoptosis. A positive correlation between cytokines stimulated apoptosis and PGE2 generation was found (RSpearman=0,7; p<0,05). Additionally, in FLS cultures that generated more than 0,03ng/ml PGE2 the percentage of apoptotic cells was twice as high as compared to FLS with basal PGE2 generation lower than 0,03ng/ml (p<0,05). Similarly, in FLS cultures expressing more than 50% COX-2-positive cells the intensity of apoptosis was twice higher than in cultures with low COX-2 expression (p<0,05).Conclusion: Our findings suggest involvement of COX-2 and PGE2 in regulation of fibroblast-like synovial cells apoptosis.Citation: , volume , supplement , year 2003, page Session: Cytokines and inflammatory mediators