The Effect of Fas stimulation on skin synovial fibroblasts

2. SUMMARY Fas mediated apoptosis is considered to be involved in the pathogenesis of RA. Although it is generally suggested that the Fas antigen transmits apoptotic signals to fibroblasts, it has been also reported that it may play a role in proliferation. In this study cultured fibroblasts from 2n"- to 9tn passage obtained from skin and normal or rheumatoid synovium were stimulated by anti-Fas-IgM (CH11) and it was tested whether this stimulation leads to apoptosis or proliferation. For this reason, fibroblasts were exposed to anti-Fas at different concentrations (100 ng/ml - 10 ug/ml) for 24-72 h. Detection of apoptosis was done morphologically by inverted phase contrast microscopy, PI staining, anneksin-V-FITC binding measured by flow cytometry. To detect proliferation, ~H? thymidine uptake assay was used. In this assay low count measurements are also an indication of apoptosis. All fibroblasts from any source have been shown to express Fas antigen by flow cytometry. Although anti-Fas-IgM induced apoptosis in Jurkat control cells, none of the methods revealed increased apoptosis in any type of fibroblasts after Fas stimulation. Instead, it resulted in increased proliferation as detected by H-* thymidine uptake assay. In further experiments we treated the fibroblasts for 24-72 h in appropriate concentrations with different cytokines and growth factors (IL-la, IL-1(3, IL-6, IL-10, IL- 17, TNF-a, IFN-y, TGF-0, GM-CSF, PDGF-AA, FGF) present in the rheumatoid synovium. Neither TNF-oc nor any other cytokine alone led to apoptosis. TNF-a, IFN-y and GM-CSF increased Fas expression markedly as measured by FC. However, stimulation of fibroblasts by anti-Fas even after pretreatment with these cytokines did not result in sensitivity to apoptosis. We conclude that anti-Fas IgM is not a reliable Fas inducing agent in human fibroblasts and that Fas receptor may play a role in transmitting growth signals as well as death signals to fibroblasts. Whether increased Fas expression by some cytokines results in higher sensitivity to proliferation by Fas stimulation is a subject of further studies. II