Characterization of CD95 ligand (CD95L)-induced apoptosis in human tenon fibroblasts

Toxic side effects of cytotoxic agents such as 5-fluorouracil or mitomycin-C in glaucomatous filtering procedures call for alternative approaches to control fibroblast proliferation. CD95L is a death ligand that triggers apoptosis in susceptible target cells. Apoptosis allows for the safe disposal of cells without damaging the surrounding tissue. The goal of this study was to characterize and to evaluate the CD95L induced cell death in cultured Tenon fibroblasts. Human Tenon fibroblasts were treated with different concentrations of CD95L. For comparison, murine NIH 3T3 fibroblasts were used. Immunohistochemistry and Western blot were used to investigate the CD95 and CD95L expression. Cytotoxicity was measured by crystal violet assay. Apoptosis was investigated using in situ DNA end labelling (TUNEL). DEVD-AMC caspase 3 like activity was measured and caspase 3 processing was studied by immunoblot and the use of the caspase inhibitor DEVD-CHO in cell culture assays. Tenon and NIH 3T3 fibroblasts express CD95 and CD95L. The authors found concentration dependent inhibition of proliferation after CD95L treatment. Tenon fibroblasts, but not NIH 3T3 fibroblasts, show synergy when combined with actinomycin D or cyclohexamide. CD95L treatment did not alter total protein or RNA synthesis. Cell death induced by CD95L was apoptotic and activated caspase 3, as TUNEL positive cells and the active fragment of caspase 3 were found. CD95L induced cell death could be inhibited by the caspase-inhibitor.Here, it is demonstrated that the CD95L induced cell death in cultured human Tenon fibroblasts is apoptotic and possibly mediated by the caspase 3 pathway. These results suggest that it may be possible to use CD95L in glaucomatous filtering procedures. In vivo studies are necessary for further evaluation.