Purpose: To investigate whether Fas ligand (FasL) and the Fas receptor system mediates apoptosis in cultured human retinal pigment epithelial (hRPE) cells and contributes to oxidant-induced death of hRPE cells.
Methods: Expression of FasL and Fas in cultured hRPE cells was examined by Western blot analysis and flow cytometry. The susceptibility of hRPE cells to Fas-mediated apoptosis was determined by incubating cells with recombinant soluble Fas ligand (sFasL). Characteristics of apoptosis assessed included chromatin condensation, DNA cleavage, and phosphatidylserine exposure. To investigate the possible involvement of Fas-mediated apoptosis in oxidative killing of hRPE cells, the effects of the oxidant tert-butylhydroperoxide (tBH) on the expression of FasL and Fas were studied. The specificity of effects of oxidant was examined using the antioxidants glutathione and N-acetyl-L-cysteine (NAC). The requirement for the Fas pathway in tBH-induced apoptosis was investigated using an antagonistic anti-Fas antibody ZB4 that blocks the interaction between FasL and Fas.
Results: Cultured hRPE cells constitutively expressed FasL and Fas. Ligation of Fas receptor with recombinant sFasL triggered apoptosis in hRPE cells. tBH treatment of hRPE cells resulted in increased expression of FasL and Fas. Glutathione and NAC completely abrogated tBH-induced increase in FasL and Fas expression and apoptosis. Blocking FasL and Fas interaction by ZB4 inhibited tBH-induced apoptosis, but only partially.
Conclusions: A functional Fas-mediated apoptotic pathway is present in cultured hRPE cells and can be activated with sFasL or by upregulation of FasL and Fas expression with an oxidant. The incomplete inhibition by blocking antibody indicates that the Fas pathway is involved in oxidant-induced apoptosis, but other triggering mechanisms are also important.