Human petal retinal pigment epithelium-induced cell cycle arrest, loss of mitochondrial membrane potential and apoptosis

PURPOSE. To investigate the mechanism of action of the soluble immune suppr essive product secreted by human fetal retinal pigment epithelial (HFRPE) c ells in a model system using the human T-cell line Jurkat (Jkt). METHODS. Pure HFRPE cells were isolated and cultured. The supernatants of b oth nonactivated and IFN-gamma -activated HFRPE cells were isolated. Cells from the human T-cell line Jkt were incubated either in standard culture me dium or in the supernatant isolated from HFRPE cells. in the first assay Jk t cell proliferation was measured by [H-3]thymidine incorporation. In the s econd assay Jkt cell apoptosis was examined for annexin V staining by now c ytometry. In the third assay Jkt cell division was evaluated with carboxyfl uorescein succinimidyl ester (CFSE) fluorescent dye. In the last assay the mitochondrial transmembrane potential of Jkt cells was measured with the ca tionic lipophilic fluorochrome 3,3'-dihexyloxacarbocyanine iodide [DiOC(6)] . In all the assays the effect of supernatants isolated from both nonactiva ted and IFN-gamma -activated HFRPE cells were compared with standard cultur e medium. The involvement of antiapoptotic human gene bcl-x(L) was determin ed by using a Jkt cell line that was stably transfected with bcl-x(L). RESULTS. The supernatant isolated from HFRPE cells significantly suppressed the cell division in Jkt cells and induced apoptosis. These effects were s tronger when the supernatant was isolated from IFN-gamma -activated HFRPE c ells. The apoptosis pathway induced by the secreted product of HFRPE cells involved the early disruption of mitochondrial transmembrane potential. Alt hough the overexpression of bcl-x(L), gene rescued the Jkt cells from super natant-induced apoptosis, it could not restore the proliferation of Jkt cel ls. CONCLUSIONS. These data suggest that HFRPE cells secrete a product that ini tiates an early cell cycle arrest in the human T-cell line Jkt, which is fo llowed by the activation of an apoptotic pathway that involves the loss of mitochondrial membrane potential. The latter could be prevented by bcl-x(L) overexpression. Also these data suggest that the HFRPE-induced T-cell apop tosis may play a significant role in maintaining the immune privilege in th e subretinal space.