The effect of type I and II interferons on human fetal retinal pigment epithelium-induced apoptosis in Jurkat T cells.

PURPOSE: To examine the regulatory effects of interferon (IFN)-alpha, IFN-gamma, transforming growth factor (TGF)-beta, and tumor necrosis factor (TNF)-alpha on human fetal retinal pigment epithelial (HFRPE) cell-induced apoptosis of human Jurkat T (Jkt) cells. METHODS: Pure cultures of HFRPE cells were isolated. The cells were precultured with medium alone or with addition of IFN-alpha, IFN-gamma, TNF-alpha, or TGF-beta for 72 hours. Thereafter, HFRPE cells were extensively washed before they were cocultured jointly with Jkt cells (standard) or cultured alone for another 48 hours to accumulate conditioned medium that is collected and added as cell-free conditioned medium to Jkt cell cultures (supernatant). Jkt cells were cocultured under the two culture conditions for 48, 72, and 96 hours. The rate of apoptosis in Jkt T cells was determined with annexin V staining and flow cytometry. RESULTS: Both IFN-alpha and -gamma upregulated HFRPE-induced apoptosis in Jkt T cells. However, the apoptosis induced by IFN-alpha-activated HFRPE cells was significant only in the absence of cell-cell contact (supernatant). The supernatant induced a higher rate of apoptosis in Jkt T cells when compared to the direct coculture of the cells. TGF-beta and TNF-alpha did not upregulate HFRPE-induced apoptosis in Jkt T cells. CONCLUSIONS: These results indicate that type I and type II IFNs can upregulated HFRPE-induced apoptosis in Jkt T cells, IFN-gamma being the more effective cytokine. Neither, TGF-beta nor TNF-alpha upregulated the HFRPE-induced apoptosis in Jkt T cells. Although HFRPE-induced apoptosis was mediated in a cell-cell-contact-independent pathway, HFRPE cells may also express membrane-bound antiapoptotic molecules. These findings may help us to understand better the modulatory effects of pro- and anti-inflammatory cytokines on immune suppressive characteristics of RPE cells.

Indocyanine green induces apoptosis in human retinal pigment epithelial cells.

PURPOSE: To examine whether indocyanine green (ICG) dye induces apoptosis in human retinal pigment epithelial (RPE) cells. DESIGN: Laboratory investigation. METHODS: Pure cultures of human RPE cells were isolated. Retinal pigment epithelial cells were incubated with different concentrations of ICG dye (1 mg/ml, 5 mg/ml, or 20 mg/ml) for 30 minutes. The rate of RPE cell apoptosis was assessed with Annexin V-FITC staining and propidium iodide (PI) by flow cytometry. RESULTS: Retinal pigment epithelial cells maintained their monolayer morphology after incubation with ICG dye. However, ICG induced a statistically significant amount of apoptosis in RPE cells at all the concentrations (1 mg/ml, 5 mg/ml, and 20 mg/ml) after 30 minutes of incubation (P <.05). The solvent solution alone (without the ICG dye) did not induce any significant apoptosis in RPE cells, when compared with culture medium. CONCLUSIONS: The incubation of RPE cells with ICG dye increased the number of apoptotic RPE cells in vitro. Our findings indicate that the decision over the intravitreal application of ICG dye needs to be made with caution.

Trypan blue induces apoptosis in human retinal pigment epithelial cells.

PURPOSE: To examine whether trypan blue dye induces apoptosis in human retinal pigment epithelium cells. DESIGN: Laboratory investigation. METHODS: Pure cultures of human retinal pigment epithelium cells were isolated. The cells were incubated with different concentrations of trypan blue (0.5%, 0.10%, and 0.05%) for either 5 or 30 minutes. The rate of retinal pigment epithelium cell apoptosis was assessed with Annexin V-PE staining and flow cytometry. RESULTS: Trypan blue induced a statistically significant amount of apoptosis in retinal pigment epithelium cells at all the concentrations (0.5%, 0.10%, and 0.05%) (P <.05). The increase in incubation time (from 5 to 30 minutes) led to an increase in the number of apoptotic retinal pigment epithelium cells. CONCLUSION: The incubation of retinal pigment epithelium cells with trypan blue increased the number of apoptotic retinal pigment epithelium cells in vitro. Our results suggest that decisions regarding the intravitreal application of trypan blue dye need to be made with caution.

Human fetal retinal pigment epithelium induces apoptosis in human T-cell line Jurkat which is independent from its expression of TRAIL.

PURPOSE: To evaluate whether human fetal retinal pigment epithelial (HFRPE) cells express TRAIL (tumor necrosis factor related apoptosis inducing ligand). The role of TRAIL in HFRPE induced apoptosis was evaluated. METHODS: Pure cultures of HFRPE cells were isolated. The expression of TRAIL protein and mRNA in non-activated and IFN-gamma activated HFRPE cells was evaluated with RT-PCR. The role of TRAIL in HFRPE induced apoptosis was assessed by incubating HFRPE cells with human T-cell leukemia line Jurkat (Jkt) in the presence or absence of neutralizing TRAIL antibodies. Cultures were pulsed with [(3)H]-thymidine to measure Jkt cell proliferation. The role of TRAIL was further examined by western blott evaluating the cleavage of caspases 8 and 10 in Jkt cells after their incubation with HFRPE cells. RESULTS: HFRPE cells expressed TRAIL mRNA. The expression of TRAIL mRNA and protein was up-regulated by IFN-gamma activation. However, anti-TRAIL antibodies were not able to prevent the HFRPE induced suppression of Jkt cell proliferation. The caspases 8 and 10 were also not cleaved in Jkt cells after their incubation with IFN-gamma activated HFRPE cells. CONCLUSIONS: Although HFRPE cells express TRAIL and its expression is upregulated by IFN-gamma activation, TRAIL is not involved in HFRPE induced apoptosis in Jkt cells. Currently the role of TRAIL in HFRPE cells is under investigation.