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J Yang, G Peng, N Agarwal, RV Patil, MB Wax; Apoptosis of Retinal Ganglion Cells Induced by hsp60 or hsp27-activated T lymphocytes in rat . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3384.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We sought to investigate the effects of heat shock protein antigen-activated rat T cells on the retinal ganglion cell (RGC) death using the co-cultures of the rat RGCs with hsp60 or hsp27 -activated T cells. Methods:: An immortalized cell line of rat RGCs (clone RGC-5) was grown in DMEM. The hsp27 or hsp60 specific T cell lines were obtained from draining lymph nodes in Lewis rats immunized with hsp27 or hsp60 antigen and incomplete Freund's adjuvant (IFA) one month prior to sacrifice. Isolated T cells were subsequently stimulated by hsp60 or hsp27, respectively, for several cycles in suspension culture. T cells and RGCs were then co-cultured in the presence of hsp antigens for 12, 24, 48 hours in DMEM. Apoptotic cell death was assessed in the RGCs grown in the co-cultures by DNA ladder formation using agarose gel electrophoresis , and also apoptotic cells were stained by APO-BRDU TM kit and analyzed by Flow Cytometry. Results:The clear DNA ladder formation characteristic of apoptotic cell death was observed in RGCs co-cultured for 24h with T cells activated by hsp60 or hsp27, and the apoptotic cells were 10% and 6% respectively by FACS. However, control groups (activated with BSA and IFA) did not induce apoptosis of RGCs. In addition, supernatant from hsp60-activated T cells induced RGC apoptosis, but supernatants from hsp27-activated T cells and the control groups did not. No DNA ladder formations or apoptotic cells were found when selected cytokines were used to stimulate RGCs alone. Conclusion:Hsp60 or hsp27 activated T cells induced apoptosis of RGC cells by direct interaction, however, the supernatant from hsp60-activated T cells also induced apoptosis of RGC cells. Further studies are in progress to identify the specific pathways involved in RGC apoptosis mediated by hsp activated T cells.
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