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Invest Ophthalmol Vis Sci 2002;43: E-Abstract 3384.
© 2002 ARVO

Apoptosis of Retinal Ganglion Cells Induced by hsp60 or hsp27-activated T lymphocytes in rat

J Yang1, G Peng2, N Agarwal3, RV Patil4 and MB Wax5

1 Dept of Ophthalmology & Visual Sciences Washington University School of Medicine St Louis MO
2 Dept of Ophthalmology & Visual Sciences Washington University School of Medicine St louis MO
3 Dept of Pathology and Anatomy UNT Health Science Center Fort Worth TX
4 Dept of Ophthalmology University of Nebraska Medical Center Omaha NE
5 Dept of Ophthalmology & Visual Sciences Washington University School of Medicine St Louis MO

Commercial Relationships:  J. Yang, None; G. Peng, None; N. Agarwal, None; R.V. Patil, None; M.B. Wax, None.
Grant Identification: EY12314(MBW)

Abstract

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.

Keywords: 415 ganglion cells • 435 immunomodulation/immunoregulation • 323 apoptosis/cell death

© 2002, The Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any part of this abstract, contact the ARVO Office at arvo{at}arvo.org.




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