Abstract
Purpose: :
Our previous results showed that both caspase-dependent and caspase-independent apoptosis are involved in retinal damage during MCMV retinitis and that TNF- is one of the apoptotic inducers. The aim of this study was to determine viral replication and apoptosis in immunosuppressed (IS) TNF--/-mice.
Methods: :
IS (2.0 mg methylprednisolone acetate every 3 days beginning on day -2 ) TNF--/- mice or wild type C57BL/6 mice were inoculated with 5 x 104 PFU of MCMV k181 via the supraciliary route. Injected eyes were collected at several times post inoculation, examined by plaque assay for replicating virus, real time RT-PCR for apoptotic genes, western blot for caspase 3, caspase 8, caspase 12, Bid, c-flip, and iNOS, and by staining for MCMV early antigen (EA) or for apoptosis by TUNEL.
Results: :
Although the titer of MCMV was similar in both groups, significantly more apoptotic cells were observed in the retina of IS TNF--/- mice than in that of wild type mice. The level of active caspase 3 protein detected by western blot in the injected eyes of both groups was also similar; however, more active proteins for genes involved in the mitochondrial pathway (cleaved caspase 8, tBid) and less anti-apoptotic protein, c-Flip, were detected in the MCMV infected TNF--/- eyes compared to the level of proteins in the eyes of wild type mice. In addition, the levels of caspase 12 (both mRNA and active proteins) and iNOS protein were higher in the injected eyes of IS TNF--/- mice than in those of IS wild type mice.
Conclusions: :
During MCMV retinitis, TNF- appears to be an inducer, as well as an inhibitor, of apoptosis. These results suggest the following anti-apoptotic mechanism: TNF- activation of NF-ΚB promotes production of the anti-apoptosis gene, c-flip, which, in turn, inhibits activation of caspase 8 and the mitochondrial pathway. TNF- also may prevent overexpression of iNOS which suppresses nitric oxide-induced endoplasmic reticulum stress and subsequent activation of caspase 12.
Keywords: apoptosis/cell death • cytomegalovirus • retinitis