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042 HEPADNAVIRAL INFECTION ENHANCES HEPATOCYTE-MEDIATED CELL KILLING DEPENDENT UPON BOTH CD95 LIGAND AND PERFORIN PATHWAYS CS Guy, SL Rankin, J Wang, TI Michalak We have recently shown that normal primary and cultured hepatocytes display CD95 ligand (CD95L) and can eliminate other cells via the CD95L-CD95 pathway, which is modulated by IFNgamma or TNFalpha. However, it was unknown whether hepatocytes can also eliminate other cells via the perforin/granzyme B mechanism or if hepadnaviral infection can modify hepatocyte-mediated cell killing. For this purpose, perforin and granzyme B mRNA expression was evaluated in freshly isolated woodchuck and human hepatocytes and in liver cell lines, while perforin protein was probed by Western blotting. In bioassays, hepatocytes were used as cytotoxic effectors against CD95-deficient K562 cells or CD95-bearing P815 targets. The results showed that hepatocytes constitutively transcribe perforin and granzyme B, display perforin protein, and mediate lysis of CD95L-deficient K562 cells which was calcium and microtubule-dependent, all indicative of the existence of a perforin-dependent cell killing pathway. In contrast with CD95L, perforin-dependent killing was unaffected by IFNgamma or TNFalpha. Hepatocytes from woodchucks recovered from acute WHV infection or with chronic hepatitis were more cytotoxic than those from healthy animals. In culture, hepatocytes were able to kill other hepatocytes, but those transcribing the complete WHV genome or the S gene, but not C or X genes, were significantly more resistant to hepatocyte-mediated killing. The data suggest that hepatocytes can induce death of other cells by both CD95L and perforin dependent mechanisms which are differentially regulated by cytokines and hepadnaviral infection. Hepadnaviral proteins may protect infected hepatocytes from fratricidal or autocidal cell lysis.
Molecular Virology and Hepatology Research, Faculty of Medicine, Memorial University, St John's, Newfoundland