Perforin and Fas/Fas ligand-mediated cytotoxicity in acute and chronic woodchuck viral hepatitis

The Fas ligand (FasL)/Fas and the perforin-granzyme cytotoxic pathways pres umably play a central role in the development of hepatocellular injury in v iral hepatitis. To recognize the potential contribution of Fast and perfori n-based cell killing in hepadnaviral infection, we adopted a cytotoxic assa y using murine Fas(+) P815 and human Fas(-) K562 cells as targets. Freshly isolated peripheral blood mononuclear cells (PBMC) from woodchucks with new ly acquired woodchuck hepatitis virus (WHV) infection (n = 6), with chronic WHV hepatitis (n = 9), and from healthy animals (n = 11) were used as effe ctor cells. We have found that woodchuck lymphoid cells kill cell targets v ia both the FasL/Fas and the perforin death pathways. The contribution of F as-dependent cytolysis was ascertained in blocking experiments with anti-Fa s antibody and by incubation of PBMC with cyclohexamide to prevent de novo synthesis of Fast. The involvement of the perforin pathway was confirmed by treatment of K562 cells with colchicine to inhibit the microtubule-depende nt perforin release. Comparative analysis showed that peripheral lymphoid c ells from acute WHV hepatitis, but not those from chronic WHV infection, ar e more cytotoxic and that this increase seems to be entirely due to activat ion of perforin-mediated killing. The data indicate that acute infection in woodchucks is associated with the augmented capacity of lymphoid cells to elicit perforin-dependent killing, but in chronic infection, independent of the severity of liver disease and duration of chronicity, these cells have the same or lower cytotoxic potential as PBMC from healthy controls. These findings suggest a role for non-specific cellular immunity, presumably nat ural killer (NK) cells, in the control of early WHV infection and in the pr ogression of chronic hepatitis.