B cell lymphoma-2 transfected P815 cells resist reactive nitrogen intermediate-mediated macrophage-dependent cytotoxicity

Abstract

Activated murine peritoneal macrophage cytotoxicity against P815 tumor cells has been shown to be mediated by the reactive nitrogen intermediates (RNI) produced by macrophages from L-arginine through nitric oxide (NO) synthase. Previous results from this laboratory indicated that NO-dependent killing of P815 fulfilled the criteria for apoptotic death. Work by others, in turn, demonstrated that the product of the bcl-2 gene confers protection against various inducers of apoptosis, including reactive oxygen intermediates. Experiments were performed to determine whether Bcl-2 could equally protect sensitive cells from RNI-dependent apoptosis within the context of a relevant biologic system such as the delivery of such RNI by activated macrophages. Results demonstrated that transfection of P815 cells with the human bcl-2 gene confers immunity from RNI-dependent, macrophage-mediated cytotoxicity. In contrast with wild-type or mock-transfected P815 cells, which do not contain detectable Bcl-2, bcl-2-transfected cells showed minimal DNA fragmentation and cell membrane failure when cocultured with activated macrophages. Additional findings indicate that Bcl-2 affords the transfected cells almost complete resistance to the DNA-fragmenting effects of chemically generated NO or H202 and partial protection from their cytolytic effects. These findings are consistent with the hypothesis that tumor cells expressing bcl-2 may escape destruction by macrophage-dependent immune surveillance mechanisms.