Cisplatin and interferon-gamma treated murine macrophages induce apoptosis in tumor cell lines

Macrophages, treated in vitro with a combination of cisplatin and interferon (IFN)-gamma, have been shown to develop enhanced tumoricidal activity against a number of tumor cell types, through mechanisms which remain largely unknown. In the present study, we have investigated the mechanism involved in the tumor cell cytotoxicity mediated by cisplatin and lFN-gamma treated macrophages, and the effector molecules involved therein. Peritoneal macrophages treated with cisplatin and IFN-gamma, when co-cultured with different tumor cell types, caused tumor cell death by induction of apoptosis. Evidence for this was provided by percent specific DNA fragmentation assay, by specific pattern of internucleosomal DNA fragmentation detected by agarose gel electrophoresis and by microscopic examination of the cells, which revealed nuclear alterations, characteristic of apoptosis. The time kinetics studies of DNA fragmentation, loss in cell viability and apoptotic cell population showed linearity with time; most of the cells that underwent apoptosis were found to be viable even after 24 h co-culture. Macrophages induced apoptosis in tumor targets even in the absence of cell-to-cell contact, i.e. via diffusible effector molecules. In P815 cells, NO produced by cisplatin and IFN-gamma treated macrophages was found to induce apoptosis as addition of N(G)-monomethyl L-arginine (L-NMMA), a specific inhibitor of NO synthase to the co-culture, prevented apoptosis in P815 cells. Further, direct treatment of P815 cells with the NO donor, sodium nitroprusside (SNP), resulted in apoptosis. In L929 cells, the effector molecule was found to be tumor necrosis factor (TNF)-alpha as apoptosis was blocked by the addition of anti-TNF-alpha antibodies to the co-culture but the addition of L-NMMA or SNP had no effect. The study thus shows that cisplatin and IFN-gamma treated macrophages can kill tumor cells by extracellular release of effector molecules which act by inducing apoptosis in a target cell-specific manner.