Direct Activation of Murine Peritoneal Macrophages for Nitric Oxide Production and Tumor Cell Killing by Interferon-γ

Abstract

Interferon-gamma (IFN-gamma) is known to prime macrophages for tumor cell lysis and nitric oxide (NO) production as measured by enhanced sensitivity to lipopolysaccharide (LPS). In the present study, the ability of IFN-gamma to directly activate peritoneal macrophages from C57BL/6 and Balb/c mice for tumor cytotoxicity and NO production was evaluated. Macrophage-mediated tumor cell killing was measured by an 18 h 51Cr release assay using P815 mastocytoma cells as targets. Concurrent NO production was measured as nitrite in the supernatants of macrophage cultures. Incubation of macrophages with IFN-gamma resulted in activation of macrophages for tumor cell lysis. IFN-gamma alone also activated macrophages for NO production under identical conditions. Addition of LPS along with IFN-gamma resulted in synergism in the activation of macrophages for both cytolysis and NO production. LPS contamination of the IFN-gamma preparation was absent as evidenced by the following criteria: (1) the IFN-gamma preparation as well as the reagents used were shown to be free of LPS contamination based on LAL endotoxin tests (sensitivity 25 pg/ml), (2) the ability of IFN-gamma to activated macrophages was not abrogated by prior treatment of the cytokine with polymyxin B, whereas the effect of LPS was inhibited (70-100%) under similar conditions, (3) pretreatment of the IFN-gamma preparation with a specific endotoxin neutralizing protein did not abrogate the ability of IFN-gamma to induce macrophage activation, and (4) heat treatment of solutions containing IFN-gamma alone or IFN-gamma+LPS abolished only the effect of IFN-gamma, not that of LPS.(ABSTRACT TRUNCATED AT 250 WORDS)