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

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Abstract
Interferon-γ (IFN-γ) 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-γ 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 5lCr 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-γ resulted in activation of macrophages for tumor cell lysis. IFN-γ alone also activated macrophages for NO production under identical conditions. Addition of LPS along with IFN-γ resulted in synergism in the activation of macrophages for both cytolysis and NO production. LPS contamination of the IFN-γ preparation was absent as evidenced by the following criteria: (1) the IFN-γ 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-γ to activate 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-γ preparation with a specific endotoxin neutralizing protein did not abrogate the ability of IFN-γ to induce macrophage activation, and (4) heat treatment of solutions containing IFN-γ alone or IFN-γ + LPS abolished only the effect of IFN-γ, not that of LPS. A comparison of the IFN-γ responsiveness in the peritoneal macrophages from C57BL/6 and Balb/c macrophages revealed that C57BL/6 macrophages are more responsive to IFN-γ. These results demonstrate that IFN-γ alone is capable of stimulating macrophages for tumor cell killing and NO production and that the magnitude of IFN-γ responsiveness varies with the strain of mice studied.

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