ACTIVATED MACROPHAGE CONDITIONED MEDIUM - IDENTIFICATION OF THE SOLUBLE FACTORS INDUCING CYTOTOXICITY AND THE L-ARGININE DEPENDENT EFFECTOR MECHANISM

Conditioned medium (CM) from cultures of cytotoxic activated macrophages causes inhibition of mitochondrial respiration, DNA synthesis, and aconitase activity in murine EMT-6 mammary adenocarcinoma cells by an L-arginine dependent effector mechanism. CM induces cytotoxicity and nitrite synthesis in EMT-6 cells in a dose dependent manner. We have identified the soluble factors in CM that induce cytotoxicity and synthesis of inorganic nitrogen oxides from L-arginine by EMT-6 cells. Using functional inhibition experiments, the activity of lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma) in CM was investigated. The LPS inhibitor polymyxin B and TNF-alpha antibody produced a modest decrease in nitrite production, while IFN-gamma antibody markedly inhibited both nitrite production and cytostasis. Simultaneous treatment with polymyxin B, TNF-alpha antibody, and IFN-gamma antibody reduced EMT-6 cell nitrite production by 81%, and cytostasis by 74%. By Western blot, IFN-gamma and TNF-alpha were shown to be present in CM. When CM was subjected to hydrophobic interaction chromatography, a single peak of activity was eluted, and Western blot showed that the active fractions contained IFN-gamma. Furthermore, IFN-gamma antibody neutralized the activity in these chromatographic fractions. We conclude that induction of inorganic nitrogen oxide synthesis from L-arginine by the synergistic combination of IFN-gamma, TNF-alpha, and LPS accounts for most of the biologic activity of CM, and that IFN-gamma is the major priming factor.