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.