Read R. Taintor

Nitric oxide: A cytotoxic activated macrophage effector molecule

The experiments reported here identify nitric oxide as a molecular effector of activated macrophage induced cytotoxicity. Cytotoxic activated macrophages synthesize nitric oxide from a terminal guanidino nitrogen atom of L-arginine which is converted to L-citrulline without loss of the guanidino carbon atom. In addition, authentic nitric oxide gas causes the same pattern of cytotoxicity in L10 hepatoma cells as is induced by cytotoxic activated macrophages (iron loss as well as inhibition of DNA synthesis, mitochondrial respiration, and aconitase activity). The results suggest that nitric oxide is the precursor of nitrite/nitrate synthesized by cytotoxic activated macrophages and, via formation of iron-nitric oxide complexes and subsequent degradation of iron-sulfur prosthetic groups, an effector molecule.

Iron depletion: Possible cause of tumor cell cytotoxicity induced by activated macrophages

The experiments reported here provide a possible molecular mechanism for the activated macrophage cytotoxic effect. Tumor cells that develop cytostasis and inhibition of mitochondrial respiration in response to cocultivation with activated macrophages release a significant fraction of their intracellular iron-59 content. Kinetic studies show that specific release of iron-59 from target cells begins 4-6 hours after initiating cocultivation which is the time point that inhibition of DNA synthesis is first detected. Treatment of tumor cells with metabolic inhibitors causing inhibition of respiration, protein synthesis, RNA synthesis, and DNA synthesis to a similar or greater extent than that caused by activated macrophages does not induce release of intracellular iron-59. It is significant that mitochondrial respiration and DNA replication, both strongly inhibited in target cells by activated macrophages, are metabolic pathways with enzymatic activity vulnerable to inhibition by depletion of intracellular iron.

Cytokines induce an L-arginine-dependent effector system in nonmacrophage cells

Treatment of EMT‐6 mammary adenocarcinoma cells with gamma interferon (rMuIFNγ) plus tumor necrosis factor (rMuTNFα) and/or interleukin‐1 (rHuIL‐1α) causes release of iron‐55 label, inhibition of DNA replication, and inhibition of aconitase activity. In addition, the same combinations of cytokines induce EMT‐6 cells to synthesize L‐citrulline, nitrite, and nitrate directly from L‐arginine. Lipopolysaccharide (LPS) can act as a cofactor in the induction of these metabolic effects when added to EMT‐6 cells in the presence of rMuIFNγ. The results show that increased levels of cytokines in the microenvironment can induce a novel effector pathway in somatic cells not specialized for host defense, resulting in specific metabolic effects as well as the inhibition of cellular proliferation.

The L-arginine dependent effector mechanism is induced in murine adenocarcinoma cells by culture supernatant from cytotoxic activated macrophages.

Culture medium conditioned by incubation with murine cytotoxic activated macrophages causes release of iron‐55 label from viable murine EMT‐6 tumor cells as well as inhibition of DNA replication and aconitase activity. These metabolic changes occur in parallel with L‐citrulline, nitrite, and nitrate synthesis from L‐arginine by EMT‐6 cells. Protein synthesis is required for activation of this effector mechanism. Once the effector pathway is induced in EMT‐6 cells in the presence of amino acids, L‐arginine is the only amino acid required for its function. Arginase inhibits the effector mechanism, which is additional evidence for its specific L‐arginine requirement. The results show induction, in a non‐macrophage cell line, of a novel effector pathway which, in addition to other effects, inhibits cellular proliferation.

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 (TNFα), and interferon gamma (IFNγ) in CM was investigated. The LPS inhibitor polymyxin B and TNFα antibody produced a modest decrease in nitrite production, while IFNγ antibody markedly inhibited both nitrite production and cytostasis. Simultaneous treatment with polymyxin B, TNFα antibody, and IFNγ antibody reduced EMT‐6 cell nitrite production by 81%, and cytostasis by 74%. By Western blot, IFNγ and TNFα 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 IFNγ. Furthermore, IFNγ 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 IFNγ, TNFα, and LPS accounts for most of the biologic activity of CM, and that IFNγ is the major priming factor.

[34] Activated macrophage-mediated cytotoxicity: Use of the in Vitro cytotoxicity assay for study of bioenergetic and biochemical changes that develop in tumor target cells

Publisher Summary This chapter discusses the use of in vitro cytotoxicity assay for the study of bioenergetic and biochemical changes that develop in tumor target cells. For macrophages to induce a cytotoxic effect on target cells, they must undergo functional change. This functional change is described as “activation.” Macrophage activation is a process of differentiation resulting from a complex interaction of environmental chemical signals that either increase or decrease the probability of expression of macrophage-mediated cytotoxicity for target cells. Cytotoxic activated macrophages do not cause rapid or global metabolic disorganization in tumor target cells. The target cells of cytotoxic activated macrophages slowly develop metabolic inhibition, and the metabolic inhibition in many tumor cell lines is completely reversible. As it is possible to remove target cells from the monolayers of cytotoxic activated macrophages, the target cells can be used for bioenergetic or biochemical analysis. Macrophages representing different functional states can be isolated from appropriate groups of mice and can also be generated in vitro by exposing the macrophages to appropriate stimuli.