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Previous studies have shown that cytotoxic activated macrophages cause inhibition of DNA synthesis, of mitochondrial respiration, and of aconitase activity in tumor target cells. An L-arginine-dependent biochemical pathway synthesizing L-citrulline and nitrite, coupled to an effector mechanism, is now shown to cause this pattern of metabolic inhibition. Murine cytotoxic activated macrophages synthesize L-citrulline and nitrite in the presence of L-arginine but not D-arginine. L-Citrulline and nitrite biosynthesis by cytotoxic activated macrophages is inhibited by NG-monomethyl-L-arginine, which also inhibits this cytotoxic effector mechanism. This activated macrophage cytotoxic effector system is associated with L-arginine deiminase activity, and the imino nitrogen removed from the guanido group of L-arginine by the deiminase reaction subsequently undergoes oxidation to nitrite. L-Homoarginine, an alternative substrate for this deiminase, is converted to L-homocitrulline with concurrent nitrite synthesis and similar biologic effects.

An interferon-'y, tumor necrosis factor, and interleukin-1-inducible, high-output pathway synthesizing nitric oxide (NO) from L-arginine was recently identified in rodents. High-dose interleukin-2 (IL-2) therapy is known to induce the same cytokines in patients with advanced cancer. Therefore, we examined renal cell carcinoma (RCC; n = 5) and malignant melanoma (MM; n = 7) patients for evidence of cytokine-inducible NO synthesis. Activity of this pathway was evaluated by measuring serum and urine nitrate (the stable degradation product of NO) during IL-2 therapy. IL-2 administration caused a striking increase in NO generation as reflected by serum nitrate levels (10-and 8-fold increase IP < 0.001, P < 0.0031 for RCC and MM patients, respectively) and 24-h urinary nitrate excretion (6.5-and 9-fold increase [both P < 0.0011 for RCC and MM patients, respectively). IL-2-induced renal dysfunction made only a minor contribution to increased serum nitrate levels. Metabolic tracer studies using L-[guanidino-5N2Jarginine demonstrated that the increased nitrate production was derived from a terminal guanidino nitrogen atom of L-arginine. Our results showing increased endogenous nitrate synthesis in patients receiving IL-2 demonstrate for the first time that a cytokine-inducible, highoutput L-arginine/NO pathway exists in humans. (J. Clin. Invest. 1992. 89:867-877.) Key words: acute renal failure * malignant melanoma * nitrate * nitrite * renal cell carcinoma

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Treatment of EMT-6 mammary adenocarcinoma cells with gamma interferon (rMuIFN gamma) plus tumor necrosis factor (rMuTNF alpha) and/or interleukin-1 (rHuIL-1 alpha) 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 rMuIFN gamma. 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.

McCoy cells, murine-derived cells commonly used for propagation of chlamydiae, were found to be efficient producers of nitric oxide (NO) when primed with murine gamma interferon (IFN-y) and then exposed to the second signals provided by Escherichia coli lipopolysaccharide, human interleukin-la, murine tumor necrosis factor a, or Chlamydia trachomatis type H. Murine recombinant IFN-y over a range of 0 to 50 U/mi inhibited infectivity of C. trachomatis type H in a dose-dependent fashion in McCoy cells while simultaneously inducing NO production. Quantitation of infectious chiamydia progeny remaining in McCoy cells 48 or 72 h postinfection revealed that IFN-y-primed McCoy cells reduced chlamydial inclusion-forming units (expressed as units per milliliter) by 4 log1o units at higher IFN-y concentrations (50 U/ml) compared with control values. The magnitude of this antichlamydial effect was directly related to increased synthesis of NO, the production of which was IFN-y dose dependent. The antichlamydial effects of IFN-y were blocked in a dose-dependent manner by the addition of N-guanidino-monomethyl L-arginine (MLA), an inhibitor of nitric oxide synthesis. These results suggest that although IFN-y priming of McCoy cells is required for antichlamydial activity, nitric oxide is a necessary effector molecule involved in the mechanism(s) of IFN-y-induced inhibition of chlamydial proliferation in this murine cell line. The ability to block the potent antichlamydial effects of IFN-y by inhibition of a specific enzyme, nitric oxide synthase, may give insights into mechanisms by which IFN-y and perhaps other cytokines are able to control proliferation of chlamydiae and other intracellular pathogens.

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.

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, nitrate, 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.