Heterocytolysis by macrophages activated by bacillus Calmette-Guérin: lysosome exocytosis into tumor cells.

The cytotoxic activity of activated macrophages against tumorigenic target cells appears to be mediated by lysosomal enzymes of activated macrophage origin. Lysosomes of activated macrophages are secreted directly into the cytoplasm of susceptible target cells, which subsequently undergo heterolysis. This reaction can be inhibited by agents which prevent the exocytosis of macrophage lysosomes (hydrocortisone) or which interfere with the action of lysosomal enzymes (trypan blue).

Macrophage nonimmunologic recognition: target cell factors related to contact inhibition.

Activated mouse macrophages were not cytotoxic to contact-inhibited nontumorigenic 3T3 fibroblasts, but caused marked destruction to non-contact-inhibited, tumorigenic 3T12 and simian virus 40-transformed fibroblasts. Nonimmunologic recognition and destruction of target cells by activated macrophages is independent of altered morphology, abnormal karyotype, and ability for continuous multiplication in vitro-all characteristics of 3T3 fibroblasts. A modification e f the target cell surface that results in a high in vitro saturation density, agglutinability by plant lectins, and tumorigenicity appears to evoke a cytotoxic response by activated macrophages.

Modulation of macrophage tumoricidal capability by components of normal serum: a central role for lipid.

The tumoricidal capabilities of macrophages can be reversibly inhibited by a lipoprotein of high molecular weight, and the inhibition appears to be reproduced by enrichment of macrophage plasma membranes with cholesterol. A second serum component of lower molecular weight enhances macrophage cytotoxicity. The presence of these components in normal serums suggests a physiological role for such factors in the regulation of macrophage function.

Macrophage cytotoxicity: role for L-arginine deiminase and imino nitrogen oxidation to nitrite.

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.

Control of carcinogenesis: a possible role for the activated macrophage.

Cytotoxic activity of activated mouse macrophages against mouse embryo fibroblasts was tested before and after spontaneous transformation of the fibroblasts in vitro. Activated macrophages caused little or no destruction of untransformed fibroblasts but were markedly cytotoxic to the same fibroblasts after spontaneous transformation. The efferent limb of this cytotoxic reaction appears to be nonimmunologic and to be related to abnormal growth properties rather than to the antigenic composition of target cells.

Macrophage tumor killing: influence of the local environment.

Tumor killing by activated macrophages is not a highly determined biologic event, but a relative capability influenced by the local environment. An intrinsic macrophage cytotoxic effector system is modulated by serum and other environmental factors that can either enhance or suppress tumor killing. Activated macrophages kill tumor cells only when a regulating threshold drops to a critically low level.

Murine cytotoxic activated macrophages inhibit aconitase in tumor cells. Inhibition involves the iron-sulfur prosthetic group and is reversible.

Previous studies show that cytotoxic activated macrophages cause inhibition of DNA synthesis, inhibition of mitochondrial respiration, and loss of intracellular iron from tumor cells. Here we examine aconitase, a citric acid cycle enzyme with a catalytically active iron-sulfur cluster, to determine if iron-sulfur clusters are targets for activated macrophage-induced iron removal. Results show that aconitase activity declines dramatically in target cells after 4 h of co-cultivation with activated macrophages. Aconitase inhibition occurs simultaneously with arrest of DNA synthesis, another early activated macrophage-induced metabolic change in target cells. Dithionite partially prevents activated macrophage induced aconitase inhibition. Furthermore, incubation of injured target cells in medium supplemented with ferrous ion plus a reducing agent causes near-complete reconstitution of aconitase activity. The results show that removal of a labile iron atom from the [4Fe-4S] cluster, by a cytotoxic activated macrophage-mediated mechanism, is causally related to aconitase inhibition.

Injury of neoplastic cells by murine macrophages leads to inhibition of mitochondrial respiration.

Cytotoxic activated macrophages (CM) inhibited the growth of neoplastic L1210 cells in vitro but L1210 cell death was minimal to nonexistent. L1210 cells injured by CM were separated from macrophages and studied in an isolated system. CM-injured L1210 cells had an absolute requirement for glucose or another glycolyzable hexose (mannose or fructose) for at least 40 h after removal from macrophages. If the culture medium lacked sufficient concentration of one of these sugars, CM-injured L1210 cells died within 4 h. Uninjured L1210 cells cultured alone or with peptone-stimulated macrophages had no such requirement and maintained complete viability in hexoseless medium. The hexose requirement of CM-injured L1210 cells could not be fulfilled by other naturally occurring monosaccharides, glucose or mannose derivatives, or substrates that can be oxidized by mitochondria. The concentration requirements for glucose, mannose, and fructose by CM-injured L1210 cells correlated with the concentrations required to support maximal glycolysis of these sugars by other murine ascites cells. A concentration of 2-deoxy-D-glucose which completely inhibited L1210 cell glycolysis also complete prevented the ability of glucose or mannose to maintain viability of CM-injured L1210 cells. Interaction with CM led to inhibition of L1210 cell mitochondrial oxidative phosphorylation. This was supported by the findings that: (a) CM-injured L1210 cells had no Pasteur effect; their rate of aerobic glycolysis was the same as the rate of anaerobic glycolysis of uninjured L1210 cells, (b) Endogenous respiration of CM-injured L1210 cells was 15% of normal. Maximal inhibition of uninjured L1210 cell respiration by a specific mitochondrial poison (oligomycin) was nearly the same (13% of normal). It followed that CM-injured L1210 cells required hexose for chemical energy production via the glycolytic pathway. CM-induced mitochondrial injury occurred in five other neoplastic cell lines tested.

Metabolic fate of L-arginine in relation to microbiostatic capability of murine macrophages.

L-arginine is required for the fungistatic action of murine macrophages in vitro. To further investigate this requirement, L-arginine metabolism by macrophages was measured under conditions where fungistasis either succeeded or failed. Macrophage fungistasis correlated with metabolism of L-arginine to citrulline, nitrite, and nitrate. The metabolic rate was dependent on extracellular L-arginine concentration, reaching a maximum of 67 nmol nitrite/h per mg protein. It accounted for one-third of arginine consumed by fungistatic macrophages. Equimolar amounts of citrulline and total nitrite plus nitrate accumulated in medium. This was consistent with the hypothesis that one of the equivalent guanidino nitrogens of L-arginine was oxidized to both nitrite and nitrate leaving L-citrulline as the amino acid reaction product. The analogue, NG-mono-methyl-L-arginine, selectively inhibited nitrogen oxidation and it was shown previously that it inhibited fungistatic capability. Resident macrophages were not fungistatic and their nitrogen oxidation was low. Once macrophages began producing nitrite/nitrate, protein synthesis was not required during the next 8 h for either fungistasis or nitrogen oxidation. Two-thirds of L-arginine consumption was due to macrophage arginase yielding L-ornithine and urea, which accumulated in medium. This activity was dissociated from macrophage fungistasis. Nitrogen oxidation metabolism by macrophages is linked to a mechanism that inhibits proliferation of fungi. This may involve synthesis of an intermediate compound(s) that has antimicrobial properties.

Specific amino acid (L-arginine) requirement for the microbiostatic activity of murine macrophages.

The microbiostatic action of macrophages was studied in vitro employing peritoneal cytotoxic macrophages (CM) from mice acting against Cryptococcus neoformans cultured in Dulbecco's medium with 10% dialyzed fetal bovine serum. Fungistasis was measured using electronic particle counting after lysis of macrophages with detergent. Macrophage fungistasis failed in medium lacking only L-arginine. Complete fungistasis was restored by L-arginine; restoration was concentration dependent, maximal at 200 microM. Deletion of all other essential amino acids did not abrogate fungistasis provided that L-arginine was present. Of twenty guanido compounds, including D-arginine, only three (L-arginine, L-homoarginine, and L-arginine methylester) supported fungistasis. Known activators or mediators of macrophage cytotoxicity (endotoxin, interferon gamma, tumor necrosis factor) did not replace L-arginine for CM-mediated fungistasis. The guanido analogue NG-monomethyl-L-arginine was a potent competitive inhibitor of CM-mediated fungistasis giving 50% inhibition at an inhibitor/L-arginine ratio of 1:27. Although CM completely blocked fungal reproduction via an L-arginine-dependent mechanism, the majority of the dormant fungi remained viable. Thus, this mechanism is viewed as a microbiostatic process similar or identical to the tumoristatic effect of macrophages. This suggests the production of a broad spectrum biostatic metabolite(s) upon consumption of L-arginine by cytotoxic macrophages.

Evidence for cytokine-inducible nitric oxide synthesis from L-arginine in patients receiving interleukin-2 therapy.

An interferon-gamma, 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 [P less than 0.001, P less than 0.003] for RCC and MM patients, respectively) and 24-h urinary nitrate excretion (6.5- and 9-fold increase [both P less than 0.001] 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-15N2]arginine 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, high-output L-arginine/NO pathway exists in humans.

Assembly and regulation of NADPH oxidase and nitric oxide synthase.

Research over the past year has revealed several interesting advances in the biosynthesis of the superoxide anion and nitric oxide. Highlights include the demonstration that the G protein Rac 2 is required for NADPH oxidase activation, the finding that nitric oxide is a feedback inhibitor of nitric oxide synthase isoforms, and the discovery that the continuous catalytic activity of the immune/inflammatory nitric oxide synthase is due to strong calmodulin binding, which is independent of elevated calcium levels. Interferon-gamma primes neutrophils and macrophages for both O2- and nitric oxide synthesis. However, NADPH oxidase and immune/inflammatory nitric oxide synthase are differentially regulated such that their activities are not simultaneously induced.

Role of nitric oxide synthesis in macrophage antimicrobial activity.

Research over the past 5 years has demonstrated that immunologic activation of mouse macrophages induces the activity of nitric oxide synthase, which oxidizes a guanidino nitrogen of L-arginine, yielding citrulline and the reactive radical, nitric oxide. A review of the biochemistry and immunologic regulation of this pathway in macrophages provides a backdrop against which to evaluate its effector functions. Reports published in the past 2 years suggest that synthesis of NO mediates much of the antimicrobial activity of mouse macrophages against some fungal, helminthic, protozoal and bacterial pathogens.

Enhanced macrophage tumoricidal activity and tumor suppression or regression caused by heat-killed Candida albicans.

The growth of line-10 hepatoma in male Sewall Wright strain 2 guinea pigs was totally suppressed when tumor cells were mixed with heat-killed Candida albicans. In a significant number of animals, injection of C. albicans into established tumors 10-12 mm in diameter caused complete, rapid tumor regression. Guinea pigs whose tumors regressed or were suppressed as a result of injection of C. albicans rejected subsequent challenges at distant sites with the line-10 hepatoma, which indicated the development of systemic immunity to the tumor. Untreated control guinea pigs had positive delayed hypersensitivity reactions to intradermally injected C. albicans, which suggested prior natural exposure of the animals to C. albicans antigens. Peritoneal macrophages from mice that had received ip injections of phosphate-buffered saline (PBS) or C. albicans were not cytocidal for mouse 3T12 tumor cells in vitro. However, macrophages from the mice given injections of C. albicans, unlike those from mice given PBS, were markedly tumoricidal in the presence of 1 ng or more endotoxin/ml in vitro. These results demonstrated that heat-killed C. albicans, when inoculated into the peritoneal cavity, increased the tumoricidal potential of peritoneal macrophages.

Macrophage nitric oxide synthesis delays progression of ultraviolet light-induced murine skin cancers.

The role of macrophages in the host immune response against cancers remains uncertain. Since nitric oxide synthesis represents a significant macrophage antitumor mechanism in vitro, we evaluated whether NO was synthesized during the immune response to growing murine skin cancers. NO synthesis was readily detectable in enzymatically dissociated tumors (RD-995 and LR-298) and was inhibited by N omega-monomethyl-L-arginine (MLA) and by macrophage depletion. Nitrosylation of iron-sulfur and heme complexes was observed in these tumors using electron paramagnetic resonance spectroscopy. NO production in the presence of increasing concentrations of MLA correlated inversely with tumor cell proliferation in vitro. To elucidate the role of NO during in vivo tumor progression, tumor-bearing mice were treated with continuous infusions of the nitric oxide synthase inhibitor MLA. MLA-treated mice demonstrated increased growth and delayed rejection of the highly antigenic UV radiation-induced regressor tumor LR-298. These experiments demonstrate that macrophage-derived NO synthesis can contribute to the antitumor immune response in vivo.

N omega -monomethyl-L-arginine inhibits nitric oxide production in murine cardiac allografts but does not affect graft rejection.

Endogenous nitric oxide biosynthesis in mice receiving allogeneic heterotopic heart transplants was monitored as a function of time post-transplant. Nitric oxide production was measured by daily urine nitrate levels and by formation of paramagnetic heme-nitrosyl complexes in the cardiac tissue. Exogenous sources of urine nitrate and EPR signal were minimized by maintaining the animals on a low nitrite/nitrate diet. Urine nitrate peaked on postoperative day 7. A heme-nitrosyl EPR signal also appeared in the cardiac tissue on postoperative day 7 and remained unchanged in size until rejection on postoperative day 9 at which time the peak height of the signal nearly tripled. Some of the animals in the study were treated with the nitric oxide synthase inhibitor, N omega-monomethyl-L-arginine which caused marked inhibition of urinary nitrate excretion and prevented heme-nitrosyl complex formation in beating hearts. However, administration of the inhibitor did not increase graft survival time. Low intensity heme-nitrosyl signals were identified in inhibitor-treated allogeneic hearts after rejection. Syngeneic heart transplants did not induce urinary nitrate excretion nor EPR signal formation. These results show that cytokine induced high output nitric oxide synthesis from L-arginine is a prominent biochemical component of the cell-mediated immune response to cardiac allografts in mice. However, nitric oxide production was not essential for rejection of cardiac allografts mismatched at the major histocompatibility locus.

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, 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.

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

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.

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.

Nitric oxide production in murine leishmaniasis: correlation of progressive infection with increasing systemic synthesis of nitric oxide.

Previous studies have shown that nitric oxide (NO) production is a major effector mechanism in the control of Leishmania major infection in the BALB/c and C3H murine models. The susceptibility of mice correlates with intrinsic NO production after infection. We have previously shown that blocking of systemic NO production with oral N-Omega-monomethyl-L-arginine results in decreased NO and exacerbated infection. The C3H mice also synthesize markedly more NO than BALB/c mice shortly after initial infection. We now show that late in infection, as the lesion size is increasing, the BALB/c NO production actually exceeds that seen during the curative stages of the C3H infection. In addition, treatment with meglumine antimoniate, which ameliorates but does not cure the BALB/c mouse, results in decreased systemic parasite load with a concomitant decrease in NO production. These results imply that in vivo, systemically measured levels of NO may in some circumstances reflect ongoing parasitic load, and that NO production is not always correlated with a curative response.