Cellular models of macrophage tumoricidal effector mechanisms in vitro. Characterization of cytolytic responses to tumor necrosis factor and nitric oxide pathways in vitro

The recently described L-arginine-dependent nitric oxide (NO) pathway has been proposed to interact synergistically with the TNF pathway in murine macrophage-mediated tumor cytotoxicity in vitro. We have employed an experimental construct in which these two pathways were independently expressed by two different effector cell populations. The TNF-dependent pathway was committed by murine 3T3 cells transfected with the cDNA encoding human pro-TNF. The NO pathway was executed by the murine EMT-6 mammary adenocarcinoma cell line treated with murine rIFN-gamma and LPS. Controls for the TNF pathway committed by the transfectant included lysis of the TNF-sensitive murine L929 cell in coculture, secretion of TNF, and absence of nitrite synthesis. For the NO pathway controls included lysis of the murine P815 mastocytoma cocultured with activated EMT-6 cells that had been pretreated with murine rIFN-gamma and LPS, production of nitrite by this activated effector cell, and an absence of TNF secretion. The target cell panel included L929, EMT-6, P815, and murine B16 melanoma and TU-5 sarcoma cell lines. All targets on this panel were susceptible to lysis by LPS-triggered murine bacillus Calmette-Guérin-activated macrophages. The 3T3 transfectant caused significant lysis of cocultured L929 and TU-5 targets. The EMT-6 effector cell only caused significant lysis of the P815 target. When both effector cells were cocultured with these target cells, lysis of the P815 target was observed to be additive or superadditive; however, for all the other targets, cytotoxicity was comparable with or subadditive compared with that seen with the 3T3 transfectant effector cell alone. Thus, these two pathways do not appear to account for the broad, potent tumoricidal activity observed for activated macrophages in vitro.     

Tumor target-derived soluble factor synergizes with IFN-gamma and IL-2 to activate macrophages for tumor necrosis factor and nitric oxide production to mediate cytotoxicity of the same target.

Inflammatory mouse peritoneal macrophages were activated by IFN-gamma in synergy with IL-2 or Lipid A to mediate TNF production for autocrine generation of cytotoxic nitric oxide (NO) to kill P815 or L1210 tumor targets. It was determined that for IL-2, but not Lipid A, to effectively trigger activation of IFN-gamma-primed macrophages, the tumor targets must be also present for interaction with effector macrophages to mediate the production of TNF and NO. IFN-gamma- and IL-2-activated macrophages from syngeneic DBA/2 and allogeneic C3H mice had identical MHC-unrestricted requirements for interaction with DBA/2 mouse-derived P815 and L1210 targets to mediate production of TNF and NO for tumor cytotoxicity. To further define the mechanistic requirements for macrophage-tumor target interaction, IFN-gamma- and IL-2-activated macrophages were separated from P815 targets in culture by a semipermeable membrane. Under these conditions, both TNF and NO were produced by the macrophage, which indicated that the requirement for tumor target-macrophage interaction may be due to a soluble factor produced by the target rather than to direct physical contact. This was confirmed by experiments in which 24-h cell-free culture fluids, derived from either P815 or L1210 tumor targets, substituted for the intact tumor cells in the stimulation of TNF mRNA synthesis and secretion with NO generation of TNF mRNA synthesis and secretion with NO generation by IFN-gamma- and IL-2-activated C3H or DBA/2 macrophages. The activity in 24-h culture fluids derived from P815 and L1210 tumor targets was tentatively designated as tumor-derived recognition factor(s) (TDRF) since it was produced constitutively by the tumor targets and synergized with IFN-gamma and IL-2 to induce macrophage production of TNF and NO for death of the same targets. A variety of nontransformed human and mouse fibroblasts, mouse spleen lymphocytes, and two adherent mouse fibrosarcomas did not produce detectable TDRF activity, whereas two mouse T lymphomas, EL4 and EL4.IL-2, produced TDRF activity similar to L1210 mouse leukemia and P815 mastocytoma. The C3H/MCA, a TDRF-nonproducing mouse fibrosarcoma, was susceptible to cytotoxicity mediated by macrophages activated by IFN-gamma and Lipid A, but not by IL-2 triggering. Exogenous TDRF derived from L1210 targets reconstituted the cytotoxic activity for C3H/MCA MCA targets mediated by IFN-gamma- and IL-2-activated macrophages accompanied by the production of TNF and cytotoxic NO.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of two distinct cytolytic mechanisms among murine CD4 subsets

A TNF (TNF-alpha and TNF-beta)-sensitive target, L929, and two TNF-resistant targets, P815 and LK were used to compare the cytolytic activity among subsets of CD4+ (Th) clones. Cytolytic activity was induced with either Con A, CD3-mAb, or Ag-pulsed LK cells. Six Th1 clones are strongly cytolytic against all three targets. In contrast, Th2 clones are either noncytolytic or weakly cytolytic. Although there is an apparent correlation between TNF production, killing of L929 cells, and the killing of TNF-resistant targets, an anti-TNF serum (capable of neutralizing both TNF-alpha and TNF-beta) selectively inhibits CD4 clones to lyse L929 cells, whereas the lysis of P815 or LK cells was unaffected. The continuous presence of noncytotoxic levels of Actinomycin D (AcD) and cycloheximide, but not mitomycin C, cyclosporin A (CsA), or cholera toxin (ChT) inhibits the lysis of Ag-pulsed, Ia-bearing LK cells; indicating a requirement for de novo synthesis of RNA and protein for cytolytic activity. Although pretreatment with AcD, CsA, or ChT strongly inhibits production of IL-2, TNF and IFN-gamma, only clones pretreated with AcD lose cytolytic activity against Ag-pulsed, Ia-bearing LK cells. These observations support a model of TNF-independent killing of TNF-resistant targets. The TNF-independent cytolytic activity does not correlate with serine esterase activity released into media upon activation of CD4 clones. Moreover, the effects of metabolic inhibitors on serine esterase release do not correlate with their effects on cytolytic activity. Collectively, the data demonstrate that activated CD4 cells express two distinct cytolytic activities; a TNF (and IFN-gamma)-mediated cytotoxicity and a TNF (and IFN-gamma)-independent cytolytic activity. Both pathways require de novo synthesis of RNA and protein and appear to be independent of granule enzyme release. Only the TNF-independent cytolytic activity is resistant to CsA and ChT inhibition.     

Sensitivity of bacteria to NaNO2 and to L-arginine-dependent system in murine macrophages

Sensitivity of bacteria to NO2-/NO and to L-arginine-dependent system in murine macrophages was tested. The growth of all bacteria tested, Salmonella typhimurium, Pseudomonas aeruginosa and Staphylococcus epidermidis, was significantly inhibited by NaNO2 at the concentration of 5 to 10 mM. However, L-arginine-dependent effector mechanism of LPS-activated murine macrophages was ineffective in killing these bacteria. It seems that the concentration of NO2-/NO in phagolysosome in the activated macrophages is insufficient to kill the bacteria in situ. We concluded that L-arginine-dependent effector mechanism can hardly work on bacteria killing in activated macrophages.     

Generation of EPR-detectable nitrosyl-iron complexes in tumor target cells cocultured with activated macrophages.

After immunostimulation, murine macrophages oxidize L-arginine into nitric oxide (NO) which acts as an effector molecule. In this study, we attempted to establish whether activated macrophage-derived NO forms paramagnetic complexes in tumor target cells which do not express by themselves the L-arginine:NO pathway. Accordingly, murine L1210 leukemia cells were cocultivated with activated peritoneal macrophages from Bacillus-Calmette-Guérin-infected mice, or activated in vitro with interferon-gamma. In control experiments, macrophages were prevented from producing nitrogen oxides by incubation with NG-monomethyl-L-arginine, a specific inhibitor of the L-arginine:NO pathway. After coculture, L1210 cells were removed from adherent macrophage monolayers and analyzed by electron paramagnetic resonance at 77 K. In the L1210 cells cultured with activated macrophages, we detected a signal typical of nitrosyl-iron-sulfur complexes, with g values of 2.041 and 2.015. This signal was not present when L1210 cells were either cultured alone or cocultured with activated macrophages in the presence of NG-monomethyl-L-arginine. Mitochondria from activated macrophage-injured L1210 cells also exhibited the signal with g values of 2.041 and 2.015. These results show that when tumor target cells undergo cell-to-cell contact with activated macrophages during culture, the macrophages promote target cell nitrosylation in compartments like mitochondria.     

L-arginine is required for expression of the activated macrophage effector mechanism causing selective metabolic inhibition in target cells

L-Arginine is required for expression of the activated macrophage cytotoxic effector mechanism that causes inhibition of mitochondrial respiration, aconitase activity, and DNA synthesis in tumor target cells. This effector mechanism is active in the presence of L-arginine even when the cocultivation medium lacks all other amino acids and serum. Cytotoxic activated macrophage-induced inhibition of mitochondrial respiration in target cells is proportional to the concentration of L-arginine in the medium. L-Arginine must be present during the cocultivation period. Pretreatment of cytotoxic activated macrophages with L-arginine or posttreatment of the target cells after cocultivation is not effective. D-Arginine does not substitute for L-arginine and at high concentrations is a competitive inhibitor of the L-arginine-dependent effector mechanism. Other analogues that could not replace L-arginine include agmatine, argininic acid, arginine hydroxamate, and tosyl-L-arginine methyl ester. L-homoarginine, however, can effectively substitute for L-arginine. NG-monomethyl-L-arginine is a potent competitive inhibitor of this effector mechanism. High concentrations of lipopolysaccharide do not reverse inhibition of the L-arginine-dependent effector mechanism by NG-monomethyl-L-arginine. However, inhibition of the effector mechanism by NG-monomethyl-L-arginine can be overridden by increasing the concentration of L-arginine in the culture medium. We compared NGNG-dimethyl-L-arginine and NGN1G-dimethyl-L-arginine with NG-monomethyl-L-arginine as inhibitors of the L-arginine-dependent effector mechanism. The results show that the inhibitory effect of these guanidino methylated derivatives of L-arginine is highly determined by structure. Guanidine is a weak competitive inhibitor of the L-arginine-dependent effector mechanism. The requirement for L-arginine does not appear to be for protein synthesis, creatine biosynthesis, polyamine biosynthesis, or ADP ribosylation reactions. Bacterial lipopolysaccharide is effective as a second signal only when the cocultivation medium contains L-arginine, and this strict L-arginine dependency is not overridden by increasing the concentration of lipopolysaccharide. Bovine liver arginase, by competing for L-arginine in the cocultivation medium, inhibits the L-arginine-dependent activated macrophage cytotoxic effector mechanism.     

Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in L-arginine-dependent inhibition of mitochondrial iron-sulfur enzymes in the macrophage effector cells

Previous studies show that cytotoxic activated macrophages cause a reproducible pattern of metabolic inhibition in viable tumor target cells. This includes inhibition of DNA synthesis, two oxidoreductases of the mitochondrial electron transport chain (NADH: ubiquinone oxidoreductase and succinate: ubiquinone oxidoreductase), and the citric acid cycle enzyme aconitase. This pattern of metabolic inhibition is induced by a cytotoxic activated macrophage associated biochemical pathway with L-arginine deimination activity that synthesizes L-citrulline from L-arginine and oxygenated nitrogen derivatives from the imino nitrogen removed from the guanido group of L-arginine. Here we report that macrophages activated in vivo by infection with bacillus Calmette-Guérin or in vitro by murine rIFN-gamma or murine IFN-alpha/beta (in the presence of the second signal LPS in all cases) develop inhibition of aconitase and the same two oxidoreductases of the mitochondrial electron transport chain as was documented earlier in target cells of cytotoxic activated macrophages. In addition, this pattern of metabolic inhibition which develops in cytotoxic activated macrophages is caused by the L-arginine-dependent effector mechanism. Inhibition of mitochondrial respiration by effectors of the L-arginine-dependent cytotoxicity system results in a compensatory increase in activity of the glycolytic pathway. We speculate that the pattern of metabolic inhibition induced in cytotoxic activated macrophages by the L-arginine-dependent effector system causes changes in the macrophage intracellular environment that increases resistance to certain facultative and obligate intracellular pathogens.     

Regulation of nitric oxide production by murine peritoneal macrophages treated in vitro with chemokine monocyte chemoattractant protein 1.

Monocyte chemoattractant protein 1 (MCP-1) is an important mediator of monocyte/macrophage recruitment and activation at the sites of chronic inflammation and neoplasia. In the current study, the role of nitrogen monoxide (NO) in the activation of murine peritoneal macrophages to the tumoricidal state in response to in vitro MCP-1 treatment and the regulatory mechanisms involved therein were investigated. Murine peritoneal macrophages upon activation with MCP-1 showed a dose- and time-dependent production of NO together with increased tumoricidal activity against P815 mastocytoma cells. N-monomethyl-l-arginine (L-NMMA), a specific inhibitor of the l-arginine pathway, inhibited the MCP-1-induced NO secretion and generation of macrophage-mediated tumoricidal activity against P815 (NO-sensitive, TNF-resistant) cells but not the L929 (TNF-sensitive, NO-resistant) cells. These results indicated l-arginine-dependent production of NO to be one of the effector mechanisms contributing to the tumoricidal activity of MCP-1-treated macrophages. Supporting this fact, expression of iNOS mRNA was also detected in the murine peritoneal macrophages upon treatment with MCP-1. Investigating the signal transduction pathway responsible for the NO production by the MCP-1-activated murine peritoneal macrophages, it was observed that the pharmacological inhibitors wortmannin, H-7 (1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride), and PD98059 blocked the MCP-1-induced NO production, suggesting the probable involvement of phosphoinositol-3-kinase, protein kinase C, and p42/44 MAPkinases in the above process. Various modulators of calcium and calmodulin (CaM) such as EGTA, nifedipine, TMB-8 (3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester), A23187, and W-7 (N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide) were also found to modulate the in vitro macrophage NO release in response to MCP-1. This observation indicated the regulatory role of calcium/CaM in the process of MCP-1-induced macrophage NO production. Similarly, the role of serine/threonine and protein tyrosine phosphatases in the above pathway was suggested using the specific inhibitors of these phosphatases, okadaic acid and sodium orthovanadate.     

Tumor necrosis factor-alpha-dependent production of reactive nitrogen intermediates mediates IFN-gamma plus IL-2-induced murine macrophage tumoricidal activity.

We have previously established that IFN-gamma plus IL-2 induces murine macrophage tumoricidal activity. The purpose of this study was to identify the effector molecules that account for the IFN-gamma plus IL-2-induced macrophage cytotoxicity against P815 mastocytoma cells. ANA-1 macrophages and normal thioglycollate-elicited mouse peritoneal macrophages produced little or no detectable nitrite (NO2-) after incubation with IFN-gamma alone or IL-2 alone; however, IL-2 synergized with IFN-gamma for the production of NO2-. IFN-gamma plus IL-2 did not induce NO2- production or tumoricidal activity in ANA-1 macrophages that were cultured in medium devoid of L-arginine or in ANA-1 macrophages that were incubated with NG-monomethyl-L-arginine. As observed previously with ANA-1 macrophage tumoricidal activity, IL-4 inhibited IFN-gamma plus IL-2-induced, but not IFN-gamma plus LPS-induced, NO2- production. IL-4 also selectively decreased the ability of IFN-gamma and/or IL-2 to augment TNF-alpha mRNA expression in ANA-1 macrophages. Lastly, incubation of ANA-1 macrophages with anti-TNF mAb selectively inhibited the ability of IFN-gamma plus IL-2 to induce NO2- production and tumoricidal activity. These results indicate that IFN-gamma plus IL-2-induced tumoricidal activity is dependent upon the metabolism of L-arginine to reactive nitrogen intermediates, and they establish a role for TNF-alpha as a required intermediate for IL-2-dependent NO2- production and tumoricidal activity.     

Spontaneous cytotoxicity of macrophages against pancreatic islet cells

Activated peritoneal macrophages were found to lyse syngeneic [3H]leucine-labeled pancreatic islet cells or rat insulinoma cells after 15 h of coculture at 37 degrees C. Lysis was verified by electron microscopic analysis. Islet cell lysis was dependent on the T:E ratio and was comparable with P815 and L929 tumor cells used as targets. The cytotoxic activity was localized in the adherent fraction of Corynebacterium parvum activated peritoneal cells and was destroyed by incubation of cells with macrophage-toxic silica particles. Syngeneic thyrocytes and hepatocytes were found to be resistant to the cytolytic action of activated macrophages. It has been shown previously that macrophages contribute to pancreatic islet inflammation. The present in vitro analysis demonstrates that macrophages can function as effector cells in islet destruction.     

Microbiostatic effect of murine-activated macrophages for Toxoplasma gondii. Role for synthesis of inorganic nitrogen oxides from L-arginine

Recent studies show the importance of a single amino acid, L-arginine, as a necessary substrate for activated macrophage-mediated cytotoxic activity for tumor target cells and microbiostatic function for Cryptococcus neoformans. The present studies were carried out to determine the role of the L-arginine-dependent macrophage effector function on the microbiostatic effects of activated macrophages on the obligate intracellular protozoan, Toxoplasma gondii. A guanidino methylated derivative of L-arginine, NGmonomethyl-L-arginine (NGMMA), a competitive inhibitor of the L-arginine-dependent effector pathway, virtually abolished the normally potent microbiostatic effect of macrophages for Toxoplasma gondii after activation of the macrophages in vitro by IFN-gamma and LPS or in vivo by i.p. injection of killed Corynebacterium parvum. Addition of supplemental L-arginine to the culture medium overcame the capacity of NGMMA to block activated macrophage-mediated microbiostasis of Toxoplasma. The ability of NGMMA to inhibit the microbiostatic capacity of activated macrophages for Toxoplasma gondii correlated with almost total inhibition of synthesis of nitrite, nitrate, and L-citrulline from L-arginine. Therefore, as is the case for tumor target cells and C. neoformans, the synthesis of inorganic nitrogen oxides from a terminal guanidino nitrogen atom of L-arginine appears to be essential for murine cytotoxic activated macrophage mediated microbiostatic capacity for T. gondii.     

Cell-associated tumor necrosis factor (TNF) as a killing mechanism of activated cytotoxic macrophages

Different macrophage populations were investigated for their abilities to secrete tumor necrosis factor (TNF) and to lyse TNF-susceptible tumor cells. In this way we could demonstrate that TNF-secretion, although a feature of all activated macrophage populations, is no absolute requirement for the killing of the TNF-sensitive Wehi 164 target. Macrophage cytotoxicity against this cell but not against the TNF-resistant P815 mastocytoma, was completely inhibitable by a specific anti-TNF serum also in the absence of measurable secreted TNF. Moreover the TNF-dependent lysis of tumor cells could also be performed by activated macrophages that had been fixed with paraformaldehyde before the addition of the target cells. In the indirect radioimmunoassay, TNF could be demonstrated on the surface of fixed effector cells. Our results must be interpreted in terms of membrane-associated TNF as the lytic principle for TNF-susceptible tumor cells.     

Cytolytic activities of activated macrophages versus paraformaldehyde-fixed macrophages; soluble versus membrane-associated TNF.

Membrane-associated tumor necrosis factor (TNF) and soluble TNF were compared as to their lytic activities, and as to the kinetics of their expression by macrophages activated with LPS and/or IFN-gamma in the presence or absence of cycloheximide. EL 4 tumor cells, resistant and sensitive to lysis by recombinant TNF or membrane-associated TNF (paraformaldehyde (PF)-fixed activated macrophages) were used as targets. In the presence of cycloheximide the TNF-resistant S-EL4 cells were lysed by both TNFs. PF-fixed macrophages was cytolytic after 1 hr activation but not after 3 or more hours of activation. Their activity was totally inhibited by anti-TNF antibodies and was a composite of transmembrane (integral) TNF and soluble TNF conjugated to macrophage membrane TNF receptors. Treatment of the macrophages with glycine pH 3.0 buffer dissociated the conjugated TNF without affecting the integral membrane TNF. When macrophages were activated with LPS +/- IFN-gamma in the presence of cycloheximide or activated just with IFN-gamma their activity after fixation with paraformaldehyde was no longer detected. Nonfixed macrophages under these conditions still remained cytotoxic. Tumor cell susceptibility to membrane-associated TNF activity, in contrast to recombinant (soluble) TNF, was greatly reduced in the presence of nicotinamide, an inhibitor of ADP-ribosyltransferase, suggesting that the mechanisms of lysis by these TNFs may be different. The lytic activity of both TNFs was found to be receptor-dependent in that tumor cells, whose TNF binding sites were "down-regulated" by TPA, were rendered resistant to lysis by both membrane-associated and soluble TNFs.     

Leishmania major amastigotes initiate the L-arginine-dependent killing mechanism in IFN-gamma-stimulated macrophages by induction of tumor necrosis factor-alpha

Macrophages exposed to IFN-gamma and infected with amastigotes of Leishmania major develop the capacity to eliminate the intracellular pathogen. This antimicrobial activity of activated macrophages correlates with the initiation of nitrogen oxidation of L-arginine, yet other reports suggest that two signals are required for induction of this biochemical pathway for effector activity. In the present studies, macrophages treated with up to 100 U/ml IFN-gamma, or 100 ng LPS, or 10(7) amastigotes produced minimal quantities (less than 9 microM) of NO2- and failed to develop cytotoxic effector activities. In contrast, the combination of IFN-gamma and either LPS (greater than 0.1 ng) or amastigotes (10(6) induced high concentrations (much greater than 30 microM) of NO2- and macrophage cytotoxicity against intra- and extracellular targets. The induction of nitrogen oxidation by amastigotes could be dissociated from LPS-induced events by 1) performing the assays in the presence of polymyxin B (which blocked LPS effects, but not amastigote effects), 2) determining the threshold of IFN-gamma required to prime cells for subsequent trigger (1 U/ml for LPS trigger effects; 10-fold higher for amastigotes), and 3) determining the heat sensitivity of the two trigger agents (amastigote effects abolished at 100 degrees C; LPS effects unaffected at this temperature). Further, culture fluids from amastigote-infected macrophages did not contain detectable LPS (less than 6 pg/ml). Possible parasite and cell-associated factors that could contribute to the induction of nitrogen oxidation and cytotoxic activity of IFN-gamma treated macrophages were examined: only certain intact microorganisms, LPS from a variety of bacteria, and the cytokine TNF alpha were effective. Both NO2- production and intracellular killing were abolished by the addition of anti-TNF-alpha mAb in the assay. TNF-alpha was produced by amastigote-infected macrophages and IFN-gamma dramatically enhanced secretion of this cytokine; IFN-gamma alone had no effect. Endogenous TNF-alpha produced during infection of macrophages with L. major acted in an autocrine fashion to trigger the production of L-arginine-derived toxic nitrogen intermediates that killed the intracellular parasites.     

Alterations of ribonucleotide reductase activity following induction of the nitrite-generating pathway in adenocarcinoma cells

The murine adenocarcinoma cell line TA 3 synthesized nitrite from L-arginine upon stimulation with gamma-interferon (IFN-gamma) associated with tumor necrosis factor (TNF), and/or bacterial lipopolysaccharide (LPS), but not with IFN-gamma, TNF, or LPS added separately. Induction of the NO2(-)-generating activity caused an inhibition of DNA synthesis in TA 3 cells. This inhibition was prevented by the L-arginine analog N omega-nitro-L-arginine, which inhibited under the same conditions nitrite production by TA 3 cells. The TA 3 M2 subclone, selected for enhanced ribonucleotide reductase activity, was found to be less sensitive than the wild phenotype TA 3 WT to the cytostatic activity mediated by the NO2(-)-generating system. Cytosolic preparations from TA 3 M2 cells treated for 24 or 48 h with IFN-gamma, TNF, and LPS exhibited a reduced ribonucleotide reductase activity, compared to untreated control cells. No reduction in ribonucleotide reductase activity was observed when N omega-nitro-L-arginine was added to treated cells. Addition of L-arginine, NADPH, and tetrahydrobiopterin into cytosolic extracts from 24-h treated TA 3 M2 cells triggered the synthesis of metabolic products from the NO2(-)-generating pathway. This resulted in a dramatic inhibition of the residual ribonucleotide reductase activity present in the extracts. The inhibition was reversed by NG-monomethyl-L-arginine, another specific inhibitor of the NO2(-)-generating activity. No L-arginine-dependent inhibition of ribonucleotide reductase activity was observed using extracts from untreated cells that did not express NO2(-)-generating activity. These results demonstrate that, in an acellular preparation, molecules derived from the NO2(-)-generating pathway exert an inhibitory effect on the ribonucleotide reductase enzyme. This negative action might explain the inhibition of DNA synthesis induced in adenocarcinoma cells by the NO2(-)-generating pathway.     

Induction of nitric oxide synthase is a necessary precondition for expression of tumor necrosis factor-independent tumoricidal activity by activated macrophages.

Various bacteria and bacterial products induce in pure, lymphocyte-free bone marrow-derived mononuclear phagocytes (BMM?) the generation of tumor necrosis factor, nitric oxide (NO) synthase, NO and nitrite (NO2-), the flow of L-arginine to citrulline, and tumoricidal activity. The flow of L-arginine to citrulline and formation of NO/NO2- on the one hand and expression of tumoricidal activity were not always closely related; however, these parameters were suppressed in a dose-dependent manner by the flavoprotein inhibitor, diphenyleneiodonium (DPI) and the L-arginine analogue, NG-monomethyl-L-arginine (NMMA). The findings support the concept of a central role of the NO synthase pathway in the generation of tumor necrosis factor-independent tumoricidal activity by activated macrophages but the exact conditions which enable the transfer of the lytic principle from the effector to the target cell remain to be elucidated.     

Activated macrophages destroy intracellular Leishmania major amastigotes by an L-arginine-dependent killing mechanism

Macrophages infected with amastigotes of Leishmania major and treated with IFN-gamma in vitro develop potent antimicrobial activities that eliminate the intracellular parasite. This antileishmanial activity was suppressed in a dose dependent fashion by NG-monomethyl-L-arginine (NGMMLA), a competitive inhibitor of nitrite, nitrate, nitric oxide and L-citrulline synthesis from L-arginine. Excess L-arginine added to infected macrophage cultures reversed the inhibitory effects of NGMMLA. Addition of arginase to culture media inhibited intracellular killing by IFN-gamma-treated cells. Similar effects were seen with macrophages obtained from BCG-infected C3H/HeN mice. Increased levels of nitrite, an oxidative product of the L-arginine-dependent effector mechanism, was measured in cultures of infected IFN gamma-treated macrophages as well as infected BCG-activated macrophages. Nitrite production correlated with development of antileishmanial activity. Nitrite production and microbicidal activity both decreased when in vivo or in vitro-activated macrophages were cultured in the presence of either arginase or NGMMLA. Nitric oxide synthesized from a terminal guanidino nitrogen atom of L-arginine and a precursor of the nitrite measured, may disrupt Fe-dependent enzymatic pathways vital to the survival of amastigotes within macrophages.     

Role of tumor necrosis factor-alpha in E1A oncogene-induced susceptibility of neoplastic cells to lysis by natural killer cells and activated macrophages

NIH-3T3 cells transfected with adenovirus E1A oncogene cDNA were found to exhibit cytolytic susceptibility to murine NK cells and activated macrophages associated with a threshold level of oncogene product expression exceeding that required for morphological transformation. A similar correlation was observed between threshold levels of E1A gene product expression and target cell susceptibility to direct cytotoxicity by rTNF. Inhibition of splenic NK cell and peritoneal macrophage cytolysis by antisera specific for murine rTNF confirmed the importance of E1A-induced TNF susceptibility as one determinant of target cell cytolytic susceptibility. Anti-TNF antibody was, however, unable to block killing of E1A-expressing targets by the NK cell line, NKB61A2. These results suggest a direct link between the functions of E1A oncogene products and cellular mechanisms of action of TNF elaborated by host effector cells and indicate that E1A expression also affects target cell susceptibility to TNF-independent cytolytic mechanisms.     

Regulation of Nitric Oxide Production by Murine Peritoneal Macrophages Treated in Vitro with Chemokine Monocyte Chemoattractant Protein 1

Monocyte chemoattractant protein 1 (MCP-1) is an important mediator of monocyte/macrophage recruitment and activation at the sites of chronic inflammation and neoplasia. In the current study, the role of nitrogen monoxide (NO) in the activation of murine peritoneal macrophages to the tumoricidal state in response to in vitro MCP-1 treatment and the regulatory mechanisms involved therein were investigated. Murine peritoneal macrophages upon activation with MCP-1 showed a dose- and time-dependent production of NO together with increased tumoricidal activity against P815 mastocytoma cells. N-monomethyl- -arginine (L-NMMA), a specific inhibitor of the -arginine pathway, inhibited the MCP-1-induced NO secretion and generation of macrophage-mediated tumoricidal activity against P815 (NO-sensitive, TNF-resistant) cells but not the L929 (TNF-sensitive, NO-resistant) cells. These results indicated -arginine-dependent production of NO to be one of the effector mechanisms contributing to the tumoricidal activity of MCP-1-treated macrophages. Supporting this fact, expression of iNOS mRNA was also detected in the murine peritoneal macrophages upon treatment with MCP-1. Investigating the signal transduction pathway responsible for the NO production by the MCP-1-activated murine peritoneal macrophages, it was observed that the pharmacological inhibitors wortmannin, H-7 (1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride), and PD98059 blocked the MCP-1-induced NO production, suggesting the probable involvement of phosphoinositol-3-kinase, protein kinase C, and p42/44 MAPkinases in the above process. Various modulators of calcium and calmodulin (CaM) such as EGTA, nifedipine, TMB-8 (3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester), A23187, and W-7 (N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide) were also found to modulate the in vitro macrophage NO release in response to MCP-1. This observation indicated the regulatory role of calcium/CaM in the process of MCP-1-induced macrophage NO production. Similarly, the role of serine/threonine and protein tyrosine phosphatases in the above pathway was suggested using the specific inhibitors of these phosphatases, okadaic acid and sodium orthovanadate.     

L-arginine independent macrophage tumor cytotoxicity

We have investigated the role of L-arginine in macrophage tumor cytotoxicity in coculture. L929, EMT-6, MCA-26, and P815 targets were all susceptible to cytolysis by activated macrophages when cocultured in medium containing L-arginine. When cocultured in arginine-free medium, these targets displayed comparable or even higher levels of lysis. L1210 targets were lytically resistant under either condition. However, 59Fe release from this target did reflect strong dependence on the presence of arginine. The structural analogue, NG-monomethyl-L-arginine, was an effective inhibitor of iron-release from L1210 targets cocultured with activated macrophages, whereas it had minimal inhibitory effects on release of 51Cr from cocultured L929 cells. These results suggest that the L-arginine requiring cytotoxic pathway of activated macrophage is independent of major effector mechanisms involved in tumor cell lysis.