Effect of aflatoxins on rat peritoneal macrophages.

Phagocytosis, intracellular killing of Candida albicans, and superoxide production by rat peritoneal macrophages exposed to aflatoxins B1, B2, G1, G2, B2a, and M1 at several times and concentrations were analyzed to evaluate the intensity of a depressive effect for each mycotoxin. All aflatoxins used at very low concentrations had a depressive effect on the functions of macrophages. The biggest impairment of phagocytosis, intracellular killing, and spontaneous superoxide production was observed in macrophages exposed to aflatoxins B1 and M1.     

Effects of recombinant human gamma interferon on intracellular survival of Bordetella pertussis in human phagocytic cells

Abstract Several studies have demonstrated that Bordetella pertussis has the ability to enter and survive intracellularly within human polymorphonuclear leukocytes (PMNL) and human monocytes/macrophages. The effects of human recombinant gamma interferon (IFN-γ) on the survival of B. pertussis in PMNL and human monocytes, and on the oxidative burst activity of PMNL and human monocytes in response to B. pertussis were assessed in this study. IFN-γ partially increased intracellular killing of phagocytosed B. pertussis in human monocytes, as determined by an orange acridine-crystal violet assay. In contrast, IFN-γ did not enhance intracellular killing of B. pertussis in PMNL. No significant increase of superoxide production was noted in human monocytes in response to B. pertussis when stimulated with various concentrations of IFN-γ. The partial increase of B. pertussis killing by IFN-γ within monocytes, together with poor production of superoxide may explain how B. pertussis can survive within human phagocytic cells, and thus cause a more prolonged course of the disease.     

Effects of fibronectin on actin organization and respiratory burst activity in neutrophils,monocytes, and macrophages

Previous studies have shown that fibronectin (Fn) enhances phagocytosis and killing of antibody-coated bacteria by neutrophils and macrophages. In an attempt to understand the mechanism of this enhancement, we have investigated the effects of Fn on phagocytosis-related actin organization as well as respiratory burst activity in neutrophils, monocytes and culture-derived macrophages. Employing an NBD-phallacidin flow cytometric analysis of filamentous actin formation, we found that Fn promotes rapid actin polymerization within 30 seconds in neutrophils, monocytes, and macrophages, but not lymphocytes. Enhancement of actin polymerization by Fn was concentration-dependent and mediated by a pertussis toxin- but not cholera toxin- sensitive G protein. Inhibition of protein kinase C by sphingosine (20 μM), calcium influx by verapamil (0.1 mM), or intracellular calcium mobilization by 8-(N, N-diethyl-amino) octyl-3,4,5-trimethoxybenzoate HCI (TMB-8; 0.1 mM) did not block Fn-enhanced actin polymerization in phagocytes. Incubation of neutrophils and macrophages on microtiter plates precoated with Fn suppressed superoxide (O₂^?) production induced by IgG- and IgA- opsonized group B streptococci. In contrast, Fn significantly enhanced IgA- and IgG-mediated O₂^? production by freshly isolated monocytes. These data suggest that Fn enhances phagocytosis, presumably through G protein-coupled cytoskeleton reorganization and augments O₂^? production by circulating monocytes. In contrast, it appears to suppress O₂^? production by the active phagocytic cells, neutrophils and macrophages. This may result in enhanced phagocytosis and intracellular killing of microorganisms without damaging interstitial tissues. © 1994 Wiley-Liss, Inc.     

The synergistic anti-inflammatory effects of lycopene, lutein, β-carotene, and carnosic acid combinations via redox-based inhibition of NF-κB signaling

Inflammatory mediators and cytokines play important roles in the pathogenesis of a vast number of human diseases; therefore much attention is focused on blunting their proinflammatory modes of action. The aims of the present research were to assess the effectiveness of combinations of carotenoids and phenolics, at concentrations that can be achieved in blood, to inhibit the release of inflammatory mediators from macrophages exposed to lipopolysaccharide (LPS) and to determine what the anti-inflammatory effect of the phytonutrient combinations was in an in vivo mouse model of peritonitis. Preincubation of mouse peritoneal macrophages with lycopene (1μM) or Lyc-O-Mato (1μM) and carnosic acid (2μM), lutein (1μM), and/or β-carotene (2μM) 1h before addition of LPS for 24h caused a synergistic inhibition of NO, prostaglandin E(2), and superoxide production derived from downregulation of iNOS, COX-2, and NADPH oxidase protein and mRNA expression and synergistic inhibition of TNFα secretion. We surmise that the anti-inflammatory action of the phytonutrient combinations used probably resides in their antioxidant properties, because they caused an immediate, efficient, and synergistic inhibition of LPS-induced internal superoxide production leading to a marked decrease in ERK and NF-κB activation. The anti-inflammatory effects of the selected phytonutrient combinations were also demonstrated in a mouse model of peritonitis: their supplementation in drinking water resulted in attenuation of neutrophil recruitment to the peritoneal cavity and in inhibition of inflammatory mediator production by peritoneal neutrophils and macrophages.     

Influence of temperature cycling on the production of aflatoxins B1 and G1 by Aspergillus parasiticus.

The effect of temperature cycling on the relative productions of aflatoxins B1 and G1 by Aspergillus parasiticus NRRL 2999 was studied. The cycling of temperature between 33 and 15 degrees C favored aflatoxin B1 accumulation, whereas cycling between 35 and 15 degrees C favored aflatoxin G1 production. Cultures subjected to temperature cycling between 33 and 25 degrees C at various time intervals changed the relative productions of aflatoxins B1 and G1 drastically. Results obtained with temperature cycling and yeast extract-sucrose medium with ethoxyquin to decrease aflatoxin G1 production suggest that the enzyme system responsible for the conversion of aflatoxin B1 to G1 might be more efficient at 25 degrees C than at 33 degrees C. The possible explanation of the effect of both constant and cycling temperatures on the relative accumulations of aflatoxins B1 and G2 might be through the control of the above enzyme system. The study also showed that greater than 57% of aflatoxin B1, greater than 47% of aflatoxin G1, and greater than 50% of total aflatoxins (B1 plus G1) were in the mycelium by day 10 under both constant and cyclic temperature conditions.     

Role of superoxide anion in the fungicidal activity of murine peritoneal exudate macrophages against Penicillium marneffei.

Penicillium marneffei is an important opportunistic fungal pathogen. The mechanisms of host defense against P. marneffei are not fully understood. In the present study, we, for the first time, investigated the role of superoxide anion (O2-) in the killing of two forms of P. marneffei, yeast cells and conidia, and the role of this killing mediator in the fungicidal activity of IFN-gamma-stimulated murine peritoneal macrophages. P. marneffei yeast cells were susceptible to the killing effect of activated macrophages and chemically generated O2, while conidia were not. These results suggested that O2- played some role in the fungicidal activity of macrophages. However, an oxygen radical scavenger, superoxide dismutase (SOD), did not suppress, but rather enhanced the fungicidal activity of IFN-gamma-stimulated macrophages against P. marneffei yeast cells. This inconsistency was explained by the release of insufficient concentrations of O2- by activated macrophages as compared with the amount of O2- necessary for the killing of yeast cells, which was predicted in a chemical generating system. On the other hand, SOD enhanced the production of nitric oxide (NO) by IFN-gamma-activated macrophages, and their increased fungicidal activity was significantly inhibited by N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO synthase. Our results suggested that O2- does not function as the killing mediator of macrophages against P. marneffei, but rather plays an important role in the regulation of the NO-mediated killing system by suppressing NO production.     

Quantitation of aflatoxin B1 and aflatoxin B1 antibody by an enzyme-linked immunosorbent microassay.

A specific microtest plate enzyme immunoassay has been developed for the rapid quantitation of aflatoxin B1 at levels as low as 25 pg per assay. Multiple-site injection of rabbits with an aflatoxin B1 carboxymethyloxime-bovine serum albumin conjugate was used for the production of hyperimmune sera. Dilutions of the purified antibody were air dried onto microplates previously treated with bovine serum albumin and glutaraldehyde and then incubated with an aflatoxin B1 carboxymethyloxime-horseradish peroxidase conjugate. The amount of enzyme bound to antibody was determined by monitoring the change in absorbance at 414 nm after the addition of a substrate solution consisting of hydrogen peroxide and 2,2'-azino-di-3-ethyl-benzthiazoline-6-sulfonate. Antibody titers determined in this manner closely correlated with those determined by radioimmunoassay. Competition assays as performed by incubation of different aflatoxin analogs with the peroxidase conjugate showed that aflatoxins B1 and B2 and aflatoxicol caused the most inhibition of conjugate binding to antibody. Aflatoxins G1 and G2 inhibited the conjugate binding to a lesser degree, whereas aflatoxins M1 and B2a had no effect of the assay.     

Inhibition of aflatoxin production by selected insecticides.

The insecticide naled completed inhibition production of aflatoxins B1, B2, G1, and G2 by and growth of Aspergillus parasiticus at a 100-ppm (100 microgram/ml) concentration. The insecticides dichlorvos, Landrin, pyrethrum, Sevin, malathion, and Diazinon significantly (P = 0.05) inhibited production of aflatoxins at a 100-ppm concentration. However, at a concentration of 10 ppm, significant inhibition in production of aflatoxins was found only with naled, dichlorvos, Sevin, Landrin, and pyrethrum. Dichlorvos, Landrin, Sevin, and naled inhibited growth of A. parasiticus by 28.9 , 18.9, 15.7, and 100%, respectively, at 100 ppm. Stimulation of growth was observed when diazinon was added to cultures. Aflatoxin B1 was most resistant to inhibition by insecticides, followed by G1, G2, and B2, respectively.     

Inhibition of aflatoxin production by selected insecticides

The insecticide naled completed inhibition production of aflatoxins B1, B2, G1, and G2 by and growth of Aspergillus parasiticus at a 100-ppm (100 microgram/ml) concentration. The insecticides dichlorvos, Landrin, pyrethrum, Sevin, malathion, and Diazinon significantly (P = 0.05) inhibited production of aflatoxins at a 100-ppm concentration. However, at a concentration of 10 ppm, significant inhibition in production of aflatoxins was found only with naled, dichlorvos, Sevin, Landrin, and pyrethrum. Dichlorvos, Landrin, Sevin, and naled inhibited growth of A. parasiticus by 28.9 , 18.9, 15.7, and 100%, respectively, at 100 ppm. Stimulation of growth was observed when diazinon was added to cultures. Aflatoxin B1 was most resistant to inhibition by insecticides, followed by G1, G2, and B2, respectively.     

The influence of antibiotics on phagocytic and bacteriocidal activity of rabbit peritoneal macrophages stimulated by filtrates of cultured t-lymphocytes

The aim of the study was to determine the influence of twelve antibacterial antibiotics (various concentrations) on the activation of rabbit peritoneal macrophages. Macrophages were stimulated by filtrates of culture of lymphocytes T obtained from OVA immunized rabbits. Phagocytic activity and intracellular killing against Listeria monocytogenes were tested by fluorescence method. Penicillin G (0.4-50 mg/l), erythromycin and lincomycin (2.5-40 mg/l) used at all concentrations, were not exerting significant effects on activation of peritoneal macrophages. Cephalosporins, aminoglycosides, and rifampicin at low concentrations (0.4-5.0 mg/l) had no influence on phagocytosis and intracellular killing, also. Cephalosporins at concentration 10 mg/l (cephradine and cefamandole) and 50 mg/l (cefotaxime) inhibited intracellular killing and phagocytic activity. The same results were observed with ampicillin and ticarcillin (50 mg/l). The highest suppression effect was demonstrated using rifampicin at concentration 10 mg/l or more. Gentamicin, streptomycin and amicacin at concentrations 40 mg/l or more significantly inhibited macrophage activation in response to filtrates lymphocytes of culture. These inhibitions were more marked with gentamicin (10 mg/l) than amicacin (20 mg/l) or streptomycin (40 mg/l). All antibiotics did not stimulated the activity of peritoneal macrophages. The suppression activity of peritoneal macrophages by some antibiotics probably acts at the level of specific immune system by interfering with cytokine production.     

Tumor necrosis factor alpha stimulates mycobactericidal/mycobacteriostatic activity in human macrophages by a protein kinase C-independent pathway.

Tumor necrosis factor (TNF) is a 17-kDa protein produced by endotoxin-stimulated macrophages. We have demonstrated that recombinant human TNF activates human macrophages to kill intracellular bacteria of the Mycobacterium avium complex (MAC) in a dose-related manner. TNF also primed macrophages to produce superoxide anion (O2-) following treatment with phorbol esther PMA (0.1 micrograms/ml). To investigate the intracellular pathway involved in the TNF-mediated activation of mycobacteriostatic/mycobactericidal activity in macrophages, we used two different protein kinase C (PKC) inhibitors: H7 (10(-5)-10(7) M) and staurosporine (10(-7)-10(-9) M). Mellitin (1 and 100 mM) was used as a calmodulin inhibitor. Human peripheral blood-derived macrophages cultured for 7 days were treated with H7, mellitin, or staurosporine for 1 hr prior to incubation with TNF (10(3) U/ml). Twenty-four hours after treatment with TNF the O2- release was measured spectrophotometrically following exposure to PMA. Macrophages were infected with MAC and the viable intracellular bacilli were quantitated following 4 days of treatment with TNF. All PKC inhibitors suppressed O2- production after incubation with PMA. However, treatment with either PKC or calmodulin inhibitors did not influence the intracellular killing of M. avium by TNF-stimulated macrophages. Exposure of the macrophages to cGMP inhibitor but not to cAMP inhibitor significantly impaired the response to the stimulation with TNF. In contrast, incubation of macrophages with protein kinase A (PKA) had no effect on TNF-mediated mycobacteriostatic/mycobactericidal activity. These results suggest that the TNF-mediated mycobactericidal activity in cultured macrophages probably occurs by a PKC-independent mechanism.     

Alteration of superoxide- and nitric oxide-mediated antimicrobial function of macrophages by in vivo cocaine exposure

Cocaine is a popular drug of abuse and despite impressive advances in the understanding of its physiological, pharmacological, and toxic effects, its mechanism of immunosuppression at the cellular level is not well understood. In this paper we report the role of effector molecules like superoxide and nitric oxide in the antibacterial function of macrophages exposed to acute and chronic doses of cocaine in vivo. Bacterial killing by acute cocaine-exposed macrophages (ACE-Mphis) increased significantly, with a concomitant rise in respiratory burst and generation of superoxide and nitric oxide, compared with control macrophages. In contrast, chronic cocaine-exposed macrophages (CCE-Mphis) exhibited limited antimicrobial activity, which correlated closely with diminished respiratory burst and reduced production of superoxide and nitric oxide. Further, a killing assay was carried out in the presence of N(G)-methyl-L-arginine acetate, an inhibitor of iNOS, to evaluate the role of nitric oxide in the killing process. The results obtained indicate that while about 30% killing of input bacteria by control and ACE-Mphis was attributable to NO-mediated killing, only about 6% killing from NO was found with CCE-Mphis. The findings indicate that acute exposure to cocaine possibly caused upregulation of enzymes responsible for the generation of ROI (reactive oxygen intermediates) and RNI (reactive nitrogen intermediates), leading to enhanced antimicrobial function. On the other hand, chronic exposure to cocaine impaired the oxygen-dependent microbicidal capacity of macrophages, possibly through impaired expression of enzymes responsible for ROI and RNI formation. Proinflammatory cytokines may play a key role in cocaine-mediated immunosuppression, since exposure of macrophages to cocaine impairs the ability of the cells to produce these cytokines.     

Tumor necrosis factor, alone or in combination with IL-2, but not IFN-gamma, is associated with macrophage killing of Mycobacterium avium complex

Organisms belonging to the Mycobacterium avium complex (MAC) are the most common bacterial pathogens in patients with AIDS but factors associated with the activation of cellular defense mechanisms against this atypical mycobacterium have not been defined. Peritoneal macrophages harvested from a chronic MAC infection in C57 black mice are able to kill approximately 86% of intracellular MAC in contrast to 0 to 20% killing by unstimulated human and mouse macrophages in vitro. The availability of human rTNF-alpha, rIFN-gamma, and rIL-2 permitted evaluation of the role of each of these lymphokines/monokines, alone or in combination, in activating macrophages in vitro to kill MAC. Human monocyte-derived macrophages were cultured in vitro, stimulated with rIL-2, rIFN-gamma, or rTNF, and then infected with MAC (serovars 1 and 8). Mouse peritoneal macrophages were harvested, cultured in vitro, and stimulated with rIFN-gamma. rTNF (10(4) U/ml) was associated with a modest increase of intracellular killing of MAC (58 +/- 5%) even when utilized 24 or 48 h after macrophage infection or when administered for 5 consecutive days after infection (78.1 +/- 4%). Both human and murine IFN-gamma were associated with increased intracellular growth of MAC (32 +/- 4% for murine and 38 +/- 3% for human macrophages). However, intracellular killing (53 +/- 6% compared with control) was observed after 6 days of treatment with IFN-gamma. This latter effect was fully blocked by anti-TNF antibody, whereas rIL-2 alone did not augment the intracellular killing of MAC by human macrophages. rTNF plus either rIFN-gamma or rIL-2 triggered significant increases in superoxide anion production, but subsequent MAC killing was no greater than with rTNF alone. Treatment of macrophages with 10 U/ml of rTNF followed by rIL-2 (200 U/ml) was associated with 68% of intracellular killing. TNF seems to be an important monokine, promoting activation of mycobactericidal mechanisms in human macrophages.     

Superoxide anions are required for the inactivation ofEscherichia coli induced by high concentrations of hydrogen peroxide

The wild-type strain and mutants ofEscherichia coli lacking Mn-superoxide dismutase (Sod A) or Fe-superoxide dismutase (Sod B) are compared for their sensitivity to the H₂O₂ insult (exposure for 15 min at 37°C, in M9 salts). Whereas mode one killing is similar in superoxide dismutase mutants and wild-type cells, the latter strain appears to be more resistant than the former ones to mode two lethality. Furthermore, Sod B cells, as well as wild-type cells but unlike Sod A cells, are capable of reversing the toxicity of the oxidant (even in the presence of chloramphenicol), this effect being observed by gradually increasing the H₂O₂ concentration from 2.5 to 10 mM. It is concluded that (a) superoxide ions may not be involved in the production of mode one killing by H₂O₂, although further experiments are needed to validate or modify this hypothesis; (b) superoxide ions mediate mode two killing by H₂O₂, possibly by reducing trivalent iron to the divalent form; and (c) the intervening zone of partial resistance observed in wild-type and Sod B cells exposed to intermediate H₂O₂ concentrations is not a consequence of Mn-superoxide dismutase induction; it would appear, however, that cells lacking this superoxide dismutase isoenzyme are not proficient in this acquired response.     

Lipopolysaccharide-mediated macrophage activation: the role of calcium in the generation of tumoricidal activity

As we have reported, calcium ionophore A23187 activates macrophages for tumor cell killing, and the activated macrophages produced a soluble cytotoxic factor (M phi-CF) that is similar, if not identical, to tumor necrosis factor. Based on these observations, we have investigated whether calcium is involved in the activation mediated by another potent macrophage activator, namely lipopolysaccharide (LPS). We first showed that A23187 caused uptake of extracellular calcium-45 by macrophage monolayers, whereas LPS did not. Because in this system rapid changes would not have been detected, several other approaches also have been used. We have examined the effect of depleting extracellular calcium by using medium containing no added calcium, supplemented with 1 mM EGTA. In no case did depletion result in decreased M phi-CF production by LPS-treated macrophages. Measurements using the fluorescent intracellular calcium indicator Quin 2 have also been performed. The calcium ionophore ionomycin caused a rapid change in the intracellular Quin 2 signal. LPS, even at a concentration in vast excess of that required to activate the macrophages, caused no change in the signal during a 2-hr period. If the macrophages were loaded with high doses of Quin 2 or another intracellular chelator, TMB-8, M phi-CF production decreased and cytotoxic activity was impaired. These data indicate that one or more of the processes involved in M phi-CF production does require calcium, but that activation mediated by LPS occurs without the influx of extracellular calcium or redistribution of intracellular calcium.     

The effect of aflatoxins on mammalian acid deoxyribonucleases

1. Difference spectroscopy studies indicated that tetrahydrodeoxyaflatoxin B1 and aflatoxicol bind slightly to DNA, whereas aflatoxins B2a, G2a, G2 and aflatoxicol bind to bovine and porcine spleen DNAse II but aflatoxins B1, B2, G1 and tetrahydrodeoxyaflatoxin did not. 2. Kinetic studies showed that aflatoxins B1, G1 and B2 activated bovine and porcine spleen DNAse II while aflatoxins B2a, G2a and G2 had an inhibiting effect. 3. Dissociation constants for the enzyme: substrate-aflatoxin complexes (KAS) as well as the inhibition constants (Ki) were obtained from kinetic studies.     

Mycobacterium lepraemurium activates macrophages but fails to trigger release of superoxide anion

Mycobacterium lepraemurium failed to stimulate a normal respiratory burst when presented to mouse peritoneal or bone marrow macrophages. By comparison, Mycobacterium bovis (strain Bacillus Calmette-Guerin) or Saccharomyces cerevisiae, as expected, stimulated macrophages to release a large amount of superoxide anion (O2-). M. lepraemurium did not interfere with the response to yeast when both microbes were added together to macrophages. The low release of O2- induced by M. lepraemurium was not due to failure of M. lepraemurium to activate or prime macrophages, because exposure of macrophages to M. lepraemurium caused the expected enhancement of O2- release when the macrophages were stimulated by PMA. Similarly, macrophages taken from mice infected with M. lepraemurium were activated, as indicated by high PMA-stimulated O2- release. Macrophages primed in vitro by exposure to Escherichia coli LPS for 24 h did show a moderate O2- response when stimulated by M. lepraemurium, but macrophages primed by exposure to IFN-gamma muramyl dipeptide, or M. lepraemurium showed a weak response when subsequently challenged with M. lepraemurium. The priming effect of M. lepraemurium or LPS decreased substantially after macrophages were cultured in fresh medium for 24 h. Heat killing or opsonization of M. lepraemurium caused the M. lepraemurium to stimulate a high amount of O2- release from LPS-primed macrophages, but heat killing or opsonization of M. lepraemurium had no effect on release of O2- from unprimed macrophages. The results suggest that M. lepraemurium is taken into macrophages by a mechanism that bypasses the FcR and other receptors that are capable of triggering the production of O2-.     

Tumor necrosis factor-alpha synergizes with IFN-gamma in mediating killing of Leishmania major through the induction of nitric oxide

CBA mice develop cutaneous lesions when infected with Leishmania major. The disease development was significantly reduced by injecting into the lesion a combination of rIFN-gamma and rTNF-alpha. The doses of IFN-gamma and TNF-alpha used were suboptimal in that either cytokine alone did not have any effect. The therapeutic effect of IFN-gamma and TNF-alpha in vivo is reflected in their ability to activate macrophages to kill the intracellular parasites in vitro. The macrophage leishmanicidal activity induced by TNF-alpha and IFN-gamma can be completely inhibited by a specific inhibitor (L-NG monomethyl arginine) of nitric oxide synthesis. There was a direct correlation between the intracellular killing of the parasites and the production of nitric oxide by the macrophages. In contrast, there was no correlation between leishmanicidal activity and superoxide production by macrophages.     

Fate of Legionella pneumophila in macrophages of C57BL/6 chronic granulomatous disease mice

We compared the intracellular survival and growth of Legionella pneumophila Philadelphia-1 in peritoneal macrophages obtained from A/J, C57BL/6, and X-linked chronic granulomatous disease (CGD) mice produced from C57BL/6 strain. The initial killing was observed in A/J and C57BL/6 macrophages at 2, 4 and 6 hr after in vitro phagocytosis, but not in the CGD macrophages. Thereafter, there was a 10-fold increase of CFU in A/J macrophages. The bacteria, however, did not proliferate in C57BL/6 and CGD macrophages at 24 or 48 hr after in vitro phagocytosis. These results suggest that effector molecules for the initial killing are a superoxide anion and its metabolites, and Lgn1 gene product inhibits the intracellular growth of L. pneumophila independently of NADPH oxidase.     

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.