Interleukin-4 and interleukin-10 inhibit nitric oxide-dependent macrophage killing of Candida albicans

Mouse peritoneal and splenic macrophages treated with interferon-γ (IFN-γ) and infected with the yeast Candida albicans expressed high fungicidal activity in vitro that correlated with increased nitrite concentrations in culture supernatants. Both effects were reduced by an inhibitor of nitric oxide (NO) synthesis which, in vivo, impaired the animals' ability to mount a footpad reaction and clear the fungus from infected organs. Because T helper type-2 (Th2) cytokines in candidiasis are known to limit the expression of protective Th1 functions, we tested the effect of interleukin (IL)-4 and IL-10 on candidacidal activity and NO production of IFN-γ-activated macrophages. Fungal killing and NO secretion were inhibited, in a dose-dependent manner, by the two cytokines either separately or in combination. Impaired candidacidal activity was also demonstrable in the presence of monoiodoacetic acid, an inhibitor of phagocytosis. These data demonstrate that NO is involved in macrophage killing of C. albicans and support the notion that regulation of Th1 effector function by IL-4 and IL-10 might involve modulation of NO synthesis.     

Nitric oxide production by Leishmania-infected macrophages and modulation by prostaglandin E2

Nitric oxide (NO), produced by the nitric oxide synthase (iNOS) enzyme, is the most-important molecule responsible for the killing of Leishmania parasites by macrophages. In previous work we have demonstrated that, after activation with recombinant human interferon-γ and/or bacterial lipopolysaccharide, human macrophages infected with Leishmania infantum are able to produce nitric oxide and to express nitric oxide synthase. The arachidonate derivative prostaglandin E₂ has been shown to modulate various macrophage activities, and in particular nitric oxide production, sometimes with opposite effects, related to experimental conditions. In this work we have evaluated nitric oxide release and parasite killing by peripheral blood-derived L. infantum-infected human macrophages in vitro stimulated with lipopolysaccharide and simultaneously treated with prostaglandin E₂. Experiments were also performed in the presence of the nitric oxide synthase inhibitor l-N ^G monomethylarginine (l-NMMA) and of the cyclooxygenase inhibitor indomethacin. Nitric oxide release in supernatants of macrophage cultures was measured by the Griess reaction for nitrites. Parasite killing was microscopically evaluated by fluorescent dyes. Results demonstrated that macrophages stimulated with lipopolysaccharide and treated with prostaglandin E₂ exhibited increased nitric oxide producation and parasite killing, which were significantly reduced by either l-NMMA or indomethacin. In indomethacin-treated macrophages, nitric oxide production and leishmanicidal ability were partially restored by the addition of exogenous prostaglandin E₂. Taken together, these results indicate that prostaglandin E₂ may be involved in nitric oxide production, and possibly in the host-protective immune response against Leishmania. Moreover, the demonstration of a stimulatory role of prostaglandin E₂ on nitric oxide production induced by intracellular pathogens in humans is interesting in the light of a possible pharmacological regulation of nitric oxide by modulation of prostaglandin E₂ synthesis. Received: 27 March 2001 / Accepted: 24 August 2001     

Production of nitric oxide and superoxide by activated macrophages and killing of Leishmania major

Murine macrophages can be activated to produce nitric oxide (NO) and superoxide and these two radicals can react to form peroxynitrite, a powerful oxidant which may be involved in parasite killing. We now show that murine macrophages activated with zymosan and interferon-γ (ZYM/IFN-γ) produced both superoxide (peaking 1–2 h after stimulation, then rapidly declining) and NO (barely detectable at 6 h, peaking by 24 h). Macrophages activated with ZYM alone produced only superoxide, while stimulation with lipopolysaccharide (LPS) and IFN-γ induced NO but not superoxide. Cells stimulated with ZYM/IFN-γ or LPS/IFN-γ killed Leishmania major to a similar degree, an effect that was completely blocked by the addition of N-iminoethyl-L -ornithine. However, macrophages stimulated with ZYM alone were unable to kill L. major. S-nitroso-acetyl-penicillamine, which release NO, was highly leishmanicidal when added directly to the parasites. 3-morpholino-sydnonimine hydrochloride which releases both NO and superoxide simultaneously, was also efficient at killing L. major and this cytotoxicity was greatly enhanced by the addition of superoxide dismutase. Finally, authentic peroxynitrite failed to induce any cytotoxic effect, even at a high concentration. Thus macrophages can produce either NO, superoxide or both, depending on the stimulus. However, the killing of L. major is dependent only on the production of NO.     

Virulence of Sporothrix schenckii conidia and yeast cells, and their susceptibility to nitric oxide

The involvement of nitric oxide (NO) in macrophage (M phi) fungicidal activity against Sporothrix schenckii, and the relationship between NO susceptibility and the differential virulence of conidia and yeast cells, were investigated. Confirming a previously reported correlation between the length of time in culture and virulence of S. schenckii, conidia isolated from 12-day mycelial cultures (Ss-12) were less virulent to mice than conidia from 7-day cultures (Ss-7) or yeast cells. Indicative of NO production, infected animals showed a significant increase in serum levels of nitrite that was lower in mice infected with Ss-12 than in mice infected with Ss-7 or yeast. Stimulation of murine M phi with interferon-gamma (IFN-gamma) induced NO production and inhibition of fungal growth. The cytotoxic activity of M phi against Ss-12 was significantly greater than against Ss-7 or yeast cells, the highly virulent fungal forms. The addition of NO synthase inhibitors abrogated M phi cytotoxic activity against all fungal forms. The phagocytic activity of M phi against Ss-7 was significantly lower than against Ss-12 or yeast cells. Although the ingestion of fungal cells triggered the oxidative burst in M phi, the fungicidal activity was not altered in the presence of superoxide dismutase (SOD) and catalase. In addition, Ss-12 and yeast cells were more susceptible than Ss-7 to the direct fungicidal activity of the NO donors S-nitroso-N-acetyl-DL-penicillamine (SNAP), S-nitrosoglutathione (GSNO) and 3-morpholinosydnonimine (SIN-1). The results of this study indicate that NO is a key cytotoxic mediator involved in the murine M phi defence against S. schenckii, and that the virulence of Ss-7, Ss-12 and yeast cells may be related to a differential susceptibility to NO.     

Differential potentiation of anti-mycobacterial activity and reactive nitrogen intermediate-producing ability of murine peritoneal macrophages activated by interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α)

The anti-mycobacterial activities of IFN-γ and TNF-α-treated murine peritoneal macrophages were determined. Resident macrophages pretreated with IFN-γ or TNF-α for 2 days were infected with test organisms and subsequently cultured for up to 7 days. First, the early-phase growth of Mycobacterium tuberculosis (days 0–3) was strongly suppressed in IFN-γ-treated macrophages, and progressive bacterial elimination was subsequently observed. Although TNF-α treatment of macrophages did not affect the early phase growth of organisms, bacterial killing was observed in the later phase of cultivation. Second, although IFN-γ-treated macrophages killed M. avium during the first 3 days of culture, regrowth of the intracellular organisms was subsequently observed. TNF-α treatment of macrophages did not influence the mode of intracellular growth of M. avium. Third, IFN-γ but not TNF-α enhanced production of reactive nitrogen intermediates (RNI) by macrophages infected with M. tuberculosis or M. avium, whereas both cytokines increased macrophage release of reactive oxygen intermediates (ROI). The present findings therefore show that IFN-γ and TNF-α potentiated the anti-mycobacterial activity of murine peritoneal macrophages in different fashions. They also suggest that RNI played more important roles than did ROI in the expression of macrophage anti-mycobacterial, particularly anti-M. avium, activity.     

Mechanisms of the in vitro fungicidal effects of human neutrophils against Penicillium marneffei induced by granulocyte-macrophage colony-stimulating factor (GM-CSF)

We examined the in vitro fungicidal activity of human neutrophils against conidia and yeast cells of Penicillium marneffei. Neutrophils showed a small but significant anti-fungal effect against the yeast form of P. marneffei. Treatment of neutrophils with GM-CSF significantly augmented their anti-fungal activity. In contrast, the conidia form resisted killing even by stimulated neutrophils. Neutrophil fungicidal effect was not inhibited by superoxide dismutase (SOD), while the same treatment significantly suppressed the killing of Candida albicans by GM-CSF-stimulated neutrophils. For effective killing of P. marneffei yeast cells by GM-CSF-stimulated neutrophils, direct contact between the two was essential; interference in such interaction by separation using a 0. 45-microm-pored membrane prevented such an effect. Addition of colchicine attenuated GM-CSF-stimulated neutrophil fungicidal activity in a dose-dependent manner. This effect did not appear to be mediated by interference with neutrophil mobility toward yeast cells, because similar results were obtained when the cultures were set in round-bottomed wells which facilitate their direct contact. Finally, granular extracts derived from unstimulated neutrophils significantly suppressed the growth of microorganisms. Pretreatment of neutrophils with GM-CSF markedly enhanced this effect. The fungicidal activity of granular lysates was strongly, but not completely, reduced by heat treatment. Considered together, our results indicate that GM-CSF-stimulated neutrophils killed the yeast form of P. marneffei present in close proximity, probably in a superoxide anion-independent mechanism, but through exocytosis of granular enzymes which were largely heat-labile.     

Failure of P strain mice to respond to vaccination against schistosomiasis correlates with impaired production of IL-12 and up-regulation of Th2 cytokines that inhibit macrophage activation

In contrast to most inbred strains, P mice fail to develop significant resistance to Schistosoma mansoni infection as a result of vaccination with either radiation-attenuated cercariae or schistosome antigens plus Bacillus Calmette Guérin, and this failure correlates with defects in macrophage larvicidal activity. Supernatant fluids from antigen-treated in vitro cultures of splenocytes from vaccinated P mice demonstrate less macrophage stimulatory activity than do supernatants from cells of vaccine-responsive strains such as C57BL/6. This is not due either to diminished production of the macrophage-activating cytokine IFN-γ by P mice, or to a lesser responsiveness of macrophages from P mice to activation by IFN-γ. Rather, P splenocytes produce two-to threefold higher amounts of IL-4 and IL-10, cytokines which down-regulate the cytotoxic potential of IFN-γ-treated macrophages. Thus, the macrophage-activating potential of cytokine preparations from vaccinated P mice can be completely recovered by in vitro treatment with antibodies to IL-4 or IL-10. Moreover, lower levels of IL-12, a cytokine involved in promoting development of Th1 responses, are produced by splenocytes from P mice as compared to C57BL/6 counterparts. These studies indicate that a genetic predisposition toward an impaired production of IL-12 and an increased production of down-regulatory Th2 cytokines correlate with low response to vaccination against S. mansoni.     

Acid-induced duodenal mucosal nitric oxide output parallels bicarbonate secretion in the anaesthetized pig

We recently showed the involvement of the L -arginine/nitric oxide (NO) pathway in acid-induced duodenal mucosal bicarbonate secretion in rats. The aim of the present study was to confirm this observation in pigs by direct measurements of NO production. Experiments were performed on 16 anaesthetized pigs of both sexes treated with guanethidine (6 mg kg^?1, intravenously). A duodenal segment, devoid of pancreaticobiliary influxes, was perfused with saline and the duodenal mucosal bicarbonate secretion was calculated from continuous measurements of pH and Pco₂. The perfusate contents of NO and its oxidative product nitrite were determined by chemiluminescence, after reduction of nitrite to NO. Luminal acidification with 30 mM hydrochloric acid increased the output of bicarbonate as well as NO to the perfusate, by 195 ± 45% and 106 ± 10%, respectively. These responses to acid were markedly inhibited by adding the NO synthase inhibitor N^G-monomethyl- L -arginine (L-NMMA, 0.3 mM) to the perfusate. The inhibitory effect of L-NMMA could be reversed by administration of L-arginine (3 mM). The study presents simultaneous measurements of bicarbonate and NO outputs to a duodenal luminal perfusate. The results strongly support the view that the L -arginine/NO pathway is involved in the acid-induced duodenal mucosal bicarbonate secretory response.     

Specific lysis of Listeria monocytogenes-infected macrophages by class II-restricted L3T4+ T cells

Mice were infected with the intracellular bacterium, Listeria monocytogenes, and T cell clones from spleens, lymph nodes and peritoneal exudates were established. The capacity of L3T4⁺, Lyt2^? T-cell clones to specifically lyse L. monocytogenes-infected macrophages was analyzed. As a source of target cells, bone marrow macrophages (BMMΦ) after 9 days of culture in hydrophobic teflon bags were used. These BMMΦ were totally Ia^?; however, significant Ia-expression could be induced by interferon-γ (IFN-γ). IFN-γ-stimulated BMMΦ, after priming with live or killed L. monocytogenes organisms were effectively lysed by the vast majority of L3T4⁺ T cell clones. In the absence of either IFN-γ stimulation or antigen priming, no lysis occurred. Cytolysis was demonstrable in a conventional 4-h ⁵¹Cr-release assay and in an 18-h neutral red uptake assay and was antigen specific and class II restricted. Native T cells from L. monocytogenes-infected mice failed to lyse stimulated, L. monocytogenes-primed BMMΦ and gained their cytolytic activity after antigenic restimulation in vitro. These data demonstrate that L. monocytogenes-specific L3T4⁺ T cells could lyse MΦ presenting listerial antigens provided that Ia antigen expression had been induced. L3T4⁺ T cell clones produced IFN-γ after restimulation with antigen plus accessory cells in vitro and IFN-γ secretion could be increased by costimulation with recombinant IL 2. These T cell clones conferred significant protection upon recipient mice which was more pronounced in the liver. The possible relevance of lysis by L3T4⁺ T cells of infected MΦ to protection against and pathogenesis of intracellular bacterial infections is discussed.     

Differences in the Rate of Intracellular Killing of Catalase-Negative and Catalase-Positive Listeria monocytogenes by Normal and Interferon-Gamma-Activated Macrophages

Intracellular killing of catalase-positive bacteria by murine resident macrophages requires the presence of extracellular serum, whereas killing of catalase-negative bacteria can occur in the absence of serum. To find out whether the intracellular killing of bacteria by rIFN-γ-activated macrophages also requires serum stimulation, we investigated the handling of ingested catalase-negative and -positive Listeria monocytogenes by peritoneal macrophages of normal Swiss mice and mice injected i. p. with I × 10⁴U rIFN-γ 18 h earlier. In the absence of extracellular serum. rlFN-γ-activated macrophages killed ingested catalase-negative Listeria more efficiently (P<0.01) than normal resident macrophages. Maximal killing of catalase-negative bacteria by rlFN-γ-activated macrophages required an extracellular serum concentration of only 1.0 to 2.5% compared with the 10% needed by normal macrophages. No differences were observed in the rates of intracellular killing of catalase-positive Listeria by rlFN-γ-activated and normal resident macrophages: both populations of macrophages required 10% extracellular serum for maximal killing of these bacteria, and killing was minimal in the absence of scrum. The rIFN-γ-activated macrophages displayed enhanced O₂-consumption after stimulation with phorbol myristate acetate and heat-killed Listeria compared with macrophages from normal mice. These findings indicate that, under suboptimal stimulation by extracellular serum. rlFN-γ enhances the intracellular killing of catalase-negative Listeria which lack endogenous catalase acting as a scavenger of reactive oxygen intermediates. The mechanism underlying the enhancement is probably the amplification of the respiratory burst by IFN-γ.     

Interleukin-10 and interleukin-4 inhibit intracellular killing of Leishmania infantum and Leishmania major by human macrophages by decreasing nitric oxide generation

The host response to Leishmania infection is regulated by a specific pattern of local cytokine production. We investigated the effect of interleukin (IL)-10 and IL-4 on the leishmanicidal activity of human macrophages (M?). As with L. major, intracellular killing of L. infantum by human M? was obtained following ligation of surface CD23 or cell treatment with Interferon-γ (IFN-γ). This leishmanicidal activity required nitric oxide (NO) generation by activated M?, and it was partially mimicked by cell treatment with chemical NO donors. Addition of recombinant human IL-10 or IL-4 to CD23 mAb or IFN-γ decreased L. infantum and L. major killing by infected M?. IL-10 was more potent than IL-4 in inhibiting the leishmanicidal activity of human M?. Inhibition of Leishmania killing by IL-4 and IL-10 correlated with decreased NO generation from M?, and was reversed when exogenous NO was added to cell cultures. Therefore, IL-10 and IL-4 down-regulate leishmanicidal activity of human M?, in part by inhibiting NO generation by these cells.     

Coccidioides releases a soluble factor that suppresses nitric oxide production by murine primary macrophages

We studied the effect of the presence of Coccidioides on the production of nitric oxide (NO) by primary macrophages previously activated by IFN-γ and LPS. The fungal cells were isolated from cultures of arthroconidia that had been incubated for 24 h in a medium that supported parasitic phase growth and were co-cultured with the macrophages. These live, first-generation parasitic cells of Coccidioides, referred to as spherule initials, suppressed NO production as well as iNOS mRNA expression by activated macrophages. Phagocytosis was not required for suppression of NO. We also showed that the culture supernatant of the spherule initials was capable of suppressing NO production, and that this activity was mediated by an as yet unidentified, secreted fungal factor(s). Heat-, paraformaldehyde- or X-ray-treated spherule initials did not show this inhibitory effect. To our surprise, macrophages obtained from iNOS-deficient mice revealed phagocytic activity and killing efficiency which were comparable to that of macrophages isolated from wild type C57BL/6 mice. Although the cultured fungal pathogen can suppress NO production, this oxidative product is apparently not essential for in vitro killing of Coccidioides by activated macrophages. Our results suggest that other unidentified fungicidal mechanisms exist against Coccidioides which are apparently independent of NO production.     

Intracellular co-localization of Trypanosoma cruzi and inducible nitric oxide synthase (iNOS): evidence for dual pathway of iNOS induction

Evidence is presented from studies in vitro and in vivo for a dual pathway of inducible nitric oxide synthase (iNOS) induction during Trypanosoma cruzi infection, one of which is interferon (IFN)-γ dependent and the other not. In vitro, the IFN-γ-dependent iNOS induction decreases parasite multiplication, and is in vivo associated with protection. iNOS induced by this pathway mediated a high NO output and showed a diffuse, cytoplasmic immunostaining in IFN-γ-activated macrophages in vitro as well as in cell infiltrates or infected tissues. Surprisingly, in such tissues, iNOS co-localized with parasite nests, and by immunoelectromicroscopy, iNOS was demonstrated on the parasite surface. iNOS co-localization with parasites was also seen in tissues from T. cruzi-infected IFN-γ receptor (R) knockout mice suggesting an IFN-γ-independent pathway of induction. However, no cytoplasmic iNOS was seen in inflammatory infiltrates of these tissues. IFN-γR^−/− mice displayed a dramatically enhanced susceptibility to infection with T. cruzi, diminished accumulation of iNOS mRNA in skeletal muscle and spleen cells, and reduced release of NO and per-oxynitrite. Expression of iNOS around intracellular parasites was also observed after infection of peritoneal macrophages or L-929 fibroblasts in vitro in the absence of other exogenous stimuli. A time-dependent NO release and enhanced accumulation of iNOS mRNA also was observed in infected peritoneal cells and fibroblasts. Cultured T. cruzi amastigotes, trypomastigotes, and epimastigotes were not labeled by the anti-iNOS antibodies and contained no iNOS mRNA, indicating that the iNOS detected actually originated from the mammalian cell. A pathogenic effect of low NO levels is suggested by the arresting effect of NOS inhibitors and the enhancing consequences of low concentrations of NO donors on intracellular parasite multiplication.     

T cell response in murine Leishmanial mexicana amazonensis infection: production of interferon-γby CD8+ cells

The immune response to Leishmania major has been the subject of many investigations. However, Leishmania includes many species with different clinical manifestations. In this report, we studied the Tcell response to L. mexicana amazonensis, a New World species, in a murine model. We found that, similar to L. major, an Old World species, resistant C57BL/6 mice produced a high level of IFN-γ and a low level of IL-4. Conversely, susceptible BALB/c mice produced a much lower level of IFN-γ and higher level of IL-4. Although IFN-γ is one of the important lymphokines that mediate macrophage activation and thus the destruction of the intracellular parasites, which lymphocyte subsets are producing the IFN-γ is still a controversy. Much evidence including the isolation of protective, IFN-γ-producing, CD4⁺ cell lines have confirmed the participation of CD4⁺ Thl cells unequivocally. However, both CD4⁺ and CD8⁺ cells produced IFN-γ. Recently, an increasing body of evidence has appeared suggesting that CD8⁺ cells also play a role in the resolution of murine L. major infection. We found that in the L. m. amazonensis model, when CD8⁺ lymphocytes from resistant C57BL/6 mice were eliminated by anti-CD8 antibody and complement-mediated lysis, the IFN-γ production was reduced by 77%. This indicated that CD8⁺ cells produced a significant amount of the IFN-γ. However, our results also indicate that IFN-γ production by CD8⁺ cells was dependent on CD4⁺ cells.     

PSK and OK-432-Induced Immunomodulation of Inducible Nitric Oxide (NO) Synthase Gene Expression in Mouse Peritoneal Polymorphonuclear Leukocytes and NO-Mediated Cytotoxicity

Abstract

We investigated whether PSK (a polysaccharide from the mycelia of Coriolus versicolor) or OK-432 (a streptococcal preparation) can up-regulate inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production in mouse peritoneal polymorphonuclear leukocytes (PMNs). Six hrs after intraperitoneal injection of mice with PSK (2500 μg/mouse) or OK-432 (100 μg/mouse), mouse peritoneal PMNs were restimulated with PSK (500 μg/ml) or OK-432 (10 μg/ml) plus 100 U/ml of mouse interferon-gamma (IFN-γ) in vitro. Northern blot analysis showed strong synergism between both PSK and OK-432 and IFN-γ for the induction of iNOS gene expression. NO production by PMNs was increased up to 20 μM (2 μM/10⁶ PMNs/24 hrs) as measured by the Griess reagent method when PMNs were restimulated with PSK or OK-432 plus IFN-γ for 24 hrs, although tumor cell killing was not detected. NO concentrations of more than 80 μM were required for P815 tumor cell killing. These results suggest that PMNs produce NO after stimulation with PSK or OK-432 in combination with IFN-γ and may regulate the immune system in vivo, although the NO production induced by these agents is insufficient for tumor cell killing in vitro.     

Increased nitric oxide (NO) production by antigen-presenting dendritic cells is responsible for low allogeneic mixed leucocyte reaction (MLR) in primary biliary cirrhosis (PBC)

The levels of blastogenesis in allogeneic MLR containing T cells from one normal volunteer and irradiated dendritic cells from 29 patients with PBC, 17 patients with chronic hepatitis type C (CH-C) and 22 allogeneic normal controls were compared to see if there is any role of antigen-presenting cells (APC) in the pathogenesis of PBC. The stimulatory capacity of dendritic cells from PBC was significantly lower compared with that of dendritic cells from CH-C (P < 0.05) and normal controls (P < 0.05), which could not be attributable either to the levels of expression of surface molecules, such as HLA-DR and CD86 on dendritic cells, or to the levels of cytokines, such as IL-10 and IL-12. Significantly higher levels of NO were seen in the allogeneic MLR supernatants containing dendritic cells from PBC compared with the supernatants from cultures containing dendritic cells from CH-C (P < 0.001) or normal controls (P < 0.001). Moreover, dendritic cells from PBC produced 10 times more NO compared with dendritic cells from CH-C and normal controls (21.9 ± 2.8 μM versus 1.6 ± 0.3 μM and 1.6 ± 0.3 μM , respectively; P < 0.001). The addition of N^G-monomethyl- L -arginine monoacetate (L-NMMA), a known inhibitor of NO in allogeneic MLR containing dendritic cells from PBC, resulted in a significant decrease of NO and increase of blastogenesis. The selective impairment of dendritic cell function, increased production of NO by dendritic cells and restoration of blastogenesis using NO inhibitor in PBC have suggested a role for NO and dysfunction of dendritic cells in the pathogenesis of PBC. This inspires optimism that modulating the function of dendritic cells and controlling NO production, an improved therapeutic approach, might be planned for PBC.     

Macrophage chemotactic protein-1 and macrophage inflammatory protein-1 alpha induce nitric oxide release and enhance parasite killing in Leishmania infantum-infected human macrophages

Chemokines are a group of structurally defined small proteins that act as chemoattractants for leukocytes and are involved in many different biological activities, including leukocyte activation for antimicrobial mechanisms. We studied the effect of the chemokines monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1α on nitric oxide release and parasitocidal ability of peripheral blood-derived human macrophages in vitro infected with Leishmania infantum, zymodeme MON1. In infected human macrophages, treatment with MCP-1 or MIP-1α significantly enhanced nitric oxide production and leishmanicidal ability, compared with untreated cells, to the same levels induced by interferon-γ. Both nitric oxide release and parasitocidal ability of macrophages were significantly reduced by addition of L-N ^Gmonomethylarginine (L-NMMA), which is a competitive inhibitor of the L-arginine nitric oxide pathway. These data suggest that MCP-1 and MIP-1α mediate macrophage activation for nitric oxide release and subsequent parasite clearance, and thus may play a role in the containment of Leishmania infection. Received: 9 November 2001 / Accepted: 16 September 2002 Correspondence to O. Brandoniso     

Nitric oxide-mediated suppression of T cell responses during Trypanosoma brucei infection: soluble trypanosome products and interferon-γ are synergistic inducers of nitric oxide synthase

African trypanosome infections result in lymphocyte unresponsiveness and anemia in the mammalian host. In murine infections, these effects are mediated by suppressor macrophages releasing nitric oxide (NO). We investigated the mechanism of activation of macrophages to produce NO during trypanosomiasis in vitro. A soluble component of trypanosome lysates induced NO synthesis in peritoneal macrophage cultures only when the macrophages were co-stimulated with interferon-gamma (IFN-γ). The macrophage-activating factor was also released in a soluble form by live bloodstream-form trypanosomes, but not procyclic trypanosomes. When splenocyte cultures were exposed to IFN-γ and trypanosomes, an NO-dependent suppression of T cell proliferation occurred. This is similar to the suppression observed in the spleens of trypanosome-infected mice, suggesting that a combination of trypanosome-released macrophage-activating factors and IFN-γ are a trigger of immune dysfunction in trypanosomiasis.