Inhibition of the induction of the inducible nitric oxide synthase in murine brain microglial cells by sodium salicylate.

The induction of the inducible nitric oxide synthase (iNOS) has been proposed to play a role in a variety of inflammatory diseases. Sodium salicylate (NaSal) is the most commonly used anti-inflammatory agent. We investigated whether NaSal can diminish the induction of iNOS in murine brain microglial cells. In primary cultures, interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS) separately did not stimulate nitric oxide (NO) production, whereas IFN-gamma combined with LPS synergistically induced iNOS. NaSal inhibited both the production of NO and expression of iNOS in microglial cells. Synergy between IFN-gamma and LPS was mainly dependent on tumour necrosis factor-alpha (TNF-alpha) secretion as the increase of the induction of the iNOS by IFN-gamma plus LPS was associated with the increase of TNF-alpha secretion and IFN-gamma plus LPS-induced TNF-alpha secretion by microglial cells was decreased by the treatment with NaSal. These results suggest a possible use of NaSal in managing inflammation of the central nervous system through inhibition of the iNOS induction.     

Entamoeba histolytica modulates the nitric oxide synthase gene and nitric oxide production by macrophages for cytotoxicity against amoebae and tumour cells.

Nitric oxide (NO) is the major cytotoxic molecule produced by activated macrophages for cytotoxicity against Entamoeba histolytica trophozoites. In the present study, we determined whether E. histolytica infection and soluble amoebic proteins affected macrophage cytotoxicity against amoebae and tumour cells by modulating the inducible NO synthase gene (iNOS) and NO (measured as nitrite, NO2-) and tumour necrosis factor-alpha (TNF-alpha) production. Amoebic liver abscess-derived macrophages [days 10, 20, 30 post-infection (p.i.)] stimulated with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) showed increased cytotoxicity against L929 cells (TNF-alpha-sensitive), but were refractory for killing amoebae and P815 cells (both NO-sensitive), concomitant with low NO2- production (< 4 microM/10(6) cells). In contrast, peritoneal and spleen macrophages at 10 and 20 days p.i. activated with IFN-gamma and LPS demonstrated increased killing of amoebae, and L929 and P815 cells concomitant with high NO2- production (> 12 microM/10(6) cells). Pretreatment of mouse bone marrow-derived macrophages with amoebic proteins suppressed IFN-gamma and LPS-induced amoebicidal (33%) and tumoricidal (44-49%) activities, with a corresponding decrease in TNF-alpha (56%) and NO (41%) production as well as TNF-alpha (41%) and iNOS (27%) mRNA by Northern blot analyses as compared to untreated activated controls. Inhibition of prostaglandin E2 (PGE2) biosynthesis in abscess and naive macrophages pretreated with amoebic proteins augmented IFN-gamma- and LPS-induced killing of L929 cells and TNF-alpha production, but failed to increase killing of P815 cells and amoebae as well as iNOS mRNA levels or NO production. These results suggest that E. histolytica selectively induces dysfunction of macrophage cytotoxicity by modulating iNOS mRNA expression and NO production independent from TNF-alpha and PGE2 allowing the parasites to survive within the host by impairing host immune responses.     

Interferon-gamma alone triggers the production of nitric oxide from serum-starved BNL CL.2, murine embryonic liver cells.

A previous study has demonstrated that both interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) were needed to induce the production of nitric oxide (NO) in BNL CL.2 cells, murine embryonic liver cells. We here demonstrate that when BNL CL.2 cells were cultured with serum-free medium, they were induced to produce NO by the stimulation of IFN-gamma alone. BNL CL.2 cells were cultured with serum-free or serum-containing medium for 1-3 days and then stimulated to synthesize NO by IFN-gamma. Surprisingly, only serum-starved cells showed significant amount of nitrite accumulation and iNOS protein expression in response to IFN-gamma in dose- and time-dependent manners, but serum-supplied cells did not. When the cells were stimulated with IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), or LPS in combinations, only the combination of IFN-gamma and LPS produced more NO than that produced by IFN-gamma alone. The production of NO by the cells stimulated with IFN-gamma or IFN-gamma plus LPS was blocked by the addition of N(G)-monomethyl-L-arginine (N(G)MMA), a NO synthesis inhibitor. To address the intracellular signal pathway responsible for the production of NO by the cells stimulated with IFN-gamma aloneor IFN-gamma plus LPS, we examined the effects of several protein kinase inhibitors on the production of NO from the cells. The production of NO was significantly inhibited by protein tyrosine kinase (PTK) inhibitors, genistein and herbimycin A, but not by protein kinase A or C inhibitors. These results suggest that the deprivation of serum from BNL CL.2 cell culture medium might prime the cells to induce NO synthesis when the cells are triggered by IFN-gamma and the involvement of PTK signal transduction pathway in the expression of inducible NO synthase gene in murine hepatoma cells.     

Transforming growth factor-beta 1 primes macrophages for enhanced expression of the nitric oxide synthase gene for nitric oxide-dependent cytotoxicity against Entamoeba histolytica.

Nitric oxide (NO) produced by activated macrophages is the major cytotoxic molecule for in vitro cytotoxicity against Entamoeba histolytica trophozoites. Transforming growth factor-beta 1 (TGF-beta 1) is a potent negative regulator of several macrophage functions, including NO production. In this study, we investigated the effect of TGF-beta 1 on macrophage nitric oxide synthase (mac-NOS) mRNA expression and NO production for macrophage cytotoxicity against E. histolytica trophozoites. TGF-beta 1 by itself was incapable of inducing mouse bone marrow-derived macrophage (BMM) amoebicidal activity and NO production (as measured by nitrite). In contrast, TGF-beta 1 pretreatment (4 hr) primed BMM for an enhanced amoebicidal activity of 15% and 23% in response to (interferon-gamma) IFN-gamma+tumour necrosis factor-alpha (TNF-alpha) or IFN-gamma+lipopolysaccharide LPS, concomitant with increased NO production of 85% and 27%, respectively. TGF-beta 1 pretreatment increased NO production in response to IFN-gamma+TNF-alpha/LPS stimulation in a time- and dose-dependent manner. By Northern blot analysis, the increased NO production of TGF-beta 1-pretreated BMM was preceded by markedly enhanced expression of mac-NOS mRNA. The priming effect of TGF-beta 1 on NO production was critically dependent on both a TNF-alpha (> or = 100 U) and a LPS (> or = 100 ng) triggering dose in the presence of IFN-gamma. TGF-beta 1 pretreatment enhanced TNF-alpha mRNA expression, but had no effect on TNF-alpha production in culture supernatants after 4 hr of stimulation with IFN-gamma+TNF-alpha/LPS; however, at a later time-point (16-48 hr), even though the levels of TNF-alpha mRNA expression were unaffected, TNF-alpha production was reduced. These data demonstrate that TGF-beta 1 priming for increased mac-NOS mRNA expression for NO-dependent cytotoxicity against E. histolytica in response to IFN-gamma+TNF-alpha/LPS stimulation may be involved in the modulation of a TNF-alpha triggering signal by TGF-beta 1.     

Surfactant protein A differentially regulates IFN-gamma- and LPS-induced nitrite production by rat alveolar macrophages

Although several studies have demonstrated that the pulmonary collectins surfactant protein (SP)-A and SP-D contribute to innate immunity by enhancing pathogen phagocytosis, the role of SP-A and SP-D in regulating production of free radicals and cytokines is controversial. We hypothesized that the state and mechanism of activation of the immune cell influence its response to SP-A. The effects of SP-A and SP-D on production of nitric oxide (NO) and inducible nitric oxide synthase (iNOS) were assessed in isolated rat alveolar macrophages activated with lipopolysaccharide (LPS), interferon gamma (IFN-gamma), or both agonists. SP-A inhibited production of NO and iNOS in macrophages stimulated with smooth LPS, which did not significantly bind SP-A, or rough LPS, which avidly bound SP-A. In contrast, SP-A enhanced production of NO and iNOS in cells stimulated with IFN-gamma or INF-gamma plus LPS. Neither SP-A nor SP-D affected baseline NO production, and SP-D did not significantly affect production of NO in cells stimulated with either LPS or IFN-gamma. These results suggest that SP-A contributes to the lung inflammatory response by exerting differential effects on the responses of immune cells, depending on their state and mechanism of activation.     

Human chorionic gonadotropin induces nitric oxide synthesis by murine microglia

This study investigated the effects of human chorionic gonadotropin (hCG) on the synthesis of nitric oxide (NO) in murine neonatal microglial cells. When hCG was used in combination with interferon-gamma (IFN-gamma), there was a marked cooperative induction of NO synthesis in a dose-dependent manner. This increase in NO synthesis was reflected as an increased amount of iNOS protein. The increase of NO synthesis by IFN-gamma-plus-hCG was associated with the increase of tumor necrosis factor-alpha (TNF-alpha) secretion and hCG-induced NO production was decreased by the treatment with anti-murine TNF-alpha neutralizing antibody. This study provides evidence that hCG activates expression of iNOS protein in murine microglial cells accompanied by NO accumulation via pathway dependent on L-arginine in the culture medium, and further offers that TNF-alpha acts on the NO synthesis from IFN-gamma-primed murine microglial cells.     

Protein kinase C independent activation of inducible nitric oxide synthase by tumor necrosis factor-alpha in TM4 Sertoli cells

To investigate the nitric oxide (NO) production and its signalling mechanism in TM4 Sertoli cells, the cells were treated with recombinant tumor necrosis factor-alpha (rTNF-alpha), recombinant interleukin-1 alpha (rIL-1alpha), or lipopolysaccharide (LPS), either alone or in combination with recombinant interferon-gamma (rIFN-gamma), and NO production was measured by using the Griess method. TM4 Sertoli cells produced a small amount of NO upon treatment with rIFN-gamma. The effect of rIFN-gamma was drastically increased by cotreatment with rTNF-alpha in a dose-dependent manner. However, combination of rIL-1alpha or LPS with rIFN-gamma did not synergize to activate cells. RIFN-gamma in combination with rTNF-alpha showed marked increase of the expression of iNOS protein. Protein kinase C inhibitors did not inhibit the production of NO induced by rIFN-gamma plus rTNF-alpha. These results suggest that the role of TNF-alpha is to provide TM4 Sertoli cells with the active cofactor for NO production and TNF-alpha-induced signaling for induction of NO synthesis is not dependent on protein kinase C activation.     

Tumor necrosis factor alpha augments nitric oxide-dependent macrophage cytotoxicity against Entamoeba histolytica by enhanced expression of the nitric oxide synthase gene.

Nitric oxide (NO measured as nitrite, NO2-) is the major effector molecule produced by activated macrophages for in vitro cytotoxicity against Entamoeba histolytica trophozoites. In this study, we determine whether tumor necrosis factor alpha (TNF-alpha) produced by activated bone marrow-derived macrophages (BMM) is involved in the induction of the inducible NO synthase gene (mac-NOS) for NO-dependent amebicidal activity. TNF-alpha alone did not directly induce macrophage NO2- production to kill amebae; however, in combination with increasing concentrations of TNF-alpha and gamma interferon (IFN-gamma), BMM amebicidal activity and NO2- production progressively increased and showed a significant linear correlation. Antiserum to TNF-alpha and the NO synthase inhibitor NG-monomethyl L-arginine (L-NMMA) inhibited the synergistic effects of TNF-alpha and IFN-gamma. BMM activated with increasing concentrations of lipopolysaccharide (LPS) and IFN-gamma showed a significant linear correlation between TNF-alpha release and NO2- production. Antiserum to TNF-alpha suppressed TNF-alpha release, NO2- production, and amebicidal activity by 93, 53, and 86%, respectively. L-NMMA diminished NO2- production by 74% and macrophage amebicidal activity by 83% but had no effect on TNF-alpha release. Quantification by Northern (RNA) blot analyses demonstrated that IFN-gamma in combination with TNF-alpha or LPS increased markedly the accumulation of mac-NOS and TNF-alpha mRNAs in a time-dependent manner with a concomitant increase in NO and TNF-alpha production. Peak induction of mac-NOS occurred after 24 h, whereas TNF-alpha mRNA was rapidly expressed after 4 h and remained stable for 48 h. Taken together, these data argue that TNF-alpha augments NO-dependent macrophage cytotoxicity against E. histolytica via elevated levels of mac-NOS mRNA expression which may be associated with the accumulation of TNF-alpha mRNA.     

Lack of macrophage migration inhibitory factor in mice does not affect hallmarks of the inflammatory/immune response during the first week after stroke

Background

Activation of glial cells, including astrocytes and microglia, has been implicated in the inflammatory responses underlying brain injury and neurodegenerative diseases including Alzheimer's and Parkinson's diseases. Although cultured astrocytes and microglia are capable of responding to pro-inflammatory cytokines and lipopolysaccharide (LPS) in the induction and release of inflammatory factors, no detailed analysis has been carried out to compare the induction of iNOS and sPLA2-IIA. In this study, we investigated the effects of cytokines (TNF-alpha, IL-1beta, and IFN-gamma) and LPS + IFN-gamma to induce temporal changes in cell morphology and induction of p-ERK1/2, iNOS and sPLA₂-IIA expression in immortalized rat (HAPI) and mouse (BV-2) microglial cells, immortalized rat astrocytes (DITNC), and primary microglia and astrocytes.

Methods/Results

Cytokines (TNF-alpha, IL-1beta, and IFN-gamma) and LPS + IFN-gamma induced a time-dependent increase in fine processes (filopodia) in microglial cells but not in astrocytes. Filopodia production was attributed to IFN-gamma and was dependent on ERK1/2 activation. Cytokines induced an early (15 min) and a delayed phase (1 ~ 4 h) increase in p-ERK1/2 expression in microglial cells, and the delayed phase increase corresponded to the increase in filopodia production. In general, microglial cells are more active in responding to cytokines and LPS than astrocytes in the induction of NO. Although IFN-gamma and LPS could individually induce NO, additive production was observed when IFN-gamma was added together with LPS. On the other hand, while TNF-alpha, IL-1beta, and LPS could individually induce sPLA₂-IIA mRNA and protein expression, this induction process does not require IFN-gamma. Interestingly, neither rat immortalized nor primary microglial cells were capable of responding to cytokines and LPS in the induction of sPLA2-IIA expression.

Conclusion

These results demonstrated the utility of BV-2 and HAPI cells as models for investigation on cytokine and LPS induction of iNOS, and DITNC astrocytes for induction of sPLA2-IIA. In addition, results further demonstrated that cytokine-induced sPLA2-IIA is attributed mainly to astrocytes and not microglial cells.     

Phagocytosis enhances murine macrophage activation by interferon-gamma and tumor necrosis factor-alpha

Previously, we reported that exposure of bone marrow-derived macrophages (M phi) to a phagocytic stimulus in the simultaneous presence of interferon-gamma (IFN-gamma) induced these cells to generate nitrite (NO2-). This effect was achieved using both living (i.e. promastigotes of the protozoan parasite Leishmania enriettii) and inert (latex beads) particles. When the phagocytic stimulus was Leishmania, enhanced intracellular killing accompanied elevated NO2- secretion. As shown here, the capacity of phagocytosis to elicit NO2- production by IFN-gamma-treated M phi was inhibited by antibody to murine recombinant tumor necrosis factor-alpha (rTNF-alpha), suggesting that phagocytosis enabled IFN-gamma to activate M phi via the induction of TNF-alpha as an autocrine second signal. M phi NO2- production in response to rIFN-gamma and either exogenous TNF-alpha or Leishmania was strongly enhanced by prostaglandin E2, consistent with such a mechanism. However, addition of either Leishmania promastigotes or latex beads to M phi cultures simultaneously exposed to both IFN-gamma and exogenous murine or human rTNF-alpha further potentiated activation as measured by NO2- release. Furthermore, anti-TNF antibody failed to inhibit M phi responses to rIFN-gamma and bacterial lipopolysaccharide (LPS) in the presence or absence of Leishmania; also exogenous rTNF-alpha did not significantly affect NO2- production by IFN-gamma/LPS cultures despite a strong enhancement by Leishmania. These results suggest that phagocytosis enhances M phi responses by a process more complex than the sole induction of TNF-alpha. Phagocytosis also increased M phi NO2- production elicited by IFN-gamma plus TNF-alpha in L-arginine-deficient media. These results indicate that phagocytosis may be an important mechanism of up-regulating M phi microbicidal activity, and could be particularly relevant upon arginine depletion which occurs during an inflammatory response.     

Induction of nitric oxide production by bovine alveolar macrophages in response toPasteurella haemolyticaA1

We assessed the kinetics of inducible nitric oxide synthase (iNOS) mRNA expression and production of nitric oxide (NO) in bovine alveolar macrophages (AMs) stimulated with purified lipopolysaccharide (LPS) fromPasteurella haemolyticastrain 12296. The effect of LPS on iNOS gene expression was dose-dependent and was expressed maximally at 24 h after stimulation with 10 μg/ml of LPS. Production of NO measured as secreted nitrite in supernatants took place in a time and dose-dependent manner with peak production at 24 h after LPS stimulation. Recombinant bovine gamma interferon (rbγIFN) augmented the LPS-induced iNOS gene expression and production of NO. The ability of LPS to induce iNOS gene expression and NO production either alone or in combination with rbγIFN was significantly abrogated by polymyxin B. In addition, the iNOS inhibitor N^G-monomethyl-Larginine (L-NMMA) significantly inhibited LPS and rbγIFN+LPS induced NO production. Our results also demonstrated that NO produced from an exogenous NO donor sodium nitroprusside (SNP), and NO generated from LPS-stimulated AMs (endogenous) caused cytotoxic injury to bovine pulmonary artery endothelial cells in a dose-dependent manner. The cytotoxic injury caused by NO generated from LPS stimulated AMs was inhibited by polymyxin B or L-NMMA. There was a markedly increased concentration of nitrite in the lung lavage fluids of calves followingP. haemolyticainfection. These findings support a role for NO in the pathogenesis of lung injury in bovine pneumonic pasteurellosis.     

The roles of Nramp1 and Tnfa genes in nitric oxide production and their effect on the growth of Salmonella typhimurium in macrophages from Nramp1 congenic and tumor necrosis factor-alpha-/- mice.

The macrophages from Nramp1 congenic mice and tumor necrosis factor (TNF)-alpha(-/-) mice were used to examine the functions of Nramp1 and Tnfa genes in nitric oxide (NO) production and Salmonella typhimurium infection. It was confirmed that the level of inducible NO synthase (iNOS)-mediated NO production in Nramp1(r) peritoneal macrophages was generally higher than that of Nramp1(s) macrophages after stimulation by interferon-gamma (IFN-gamma), lipopolysaccharide (LPS), and tumor necrosis factor-alpha (TNF-alpha) alone or in combination. Nramp1 mRNA expression in both Nramp1 congenic macrophages was constitutive notwithstanding cytokine stimulation. During infection with S. typhimurium strain 6203, Nramp1(r) macrophages produced a lower amount of NO because of an initial strong reaction and unsustained iNOS gene expression as compared with Nramp1(s) macrophages. An inhibitory effect of the Nramp1(r) gene on bacterial replication was also observed during the early stage of S. typhimurium infection, whereas the effect of TNF-alpha occurred later. NO production and iNOS expression in TNF-alpha(-/-) macrophages were not detected from the start of the bacterial infection or at 24 h after infection. We also observed that S. typhimurium strain 6203 grew more profoundly without TNF-alpha, especially in Nramp1(s) macrophages. These data, therefore, demonstrate that there is cooperation of the Nramp1 and Tnfa genes in NO production and a growth inhibitory effect in response to S. typhimurium infection.     

CpG ODN activates NO and iNOS production in mouse macrophage cell line (RAW 264.7)

Synthetic CpG containing oligodeoxynucleotide (CpG ODN) is recognized for its ability to activate cells to produce several cytokines, such as IL-12 and TNF-alpha. In the present study we have demonstrated that CpG ODN 1826, known for its immunostimulatory activity in the mouse system could, by itself, induce nitric oxide (NO) and inducible nitric oxide synthase (iNOS) production from mouse macrophage cell line (RAW 264.7). Neutralizing antibody against TNF-alpha was not able to inhibit NO or iNOS production from the CpG ODN 1826-activated macrophages, suggesting that although the TNF-alpha was also produced by CpG ODN-activated macrophages, the production of iNOS was not mediated through TNF-alpha. Although both CpG ODN 1826 and lipopolysaccharide (LPS) were able to stimulate NO and iNOS production, the exposure time required for maximum production of NO and iNOS for the CpG ODN 1826-activated macrophages was significantly longer than those activated with LPS. These results were due probably to a delay of NF-kappaB translocation, as indicated by the delay of IkappaBalpha degradation. Moreover, the fact that chloroquine abolished NO and iNOS production from the cells treated with CpG ODN 1826 but not from those treated with LPS suggested that the induction of NO and iNOS production from the cells stimulated with CpG ODN (1826) also required endosomal maturation/acidification.     

Role of IFN-gamma on dissociation between nitric oxide and TNF/IL-6 production by murine peritoneal cells after restimulation with bacterial lipopolysaccharide

Murine peritoneal exudate cells (PEC) pre-exposed to bacterial lipopolysaccharide (LPS) show augmented nitric oxide (NO) production by LPS restimulation, in contrast to LPS tolerance with reduced production of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6). Significant amounts of interferon-gamma (IFN-gamma) were detected in the PEC cultures on LPS stimulation, and anti-IFN-gamma antibody suppressed the LPS-induced NO, but not TNF-alpha and IL-6, production. Addition of anti-IFN-gamma antibody to the cultures in the LPS pre-exposure step strongly suppressed the augmented NO production on LPS restimulation. Anti-IL-12 antibody, which suppressed the LPS-induced IFN-gamma production, also suppressed the augmented NO production, as did anti-IFN-gamma antibody. Taken together, we propose the following mechanisms: (1) T and NK cells in PEC produce IFN-gamma by the action of IL-12, which is derived from LPS-stimulated macrophages, and (2) the de novo-produced IFN-gamma activates macrophages to augment NO production on LPS restimulation.     

Role of endogenous IFN-gamma in macrophage programming induced by IL-12 and IL-18.

Besides the established role of interleukin-12 (IL-12) and IL-18 on interferon-gamma (IFN-gamma) production by natural killer (NK), T, and B cells, the effects of these cytokines on macrophages are largely unknown. Here, we investigated the role of IL-12/IL-18 on nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by CD11b(+) adherent peritoneal cells, focusing on the involvement of endogenously produced IFN-gamma. C57BL/6 cells released substantial amounts of NO when stimulated with IFN-gamma or lipopolysaccharide (LPS), but failed to respond to IL-12 or IL-18 or both. However, IL-12/IL-18 pretreatment was able to program these cells to release 6-8-fold more NO and TNF-alpha in response to LPS or Trypanosoma cruzi stimulation, with NO levels directly correlating with macrophage resistance to intracellular parasite growth. Analysis of IL-12/IL-18-primed cells from mice deficient in IFN-gamma, IFNGR, and IFN regulatory factor-1 (IRF-1) revealed that these molecules were essential for LPS-induced NO release, but TNF-alpha production was IFN-gamma independent. Conversely, the myeloid differentiation factor 88 (MyD88)-dependent pathway was indispensable for IL-12/IL-18-programmed LPS-induced TNF-alpha production, but not for NO release. Contaminant T and NK cells largely modulated the IL-12/IL-18 programming of LPS-induced NO response through IFN-gamma secretion. Nevertheless, a small population of IFN-gamma(+) cells with a macrophage phenotype was also identified, particularly in the peritoneum of chronically T. cruzi-infected mice, reinforcing the notion that macrophages can be an alternative source of IFN-gamma. Taken together, our data contribute to elucidate the molecular basis of the IL-12/IL-18 autocrine pathway of macrophage activation, showing that endogenous IFN-gamma plays an important role in programming the NO response, whereas the TNF-alpha response occurs through an IFN-gamma-independent pathway.     

Benznidazole, a drug employed in the treatment of Chagas' disease, down-regulates the synthesis of nitrite and cytokines by murine stimulated macrophages.

Benznidazole (BZL) is a nitroheterocyclic drug employed in the chemotherapy of Chagas' disease, a protozoan disease caused by Trypanosoma cruzi. Because this parasite mostly replicates in macrophages, we investigated whether BZL was likely to modify the synthesis of macrophage mediators such as nitrite, tumour necrosis factor-alpha (TNF-alpha), IL-1beta, IL-6 and IL-10. Control and stimulated murine macrophages (lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma)) were treated with BZL and measurements were carried out in culture supernatants collected 24 h later. Synthesis of nitrite, IL-6 and IL-10 was maximal upon combined stimulation with LPS + IFN-gamma, whereas lower amounts of the three mediators were detected when both stimuli were given alone. BZL treatment significantly reduced nitrite, IL-6 and IL-10 production, to undetectable levels in some cases, particularly IL-6 and IL-10. LPS was the most potent stimulus of IL-1beta and TNF-alpha production, followed by LPS + IFN-gamma and IFN-gamma in decreasing order. BZL partly inhibited TNF-alpha synthesis, but this effect was smaller than that observed for nitrite, IL-6 and IL-10. LPS-induced production of IL-1beta was also affected by BZL. Semiquantification of gene expression for inducible nitric oxide synthase (iNOS) showed that BZL completely inhibited iNOS gene induction by IFN-gamma, and resulted in respective inhibitions of 30% and 50% with LPS- and LPS + IFN-gamma-stimulated cells. BZL was not cytotoxic on macrophage cultures, as shown by the lactate dehydrogenase activity. Besides its trypanocidal activity, BZL may also alter the balance between pro- and anti-inflammatory mediators with important consequences for the course of T. cruzi infection.     

Intracellular growth of Trypanosoma cruzi in cardiac myocytes is inhibited by cytokine-induced nitric oxide release

The effect of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) on Trypanosoma cruzi multiplication and nitric oxide (NO) production in cardiac myocytes was investigated. Cardiac myocyte cultures were obtained from neonatal Wistar rat hearts, infected with T. cruzi, and treated with IL-1beta, TNF-alpha, IFN-gamma, or N-monomethyl-L-arginine (L-NAME) for 72 h. Parasite growth was calculated from the number of infected cells in Giemsa-stained smears. Nitric oxide production was determined with the Griess reagent. Inducible nitric oxide synthase (iNOS) expression by cardiac myocytes was detected by Western blot. The results showed that the percentages of cardiac myocytes containing T. cruzi amastigotes in cytokine-treated cultures were significantly lower than in nontreated cultures. The addition of L-NAME reversed the inhibitory effect on parasite growth of IL-1beta and TNF-alpha but not of IFN-gamma. Nitrite levels released by T. cruzi-infected and noninfected cardiac myocyte cultures after 72 h of stimulation with IL-1beta were significantly higher than those produced upon treatment with TNF-alpha, IFN-gamma, or medium alone, regardless of the infection status. Nitrite levels in TNF-alpha-stimulated infected cultures were significantly higher than in untreated infected cultures and TNF-alpha-treated noninfected cultures. L-NAME inhibited IL-1beta- but not TNF-alpha-induced NO production, indicating the presence of iNOS-dependent and iNOS-independent mechanisms for NO formation in this experimental system. iNOS expression was detected in infected and noninfected cardiac myocytes stimulated with IL-1 beta and TNF-alpha but not with IFN-gamma. These results suggest an important role for cardiac myocytes and locally secreted cytokines in the control of parasite multiplication in T. cruzi-induced myocarditis.