Mycoplasma arthritidis superantigen (MAM)-induced macrophage nitric oxide release is MHC class II restricted,interferongamma dependent,and toll-like receptor 4 independent

Mycoplasma arthritidis causes arthritis in rodents that resembles human rheumatoid arthritis. It produces a superantigen (MAM) that stimulates production of cytokines by making a bridge between lymphocyte T-cell receptor with the appropriate Vbeta chain, and H-2 1-Ealpha MHC class II molecules. Here we studied MAM-induced nitric oxide (NO) production in mouse peritoneal macrophages and found that it was: (1) time and concentration dependent, (2) possibly derived from inducible NOS synthase since it was reduced significantly by amino guanidine pretreatment, (3) restricted to H-2(K) (C3H/HePas and C3H/HeJ) and H-2(d) strains (BALB/c), (4) independent of TLR4 signaling since the coisogenic strains C3H/HePas and C3H/HeJ (TLR4 deficient) produced similar levels of NO following MAM stimulation, (5) potentiated by lipopolysaccharide, and (6) dependent on the presence of nonadherent peritoneal cells. Neutralization of interferon-gamma (IFNgamma in the peritoneal cell cultures with monoclonal antibodies abolished MAM-induced NO production. Addition of rIFNgamma to the adherent cells substituted the nonadherent cells for MAM-induced NO production. A macrophage cell line, J774A.1 (H-2(d)), also produced NO upon MAM stimulation but only when BALB/c spleen lymphocytes were added. Thus, in murine macrophages, MAM induces NO production that is dependent on signaling through MHC class II molecules and IFNgamma but independent of TLR4 expression.     

Effects of centimeter waves on the immune system of mice in endotoxic shock

The effects of centimeter waves (8.15-18 GHz, 1 microW/cm2, 1 h daily for 10 days; MW) on the production of the tumor necrosis factor alpha, interleukin-lalpha, interleukin-1beta, interleukin-2, and the expression of interleukin-6, interleukin-10, interferon-gamma, nitric oxide and HSP27, HSP72 and HSP90alpha in mice irradiated before or after LPS injection were studied. An acute endotoxic model was produced by a single LPS injection. The effects of microwaves on nitric oxide, interleukin-6, tumor necrosis factor-alpha, and interferon-gamma were dependent on the functional status of exposed animals. Thus, an exposure of healthy mice to microwaves for 10 days was followed by a decrease in nitric oxide and interferon-gamma production, and an increase in the production of the tumor necrosis factor-alpha and interleukin-6. On the contrary, an exposure to MW before intoxication resulted in an increase in the synthesis of nitric oxide and interferon-gamma as well as a decrease in the concentration of the tumor necrosis factor-alpha and interleukin-6 in blood of mice in endotoxic shock. When microwave exposure was used after LPS injection, it did not provide any protective effect, and preliminary irradiation enhanced the resistance of the organism to endotoxic shock.     

Chicken cystatin stimulates nitric oxide release from interferon-gamma-activated mouse peritoneal macrophages via cytokine synthesis.

Cystatins are natural tight-binding, reversible inhibitors of cysteine proteases. We have shown that cystatins also stimulate nitric oxide (NO) production by interferon-gamma-activated mouse peritoneal macrophages [Verdot, L., Lalmanach, G., Vercruysse, V., Hartman, S., Lucius, R., Hoebeke, J., Gauthier F. & Vray, B. (1996) J. Biol. Chem. 271, 28077-28081]. The present study was undertaken to further document this new function. Macrophages activated with interferon-gamma and then stimulated with interferon-gamma plus chicken cystatin generated increased amounts of NO in comparison with macrophages only activated with interferon-gamma. Interferon-gamma-activated macrophages must be incubated with chicken cystatin for at least 8 h to upregulate NO production. NO induction was due to increased inducible nitric oxide synthase protein synthesis. Macrophages incubated with chicken cystatin alone or with interferon-gamma plus chicken cystatin produced increased amounts of both tumor necrosis factor alpha and interleukin 10. The addition of recombinant murine tumor necrosis factor alpha alone or in combination with recombinant murine interleukin-10 mimicked the effect of chicken cystatin. The addition of neutralizing anti-(tumor necrosis factor alpha) antibodies reduced sharply NO production by chicken cystatin/interferon-gamma-activated mouse peritoneal macrophages. Taken together, these data suggest that chicken cystatin induces the synthesis of tumor necrosis factor alpha and interleukin 10. In turn, these two cytokines stimulate the production of NO by interferon-gamma-activated macrophages. The findings point to a new relationship between cystatins, cytokines, inflammation and the immune response.     

Effect of lactoferrin on the production of tumor necrosis factor‐α and nitric oxide

One of the biological functions of lactoferrin is the modulation of the host defense systems, including cytokine production and immune responses. We have tested the effect of lactoferrin on the productions of tumor necrosis factor‐α and nitric oxide in some cells. Lactoferrin itself did not induce either tumor necrosis factor‐α production or nitric oxide production, but lipopolysaccharide‐stimulated tumor necrosis factor‐α production of macrophages and monocytes were inhibited by lactoferrin treatment combined with stimulant. The induction of nitric oxide synthesis in stimulated macrophages and vascular smooth muscle cells was not affected by the lactoferrin. J. Cell. Biochem. 76:30–36, 1999. © 1999 Wiley‐Liss, Inc.     

Effect of lactoferrin on the production of tumor necrosis factor-alpha and nitric oxide

One of the biological functions of lactoferrin is the modulation of the host defense systems, including cytokine production and immune responses. We have tested the effect of lactoferrin on the productions of tumor necrosis factor-alpha and nitric oxide in some cells. Lactoferrin itself did not induce either tumor necrosis factor-alpha production or nitric oxide production, but lipopolysaccharide-stimulated tumor necrosis factor-alpha production of macrophages and monocytes were inhibited by lactoferrin treatment combined with stimulant. The induction of nitric oxide synthesis in stimulated macrophages and vascular smooth muscle cells was not affected by the lactoferrin.     

Leonurus sibiricus induces nitric oxide and tumor necrosis factor-alpha in mouse peritoneal macrophages

Using mouse peritoneal macrophages, we have examined the mechanism by which Leonurus sibiricus (LS) regulates nitric oxide (NO) production. When LS was used in combination with recombinant interferon-gamma (rIFN-gamma), there was a marked cooperative induction of NO production; however, LS by itself had no effect on NO production. The increased production of NO from rIFN-gamma plus LS-stimulated cells was almost completely inhibited by pretreatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappaB. Furthermore, treatment of peritoneal macrophages with rIFN-gamma plus LS caused a significant increase in tumor necrosis factor-alpha (TNF-alpha) production. PDTC also decreased the effect of LS on TNF-alpha production significantly. Because NO and TNF-alpha play an important role in immune function and host defense, LS treatment could modulate several aspects of host defense mechanisms as a result of stimulation of the inducible nitric oxide synthase.     

PAF is involved in the Mycoplasma arthritidis superantigen-triggering pathway for iNOS and COX-2 expression in murine peritoneal cells

We investigated the capacity of Mycoplasma arthritidis mitogen (MAM) to induce (a) expression of the inducible enzymes cyclo-oxygenase (COX-2) and nitric oxide synthase (iNOS), (b) production of prostaglandin E2 (PGE2) and nitric oxide (NO), and (c) involvement of platelet-activating factor (PAF) in the MAM-induced activation pathway. Resident peritoneal cells from C3H/HePas mice were incubated with MAM in the presence or absence of a PAF-antagonist (WEB2170) or COX-2 inhibitors (nimesulide or NS398). Enzyme expression was evaluated by immunoblotting, PGE2 by EIA, and NO by Griess reaction. Following MAM-stimulation of peritoneal cells, expression of COX-2 was detected at 3 h (peak levels at 12 h) and of iNOS at 6 h (peak levels at 20 h). PGE2 increased till 20 h, decreasing thereafter, whereas NO increased with time. WEB2170 (5 x 10(-5) M) treatment caused 44% inhibition of NO output and reduced iNOS expression (48% at the peak of expression). Concomitant treatment with WEB2170 and nimesulide (10(-5) M) reversed these inhibitory effects. WEB2170 reduced COX-2 expression (43% at the peak of expression) and prevented the decline in PGE2 levels after 20 h. These results suggest the involvement of PAF in the signaling pathway triggered by MAM that leads to expression of iNOS and COX-2, and show that PAF regulates the production of NO, possibly by controlling levels of PGE2.     

Reciprocal CD40 signals through p38MAPK and ERK-1/2 induce counteracting immune responses

Macrophages play host to Leishmania major, a parasite that causes leishmaniasis in 500,000 people annually. Macrophage-expressed CD40, a costimulatory molecule, induces interleukin-12 (IL-12)-dependent and interferon-gamma (IFN-gamma)-dependent host-protective immune responses to Leishmania and other intracellular pathogens. Paradoxically, IL-10, another CD40-induced cytokine in macrophages, promotes Leishmania infection. How CD40 signaling regulates the secretion of these two counteractive cytokines remains unknown. Here we show that weak CD40 signals induce extracellular stress-related kinase-1/2 (ERK-1/2)-dependent IL-10 expression, whereas stronger signals induce p38 mitogen-activated protein kinase (p38MAPK)-dependent IL-12 production. p38MAPK and ERK-1/2 therefore have counter-regulatory actions. Leishmania skews CD40 signaling toward ERK-1/2, inducing IL-10, which inhibits activation of CD40-induced p38MAPK and expression of inducible nitric oxide synthase-2 (iNOS-2) and IL-12. ERK-1/2 inhibition or IL-10 neutralization restores CD40-induced p38MAPK activation and parasite killing in macrophages and the BALB/c mouse, a susceptible host. These data uncover a new immune evasion strategy, whereby Leishmania differentially modulates CD40-engaged, reciprocally functioning signaling modules, and provide a new conceptual framework for immune homeostasis.     

The role of heat shock proteins HSP90 in the response of immune cells to centimeter microwaves

The effects of low-level electromagnetic waves (8.15-18 GHz, 1 microW/cm2, 1 h) on the production of heat shock proteins, several cytokines, and nitric oxide in isolated mouse macrophages and lymphocytes were examined both under normal conditions and after the treatment of the cells with geldanamycin (GA), a depressor of activity of the heat shock protein 90 (Hsp90). The irradiation of cells without GA induced the production of Hsp70, nitric oxide (NO), interleukin-1beta (IL-1beta), interleukin-10 (IL-10), and the tumor necrosis factor -alpha (TNF-alpha). No changes in the production of Hsp90 in irradiated cells were observed, but intracellular locations of Hsp25 and Hsp70 altered. The preliminary treatment of cells with GA did not remove the effects of microwaves: in these conditions, the synthesis of all cytokines tested, nitric oxide, as well as total and membrane amount of Hsp70, and the amount of Hsp25 in the cytoplasm and cytoskeleton increased. Moreover, the exposure of cells incubated with GA resulted in the reduction of Hsp90-alpha production.     

A selective adenosine A2A receptor agonist, ATL-146e, prevents concanavalin A-induced acute liver injury in mice

BACKGROUND AND AIMS: Concanavalin A (Con A) activates T lymphocytes and induces CD4+ T cell-mediated hepatic injury in mice. Pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and interleukin-6 (IL-6), are critical mediators in this experimental model. Activation of adenosine A2A receptors reduces the production of various pro-inflammatory cytokines and suppresses T cell activation. A selective adenosine A2A receptor agonist (ATL-146e) has been shown to be a potent inhibitor of inflammation by increasing intracellular cyclic AMP (cAMP) in leukocytes. The aim of the present study was to determine whether ATL-146e could ameliorate Con A-induced hepatic injury, reduction of pro-inflammatory cytokine production. METHODS: Balb/c mice were injected with 25mg/kg Con A with or without a single injection of ATL-146e (0.5-50 microg/kg), 5 min prior to Con A administration. Liver enzymes, histology, and serum levels of tumor necrosis factor-alpha, interferon-gamma, and interleukin-6 were examined. We also assessed the effects of ATL-146e on pro-inflammatory cytokine production with CD4+ T cell. RESULTS: Pretreatment with ATL-146e significantly reduced serum levels of liver enzymes (P<0.001). The serum pro-inflammatory cytokines were all increased after Con A administration and reduced to near normal levels by ATL-146e. ATL-146e also inhibited CD4+ T cell pro-inflammatory cytokine production. CONCLUSION: A selective adenosine A2A receptor agonist, ATL-146e, can prevent concanavalin A-induced hepatic injury that is presumably mediated by its anti-inflammatory properties.     

Molecular Characteristics and Potent Immunomodulatory Activity of Fasciola hepatica Cystatin

Cystatin, a cysteine protease inhibitor found in many parasites, plays important roles in immune evasion. This study analyzed the molecular characteristics of a cystatin from Fasciola hepatica (FhCystatin) and expressed recombinant FhCystatin (rFhcystatin) to investigate the immune modulatory effects on lipopolysaccharide-induced proliferation, migration, cytokine secretion, nitric oxide (NO) production, and apoptosis in mouse macrophages. The FhCystatin gene encoded 116 amino acids and contained a conserved cystatin-like domain. rFhCystatin significantly inhibited the activity of cathepsin B. rFhCystatin bound to the surface of mouse RAW264.7 cells, significantly inhibited cell proliferation and promoted apoptosis. Moreover, rFhCystatin inhibited the expression of cellular nitric oxide, interleukin-6, and tumor necrosis factor-α, and promoted the expression of transforming growth factor-β and interleukin-10. These results showed that FhCystatin played an important role in regulating the activity of mouse macrophages. Our findings provide new insights into mechanisms underlying the immune evasion and contribute to the exploration of potential targets for the development of new drug to control F. hepatica infection.     

Inhibition of macrophage nitric oxide production by gangliosides derived from a spontaneous T cell lymphoma: the involved mechanisms.

Gangliosides (DLG) derived from a spontaneous T cell lymphoma (Dalton's lymphoma) have been shown to impair the ability of lipopolysaccharide-activated macrophages to produce nitric oxide (NO). However, the mechanism and nature of this effect is not known. In this investigation, we sought to (1) determine whether the inhibitory action of DLG on macrophages is through the modulation of inducible nitric oxide synthase (iNOS) expression and (2) identify the possible mechanisms and signal transduction events underlying the inhibitory action of DLG. Immunoblot analysis of DLG-treated macrophages showed a decrease in iNOS expression. DLG also inhibited the production of monokines interleukin-1 and tumor necrosis factor by macrophages. However, the DLG-induced inhibition was reversible in nature. Studies showed that DLG-induced inhibition of macrophage activation could be blocked by sodium orthovanadate, indicating a role of phosphatase activity in ganglioside-induced inhibition.     

Effect of electromagnetic waves in the centimeter range on the production of tumor necrosis factor and interleukin-3 in immunized mice

The effect of prolonged treatment with weak microwaves on the production of tumor necrosis factor and interleukin-3 in peritoneal macrophages and T cells of male NMRI mice twice immunized by affinity-purified carboanhydrase was studied. Against the back ground of a high titer of antibody production, a significant increase in the production of tumor necrosis factor in peritoneal macrophages and splenic T lymphocytes of immunized mice was revealed, and a much stronger effect was observed for irradiated immunized animals. A tendency to increased secretion of interleukin-3 for unirradiated and irradiated immunized animals was found; in the latter group of animals, the effect being more pronounced. The stimulation of production of the cytokins, especially tumor necrosis factor, by combination of antigenic stimulation and microwaves can be used in adjuvant therapy of various immune diseases.     

Selective interleukin-12 synthesis defect in 12/15-lipoxygenase-deficient macrophages associated with reduced atherosclerosis in a mouse model of familial hypercholesterolemia

Targeted gene disruption or overexpression of 12/15-lipoxygenase in mice on the genetic background of apolipoprotein E or low density lipoprotein-receptor (LDL-R) deficiency has implicated 12/15-lipoxygenase in atherogenesis. The data support indirectly a role for 12/15-lipoxygenase in the oxidative modification of low density lipoprotein. In this study we set out to explore other potential mechanisms for 12/15-lipoxygenase in atherosclerosis using apolipoprotein B mRNA editing catalytic polypeptide-1/LDL-R double-deficient mice, a model highly related to the human condition of familial hypercholesterolemia. 12/15-Lipoxygenase deficiency in this strain led to approximately 50% decrease in aortic lesions in male and female mice at 8 months on a chow diet in the absence of cholesterol differences. While studying 12/15-lipoxygenase-deficient macrophages in culture, we discovered a remarkable selective defect (75-90% decrease) in interleukin-12 production but not in tumor necrosis factor-alpha or nitric oxide release, in response to lipopolysaccharide in the presence or absence of interferon-gamma priming. The lipopolysaccharide/interferon-gamma response was associated with a 33-50% decrease in nuclear interferon consensus sequence-binding protein, which is consistent with interferon consensus sequence-binding protein containing protein complex-dependent regulation of the interleukin-12 p40 gene. The decrease in interleukin-12 production was recapitulated in vivo in mouse aortas of the triple knockout group and was reflected in a marked decrease in interferon-gamma expression. The data provide support for a novel mechanism linking the 12/15-lipoxygenase pathway to a known immunomodulatory Th1 cytokine in atherogenesis.     

Effect of Tityus serrulatus venom on cytokine production and the activity of murine macrophages

THE purpose of this study was to investigate the effects of Tityus serrulatus venom (TSV) on murine peritoneal macrophages evaluated in terms of activation. The effects of crude TSV were analysed by detection of cytokines, oxygen intermediate metabolites (H2O2) and nitric oxide (NO) in supernatants of peritoneal macrophages. Several functional bioassays were employed including an in vitro model for envenomating: cytotoxicity of TSV was assessed using the lyses percentage. Tumor necrosis factor (TNF) activity was assayed by measuring its cytotoxic activity on L-929 cells, and interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) were assayed by enzyme-linked immunosorbent assay, whereas NO levels were detected by Griess colorimetric reactions in culture supernatant of macrophages incubated with TSV and subsequently exposed to either lipopolysaccharide or IFN-gamma. Incubation of macrophages with TSV increased production of IL-6 and IFN-gamma in a dose-dependent manner. TNF production was not detected in supernatants treated with TSV at any concentration. The increase in IL-6 secretion was not associated with concentration-dependent cytoxicity of TSV on these cells. These data suggest that the cytotoxicity does not appear to be the main cause of an increased cytokine production by these cells. Although NO is an important effector molecule in macrophage microbicidal activity, the inducing potential of the test compounds for its release was found to be very moderate, ranging from 125 to 800 mM. Interestingly, NO levels of peritoneal macrophages were increased after IFN-gamma. Moreover, NO production had an apparent effect on macrophage activity. The results obtained here also shown that the TSV induces an important elevation in H2O2 release. These results combined with NO production suggest that TSV possesses significant immunomodulatory activities capable of stimulating immune functions in vitro.     

Antimicrobial peptide P18 inhibits inflammatory responses by LPS- but not by IFN-γ-stimulated macrophages

Antimicrobial peptide P18 markedly inhibited the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells, whereas magainin 2 did not inhibit these activities. P18 dose-dependently reduced nitric oxide (NO) production by LPS-stimulated RAW 264.7 macrophage cells, with complete inhibition at 20 microg P18 ml(-1). In contrast, P18 had no effect on NO production and the expression of iNOS mRNA and iNOS protein by interferon-gamma (IFN-gamma)-stimulated RAW264.7 cells, suggesting P18 selectively inhibits LPS-stimulated inflammatory responses in macrophages. An LAL assay showed that P18 has strong LPS-neutralizing activity, indicating that P18 inhibits the inflammatory responses in LPS-stimulated macrophages by direct binding to LPS. Collectively, our results indicate that P18 has promising therapeutic potential as a novel anti-inflammatory as well as antimicrobial agent.     

Thrombin-induced activation of cultured rodent microglia

Microglia are the resident immune cells of the CNS. Upon brain damage, these cells are rapidly activated and function as tissue macrophages. The first steps in this activation still remain unclear, but it is widely believed that substances released from damaged brain tissue trigger this process. In this article, we describe the effects of the blood coagulation factor thrombin on cultured rodent microglial cells. Thrombin induced a transient Ca(2+) increase in microglial cells, which persisted in Ca(2+)-free media. It was blocked by thapsigargin, indicating that thrombin caused a Ca(2+) release from internal stores. Preincubation with pertussis toxin did not alter the thrombin-induced [Ca(2+)](i) signal, whereas it was blocked by hirudin, a blocker of thrombin's proteolytic activity. Incubation with thrombin led to the production of nitric oxide and the release of the cytokines tumor necrosis factor-alpha, interleukin-6, interleukin-12, the chemokine KC, and the soluble tumor necrosis factor-alpha receptor II and had a significant proliferative effect. Our findings indicate that thrombin, a molecule that enters the brain at sites of injury, rapidly triggered microglial activation.