Enhanced nitric oxide production associated with airway hyporesponsiveness in the absence of IL-10

Interleukin (IL)-10 is an anti-inflammatory cytokine implicated in the regulation of airway inflammation in asthma. Among other activities, IL-10 suppresses production of nitric oxide (NO); consequently, its absence may permit increased NO production, which can affect airway smooth muscle contractility. Therefore, we investigated airway reactivity (AR) in response to methacholine (MCh) in IL-10 knockout (-/-) mice compared with wild-type C57BL/6 (C57) mice, in which airway NO production was measured as exhaled NO (E(NO)), and NO production was altered with administration of either NO synthase (NOS)-specific inhibitors or recombinant murine (rm)IL-10. AR, measured as enhanced pause in vivo, and tracheal ring tension in vitro were lower in IL-10(-/-) mice by 25-50%, which was associated with elevated E(NO) levels (13 vs. 7 ppb). Administration of NOS inhibitors N(G)-nitro-L-arginine methyl ester (8 mg/kg ip) or L-N(6)-(1-iminoethyl)-lysine (3 mg/kg ip) to IL-10(-/-) mice decreased E(NO) by an average of 50%, which was associated with increased AR, to levels similar to C57 mice. E(NO) in IL-10(-/-) mice decreased in a dose-dependent fashion in response to administered rmIL-10, to levels similar to C57 mice (7 ppb), which was associated with a 30% increment in AR. Thus increased NO production in the absence of IL-10, decreased AR, which was reversed with inhibition of NO, either by inhibition of NOS, or with reconstitution of IL-10. These findings suggest that airway NO production can modulate airway smooth muscle contractility, resulting in airway hyporesponsiveness when IL-10 is absent.     

Absence of functional inducible NO synthase enhances the efficacy of tolerance induced by high dose antigen feeding

Recent studies have suggested that IL-12 and IFN-gamma may impair the ability of fed Ag to induce systemic tolerance. Because both of these cytokines can function to directly or indirectly induce inducible NO synthase (iNOS) expression, we have investigated whether the functional expression of iNOS regulates oral tolerance. C57BL/6J wild-type or C57BL/6J NOS2(-/-) mice were gavaged with a single dose of 20 mg of keyhole limpet hemocyanin (KLH), followed by s.c. immunization with KLH/CFA. In the absence of feeding Ag, several parameters of the immune response were more robust in C57BL/6J NOS2(-/-) mice following KLH/CFA immunization, including the magnitude of the delayed-type hypersensitivity response, the proliferative response, and the production of IFN-gamma and IL-2 by Ag-activated draining lymph node cells. These heightened responses in the C57BL/6J NOS2(-/-) mice are still effectively inhibited by feeding KLH. Feeding KLH to the C57BL/6J NOS2(-/-) mice elicited heightened TGF-ss1 production by Ag-activated lymphocytes, as well as augmented total IgG, IgG1, and IgG2a responses to KLH/CFA compared with that seen in Ag-fed wild-type mice. Feeding Ag to the NOS2(-/-) mice suppressed proliferative responses and IFN-gamma production, while increasing IL-4 production and the IgG1/IgG2a ratio even following a booster immunization of KLH/CFA. Administrating L-N:(6)-(1-iminoethyl)-lysine. 2HCl to wild-type mice during the period of Ag feeding reproduced the high TGF-ss1 production seen in Ag-activated lymphocytes from Ag-fed NOS2(-/-) mice. Feeding KLH is followed by transient up-regulation of NOS2 mRNA expression in the Peyer's patches of wild-type mice. Selective inhibition of NOS2 may be a simple way to augment tolerogenic mucosal immune responses.     

Effects of arginase isoforms on NO Production by nNOS.

Both arginase isoforms (AI and AII) regulate high-level NO production by the inducible NOS, but whether the arginase isoforms also regulate low-level NO production by neuronal NOS (nNOS) is not known. In this study, 293 cells that stably overexpress nNOS gene (293nNOS cells) were transfected with rat AI (pEGFP-AI) or AII (pcDNA-AII) plasmids, and nitrite production was measured with or without supplemental L-arginine. Transfection with pEGFP-AI increased AI expression and activity 10-fold and decreased intracellular l-arginine by 50%. Nitrite production was inhibited by >80% when no l-arginine was supplemented but not when 1 mM L-arginine was present. The inhibition was reversed by an arginase inhibitor, N(omega)-hydroxy-L-arginine. Transfection with pcDNA-AII increased AII expression and activity but had little effect on nitrite production even if no l-arginine was added. These results suggest that, in 293nNOS cells, AI was more effective in regulating NO production by nNOS, most likely by competing for L-arginine.     

Antifungal type 1 responses are upregulated in IL-10-deficient mice

C57BL/6 mice are highly resistant to infections caused by Candida albicans and Aspergillus fumigatus. To elucidate the role of IL-10 produced by C57BL/6 mice during these infections, parameters of infection and immunity to it were evaluated in IL-10-deficient and wild-type mice with disseminated or gastrointestinal candidiasis or invasive pulmonary aspergillosis. Unlike parasitic protozoan infection, C. albicans or A. fumigatus infection did not induce significant acute toxicity in IL-10-deficient mice, who, instead, showed reduced fungal burden and fungal-associated inflammatory responses. The increased resistance to infections as compared to wild-type mice was associated with upregulation of innate and acquired antifungal Th1 responses, such as a dramatically higher production of IL-12, nitric oxide (NO) and TNF-alpha as well as IFN-gamma by CD4+ T cells. Pharmacological inhibition of NO production greatly reduced resistance to gastrointestinal candidiasis, thus pointing to the importance of IL-10-dependent NO regulation at mucosal sites in fungal infections. These results are reminiscent of those obtained in genetically susceptible mice, in which IL-10 administration increased, and IL-10 neutralization decreased, susceptibility to C. albicans and A. fumigatus infections. Collectively, these observations indicate that the absence of IL-10 augments innate and acquired antifungal immunity by upregulating type 1 cytokine responses. The resulting protective Th1 responses lead to a prompt reduction of fungal growth, thus preventing tissue destruction and lethal levels of proinflammatory cytokines.     

Reduced neuronal nitric oxide synthase expression contributes to cardiac oxidative stress and nitroso-redox imbalance in ob/ob mice.

Disruption of leptin signaling in the heart may contribute to obesity-related cardiac disease, as leptin deficient (oblob) mice display cardiac hypertrophy, increased cardiac apoptosis and reduced survival. Since leptin maintains a tonic level of neuronal nitric oxide synthase (NOS1) expression in the brain, we hypothesized that leptin deficiency would decrease NOS1 cardiac expression, in turn activating xanthine oxidoreductase (XOR) and creating nitroso-redox imbalance. We studied 2- to 6-month-old oblob (n=26) and C57Bl/6 controls (n=27). Cardiac NOS1 protein abundance (P<0.01) and mRNA expression (P=0.03) were reduced in oblob (n=10 and 6, respectively), while NOS3 protein abundance and mRNA expression were unaltered. Importantly, cardiac NOS1 protein abundance was restored towards normal in oblob mice after leptin treatment (n=3; P<0.05 vs leptin untreated oblob mice). NO metabolite (nitrite and nitrate) production within the myocardium was also reduced in oblob mice (n=5; P=0.02). Furthermore, oxidative stress was increased in oblob mice as GSH/GSSG ratio was decreased (n=4; P=0.02). Whereas XOR activity measured by Amplex Red fluorescence was increased (n=8; P=0.04), XOR and NADPH oxidase subunits protein abundance were not changed in oblob mice (n=6). Leptin deficiency did not disrupt NOS1 subcellular localization, as NOS1 co-localized with ryanodine receptor but not with caveolin-3. In conclusion, leptin deficiency is linked to decreased cardiac expression of NOS1 and NO production, with a concomitant increase in XOR activity and oxidative stress, resulting in nitroso-redox imbalance. These data offer novel insights into potential mechanisms of myocardial dysfunction in obesity.     

Non-invasive measurements of exhaled NO and CO associated with methacholine responses in mice

Background

Nitric oxide (NO) and carbon monoxide (CO) in exhaled breath are considered obtainable biomarkers of physiologic mechanisms. Therefore, obtaining their measures simply, non-invasively, and repeatedly, is of interest, and was the purpose of the current study.

Methods

Expired NO (E_NO) and CO (E_CO) were measured non-invasively using a gas micro-analyzer on several strains of mice (C57Bl6, IL-10^-/-, A/J, MKK3^-/-, JNK1^-/-, NOS-2^-/- and NOS-3^-/-) with and without allergic airway inflammation (AI) induced by ovalbumin systemic sensitization and aerosol challenge, compared using independent-sample t-tests between groups, and repeated measures analysis of variance (ANOVA) within groups over time of inflammation induction. E_NO and E_CO were also measured in C57Bl6 and IL-10-/- mice, ages 8–58 weeks old, the relationship of which was determined by regression analysis. S-methionyl-L-thiocitrulline (SMTC), and tin protoporphyrin (SnPP) were used to inhibit neuronal/constitutive NOS-1 and heme-oxygenase, respectively, and alter NO and CO production, respectively, as assessed by paired t-tests. Methacholine-associated airway responses (AR) were measured by the enhanced pause method, with comparisons by repeated measures ANOVA and post-hoc testing.

Results

E_NO was significantly elevated in naïve IL-10^-/- (9–14 ppb) and NOS-2^-/- (16 ppb) mice as compared to others (average: 5–8 ppb), whereas E_CO was significantly higher in naïve A/J, NOS-3^-/- (3–4 ppm), and MKK3^-/- (4–5 ppm) mice, as compared to others (average: 2.5 ppm). As compared to C57Bl6 mice, AR of IL-10^-/-, JNK1^-/-, NOS-2^-/-, and NOS-3^-/- mice were decreased, whereas they were greater for A/J and MKK3^-/- mice. SMTC significantly decreased E_NO by ~30%, but did not change AR in NOS-2^-/- mice. SnPP reduced E_CO in C57Bl6 and IL-10^-/- mice, and increased AR in NOS-2^-/- mice. E_NO decreased as a function of age in IL-10^-/- mice, remaining unchanged in C57Bl6 mice.

Conclusion

These results are consistent with the ideas that: 1) E_NO is associated with mouse strain and knockout differences in NO production and AR, 2) alterations of E_NO and E_CO can be measured non-invasively with induction of allergic AI or inhibition of key gas-producing enzymes, and 3) alterations in AR may be dependent on the relative balance of NO and CO in the airway.     

Spontaneously developing chronic colitis in IL-10/iNOS double-deficient mice

Mice deficient in both inducible nitric oxide synthase (iNOS) and interleukin (IL)-10 (iNOS(-/-)/IL-10(-/-)) were created to examine the role of iNOS in spontaneously developing intestinal inflammation. IL-10(-/-)/iNOS(-/-) mice were compared with IL-10(-/-) (iNOS(+/+)) littermates over 6 mo. RT-PCR, Western blot analysis, and immunohistochemistry were performed to measure iNOS message and protein levels. Plasma nitrate/nitrite (NO(x)) levels were assessed by HPLC. Damage scores (macroscopic and microscopic) and granulocyte infiltration were assessed. At 3-4 wk, IL-10(-/-) and IL-10(-/-)/iNOS(-/-) mice had no signs of colonic inflammation or granulocyte infiltration. Plasma NO(x) levels were not different from controls. By 3-4 mo, IL-10(-/-) mice had increased damage scores and granulocyte infiltration concurrent with increased mRNA and protein synthesis (restricted to the epithelium) for iNOS in intestinal tissues but not other tissues. Plasma NO(x) levels increased fivefold. Interestingly, in the absence of iNOS induction or increased plasma NO(x), iNOS(-/-)/IL-10(-/-) mice had damage and granulocyte infiltration equivalent to those observed in IL-10(-/-) littermates. These data suggest that iNOS does not impact on the development or severity of spontaneous chronic inflammation in IL-10-deficient mice.     

Role of phosphatidylinositol-3-kinase-gamma (PI3Kgamma)-mediated pathway in 17beta-estradiol-induced killing of L. mexicana in macrophages from C57BL/6 mice

We recently demonstrated that 17beta-estradiol (E2) enhances killing of Leishmania mexicana in macrophages from both male and female DBA/2 mouse by increasing nitric oxide (NO) production. Here, we analyzed the effect of E2 on leishmanicidal activity and cytokine production by bone marrow-derived macrophages (BMDMs) from male and female C57BL/6 mice in vitro, specifically examining the role of phosphatidylinositol-3-kinase-gamma (PI3Kgamma) in E2-induced parasite killing. Unlike its effect on macrophages from both male and female DBA/2 mice, E2 only increased leishmanicidal activity in macrophages from female C57BL/6 mice, which was evident by a significant reduction in both infection rates and infection levels compared to sham controls. E2-treated BMDMs from female C57BL/6 mice expressed higher levels of interferon-gammaRalpha, and also produced more interleukin (IL)-12, IL-6 and NO than both the sham controls and E2-treated male-derived macrophages. Sham-treated BMDMs from female PI3Kgamma-/- C57BL/6 mice displayed lower infection rates and infection levels compared to sham-treated wild-type (WT) macrophages. However E2, unlike its effect on macrophages from female WT C57BL/6 mice, failed to reduce infection rates and infection levels in BMDMs from female PI3Kgamma-/- mice. Interestingly, E2-treated BMDMs from female C57BL/6 mice produced significant amounts of inflammatory cytokines and NO in levels comparable to those observed in sham-treated PI3Kgamma-deficient macrophages as well as E2-treated macrophages from WT mice. These findings show that E2 exerts a distinct effect on leishmanicidal activity of macrophages from male versus female C57BL/6 mice. In addition, they suggest that PI3Kgamma is not required for E2-induced cytokine and NO production in L. mexicana-infected macrophages from female C57BL/6 mice but it may be involved in parasite clearance from these cells.     

Opposite effects of interleukin-4 and interleukin-10 on nitric oxide production in murine macrophages

Interleukin-4 (IL-4) and interleukin-10 (IL-10) were evaluated for their ability to inhibit the production of nitric oxide (NO) by interferon-gamma (IFN-gamma)- or lipopolysaccharide (LPS)-activated murine macrophages (RAW 264.7 and J774.2). Macrophages pre-treated with IL-4 and then stimulated with IFN-gamma or LPS showed significant inhibition in their ability to produce NO as measured by nitrite production. Simultaneous treatment of IL-4 pre-incubated cells with IFN-gamma and LPS together augmented nitrite accumulation. On the other hand, similar exposures of the macrophages to IL-10 followed by IFN-gamma or LPS treatments resulted in significantly increased NO production. Thus IL-10 failed to suppress IFN-gamma or LPS-induced NO production and showed opposite effects in these experiments to IL-4. We conclude that the two lymphokines have differing roles in the control of production of NO and might act to control the secretion of nitric oxide in vivo.     

Role of increased guanosine triphosphate cyclohydrolase-1 expression and tetrahydrobiopterin levels upon T cell activation

Tetrahydrobiopterin (BH(4)) is an essential co-factor for the nitric-oxide (NO) synthases, and in its absence these enzymes produce superoxide (O(2)(·-)) rather than NO. The rate-limiting enzyme for BH(4) production is guanosine triphosphate cyclohydrolase-1 (GTPCH-1). Because endogenously produced NO affects T cell function, we sought to determine whether antigen stimulation affected T cell GTPCH-1 expression and ultimately BH(4) levels. Resting T cells had minimal expression of inducible NOS (NOS2), endothelial NOS (NOS3), and GTPCH-1 protein and nearly undetectable levels of BH(4). Anti-CD3 stimulation of T cells robustly stimulated the coordinated expression of NOS2, NOS3, and GTPCH-1 and markedly increased both GTPCH-1 activity and T cell BH(4) levels. The newly expressed GTPCH-1 was phosphorylated on serine 72 and pharmacological inhibition of casein kinase II reduced GTPCH-1 phosphorylation and blunted the increase in T cell BH(4). Inhibition of GTPCH-1 with diaminohydroxypyrimidine (1 mmol/liter) prevented T cell BH(4) accumulation, reduced NO production, and increased T cell O(2)(·-) production, due to both NOS2 and NOS3 uncoupling. GTPCH-1 inhibition also promoted TH(2) polarization in memory CD4 cells. Ovalbumin immunization of mice transgenic for an ovalbumin receptor (OT-II mice) confirmed a marked increase in T cell BH(4) in vivo. These studies identify a previously unidentified consequence of T cell activation, promoting BH(4) levels, NO production, and modulating T cell cytokine production.     

(+)-Catechin inhibits tumour angiogenesis and regulates the production of nitric oxide and TNF-alpha in LPS-stimulated macrophages

The anti-angiogenic activity of (+)-catechin as well as its regulatory effect on the production of nitric oxide and TNFalpha were studied using in vivo and in vitro models. In vivo angiogenic activity was studied using B16F-10 melanoma cell-induced capillary formation in C57BL/6 mice. Administration of (+)-catechin significantly inhibited (36.09%) the number of tumour-directed capillaries induced by injecting B16F-10 melanoma cells on the ventral side of C57BL/6 mice. The cytokine profile in the serum of these animals showed a drastically increased level of proinflammatory cytokines such as IL-1 beta, IL-6, TNF-alpha, GM-CSF and the direct endothelial cell proliferating agent, VEGF. Administration of (+)-catechin could differentially regulate elevation of these cytokines. The differential elevation is further evidenced by the increased production of IL-2 and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the B16F-10 injected, (+)-catechin-treated animals. In vitro L929 bioassay revealed the inhibition of TNF-alpha production by (+)-catechin treatment. In the rat aortic ring assay, (+)-catechin inhibited the microvessel outgrowth at non-toxic concentrations. (+)-Catechin at non-toxic concentrations (5-25 microg/ml) showed significant inhibition in the proliferation, migration and tube formation of endothelial cells, which are the key events in the process of angiogenesis. (+)-Catechin also showed inhibitory effect on VEGF mRNA levels in B16F-10 melanoma cells. (+)-Catechin inhibited the production of NO and TNF-alpha in LPS-stimulated primary macrophages. Taken together, these results demonstrate that (+)-catechin inhibits tumour-specific angiogenesis by regulating the production of pro- and anti-angiogenic factors such as pro-inflammatory cytokines, nitric oxide, VEGF, IL-2 and TIMP-1. These results also suggest that (+)-catechin could significantly inhibit nitrite and TNF-alpha production in LPS-stimulated macrophages.     

Specific inhibition of nitric oxide synthases at different time points in a murine model of pulmonary sepsis

Excessive production of nitric oxide (NO) by NO synthase (NOS) and a subsequent oxidative stress reaction are thought to be critically involved in the pathophysiology of sepsis. Previous studies suggested that NO production by neuronal NOS (nNOS) and inducible NOS (iNOS) is implemented in the disease process at different time points after the injury. Here we tested the roles of selective pharmacological inhibition of nNOS and iNOS at different time points in a murine model of pulmonary sepsis. The injury was induced by intranasal administration of live Pseudomonas aeruginosa (3.2 × 10⁷ colony-forming units) in C57BL/6 wild-type mice. The animals received no treatment (control) or treatment with a specific nNOS inhibitor (4 or 8 h), iNOS inhibitor (4 or 8 h), or non-specific NOS inhibitor (4 or 8 h). In controls, the injury was associated with excessive releases of pro-inflammatory cytokines in the plasma, enhanced tissue lipid peroxidation, and decreased survival. Non-specific NOS inhibition at either time point did not influence survival and was not further investigated. While nNOS inhibition at 4 h was associated with a trend toward improved survival and significantly reduced contents of lung nitrite/nitrate (NO_x) and liver malondialdehyde, the blockade of nNOS at 8 h had no effect on these parameters. In contrast, early iNOS inhibition was associated with a trend toward decreased survival and no effects on lung NO_x and liver malondialdehyde contents, whereas later iNOS blockade was associated with decreased malondialdehyde content in liver homogenates. In conclusion, pulmonary sepsis in mice may be beneficially influenced by specific pharmacological nNOS inhibition at an earlier time point and iNOS inhibition at a later time points post-injury. Future investigations should identify the time changes of the expression and activation of NOS isoforms.     

Helicobacter pylori-induced mucosal inflammation is Th1 mediated and exacerbated in IL-4, but not IFN-gamma, gene-deficient mice

To elucidate the pathogenesis of Helicobacter pylori-associated gastritis, we studied immune responses of C57BL/6J wild-type (WT), SCID, and gene deficient (IFN-gamma-/- and IL-4-/-) mice following infection with a pathogenic isolate of H. pylori (SPM326). During early infection in WT mice, mononuclear and polymorphonuclear cells accumulated in the gastric lamina propria, and the numbers of cells in the inflamed mucosa expressing IFN-gamma, but not IL-4, mRNA rose significantly (p < 0.005), consistent with a local Th1 response. Splenic T cells from the same infected WT mice produced high levels of IFN-gamma, no detectable IL-4, and low amounts of IL-10 following in vitro H. pylori urease stimulation, reflecting a systemic Th1 response. Infected C57BL/6J SCID mice did not develop gastric inflammation despite colonization by many bacteria. Infected C57BL/10J and BALB/c mice also did not develop gastric inflammation and displayed a mixed Th1/Th2 splenic cytokine profile. These data imply a major role for the Th1 cytokine IFN-gamma in H. pylori-associated gastric inflammation in C57BL/6J mice. Compared with WT animals, infected IL-4-/- animals had more severe gastritis and higher levels of IFN-gamma production by urease-stimulated splenocytes (p < 0.01), whereas IFN-gamma-/- mice exhibited no gastric inflammation and higher levels of IL-4 production by stimulated splenocytes. These findings establish C57BL/6J mice as an important model for H. pylori infection and demonstrate that up-regulated production of IFN-gamma, in the absence of the opposing effects of IL-4 (and possibly IL-10), plays a pivotal role in promoting H. pylori-induced mucosal inflammation.     

Inhibition by nitric oxide of cytochrome P450 4A activity contributes to endotoxin-induced hypotension in rats.

Nitric oxide (NO) production during endotoxemia is associated with decreased total CYP content, CYP 1A1/2, 2B1/2, 2C6, 2C11, 3A1, and 3A2 mRNA, protein expression or activity which is prevented by NO synthase (NOS) inhibitors in rats. This study was conducted to determine if endotoxin-induced hypotension caused by NO production is mediated by inhibition of renal CYP 4A protein expression and activity. In conscious male Sprague-Dawley rats, endotoxin (10 mg/kg, ip) reduced mean arterial pressure (MAP), increased serum and renal nitrite levels, and inducible NOS (iNOS), and decreased renal CYP 4A1/A3 protein and CYP 4A activity. The selective iNOS inhibitor 1,3-PBIT (10 mg/kg, ip; 1h after endotoxin) prevented endotoxin-induced decrease in MAP, renal CYP 4A1/A3 protein level and CYP 4A activity and increase in systemic and renal nitrite production. The selective constitutive NOS (cNOS) inhibitor N(G)-nitro-L-arginine (L-NNA; 20 mg/kg, ip; 1 h after endotoxin) partially attenuated endotoxin-induced decrease in MAP. The selective CYP 4A inhibitor, aminobenzotriazole (50 mg/kg, ip; 1 h after endotoxin) diminished CYP 4A1/A3 protein level and CYP 4A activity. Aminobenzotriazole did not alter the endotoxin-induced decrease in MAP, but it reversed the effect of 1,3-PBIT in preventing endotoxin-induced fall in MAP and CYP 4A activity. These data suggest that the endotoxemia-induced increase in NO production primarily via iNOS suppresses renal CYP 4A expression and activity, and inhibition of iNOS with 1,3-PBIT restores renal CYP 4A protein and activity and MAP presumably due to increased production of arachidonic acid metabolites derived from CYP 4A.     

Nitric oxide and the Th2 response combine to prevent severe hepatic damage during Schistosoma mansoni infection.

During infection with Schistosoma mansoni, NO production increases following the deposition of parasite eggs in the liver. In wild-type C57BL/6 mice, NO levels peak during the sixth week of infection and are subsequently down-regulated. Inducible NO synthase (iNOS) mRNA was found in diseased liver tissue along with TNF-alpha and IFN-gamma, which are known promoters of iNOS expression. Mice treated with aminoguanidine, a selective inhibitor of iNOS, exhibited cachexia and exacerbated liver pathology, suggesting that NO limits hepatocyte damage when the liver is first exposed to eggs. Hepatic iNOS is up-regulated in SCID mice, indicating that NO production is part of an innate response. Studies with infected highly susceptible IL-4-/- mice revealed that prolonged NO production is in itself deleterious and that a major function of the Th2 response, which is severely compromised in the absence of IL-4, is to regulate NO production. In these animals, plasma NO levels are high compared with those in infected wild-type mice and remain elevated until death. Nevertheless, the underlying importance of NO is illustrated by the finding that aminoguanidine treatment leads to more severe liver disease and reduced time to death in infected IL-4-/- mice.     

TGF-beta and IL-10 regulation of IFN-gamma produced in Th2-type schistosome granulomas requires IL-12

Interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) regulate CD4+ T cell interferon-gamma (IFN-gamma) secretion in schistosome granulomas. The role of IL-12 was determined using C57BL/6 and CBA mice. C57BL/6 IL-4-/- granuloma cells were stimulated to produce IFN-gamma when cultured with IL-10 or TGF-beta neutralizing monoclonal antibody. In comparison, C57BL/6 wild-type (WT) control granuloma cells produced less IFN-gamma. IL-12, IL-18, and soluble egg antigen stimulated IFN-gamma release from C57BL/6 IL-4-/- and WT mice. IFN-gamma production in C57 IL-4-/- and WT granulomas was IL-12 dependent, because IL-12 blockade partly abrogated IFN-gamma secretion after stimulation. All granuloma cells released IL-12 (p70 and p40), and IL-12 production remained constant after anti-TGF-beta, anti-IL-10, recombinant IL-18, or antigen stimulation. C57 WT and IL-4-/- mouse granuloma cells expressed IL-12 receptor (IL-12R) beta1-subunit mRNA but little beta2 mRNA. TGF-beta or IL-10 blockade did not influence beta1 or beta2 mRNA expression. CBA mouse dispersed granuloma cells released no measurable IFN-gamma, produced IL-12 p70 and little p40, and expressed IL-12R beta2 and little beta1 mRNA. In T helper 2 (Th2) granulomas of C57BL/6 WT and IL-4-/- mice, cells produce IL-12 (for IFN-gamma production) and IL-10 and TGF-beta modulate IFN-gamma secretion via mechanisms independent of IL-12 and IL-12R mRNA regulation. We found substantial differences in control of granuloma IFN-gamma production and IL-12 circuitry in C57BL/6 and CBA mice.     

Inhibition of inducible NO synthase by TH2 cytokines and TGF beta in rheumatoid arthritic synoviocytes: effects on nitrosothiol production.

The aim of this study was to compare the effects on NO production of IL-4, IL-10, and IL-13 with those of TGF-beta. RA synovial cells were stimulated for 24 h with IL-1 beta (1 ng/ml), TNF-alpha (500 pg/ml), IFN-gamma (10(-4)IU/ml) alone or in combination. Nitrite was determined by the Griess reaction, S-nitrosothiols by fluorescence, and inducible NO synthase (iNOS) by immunofluorescence and fluorescence activated cell sorter analysis (FACS). In other experiments, IL-4, IL-10, IL-13, and TGF beta were used at various concentrations and were added in combination with proinflammatory cytokines. The addition of IL-1 beta, TNF-alpha, and IFN-gamma together increased nitrite production: 257.5 +/- 35.8 % and S-nitrosothiol production : 413 +/- 29%, P < 0.001. None of these cytokines added alone had any significant effect. iNOS synthesis increased with NO production. IL-4, IL-10, IL-13, and TGF beta strongly decreased the NO production caused by the combination of IL-1 beta, TNF-alpha, and IFN-gamma. These results demonstrate that stimulated RA synoviocytes produce S-nitrosothiols, bioactive NO* compounds, in similar quantities to nitrite. IL-4, IL-10, IL-13, and TGF-beta decrease NO production by RA synovial cells. The anti-inflammatory properties of these cytokines may thus be due at least in part to their effect on NO metabolism.     

Nitric oxide is neither necessary nor sufficient for resolution of Plasmodium chabaudi malaria in mice

Malaria is a life-threatening re-emerging disease, yet it is still not clear how bloodstage malarial parasites are killed. Nitric oxide (NO), which has potent anti-microbial activity, may represent an important killing mechanism. The production of NO during descending Plasmodium chabaudi parasitemia, a period when parasites are killed by the immune response, supports this concept. However, NOS20/0 and NOS30/0 mice as well as mice treated with NO synthase 2 (NOS2) inhibitors do not develop exacerbated malaria, indicating that NO production is not necessary for the suppression of P. chabaudi parasitemia. It is possible due to the plasticity in the immune response during malaria that Ab-mediated immunity is enhanced in the absence of NO, thereby explaining the lack of exacerbated malaria in NOS-deficient mice even though NO may function in protection. However, NOS2- and B cell-deficient mice, which cannot use Ab-mediated immunity, suppress their parasitemia with a similar time course as B cell-deficient controls. C57BL/6 mice treated with Propionibacterium acnes to elicit high levels of macrophage-derived NO have a similar time course of P. chabaudi parasitemia as P. acnes-treated NOS20/0 mice, which do not produce NO; this indicates that NO is not sufficient for parasite killing. Collectively, these results indicate that NO is not necessary or sufficient to resolve P. chabaudi malaria.     

Role of neuronal and endothelial nitric oxide synthase in nitric oxide generation in the brain following cerebral ischemia.

Nitric oxide (NO) plays an important role in the pathogenesis of neuronal injury during cerebral ischemia. The endothelial and neuronal isoforms of nitric oxide synthase (eNOS, nNOS) generate NO, but NO generation from these two isoforms can have opposing roles in the process of ischemic injury. While increased NO production from nNOS in neurons can cause neuronal injury, endothelial NO production from eNOS can decrease ischemic injury by inducing vasodilation. However, the relative magnitude and time course of NO generation from each isoform during cerebral ischemia has not been previously determined. Therefore, electron paramagnetic resonance spectroscopy was applied to directly detect NO in the brain of mice in the basal state and following global cerebral ischemia induced by cardiac arrest. The relative amount of NO derived from eNOS and nNOS was accessed using transgenic eNOS(-/-) or nNOS(-/-) mice and matched wild-type control mice. NO was trapped using Fe(II)-diethyldithiocarbamate. In wild-type mice, only small NO signals were seen prior to ischemia, but after 10 to 20 min of ischemia the signals increased more than 4-fold. This NO generation was inhibited more than 70% by NOS inhibition. In either nNOS(-/-) or eNOS(-/-) mice before ischemia, NO generation was decreased about 50% compared to that in wild-type mice. Following the onset of ischemia a rapid increase in NO occurred in nNOS(-/-) mice peaking after only 10 min. The production of NO in the eNOS(-/-) mice paralleled that in the wild type with a progressive increase over 20 min, suggesting progressive accumulation of NO from nNOS following the onset of ischemia. NOS activity measurements demonstrated that eNOS(-/-) and nNOS(-/-) brains had 90% and < 10%, respectively, of the activity measured in wild type. Thus, while eNOS contributes only a fraction of total brain NOS activity, during the early minutes of cerebral ischemia prominent NO generation from this isoform occurs, confirming its importance in modulating the process of ischemic injury.