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.     

Differential cytokine expression in skin graft healing in inducible nitric oxide synthase knockout mice

Inducible nitric oxide synthase (iNOS) and its product, nitric oxide, have been shown to play important roles in wound biology. The present study was performed to investigate the role of iNOS in modulating the cytokine cascade during the complex process of skin graft wound healing.Fifteen iNOS-knockout mice and 15 wild-type C57BL/6J mice were subjected to autogenous 1-cm2 intrascapular full-thickness skin grafts. Three animals in each group were killed on postoperative days 3, 5, 7, 10, and 14. Specimens were then analyzed using nonisotopic in situ hybridization versus mRNA of tumor growth factor-beta1, vascular endothelial growth factor, iNOS, endothelial nitric oxide synthase (eNOS), tumor necrosis factor-alpha, and basic fibroblast growth factor, as well as positive and negative control probes. Positive cells in both grafts and wound beds were counted using a Leica microgrid. Scar thickness was measured with a Leica micrometer. Data were analyzed using the unpaired Student's t test.Expression of iNOS was 2- to 4-fold higher in knockout mice than in wild-type mice on postoperative days 5, 7, and 14. Expression of eNOS was 2- to 2.5-fold higher in knockout mice than in wild-type mice on postoperative days 5 and 7. Tumor necrosis factor-alpha expression was 2- to 7-fold higher in knockout mice than in wild-type mice on all postoperative days. In contrast, expression levels of angiogenic/fibrogenic cytokines (vascular endothelial growth factor, basis fibroblast growth factor, and tumor growth factor-beta1) were 2.5- to 4-fold higher in wild-type mice than in knockout mice. Scars were 1.5- to 2.5-fold thicker in knockout mice than in wild-type mice at all time points. All of the above results represent statistically significant differences (p < 0.05).Significantly different patterns of cytokine expression were seen in knockout and wild-type mice. Although the scar layer was thicker in knockout mice, it showed much greater infiltration with inflammatory cells. These data further delineate the modulatory effect of iNOS and nitric oxide in healing skin grafts.     

Proinflammatory agents,IL-8 and IL-10, upregulate inducible nitric oxide synthase expression and nitric oxide production in avian osteoclast-like cells

Nitric oxide synthase (NOS) isoenzymes generate nitric oxide (NO), a sensitive multifunctional intercellular signal molecule. High NO levels are produced by an inducible NOS (iNOS) in activated macrophages in response to proinflammatory agents, many of which also regulate local bone metabolism. NO is a potent inhibitor of osteoclast bone resorption, whereas inhibitors of NOS promote bone resorption both in vitro and in vivo. The possibility that osteoclasts, like macrophages, express a regulated iNOS and produce NO as a potential autocrine signal following inflammatory stimulation was investigated in well-characterized avian marrow-derived osteoclast-like cells. NO production (reflected by medium nitrite levels) was markedly elevated in these cells by the proinflammatory agents lipopolysaccharide (LPS) and the synergistic action of IL-1α, TNFα, and IFNγ. Inhibitors of NOS activity (aminoguanidine, L-NAME) or iNOS induction (dexamethasone, TGFβ) reduced LPS-stimulated nitrite production. LPS also increased the NOS-associated diaphorase activity of these cells and their reactivity with anti-iNOS antibodies. RT-PCR cloning, using avian osteoclast-like cell RNA and human iNOS primers, yielded a novel 900 bp cDNA with high sequence homology (76%) to human, rat, and mouse iNOS genes. In probing osteoclast-like cell RNA with the PCR-derived iNOS cDNA, a 4.8 kb mRNA species was detected whose levels were greatly increased by LPS. Induction of iNOS mRNA by LPS, or by proinflammatory cytokines, occurred prior to the rise of medium nitrite in time course studies and was diminished by dexamethasone. Moreover, osteoclast-like cells demonstrated an upregulation of NO production and iNOS mRNA by IL-8 and IL-10, regulatory mechanism's not previously described. It is concluded that osteoclast-like cells express a novel iNOS that is upregulated by inflammatory mediators, leading to NO production. Therefore, NO may serve as both a paracrine and autocrine signal for modulating osteoclast bone resorption. © 1996 Wiley-Liss, Inc.     

Alterations in nitric oxide and cytokine production with airway inflammation in the absence of IL-10

IL-10 is an anti-inflammatory cytokine that suppresses NO synthase (NOS) and production of NO; its lack may promote NO production and alterations in cytokines modulated by NO with allergic airway inflammation (AI), such as IL-18 and IL-4. Therefore, we induced AI in IL-10 knockout ((-/-)) and IL-10-sufficient C57BL/6 (C57) mice with inhaled OVA and measured airway NO production, as exhaled NO (E(NO)) and bronchoalveolar lavage fluid nitrite levels. E(NO) and nitrite levels were elevated significantly in naive IL-10(-/-) mice as compared with C57 mice. With AI, E(NO) and nitrite levels increased in C57 mice and decreased in IL-10(-/-) mice. IL-18 production fell with both AI and addition of S-nitroso-N-acetyl-d,l-penicillamine (a NO donor) but was not significantly increased by chemical NOS inhibition by l-N(5)-(1-iminoethyl)-ornithine. IL-4 AI was increased significantly (up to 10-fold greater) in the absence of IL-10 but was reduced significantly with chemical inhibition of NOS. Airway responsiveness was lower in IL-10(-/-) mice and was associated with alteration in production of NO and IL-4. Thus, IL-4 production was increased, and likely decreased NO production, in a way not predicted by the absence of IL-10. Inhibition of IL-4 production, with inhibition of NOS in the absence of IL-10, demonstrated the importance of a NO and IL-4 feedback mechanism regulating this interaction.     

Deficiency in inducible nitric oxide synthase results in reduced atherosclerosis in apolipoprotein E-deficient mice

Inducible NO synthase (iNOS) present in human atherosclerotic plaques could contribute to the inflammatory process of plaque development. The role of iNOS in atherosclerosis was tested directly by evaluating the development of lesions in atherosclerosis-susceptible apolipoprotein E (apoE)-/- mice that were also deficient in iNOS. ApoE-/- and iNOS-/- mice were cross-bred to produce apoE-/-/iNOS-/- mice and apoE-/-/iNOS+/+ controls. Males and females were placed on a high fat diet at the time of weaning, and atherosclerosis was evaluated at two time points by different methods. The deficiency in iNOS had no effect on plasma cholesterol, triglyceride, or nitrate levels. Morphometric measurement of lesion area in the aortic root at 16 wk showed a 30-50% reduction in apoE-/-/iNOS-/- mice compared with apoE-/-/iNOS+/+ mice. Although the size of the lesions in apoE-/-/iNOS-/- mice was reduced, the lesions maintained a ratio of fibrotic:foam cell-rich:necrotic areas that was similar to controls. Biochemical measurements of aortic cholesterol in additional groups of mice at 22 wk revealed significant 45-70% reductions in both male and female apoE-/-/iNOS-/- mice compared with control mice. The results indicate that iNOS contributes to the size of atherosclerotic lesions in apoE-deficient mice, perhaps through a direct effect at the site of the lesion.     

Dietary nitrite restores NO homeostasis and is cardioprotective in endothelial nitric oxide synthase-deficient mice

Endothelial production of nitric oxide (NO) is critical for vascular homeostasis. Nitrite and nitrate are formed endogenously by the stepwise oxidation of NO and have, for years, been regarded as inactive degradation products. As a result, both anions are routinely used as surrogate markers of NO production, with nitrite as a more sensitive marker. However, both nitrite and nitrate are derived from dietary sources. We sought to determine how exogenous nitrite affects steady-state concentrations of NO metabolites thought to originate from nitric oxide synthase (NOS)-derived NO as well as blood pressure and myocardial ischemia-reperfusion (I/R) injury. Mice deficient in endothelial nitric oxide synthase (eNOS-/-) demonstrated decreased blood and tissue nitrite, nitrate, and nitroso proteins, which were further reduced by low-nitrite (NOx) diet for 1 week. Nitrite supplementation (50 mg/L) in the drinking water for 1 week restored NO homeostasis in eNOS-/- mice and protected against I/R injury. Nitrite failed to alter heart rate or mean arterial blood pressure at the protective dose. These data demonstrate the significant influence of dietary nitrite intake on the maintenance of steady-state NO levels. Dietary nitrite and nitrate may serve as essential nutrients for optimal cardiovascular health and may provide a novel prevention/treatment modality for disease associated with NO insufficiency.     

Uterine artery remodeling and reproductive performance are impaired in endothelial nitric oxide synthase-deficient mice

The progressive rise in uterine blood flow during pregnancy is accompanied by outward hypertrophic remodeling of the uterine artery (UA). This process involves changes of the arterial smooth muscle cells and extracellular matrix. Acute increases in blood flow stimulate endothelial production of nitric oxide (NO). It remains to be established whether endothelial NO synthase (eNOS) is involved in pregnancy-related arterial remodeling. We tested the hypothesis that absence of eNOS results in a reduced remodeling capacity of the UA during pregnancy leading to a decline in neonatal outcome. UA of nonpregnant and pregnant wild-type (Nos3+/+) and eNOS-deficient (Nos3-/-) mice were collected and processed for standard morphometrical analyses. In addition, cross sections of UA were processed for cytological (smoothelin, smooth muscle alpha-actin) and proliferation (Ki-67) immunostaining. We compared the pregnancy-related changes longitudinally and, together with the data on pregnancy outcome, transversally by analysis of variance with Bonferroni correction. During pregnancy, the increases in radius and medial cross sectional area of Nos3-/- UA was significantly less than those of Nos3+/+ UA. Smooth muscle cell dedifferentiation and proliferation were impaired in gravid Nos3-/- mice as deduced from the lack of change in the expression of smoothelin and smooth muscle alpha-actin, and the reduced Ki-67 expression. Until 17 days of gestation, litter size did not differ between both genotypes, but at birth the number of viable newborn pups and their weights were smaller in Nos3-/- than in Nos3+/+ mice. We conclude that absence of eNOS adversely affects UA remodeling in pregnancy, which may explain the impaired pregnancy outcome observed in these mice.     

Detection of inducible nitric oxide synthase in Schistosoma japonicum and S. mansoni

Schistosoma japonicum and S. mansoni were tested for reactivity with an anti-inducible nitric oxide (iNOS) antibody and the distribution of iNOS was studied by immunofluorescent tests in different stages of the parasites. Reactivity was associated with the tegument in both larval schistosomes (sporocysts and cercariae) and eggs. With adult worms, the majority of the immunofluorescence was predominantly subtegumental in S. japonicum and parenchymal in S. mansoni. Fluorescence was also observed in host tissues (snails and mouse liver). In Western blots, the enzyme of S. japonicum had an apparent molecular weight of about 210 kDa. The possible role of worm and host iNOS in the parasite-host interrelation remains to be clarified.     

DNA damage and nitric oxide production in mice following infection with L. chagasi

Leishmania chagasi, which causes visceral leishmaniasis in South America, is an obligate intracellular protozoan. Production of nitric oxide by macrophages during the inflammatory response is one of the main microbicidal mechanisms against this parasite. The goal of this study was to evaluate whether L. chagasi infection causes DNA damage in peripheral blood and spleen cells of Balb/c mice and whether such damage may be related to NO production. Balb/c mice were either infected with L. chagasi or maintained as controls. The single-cell gel electrophoresis (comet) assay was used to measure DNA damage in peripheral blood and spleen cells, and the Griess reaction was used to measure NO production in the spleen. L. chagasi infection induced DNA damage in peripheral blood and spleen cells of infected mice. Macrophages from the control group, challenged with L. chagasi, showed significantly (p<0.05) greater NO production, compared to non-challenged cells. Treatment of spleen cells with N(G)-monomethyl-l-arginine (LNMMA) caused a significant reduction of NO production and DNA damage (p<0.05). Our results indicate that L. chagasi induces DNA damage in the peripheral blood and spleen cells and that NO not only causes killing of the parasite but also induces DNA damage in adjacent cells.     

Testosterone production in mice lacking inducible nitric oxide synthase expression is sensitive to restraint stress

Immobilization stress (IMO) induces a rapid increase in glucocorticoid secretion [in rodents, corticosterone CORT)] and this is associated with decreased circulating testosterone (T) levels. Nitric oxide (NO), a reactive free radical and neurotransmitter, has been reported to be produced at higher rates in tissues such as brain during stress. The biosynthesis of T is also known to be dramatically suppressed by NO. Specifically, the inducible isoform of nitric oxide synthase (iNOS) was directly implicated in this suppression. To assess the respective roles of CORT and NO in stress-mediated inhibition of T production, adult wild-type (WT) and inducible nitric oxide synthase knockout (iNOS(-/-)) male mice were evaluated. Animals of each genotype were assigned to either basal control or 3-h IMO groups. Basal plasma and testicular T levels were equivalent in both genotypes, whereas testicular weights of mutant mice were significantly higher compared with WT animals. Exposure to 3-h IMO increased plasma CORT and decreased T concentrations in mice of both genotypes. Testicular T levels were also affected by stress in WT and mutant males, being sharply reduced in both genotypes. However, the concentrations of nitrite and nitrate, the stable metabolites of NO measured in testicular extracts, did not differ between control and stressed WT and iNOS(-/-) mice. These results support the hypothesis that CORT, but not NO, is a plausible candidate to mediate rapid stress-induced suppression of Leydig cell steroidogenesis.     

Oltipraz inhibits inducible nitric oxide synthase in vitro and inhibits nitric oxide production in activated microglial cells

The clinically relevant drug oltipraz (OPZ) has previously been shown to inhibit cytochrome P450 enzymes [Chem. Res. Toxicol. 13 (2000) 245]. The current study reveals that OPZ is also able to inhibit *NO formation by purified inducible nitric oxide synthase (iNOS) but not by neuronal nitric oxide synthase in hemoglobin assays. The inhibition of iNOS by OPZ is reversible and competitive with an IC(50) of 5.9 microM and Ki of 0.6 microM. In murine BV-2 microglial cells, an immortalized cell line that produces *NO in response to lipopolysaccharide (LPS), OPZ is able to block the formation of nitrite in LPS treated cells. The inhibitory effect of OPZ on LPS treated cells is not due to cell toxicity. Finally, treatment of cells with OPZ does not induce or suppress expression of iNOS protein as shown by Western blot analysis.     

Effect of myrrh and thyme on Trichinella spiralis enteral and parenteral phases with inducible nitric oxide expression in mice

Trichinellosis is a serious disease with no satisfactory treatment. We aimed to assess the effect of myrrh (Commiphora molmol) and, for the first time, thyme (Thymus vulgaris L.) against enteral and encysted (parenteral) phases of Trichinella spiralis in mice compared with albendazole, and detect their effect on inducible nitric oxide synthase (iNOS) expression. Oral administration of 500 mg/kg of myrrh and thyme led to adult reduction (90.9%, 79.4%), while 1,000 mg/kg led to larvae reduction (79.6%, 71.3%), respectively. Administration of 50 mg/kg of albendazole resulted in adult and larvae reduction (94.2%, 90.9%). Positive immunostaining of inflammatory cells infiltrating intestinal mucosa and submucosa of all treated groups was detected. Myrrh-treated mice showed the highest iNOS expression followed by albendazole, then thyme. On the other hand, both myrrh and thyme-treated groups showed stronger iNOS expression of inflammatory cells infiltrating and surrounding encapsulated T. spiralis larvae than albendazole treated group. In conclusion, myrrh and thyme extracts are highly effective against both phases of T. spiralis and showed strong iNOS expressions, especially myrrh which could be a promising alternative drug. This experiment provides a basis for further exploration of this plant by isolation and retesting the active principles of both extracts against different stages of T. spiralis.     

Reduced inotropic heart response in selenium-deficient mice relates with inducible nitric oxide synthase

Atria from mice fed a selenium-deficient (Se(-)) diet have a diminished beta-adrenoceptor-inotropic cardiac response to isoproterenol or norepinephrine compared with atria from mice fed the same diet supplemented with 0.2 mg/kg Se as sodium selenite (Se(+)). This diminished response could be reversed by feeding Se(-) mice the Se(+) diet for 1 wk or by pretreatment with nitric oxide synthase (NOS) inhibitors such as N(G)-monomethyl-l-arginine or aminopyridine. Elevated serum concentrations of nitrite/nitrate as well as a threefold increase in the atrial NOS activity were seen in the Se(-) versus Se(+) mice. Western blotting and indirect immunofluorescence indicated an enhanced expression of inducible NOS in hearts from Se(-) mice. Increased expression and activity of NOS and increased nitrite/nitrate levels from Se(-) mice correlated with an impaired response to beta-adrenoceptor inotropic cardiac stimulation. Elevated nitric oxide levels may account for some of the pathophysiological effects of Se deficiency on the heart.     

Phenotype of transgenic mice overexpressed with inducible nitric oxide synthase in the retina

Background

Unlike its constitutive isoforms, including neuronal and endothelial nitric oxide synthase, inducible nitric oxide synthase (iNOS) along with a series of cytokines are generated in inflammatory pathologic conditions in retinal photoreceptors. In this study, we constructed transgenic mice overexpressing iNOS in the retina to evaluate the effect of sustained, intense iNOS generation in the photoreceptor damage.

Methods

For construction of opsin/iNOS transgene in the CMVSport 6 expression vector, the 4.4 kb Acc65I/Xhol mouse rod opsin promoter was ligated upstream to a 4.1 kb fragment encoding the complete mouse cDNA of iNOS. From the four founders identified, two heterozygote lines and one homozygote line were established. The presence of iNOS in the retina was confirmed and the pathologic role of iNOS was assessed by detecting nitrotyrosine products and apoptosis. Commercial TUNEL kit was used to detect DNA strand breaks, a later step in a sequence of morphologic changes of apoptosis process.

Results

The insertion and translation of iNOS gene were demonstrated by an intense single 130 kDa band in Western blot and specific immunolocalization at the photoreceptors of the retina. Cellular toxicity in the retinas of transgenic animals was detected by a post-translational modification product, tyrosine-nitrated protein, the most significant one of which was nitrated cytochrome c. Following the accumulation of nitrated mitochondrial proteins and cytochrome c release, marked apoptosis was detected in the photoreceptor cell nuclei of the retina.

Conclusions

We have generated a pathologic phenotype with sustained iNOS overexpression and, therefore, high output of nitric oxide. Under basal conditions, such overexpression of iNOS causes marked mitochondrial cytochrome c nitration and release and subsequent photoreceptor apoptosis in the retina. Therefore, the modulation of pathways leading to iNOS generation or its effective neutralization can be of significant therapeutic benefit in the oxidative stress-mediated retinal degeneration, a leading cause of blindness.     

Inducible nitric oxide synthase-deficient mice show exacerbated inflammatory process and high production of both Th1 and Th2 cytokines during paracoccidioidomycosis

Paracoccidioidomycosis, the major systemic mycosis in Latin America, is caused by fungus Paracoccidioides brasiliensis. To analyze the influence of inducible nitric oxide synthase (iNOS) in this disease, iNOS-deficient (iNOS^−/−) and wild-type (WT) mice were infected intravenously with P. brasiliensis 18 isolate. We found that, unlike WT mice, iNOS^−/− mice did not control fungal proliferation, and began to succumb to infection by day 50 after inoculation of yeast cells. Typical inflammatory granulomas were found in WT mice, while, iNOS^−/− mice presented incipient granulomas with intense inflammatory process and necrosis. Additionally, splenocytes from iNOS^−/− mice did not produce nitric oxide, however, their proliferative response to Con-A was impaired, just like infected WT mice. Moreover, infected iNOS^−/− mice presented a mixed pattern of immune response, releasing high levels of both Th1 (IL-12, IFN-γ and TNF-α) and Th2 (IL-4 and IL-10) cytokines. These data suggest that the enzyme iNOS is a resistance factor during paracoccidioidomycosis by controlling fungal proliferation, by influencing cytokines production, and by appeasing the development of a high inflammatory response and consequently formation of necrosis. However, iNOS-derived nitric oxide seems not being the unique factor responsible for immunosuppression observed in infections caused by P. brasiliensis.     

Concurrent infections with Trypanosoma brucei and Nippostrongylus brasiliensis in mice deficient in inducible nitric oxide

Concurrent infection with Trypanosoma brucei (Tb) delays the normal protective responses of mice to the gastrointestinal parasite Nippostrongylus brasiliensis (Nb). The course of such infections was followed in mice genetically deficient in inducible nitric oxide synthase (INOS) to assess the role of nitric oxide (NO) in this effect. The time course of trypanosome infection in INOS deficient (INOS-/-) mice was similar to that in wild type (WT) and heterozygote (INOS+/-) mice but did not result in NO production. Although concurrent infection with Tb increased initial susceptibility to Nb in INOS-/- mice, the immune-mediated loss of N. brasiliensis and the associated decline in faecal egg output occurred more rapidly then in WT and INOS+/- littermates. Concurrent infection with trypanosomes markedly suppressed Concanavalin A (ConA)-induced in vitro proliferation of splenic lymphocytes in all groups, but had little effect on the responses of mesenteric node lymphocytes. Trypanosome infection was also associated with increased early release of interferon-gamma and reduced IL-5 from lymphocytes stimulated in vitro with ConA, but did not affect later release of IL-5. The overall similarity of proliferative and cytokine responses in WT, INOS+/- and INOS-/- mice suggest that the suppressive effects of T. brucei on N. brasiliensis infection do not simply reflect depressed lymphocyte responsiveness or altered cytokine profiles. NO appears to be involved in suppression only of the later phases of the host responses to Nb.     

Indomethacin prevents the induction of inducible nitric oxide synthase in murine peritoneal macrophages and decreases their nitric oxide production

Indomethacin (0.14-.5 mM concentration) inhibits nitric oxide production in murine peritoneal macrophages. This was evidenced by measuring both nitrite production or 14C-L-citrulline formation. The inhibition was caused by the diminution of de novo inducible nitric oxide synthase production as demonstrated by Western blotting experiment. The effect of indomethacin after 4 h treatment was irreversible. NO synthase and arginase activities and the uptake of arginine were not directly affected by the drug. Indomethacin also decreased uridine incorporation in macrophages. The effect of indomethacin on the induction of other enzymes (i.e. arginase) was weaker.     

Augmented inducible nitric oxide synthase expression and increased NO production reduce sepsis-induced lung injury and mortality in myeloperoxidase-null mice

The myeloperoxidase (MPO)-hydrogen peroxide-halide system is an efficient oxygen-dependent antimicrobial component of polymorphonuclear leukocyte (PMN)-mediated host defense. However, MPO deficiency results in few clinical consequences indicating the activation of compensatory mechanisms. Here, we determined possible mechanisms protecting the host using MPO(-/-) mice challenged with live gram-negative bacterium Escherichia coli. We observed that MPO(-/-) mice unexpectedly had improved survival compared with wild-type (WT) mice within 5-12 h after intraperitoneal E. coli challenge. Lungs of MPO(-/-) mice also demonstrated lower bacterial colonization and markedly attenuated increases in microvascular permeability and edema formation after E. coli challenge compared with WT. However, PMN sequestration in lungs of both groups was similar. Basal inducible nitric oxide synthase (iNOS) expression was significantly elevated in lungs and PMNs of MPO(-/-) mice, and NO production was increased two- to sixfold compared with WT. Nitrotyrosine levels doubled in lungs of WT mice within 1 h after E. coli challenge but did not change in MPO(-/-) mice. Inhibition of iNOS in MPO(-/-) mice significantly increased lung edema and reduced their survival after E. coli challenge, but iNOS inhibitor had the opposite effect in WT mice. Thus augmented iNOS expression and NO production in MPO(-/-) mice compensate for the lack of HOCl-mediated bacterial killing, and the absence of MPO-derived oxidants mitigates E. coli sepsis-induced lung inflammation and injury.