Expression of inducible nitric oxide synthase in rat pulmonary Cryptococcus neoformans granulomas.

Rats, like humans, have extremely effective immune mechanisms for controlling pulmonary Cryptococcus neoformans infection. The mechanism(s) responsible for efficient immunity in rat experimental infection is unknown. Recently, induction of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) have been implicated as an important microbicidal mechanism by which activated macrophages effect cytotoxicity against microbes. In this report, we investigated the expression of iNOS in rat pulmonary cryptococcosis. Localization and regulation of NO production was studied by immunohistochemistry for iNOS in conjunction with immunohistochemistry for cell markers, cytokines, and cryptococcal capsular polysaccharide. iNOS immunoreactivity was detected in macrophages, neutrophils, vascular endothelium, and respiratory epithelium. Double-immunolabeling studies revealed that the most prominent iNOS immunoreactivity was localized to epithelioid macrophages (CD11b/c+) within granulomas; CD4+ and CD8+ T cells were numerous around granulomas but did not express iNOS. iNOS immunoreactivity was detected in a selective population of epithelioid macrophages within some granulomas but not others. iNOS- granulomas were identical to iNOS+ granulomas with respect to morphology and immunohistochemical profiles. Macrophage iNOS immunoreactivity was detected 1 week after infection in one out of four rats and was strongly expressed in all rats at 2 weeks (in up to 50 percent of the granulomas) but declined considerably by 25 days. iNOS expression coincided with granuloma formation and preceded a decrease in lung fungal burden, suggesting an anticryptococcal role for NO. By double labeling, cytokines that have been shown to promote (interferon-gamma, granulocyte/macrophage colony-stimulating factor) and inhibit (transforming growth factor-beta) macrophage iNOS expression were detected around iNOS+ granuloma. iNOS immunoreactivity was expressed in selected neutrophils (1 and 2 weeks) and endothelial cells (1 and 2 weeks and 25 days) in the inflamed lung. Airway iNOS immunoreactivity was limited to the luminal border of rare bronchiolar epithelial cells. iNOS immunoreactivity was not detected in uninfected rats. The present study provides the first evidence for association of iNOS expression with protective cellular responses to cryptococcal infection in vivo.     

Differing requirement for inducible nitric oxide synthase activity in clearance of primary and secondary Cryptococcus neoformans infection.

The role of nitric oxide in resistance to cryptococcal infection was investigated. Mice deficient in inducible nitric oxide synthase (INOS) did not survive a primary intratracheal infection as did INOS-replete control mice. Despite adequate recruitment of host cells and generation of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha at the site of infection, INOS-deficient mice failed to clear yeast from their lungs by five weeks of infection, in contrast to wild-type mice. INOS-deficient mice also had higher yeast brain burdens than did control mice after a primary intracerebral infection. Therefore, generation of nitric oxide is required for resistance to primary cryptococcal infection. However, INOS-deficient mice vaccinated subcutaneously and rechallenged intravenously had lung and brain yeast burdens equivalent to those of vaccinated controls, and therefore expressed effective acquired immunity to Cryptococcus neoformans. Cells harvested from infected INOS-deficient mice by bronchoalveolar lavage acted as anti-cryptococcal effectors in vitro at an effector:target ratio of 100:1, provided IFN-gamma was present, but did not inhibit yeast proliferation at a 10:1 effector:target ratio as cells from wild-type mice did. Therefore, INOS activity is important for anti-cryptococcal function of effectors of immunity during the primary response, but not for the generation or expression of secondary immunity to C. neoformans.     

Expression of cytokines and inducible nitric oxide synthase mRNA in the lungs of mice infected with Cryptococcus neoformans: effects of interleukin-12.

We have recently established a murine model of pulmonary and disseminated infection with a highly virulent strain of Cryptococcus neoformans and demonstrated that administration of interleukin-12 (IL-12) protected the animals against infection. In this study, we extended these studies by investigating the host defense mechanisms. In particular, we examined the expression of mRNA for helper T-cell 1 (Th1) cytokines (IL-2, lymphotoxin, and gamma interferon [IFN-gamma]), Th2 cytokines (IL-4, -6, and -10), macrophage-derived cytokines (tumor necrosis factor alpha [TNF-alpha], IL-1beta, transforming growth factor beta [TGF-beta, IL-12p40, and IFN-gamma-inducing factor [IGIF]), and inducible nitric oxide synthase (iNOS) in the lungs on days 1, 3, 7, and 14 after infection and following treatment with IL-12. There was little or no expression of mRNAs for Th1 cytokines, TNF-alpha, IL-12p40, IGIF, and iNOS in the infected mice, but expression increased markedly after treatment with IL-12. In contrast, the mRNAs for Th2 cytokines, IL-1beta, and TGF-beta were detected at considerable levels during the early stages of infection, and, interestingly, expression was not suppressed by IL-12 but rather augmented, particularly during the late stage. Similar results were also obtained for IFN-gamma, IL-4, IL-10, and TNF-alpha measured in the lung homogenates by enzyme-linked immunosorbent assay. These results suggest that the predominance of expression of Th2 cytokines and TGF-beta over Th1 cytokines, TNF-alpha, IL-12p40, IGIF, and iNOS is associated with severe lethal infection in mice and that administration of IL-12 protects infected animals by stimulating Th1 cytokines.     

内皮型、诱导型一氧化氮合酶在乳腺癌中的表达

目的 :研究内皮型一氧化氮合酶 (eNOS)、诱导型一氧化氮合酶 (iNOS)在乳癌中表达及与淋巴结转移的关系。方法 :采用免疫组化S P法检测 60例乳癌中eNOS和iNOS的表达。结果 :eNOS和iNOS阳性在乳癌中表达率分别为 75 0 %和71 7%。在淋巴结转移组和无淋巴结转移组中eNOS阳性表达率分别为 66 7%和 83 3 % ,两组间差异无统计学意义 (χ2 =2 2 2 ,P >0 0 5) ,而iNOS在淋巴结转移和无转移组中阳性表达率分别为 53 3 %和 90 0 % ,两组间差异有统计学意义 (χ2 =9 93 ,P <0 0 1 )。结论 :内皮型、诱导型一氧化氮合酶在乳腺癌中高表达 ;iNOS的表达与乳腺癌的淋巴转移相关     

一氧化氮/诱导型一氧化氮合酶在动脉粥样硬化过程中的作用

 目的：探讨一氧化氮（NO）/诱导型一氧化氮合酶（iNOS）在动脉粥样硬化（atherosclerosis,AS）过程中的动态变化,分析其对动脉粥样硬化形成过程的影响。方法：将60只SD大鼠随机分成2组：对照组及AS组,每组30只。AS组采用维生素D3腹腔注射联合高脂饲料饲养的方法构建动脉粥样硬化模型。用相关生化方法检测血清各项生化指标：总胆固醇、甘油三酯、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇、空腹血糖和钙离子,比色法检测血清NO浓度,并对主动脉行HE染色,免疫组化技术检测iNOS蛋白表达,将所得数据进行统计分析,用简单线性相关分析NO与钙离子及动脉粥样硬化指数的相关性。结果：90 d后成功构建了主动脉中膜钙化型动脉粥样硬化模型。血清NO浓度在动脉粥样硬化过程中逐步下降,各组间差异均有统计学意义（均P<0.05）。动脉粥样硬化过程中动脉粥样硬化指数与钙离子呈正相关,与NO呈负相关。在90 d的AS组粥样斑块区免疫组化技术检测到iNOS蛋白表达。结论:在动脉粥样硬化形成过程中,主动脉粥样斑块区iNOS蛋白高表达,但血清NO浓度逐渐降低,NO抗动脉粥样硬化作用减弱。     

Persistent Cryptococcus neoformans pulmonary infection in the rat is associated with intracellular parasitism, decreased inducible nitric oxide synthase expression, and altered antibody responsiveness to cryptococcal polysaccharide

Fungal pathogens are notorious for causing chronic and latent infections, but the mechanism by which they evade the immune response is poorly understood. A major limitation in the study of chronic fungal infection has been the lack of suitable animal models where the infection is controlled and yet persists. Pulmonary Cryptococcus neoformans infection in rats results in a diffuse pneumonitis that resolves without dissemination or scarring except for the persistence of interstitial and subpleural granulomas that harbor viable cryptococci inside macrophages and epithelioid cells. Infected rats are asymptomatic but remain infected for as long as 18 months after inoculation with C. neoformans. Containment of infection is associated with granuloma formation that can be partially abrogated by glucocorticoid administration. Using this model, we identified several features associated with persistent infection in the rat lung, including (i) localization of C. neoformans to discrete, well-organized granulomas; (ii) intracellular persistence of C. neoformans within macrophages and epithelioid cells; (iii) reduced inducible nitric oxide synthase expression by granulomas harboring C. neoformans; and (iv) reduced antibody responses to cryptococcal polysaccharide. The results show that maintenance of persistent infection is associated with downregulation of both cellular and humoral immune responses.     

Production of nitric oxide by rat alveolar macrophages stimulated by Cryptococcus neoformans or Aspergillus fumigatus.

Cryptococcus neoformans and Aspergillus fumigatus are airborne fungi and the alveolar macrophages (AM) constitute a first line of host defence against both pathogens. We investigated the ability of rat AM to produce nitric oxide (NO) when challenged in vitro with C. neoformans, A. fumigatus conidia or inert silica particles alone and together with interferon gamma (IFN-Gamma). The role of NO in the killing of C. neoformans as well as the relationship between phagocytosis of the yeast or A. fumigatus conidia and NO production by AM were studied. Both fungi, but not the inert particles induced a small but significant increase in NO production by AM. A synergistically enhanced NO production by AM was observed when each fungus, but not silica particles, were incubated together with IFN-Gamma. AM treated with IFN-Gamma and challenged with C. neoformans showed higher killing activity than untreated AM, a finding that correlated with increased NO production by AM. Both effects were reduced by an inhibitor of NO synthesis. Increased NO production by IFN-Gamma activated AM was found together with an increased accumulated attachment of A. fumigatus conidia and serum opsonized, but not unopsonized C. neoformans. The IFN-Gamma dependent increase in accumulated attachment of the fungi might be responsible for the synergistic effect of the fungi and IFN-Gamma on the NO production. Our data suggest that activated rat AM might efficiently use the antimicrobial nitric oxide system in the defence against these pathogens in the normal host.     

Repeated induction of nitric oxide synthase and leishmanicidal activity in murine macrophages

Murine macrophages express high levels of nitric oxide (NO) synthase and produce large amounts of NO when stimulated with interferon-y plus lipopolysaccharide in vitro. The expression of NO synthase peaks at 12 h after stimulation and declines rapidly to the background level by 72 h. These macrophages can be repeatedly reactivated to express similar levels of NO synthase. The reactivation is not due to newly divided cells since peritoneal macrophages which do not divide in vitro and J774 cells cultured in the presence of colchicine can also be restimulated to express NO synthase. The reactivation is accompanied by re-expression of NO synthase mRNA, as assessed by polymerase chain reaction analysis. Furthermore, the reactivated macrophages are fully capable of killing the intracellular protozoan parasite Leishmania major.     

Expression of inducible nitric oxide synthase by stimulated macrophages correlates with their antihistoplasma activity.

The antihistoplasma activity of recombinant murine gamma interferon (rMuIFN-gamma)-treated macrophages of the RAW 264.7 cell line depends on the generation of nitric oxide (NO.) from L-arginine. Macrophages of the P388D1 cell line treated with rMuIFN-gamma do not produce NO. or inhibit the intracellular growth of Histoplasma capsulatum. NO. is generated by the inducible enzyme nitric oxide synthase (iNOS) formed by stimulated macrophages. Northern (RNA) blot analysis of RAW 264.7 cells revealed the expression of iNOS mRNA after exposure to rMuIFN-gamma. In contrast, rMuIFN-gamma-treated P388D1 cells did not produce detectable levels of iNOS. These data suggest that the failure of P388D1 cells to generate NO. and to restrict the intracellular growth of H. capsulatum is due to a lack of expression of iNOS following treatment with rMuIFN-gamma.     

Expression of inducible nitric oxide synthase and its involvement in pulmonary granulomatous inflammation in rats.

Two types of pulmonary granulomatosis were produced in rats by intratracheal instillation of zymosan or silica. In both models, immunostaining with anti-rat monoclonal antibody for inducible nitric oxide synthase (iNOS), ANOS11, showed that the intensity of iNOS immunoreactivity in the inflammatory lesions peaked at 3 days and declined thereafter. Immunohistochemical double staining and in situ hybridization demonstrated the expression of iNOS in neutrophils, monocyte-derived macrophages, and bronchiolar epithelial cells in the pulmonary lesions. Electron spin resonance spectroscopy revealed the production of an excessive amount of nitric oxide (NO) in the pulmonary lesions. Immunostaining with a polyclonal antibody against nitrotyrosine indicated the formation of nitrotyrosine residues in the granulomatous lesions, particularly in the periphery of the lesions, providing indirect evidence for the generation of peroxynitrite anion in the zymosan- or silica-instilled lungs. Administration of N omega-nitro-L-arginine methyl ester or S-methylisothiourea sulfate, which significantly suppressed NO production, resulted in marked reduction of monocyte/macrophage infiltration as well as in inhibition of induction of monocyte chemoattractant protein-1 in the lesions. These data indicate that NO and its more reactive product peroxynitrite anion may be important mediators of granuloma formation in the lung.     

TNF-α对精氨酸加压素诱导下大鼠心肌成纤维细胞诱导型一氧化氮合酶-一氧化氮系统活性的影响

目的：探讨肿瘤坏死因子-α(TNF-α)对精氨酸加压素(AVP)诱导下大鼠心肌成纤维细胞(CFs)诱导型一氧化氮合酶(iNOS)-一氧化氮(NO)系统活性的影响。方法： 胰酶消化法分离培养Sprague-Dawley仔鼠CFs，应用硝酸还原酶法、分光光度法和逆转录-聚合酶链式反应(RT-PCR)测定不同浓度TNF-α与AVP联合作用下CFs的NO含量、NOS活性和iNOS mRNA表达。 结果： AVP诱导CFs iNOS mRNA表达上调，NOS活性提高，NO合成增加。一定浓度下TNF-α可与AVP联合作用，剂量依赖性地增加AVP对CFs iNOS-NO系统活性的提高作用。但当TNF-α浓度过高时，CFs的iNOS-NO系统活性不继续升高，反而有所下降。 结论： TNF-α可与AVP联合提高CFs的iNOS-NO系统活性，NO合成增加可拮抗TNF-α和AVP的促心脏重构作用。     

Time-course of neuropathic pain in mice deficient in neuronal or inducible nitric oxide synthase

Nitric oxide which is synthesised by nitric oxide synthase (NOS) is involved in processes related to regeneration after nerve injury and neuropathic pain. Here we investigated functional aspects of the nociceptive system. For that purpose, the chronic constriction injury (CCI) model induced by loose ligation of the sciatic nerve was employed in C57Bl/6J wild-type (WT), nNOS and iNOS knock-out (−/−) mice. Their thermal and mechanical pain thresholds were then measured over a period of six weeks. In addition, ³H-DAMGO, ³H-CP 55.940, and ³H-l-glutamate binding, and neuronal (NeuN-immunostained) and astroglial (GFAP-immunostained) cell composition were studied. There were no significant differences in cell composition between the three strains used. Significant differences between CCI and sham-operated animals were found in nNOS−/− after day 6, in WT mice after day 10, and in iNOS−/− after day 17 post surgery. The mechanical pain threshold was normalised after day 45 post surgery in WT mice only. There were no changes in DAMGO and glutamate binding. However, we found significant differences in CP 55.940 binding in the spinal cord. It was concluded that NOS–cannabinoid interaction contributes to differences in nociceptive behaviour.     

Endothelial and inducible nitric oxide synthase expression in Venezuelan patients with pre-eclampsia

Preeclampsia (PE) is a disease that onsets in the second half of pregnancy. This condition is characterized by hypertension, proteinuria and, frequently, intrauterine growth restriction (IUGR). Nitric oxide (NO) regulates blood flow in the human placenta, it induces vasodilatation, inhibition of platelet aggregation and prevents adhesion of platelets to endothelial cells. In this work, nitrite levels were evaluated in the sera of peripheral blood of normal pregnant women (n = 46) and women with PE (n = 50); additionally, the expression of endothelial constitutive nitric oxide and inducible synthases (eNOS and iNOS, respectively) of placental tissues, were determined. An increased concentration of serum nitrites from patients with PE, in relation to normal pregnant women (150.64 +/- 8.94 vs 40.62 +/- 1.65 microM, p < 0.00001) was observed. An increased expression of nitric oxide synthases (eNOS and iNOS), in the placental tissues of (PE) patients, as compared to that of normal pregnant women (iNOS 4.29 +/- 1.51 vs 0.59 +/- 0.13; eNOS 1.78 +/- 0.74 vs 0.46 +/- 0.22, p < 0.005) was also observed. Our results show that there exists a relationship between serum nitrites concentration and the expression of eNOS and iNOS, as analyzed in protein extracts of placental tissues.     

Activation of nuclear factor kappaB and induction of inducible nitric oxide synthase by Ureaplasma urealyticum in macrophages

Chronic lung disease (CLD) of prematurity is an inflammatory disease with a multifactorial etiology. The importance of Ureaplasma urealyticum in the development of CLD is debated, and steroids produce some improvement in neonates with this disease. In the present study, the capability of U. urealyticum to stimulate rat alveolar macrophages to produce nitric oxide (NO), express inducible nitric oxide synthase (iNOS), and activate nuclear factor kappaB (NF-kappaB) in vitro was characterized. The effect of NO on the growth of U. urealyticum was also investigated. In addition, the impact of dexamethasone and budesonide on these processes was examined. We found that U. urealyticum antigen (> or =4 x 10(7) color-changing units/ml) stimulated alveolar macrophages to produce NO in a dose- and time-dependent manner (P<0.05). This effect was further enhanced by gamma interferon (100 IU/ml; P<0.05) but was attenuated by budesonide and dexamethasone (10(-4) to 10(-6) M) (P<0.05). The mRNA and protein levels of iNOS were also induced in response to U. urealyticum and inhibited by steroids. U. urealyticum antigen triggered NF-kappaB activation, a possible mechanism for the induced iNOS expression, which also was inhibited by steroids. NO induced by U. urealyticum caused a sixfold reduction of its own growth after infection for 10 h. Our findings imply that U. urealyticum may be an important factor in the development of CLD. The host defense response against U. urealyticum infection may also be influenced by NO. The down-regulatory effect of steroids on NF-kappaB activation, iNOS expression, and NO production might partly explain the beneficial effect of steroids in neonates with CLD.     

Human monocytic U937 cells transfected with human hepatic inducible nitric oxide synthase exhibit leishmanicidal activity

In mice, the high inducible synthesis of nitric oxide (NO) resulting from inducible NO synthase (iNOS, NOS2) expression by macrophages (Mphi) is considered an essential component of the protective immune response against infection by intracellular pathogens. Conversely, in humans, the question of a role for NO as an antimicrobial defense mechanism has been the subject of much debate. Recently, however, iNOS expression by human Mphi and formation of NO or its derivatives have been reported both in vivo and in vitro, strongly suggesting that human Mphi are indeed capable of inducible NO synthesis. However, the conditions allowing NO production by human Mphi in culture remain poorly defined, rendering more difficult the study of the effector functions of NO in these cells. To alleviate this problem, cells of the U937 monocytoid line were engineered to express iNOS by transfection with human hepatic iNOS (DFGiNOS), leading to production of NO on supplementation with the cofactor tetrahydrobiopterin. We report that U937 cells, when differentiated with 1,25-dihydroxyvitamin D3 and retinoic acid, acquire a phenotype allowing infection by Leishmania parasites and maintain viable intracellular microorganisms up to 72 h post-infection. Leishmania survival in DFGiNOS cells is strongly decreased when the cells are treated with tetrahydrobiopterin. Intracellular killing is evident by 24 h and increases up to 72 h post-infection, and is inhibited by L-N5-(1-iminoethyl)ornithine, an inhibitor of NO synthesis. In contrast, superoxide anion does not appear to play a role in the killing of Leishmania by DGFiNOS U937 cells. The relevance of this model to the study of the mechanisms of intracellular killing by human macrophages is discussed.     

Cell-specific effects of pentoxifylline on nitric oxide production and inducible nitric oxide synthase mRNA expression.

Cytokine-stimulated astrocytes and macrophages are potent producers of nitric oxide (NO), a free radical proposed to play an important role in organ-specific autoimmunity, including demyelinating diseases of the central nervous system. The aim of this study was to investigate effects of pentoxifylline (PTX), a phosphodiesterase inhibitor with immunomodulatory properties, on NO production and inducible NO synthase (iNOS) mRNA expression in rat astrocytes and macrophages. We have shown that PTX affects cytokine (interferon-gamma, IFN-gamma; interleukin-1, IL-1; tumour-necrosis factor-alpha, TNF-alpha)-induced NO production in both cell types, but in the opposite manner--enhancing in astrocytes and suppressive in macrophages. While PTX did not have any effect on enzymatic activity of iNOS in activated cells, expression of iNOS mRNA was elevated in astrocytes and decreased in macrophages treated with cytokines and PTX. Treatment with PTX alone affected neither NO production nor iNOS mRNA levels in astrocytes or macrophages. This study indicates involvement of different signalling pathways associated with iNOS induction in astrocytes and macrophages, thus emphasizing complexity of regulation of NO synthesis in different cell types.     

Functional significance of inducible nitric oxide synthase induction and protein nitration in the thermally injured cutaneous microvasculature

Increased nitric oxide (NO) production after burn injury is well established. However, there is little information relating to the reactions that occur as a consequence of NO generation under such circumstances. We have investigated the synthesis and function of NO in a rat model of local cutaneous thermal injury. We show that NO levels are elevated from 3 hours after injury with a concomitant increase in protein nitration. A selective inducible nitric oxide synthase (iNOS) inhibitor (1400W) significantly attenuated NO synthesis, protein nitration, and neutrophil accumulation in this model, but had no effect on edema formation. The results also indicate that NO synthesis and protein nitration occurred independently of neutrophil accumulation because these parameters were unaffected by depletion of circulating neutrophils. 3-Chlorotyrosine, a marker of neutrophil/myeloperoxidase-mediated protein damage was significantly increased from 1 hour after burn. Our observations provide evidence for the involvement of reactive species in the inflammatory response after burn. The use of selective iNOS inhibitors may represent a novel approach for the management of human burn injuries.     

Survivin and inducible nitric oxide synthase production during 4NQO-induced rat tongue carcinogenesis: a possible relationship

This study was undertaken to investigate, by immunohistochemistry, the expression of survivin and inducible nitric oxide synthase during 4NQO-induced rat tongue carcinogenesis. Male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution through their drinking water for 4, 12, and 20 weeks. Ten animals were used as negative control. Although no histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure, survivin and iNOS were expresssed (p<0.05) in some cells of the 'normal' oral epithelium. In pre-neoplastic lesions at 12 weeks following carcinogen exposure, the levels of survivin and iNOS were increased (p<0.05) when compared to negative control, being the strongest effect observed to iNOS. In well-differentiated squamous cell carcinoma induced after 20 weeks of treatment with 4NQO, survivin and iNOS were expressed in some tumor cells. Lack of immunoreactivity for both markers was observed in the negative control group. Taken together, our results support the belief that expression of survivin and iNOS are early events during malignant transformation and conversion of the oral mucosa.