Nitric oxide production and mononuclear cell nitric oxide synthase activity in malaria-tolerant Papuan adults

Individuals living in regions of intense malaria transmission exhibit natural immunity that allows them to be without fever and other symptoms for most of the time despite frequent parasitization. Although this tolerance of parasitemia appears to be more effective in children than in adults (as evidenced by lower parasitemia fever thresholds with age), adults do exhibit a degree of tolerance but the mechanism(s) underlying this are unclear. Asymptomatic malaria-exposed children have higher levels of nitric oxide (NO) than children with severe disease, and NO has been proposed as a mediator of malarial tolerance. However, the ability of highly malaria-exposed asymptomatic adults to generate high-level basal NO is unknown, as is the relationship between NO and malaria tolerance in adults. The relationship between NO and malaria parasitemia was therefore determined in asymptomatic adults from Papua, Indonesia. Adults with Plasmodium falciparum parasitemia had markedly increased basal systemic NO production relative to aparasitemic Papuan controls, who in turn produced more NO than healthy controls from a region without malaria. Immunoglobulin E levels were universally elevated in malaria-exposed Papuan subjects, suggesting that the prevalence of intestinal parasitosis may be high and that nonmalarial infection may also contribute to high basal NO production. Basal peripheral blood mononuclear cell (PBMC) NO synthase activity was elevated in Papuans but poorly correlated with systemic NO production, suggesting that NO production in this setting arises not only from PBMCs but also from other tissue and cellular sources. NO production was associated with and may contribute to malaria tolerance in Papuan adults.     

Nitric oxide synthase and nitric oxide production in in vivo-derived mast cells

Nitric oxide (NO) is a potent mediator synthesized by a variety of cells involved in inflammatory reactions. We investigated the expression of NO synthase (NOS) in rat peritoneal mast cells (PMC). Small amounts of eNOS mRNA were detected basally, whereas neither mRNA for iNOS nor nNOS was detected in unstimulated PMC. Following stimulation by antigen, interferon-gamma (IFN-gamma), or anti-CD8 antibody, PMC up-regulated iNOS mRNA expression. In situ RT-PCR confirmed that iNOS mRNA originated from PMC. Production of iNOS protein was confirmed in stimulated PMC by immunohistochemistry. Upon stimulation with antigen, IFN-gamma, or anti-CD8, nitrite production was increased significantly (8.4+/-0.6, 7.6+/-0.9, and 6.6+/-0.9 microM/2x10(5) cells/48 h NO2-, respectively; P<0.01), whereas unstimulated PMC released 2.1 +/- 0.3 microM/2 x 10(5) cells/48 h NO2-. These findings demonstrate that in vivo-derived PMC transcribe and translate mRNA for NOS and produce NO.     

Effect of N-nitrosodimethylamine on inducible nitric oxide synthase expression and production of nitric oxide by neutrophils and mononuclear cells: the role of JNK signalling pathway

In neutrophils (PMN) and mononuclear cells (PBMC), one of the enzymes responsible for nitric oxide (NO) synthesis is inducible nitric oxide synthase (iNOS). Changes in iNOS expression result from various signalling pathways including the mitogen-activated protein kinase (MAPK) pathway activated by endogenic and exogenic factors. N-nitrosodimethylamine (NDMA) is a xenobiotic with widespread occurrence in human environment and has an effect on cells of the immune system. The aim of this study was to determine the effect of NDMA on iNOS expression and NO production by human PMN and PBMC cells in the light of superoxide anion production by PMN cells. Moreover, the role of JNK and p38 pathways in NO production with involvement of iNOS was studied. Additionally, the function of JNK pathway in generation of superoxide anion was determined. Moreover, nitrotyrosine expression was studied in PMN and PBMC cells in the presence of NDMA. This work shows that NDMA increases iNOS expression and NO production by PMN and PBMC cells. In addition, elevated expression of phospho-JNK and phospho-p38, which are markers of JNK and p38 MAPK pathways activation, were observed. Lower iNOS expression and NO production by neutrophils exposed to extended action of NDMA were observed after application of inhibitor of JNK and p38 pathways. Lower phospho-p38 expression in PMN stimulated by NDMA was found as a result of arresting JNK pathway, whereas, application of inhibitor of p38 pathway resulted in enhanced phospho-JNK expression in PMN and PBMC cells. Increased ability to release superoxide anion by NDMA-stimulated PMN cells was observed. This ability was reduced after the application of inhibitor of JNK pathway. In PMN and PBMC cells exposed to NDMA, an increased expression of nitrotyrosine, which is dependant on JNK and p38 pathways that are activated by this particular xenobiotic, was observed. Generally, increased induction of iNOS related to elevated production of NO by PMN and PBMC cells exposed to NDMA may result in dysfunction of regulation of immunity responses controlled by this molecule in various conditions. Increased expression of nitrotyrosine in PMN and PBMC cells exposed to NDMA may affect their functions in an auto- and/or a paracrine way. Mutual interactions of JNK and p38 MAPK during the induction of iNOS expression in cells exposed to NDMA indicate complex mechanism of induction of iNOS synthase.     

Elevated production of GM-CSF by peripheral blood mononuclear cells stimulated with mite antigens in children with bronchial asthma]

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine which can stimulate the proliferation and functions of eosinophils and neutrophils. We have studied whether peripheral blood mononuclear cells (PBMC), obtained from children with stable asthma, can produce GM-CSF through stimulation with crude Dermatophagoides farinae (Df) antigen in vitro. The patients had strongly positive RAST results for house dust mite Df. Levels of GM-CSF in PBMC culture supernatant were assayed by an enzyme immunoassay. When stimulated with Df antigen, patient's PBMC released significantly increased levels of GM-CSF compared with unstimulated ones. PBMC of non-allergic controls, however, did not. The addition of polymyxin B, an inactivator of endotoxin, did not suppress the Df-antigen-induced production of GM-CSF. Levels of GM-CSF produced by PBMC stimulated with Df antigens were significantly higher in the patients than in the controls. These results indicate that PBMC of children with Df-allergic asthma, unlike those of non-allergic children, can produce elevated levels of GM-CSF by stimulation with Df antigens.     

Blastomyces dermatitidis yeast cells inhibit nitric oxide production by alveolar macrophage inducible nitric oxide synthase

The ability of pathogens to evade host antimicrobial mechanisms is crucial to their virulence. The dimorphic fungal pathogen Blastomyces dermatitidis can infect immunocompetent patients, producing a primary pulmonary infection that can later disseminate to other organs. B. dermatitidis possesses a remarkable ability to resist killing by alveolar macrophages. To date, no mechanism to explain this resistance has been described. Here, we focus on macrophage production of the toxic molecule nitric oxide as a potential target of subversion by B. dermatitidis yeast cells. We report that B. dermatitidis yeast cells reduce nitric oxide levels in the supernatants of activated alveolar macrophages. This reduction is not due to detoxification of nitric oxide, but rather to suppression of macrophage nitric oxide production. We show that B. dermatitidis yeast cells do not block upregulation of macrophage inducible nitric oxide synthase (iNOS) expression or limit iNOS access to its arginine substrate. Instead, B. dermatitidis yeast cells appear to inhibit iNOS enzymatic activity. Further investigation into the genetic basis of this potential virulence mechanism could lead to the identification of novel antifungal drug targets.     

Microenvironmental regulation of inducible nitric oxide synthase expression and nitric oxide production in mouse bone marrow-derived mast cells

In addition to its well-known role in relaxation of vascular smooth muscle, NO modulates immune responses in a concentration- and location-specific manner. For MC, it is well accepted that exogenous NO regulates their function. However, there are inconsistencies in the literature of whether MC express NOS and make NO. MC progenitors mature in peripheral tissues, but the factors that influence MC maturation and their specific phenotype, such as whether they express NOS, are not well understood. To study microenvironmental conditions that could be "permissive" for NOS expression, we cultured BMMC in various conditions--BMMC(IL-3), BMMC(SCF/IL-3), or BMMC(SCF/IL-4)-for >3 weeks and examined NOS expression. We detected Nos2 mRNA in BMMC(SCF/IL-4) but not BMMC(IL-3) or BMMC(SCF/IL-3). After stimulation with IFN-γ and/or LPS, NOS2 expression and NO production were detected in BMMC(SCF/IL-4) but rarely detected in BMMC cultured with other conditions. Confocal microscopic analysis showed that NOS2 expression induced by IFN-γ colocalized in CD117(+) BMMC. NO production, after activation with IFN-γ and LPS in BMMC(SCF/IL-4), was abrogated by pretreatment with the NOS2-specific inhibitor. In addition to NOS2 expression, BMMC(SCF/IL-4) were distinguished from BMMC(IL-3) in heparin and MMCP expression. Thus, MC progenitors that develop in SCF + IL-4 can be induced to express NOS2 after receiving appropriate signals, such as IFN-γ, and subsequently produce NO. Microenvironmental conditions during their development can influence whether MC are capable of NOS expression and of NO production.     

Inducible nitric oxide synthase mediates prostaglandin h2 synthase nitration and suppresses eicosanoid production

Nitric oxide (NO) modulates the biological levels of arachidonate-derived cell signaling molecules by either enhancing or suppressing the activity of prostaglandin H(2) isoforms (PGHS-1 and PGHS-2). Whether NO activates or suppresses PGHS activity is determined by alternative protein modifications mediated by NO and NO-derived species. Here, we show that inducible NO synthase (iNOS) and PGHS-1 co-localize in atherosclerotic lesions of ApoE(-/-) mouse aortae. Immunoblotting and immunohistochemistry revealed Tyr nitration in PGHS-1 in aortic lesions but markedly less in adjacent nonlesion tissue. PGHS-2 was also found in lesions, but 3-nitrotyrosine incorporation was not detected. 3-Nitrotyrosine formation in proteins is considered a hallmark reaction of peroxynitrite, which can form via NO-superoxide reactions in an inflammatory setting. That iNOS-derived NO is essential for 3-nitrotyrosine modification of PGHS-1 was confirmed by the absence of 3-nitrotyrosine in lesions from ApoE(-/-)iNOS(-/-) mice. Mass spectrometric studies specifically identified the active site residue Tyr385 as a 3-nitrotyrosine modification site in purified PGHS-1 exposed to peroxynitrite. PGHS-mediated eicosanoid (PGE(2)) synthesis was more than fivefold accelerated in cultured iNOS(-/-) versus iNOS-expressing mouse aortic smooth muscle cells, suggesting that iNOS-derived NO markedly suppresses PGHS activity in vascular cells. These results further suggest a regulatory role of iNOS in eicosanoid biosynthesis in human atherosclerotic lesions.     

Tissue-engineered blood vessels with endothelial nitric oxide synthase activity

Nondegradable synthetic polymer vascular grafts used in cardiovascular surgery have shown serious shortcomings, including thrombosis, calcification, infection, and lack of growth potential. Tissue engineering of vascular grafts with autologous stem cells and biodegradable polymeric materials could solve these problems. The present study is aimed to develop a tissue-engineered vascular graft (TEVG) with functional endothelium using autologous bone marrow-derived cells (BMCs) and a hybrid biodegradable polymer scaffold. Hybrid biodegradable polymer scaffolds were fabricated from poly(lactide-co-epsilon-caprolactone) (PLCL) copolymer reinforced with poly(glycolic acid) (PGA) fibers. Canine bone marrow mononuclear cells were induced in vitro to differentiate into vascular smooth muscle cells and endothelial cells. TEVGs (internal diameter: 10 mm, length: 40 mm) were fabricated by seeding vascular cells differentiated from BMCs onto PGA/PLCL scaffolds and implanted into the abdominal aorta of bone marrow donor dogs (n = 7). Eight weeks after implantation of the TEVGs, the vascular grafts remained patent. Histological and immunohistochemical analyses of the vascular grafts retrieved at 8 weeks revealed the regeneration of endothelium and smooth muscle and the presence of collagen. Western blot analysis showed that endothelial nitric oxide synthase (eNOS) was expressed in TEVGs comparable to native abdominal aortas. This study demonstrates that vascular grafts with significant eNOS activity can be tissue-engineered with autologous BMCs and hybrid biodegradable polymer scaffolds.     

Identification of CpG oligodeoxynucleotide motifs that stimulate nitric oxide and cytokine production in avian macrophage and peripheral blood mononuclear cells

Unmethylated CpG dinucleotides within specific flanking bases (referred to as CpG motif) are relatively abundant in bacterial DNA and are known to stimulate innate immune responses. In this study, synthetic CpG containing oligodeoxydinucleotides (CpG-ODNs) were evaluated for their ability to stimulate nitric oxide (NO), interleukin-1beta (IL-1beta), and interferon-gamma (IFN-gamma) production using an avian macrophage cell line (HD11) and peripheral blood mononuclear cells (PBMC). Results showed ODNs containing the CpG motif can activate the HD11cells and induce NO production. The optimal CpG-ODN motif for NO induction was GTCGTT. Increasing GTCGTT motifs in CpG-ODN significantly enhanced the stimulatory effect. Deviation of flanking bases of the CpG dinucleotide diminished the stimulatory activity. We also found CpG-ODN differentially stimulated expression of cytokine genes. The most active CpG motif for NO induction was also a strong stimulant for the IL-1beta gene expression in the HD11 cells, whereas different CpG motifs were found to induce IFN-gamma gene expression in PBMC.     

Ischemic damage increases nitric oxide production via inducible nitric oxide synthase in the cochlea

The present study was designed to elucidate the dynamic changes of nitric oxide (NO) production in the perilymph and to investigate the immunostaining for inducible nitric oxide synthase (iNOS) in the cochlea for 7 days after transient cochlear ischemia. Moreover, aminoguanidine, which is a selective iNOS inhibitor, was administrated immediately following ischemia and every 24h thereafter for 7 days to investigate whether the production of NO is dependent on the iNOS pathway. Significant increases in the oxidative NO metabolites, nitrite (NO(2)(-)) and nitrate (NO(3)(-)), were measured on day 1 using an in vivo microdialysis and on-line high performance liquid chromatography (HPLC) system. The immunostaining for iNOS was strongly expressed on days 1 and 4 and returned to normal on day 7 after the ischemia. The administration of aminoguanidine reduced the oxidative NO metabolites on day 1 and suppressed the expression of iNOS. These findings suggest that transient ischemia causes a remarkable increase in NO production in the perilymph, which might be attributable to the iNOS pathway.     

Staphylococcal enterotoxin A acts through nitric oxide synthase mechanisms in human peripheral blood mononuclear cells to stimulate synthesis of pyrogenic cytokines

The pyrogenic response to supernatant fluids obtained from human peripheral blood mononuclear cells (PBMC) stimulated with staphylococcal enterotoxin A (SEA) was characteristic of a response to an endogenous pyrogen in that it was brief and monophasic and was destroyed by heating supernatant fluids at 70 degrees C for 30 min. The febrile responses were in parallel with the levels of interleukin-1 (IL-1), tumor necrosis factor (TNF), interferon-gamma (IFN-gamma), IL-2, and IL-6 in supernatant fluids obtained from PBMC treated with SEA. Both the pyrogenicity and the levels of IL-1, TNF, IFN-gamma, IL-2, and IL-6 in supernatant fluids started to rise at 6 to 18 h and reached their peak levels at 24 to 96 h after SEA incubation. Both the fever and the increased levels of IL-1, TNF, IFN-gamma, IL-2, and IL-6 in supernatant fluids obtained from the SEA-stimulated PBMC were decreased by incubating SEA-PBMC with anisomycin (a protein synthesis inhibitor), aminoguanidine (an inhibitor of inducible nitric oxide synthase [NOS]), or dexamethasone (an inhibitor of NOS). The febrile response to supernatant fluids obtained from the SEA-stimulated PBMC was attenuated by adding either anti-IL-1beta, anti-TNF-alpha, or anti-IFN-gamma monoclonal antibody (MAb) to supernatant fluids. The antipyretic effects exerted by anti-IL-1beta MAb were greater than those exerted by anti-TNF-alpha or anti-IFN-gamma MAb. The data suggest that SEA acts through the NOS mechanisms in PBMC to stimulate synthesis of pyrogenic cytokines (in particular, the IL-1beta).     

Induction of nitric oxide in bovine peripheral blood mononuclear cells by bovine herpesvirus 1

Bovine peripheral blood mononuclear cells (PBMCs) and monocytes were stimulated with bovine herpesvirus 1 (BHV-1), LPS and concanavalin A (Con A) to produce L-arginine-dependent nitric oxide (NO) in vitro. NO was detected as early as 12 hrs and up to 72 hrs post stimulation (p.s.). The NO from lipopolysacharide (LPS)-stimulated PBMCs and monocytes was found to exhibit antiviral effect against BHV-1. The anti-BHV-1 effect was inhibited with N(omega)-methyl L-arginine indicating involvement of inducible NO synthase (iNOS) in NO production.     

Regulation of type 2 nitric oxide synthase by type 1 interferons in macrophages infected with Leishmania major

We recently reported that the infection of macrophages with Leishmania major led to the release of type 1 interferons (IFN-alpha /beta ). Moreover, at day 1 of infection of mice with L. major, IFN-alpha /beta was required for the expression of type 2 (inducible) NO synthase (NOS2 or iNOS) which, however, was restricted to a few macrophages in the dermis. Here, we further characterized the regulation of NOS2 by IFN-alpha /beta. Macrophages that were either simultaneously or sequentially exposed to L. major promastigotes and IFN-alpha /beta expressed NOS2 and anti-leishmanial activity. In contrast, when high amounts of IFN-alpha /beta were used or when IFN-alpha /beta was added to the macrophages 2 h prior to the parasites, almost no induction of NOS2 was observed. After pretreatment with IFN-alpha /beta, tyrosine phosphorylation and nuclear DNA binding of Stat1alpha, the degradation of the NF-kappaB inhibitor (IkappaBalpha and beta), and the nuclear translocation of NF-kappaB were strongly impaired compared with macrophages exposed to IFN-alpha /beta and L. major simultaneously. Thus, IFN-alpha /beta exerts agonistic or antagonistic effects on the expression of NOS2 in macrophages infected with a microbial pathogen, depending on the sequence of the stimuli and the amount of IFN-alpha /beta added. The limited number of NOS2-positive macrophages at day 1 of infection in vivo might result from a blockage of non-infected macrophages by IFN-alpha /beta that is released by neighboring infected cells.     

Elevated glucose attenuates agonist- and flow-stimulated endothelial nitric oxide synthase activity in microvascular retinal endothelial cells.

Impaired vasoactive release of opposing vasodilator and vasoconstrictor mediators due to endothelial dysfunction is integral to the pathogenesis of diabetic retinopathy. The aim of this study was to determine the effect of hyperglycemia on the expression of endothelial nitric oxide synthase (eNOS) and the release of nitric oxide (NO) in bovine microvascular retinal endothelial cells (BRECs) under both static (basal and acetylcholine stimulated) and flow (laminar shear stress [10 dynes/cm2 and pulsatile flow 0.3 to 23 dynes/cm2) conditions using a laminar shear apparatus and an in vitro perfused transcapillary culture system. The activity and expression of eNOS, measured by nitrate levels and immunoblot, respectively, were determined following exposure of BRECs to varying concentrations of glucose and mannitol (0 to 25 mM). Under static conditions the expression of eNOS decreased significantly following exposure to increasing concentrations of glucose when compared to osmotic mannitol controls and was accompanied by a significant dose-dependent decrease in nitrate levels in conditioned medium. The acetylcholine stimulated increase in NO release (2.0 +/- 0.3-fold) was significantly reduced by 55% +/- 5% and 65% +/- 4.5% following exposure to 16 and 25 mM glucose, respectively, when compared to osmotic controls. In parallel studies, glucose significantly inhibited both laminar shear stress and pulsatile flow-induced activity when compared to mannitol. We conclude that hyperglycemia impairs agonist- and flow-dependent release of NO in retinal microvascular endothelial cells and may thus contribute to the vascular endothelial dysfunction and impaired autoregulation of diabetic retinopathy.     

Effect of skin sensitizers on inducible nitric oxide synthase expression and nitric oxide production in skin dendritic cells: role of different immunosuppressive drugs

Nitric oxide (NO) is involved in the pathogenesis of acute and chronic inflammatory conditions, namely in allergic contact dermatitis (ACD). However, the mechanism by which NO acts in ACD remains elusive. The present study focuses on the effects of different contact sensitizers (2,4-dinitrofluorbenzene, 1,4-phenylenediamine, nickel sulfate), the inactive analogue of DNFB, 2,4-dichloronitrobenzene, and two irritants (sodium dodecyl sulphate and benzalkonium chloride) on the expression of the inducible isoform of nitric oxide synthase (iNOS) and NO production in skin dendritic cells. It was also studied the role of different immunosuppressive drugs on iNOS expression and NO production. Only nickel sulfate increased the expression of iNOS and NO production being these effects inhibited by dexamathasone. In contrast, cyclosporin A and sirolimus, two other immunosuppressive drugs tested, did not affect iNOS expression triggered by nickel.     

脑挫伤后一氧化氮合酶阳性细胞及一氧化氮含量的变化

目的　探讨脑挫伤后一氧化氮合酶 (NOS)阳性细胞和一氧化氮 (NO)的变化和意义。方法　采用自由落体法致Wistar大鼠顶叶皮质挫裂伤动物模型。伤后 2 4h、72h和 7d取脑 ,制作冰冻切片 ,采用NADPH组织化学染色 ,显示脑挫伤区NOS阳性细胞。用硝酸还原酶法测定血液和脑组织中NO含量。结果　脑挫伤后 72h ,NOS阳性细胞数密度 (Nv)和面密度(Sv)明显增高 (P <0 0 5 ) ,而且 7d时仍无明显下降。血液和脑中NO含量也增高 ,并与NOS细胞呈平行关系。结论　脑挫伤后不同时间NOS细胞数目和NO含量有明显改变 ,提示NOS和NO参与了脑挫伤的病理过程     

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     

Nitric oxide in the human hair follicle: constitutive and dihydrotestosterone-induced nitric oxide synthase expression and NO production in dermal papilla cells

The free radical nitric oxide, generated by different types of epidermal and dermal cells, has been identified as an important mediator in various physiological and pathophysiological processes of the skin, such as regulation of blood flow, melanogenesis, wound healing, and hyperproliferative skin diseases. However, little is known about the role of NO in the human hair follicle and in hair cycling processes. Here we demonstrate for the first time that dermal papilla cells derived from human hair follicles spontaneously produce NO by measuring nitrate and nitrite levels in culture supernatants. This biomolecule is apparently formed by the endothelial isoform of nitric oxide synthase, which was detected at the mRNA and protein levels. Remarkably, basal NO level was enhanced threefold by stimulating dermal papilla cells with 5alpha-dihydrotestosterone (DHT) but not with testosterone. Addition of N-[3-(aminomethyl)benzyl]acetamidine (1400W), a highly selective inhibitor of inducible nitric oxide synthase, restrained the elevation in NO level induced by DHT. Analyses of DHT-stimulated cells at the mRNA and protein levels confirmed the expression of inducible nitric oxide synthase. These findings suggest NO as a signaling molecule in human dermal papilla cells and implicate basal and androgen-mediated NO production to be involved in the regulation of hair follicle activity.