Tumor necrosis factor-alpha and nitric oxide in vertically HIV-1-infected children: implications for pathogenesis

We performed a cross-sectional study to investigate the plasma TNF-alpha and nitric oxide (NO) production in 44 vertically HIV-1-infected children, and the relationship with immunological status and viral replication. As a control group, 36 healthy, uninfected children were studied. Plasma TNF-alpha and NO levels were determined by ELISA. Viral load was quantified using standard assays. Cell proliferation, apoptosis and viral replication were evaluated in vitro by incorporation of (3H)-thymidine, flow cytometry and p24 antigen, respectively. Higher plasma TNF-alpha and NO levels were observed in HIV-1-infected children compared with healthy controls. We found a very strong correlation between plasma TNF-alpha and NO levels in HIV-1-infected children (r = 0.98; p < 0.001). Moreover, HIV-1-infected children with higher viral load (> 4.7 log10) showed higher TNF-alpha and NO levels than those with viral load below this threshold. Interestingly, we detected inducible nitric oxide synthase (iNOS) mRNA in T-lymphocytes from HIV-1-infected children. To address their possible patho-physiological significance, we tested the in vitro effects of NO and TNF-alpha in HIV-1 replication. Addition of TNF-alpha and NO donors to mitogen-activated, HIV-1-infected PBMC cultures produced a significant increase in viral replication. Moreover, HIV-1 replication in mitogen-stimulated, PBMC cultures was partially inhibited by iNOS specific inhibitors, and a neutralising, anti-TNF-alpha monoclonal antibody. Our results indicate that TNF-alpha and NO correlated with high viral load in HIV-1-infected children and favoured HIV-1 in vitro replication. These data suggest a detrimental role of NO in HIV-1 infection, and that NOS inhibitors may have some therapeutic benefit in HIV-1-infection.     

Saccharated colloidal iron enhances lipopolysaccharide-induced nitric oxide production in vivo

We investigated the effects of iron on the production of nitric oxide (NO), inducible NO synthase (iNOS), and plasma cytokines induced by lipopolysaccharide (LPS) in vivo. Male Wistar rats were preloaded with a single intravenous injection of saccharated colloidal iron (Fesin, 70 mg iron/kg body weight) or normal saline as a control, and then given an intraperitoneal injection of LPS (5.0 mg/kg body weight). Rats, preloaded with iron, had evidence of both iron deposition and strong iNOS induction in liver Kupffer cells upon injection of LPS; phagocytic cells in the spleen and lung had similar findings. LPS-induced NO production in iron-preloaded rats was significantly higher than control rats as accessed by NO-hemoglobin levels measured by ESR (electron spin resonance) and NOx (nitrate plus nitrite) levels. Western blot analysis showed that iron preloading significantly enhanced LPS-induced iNOS induction in the liver, but not in the spleen or lung. LPS-induced plasma levels of IL-6, IL-1beta, and TNF-alpha were also significantly higher in iron-preloaded rats as shown by ELISA, but IFN-gamma levels were unchanged. We conclude that colloidal-iron phagocytosed by liver Kupffer cells enhanced LPS-induced NO production in vivo, iNOS induction in the liver, and release of IL-6, IL-1beta, and TNF-alpha.     

Mixed exhaled nitric oxide and plasma nitrites and nitrates in newborn infants.

Plasma nitrite (NO2-) and nitrate (NO3-) are the stable end-products of endogenous nitric oxide (NO) metabolism. NO is present in the exhaled air of humans, but it is not clear if exhaled NO may be an indicator of the systemic endogenous NO production. The aims of the study were to determine the levels of exhaled NO and plasma NO2-/NO3- in healthy term and preterm newborns, and to assess if exhaled NO correlates with plasma NO2-/NO3- at birth. After the stabilization of the newborn, we measured by chemiluminescence the concentration of NO in the mixed expired breath of 133 healthy newborns. Measurement of exhaled NO was repeated after 24 and 48 hours. Plasma NO2-/NO3- levels at birth were measured by the Griess reaction. NO concentrations were 8.9 (CI 8.1-9.8) parts per billion (ppb), 7.7 (CI 7.2-8.3) ppb and 9.0 (CI 8.4-9.6) ppb at birth, 24 and 48 hours, respectively. At birth, exhaled NO was inversely correlated with gestational age (p=0.008) and birth weight (p<0.001). Plasma NO2-/NO3- level was 27.30 (CI 24.26-30.34) micromol/L. There was no correlation between exhaled NO and plasma NO2-/NO3- levels at birth (p=0.88). We speculate that the inverse correlation between exhaled NO and gestational age and birth weight may reflect a role of NO in the postnatal adaptation of pulmonary circulation. At birth, exhaled NO does not correlate with plasma NO2-/NO3- and does not seem to be an index of the systemic endogenous NO production.     

The effect of weight loss on serum concentrations of nitric oxide, TNF-alpha and soluble TNF-alpha receptors

INTRODUCTION: The aims of the present study were to evaluate the effect of weight-loss treatment on serum concentrations of NO and TNF-alpha and to examine whether there is an association between TNF-system activity and serum concentrations of NO after weight loss. MATERIAL AND METHODS: The study group involved 43 obese women (aged 41.8 +/- 11.9 years, weight 95.2 +/- 15.0 kg, BMI 36.5 +/- 4.6 kg/m(2)). The women were subjected to three-month complex weight-loss treatment. Patients were advised to keep to a 1000-1200 kcal diet and to exercise regularly. Pharmacological treatment was not administered. Serum concentrations of nitric oxide metabolites, TNF-alpha and its soluble receptors (sTNFR1, sTNFR2) were measured by ELISA kits; insulin was measured by RIA and glucose, cholesterol, HDL cholesterol and triglicerydes by an enzymatic procedure before and after weight loss. Body composition was determined by impedance analysis using Bodystat. RESULTS: The mean weight loss during treatment was 8.3 +/- 4.3 kg. The serum concentrations of TNF-alpha decreased significantly (p < 0.000) and both receptors sTNFR1 and sTNFR2 increased significantly (p < 0.000) after weight loss. No significant changes in serum concentrations of NO were observed after weight loss. A multiple regression analysis was performed using DeltaTNF-alpha, DeltasTNFR1, DeltaTNFR2 and DeltaNO as dependent variables. A significant correlation was observed between DNO and initial plasma concentrations of TNF-alpha, sTNFR1 and sTNFR2. CONCLUSIONS: This study demonstrates a decrease in serum TNF-alpha concentration as well as an increase in plasma concentration of both TNF receptors but does not show any change in serum concentrations of NO after weight-loss treatment in obese women. It seems that changes in TNF-system activity may be a counter-regulating mechanism, which inhibits further body mass loss. We did not observe any association between changes in TNF-system activity and serum concentrations of NO after weight loss.     

Th1/Th2 cytokine balance and nitric oxide in cerebrospinal fluid and serum from patients with multiple sclerosis

Tumor necrosis factor (TNF-alpha) and IL-10 are key regulators of the T helper (Th)1/Th2 balance, which is critically skewed in many pathological conditions including immune-mediated inflammatory diseases of central nervous system (CNS) such as multiple sclerosis (MS). Nitric oxide (NO) has been reported to have dual effects on CNS pathology, and to play an important role in MS. We performed a cross-sectional study in 17 randomly selected patients during MS flare-up, and compared levels of TNF-alpha, IL-10 and NO in serum and cerebrospinal fluid (CSF) with the serum values of these mediators in two different control groups, healthy subjects and HIV-infected untreated patients. Serum and CSF values of TNF-alpha, IL-10 and NO were higher in MS patients than in the serum of healthy controls. Two MS patients showed increased levels of NO in CSF, with inversion of the NO(SERUM)/NO(CSF) quotient, which is clearly indicative of an intrathecal production of NO. No correlation among the values of both cytokines and NO, and the laboratory parameters analysed in MS patients (IgG index, presence of IgG oligoclonal bands and albumin quotient) was found. The high levels of TNF-alpha and IL-10 (both in serum and CSF) accompanying an MS attack suggest a simultaneous expression of Th1 and Th2 cytokines as opposed to sequential expression of Th1 followed by Th2 as described in the models of experimental autoimmune encephalomyelitis (EAE). Globally, our results support the inherent heterogeneity of the disease.     

Inducible nitric oxide synthase mediates cytokine release: the time course in conscious and septic rats

Nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), and interleukin 1-beta (IL-1beta) are postulated to play a key pathophysiologic role during sepsis. In this study, we examined the time course of inducible NO synthase (iNOS) mRNA expression and the plasma TNF-alpha and IL-1beta in lipopolysaccharide (LPS)-treated conscious rats. The hemodynamic pattern in septic shock is more similar to clinical conditions without anesthesia. The data showed that a significant increase in iNOS mRNA levels was found in the spleen, lung, liver, with slight elevation in the heart and kidney at 3 h after LPS administration. However, iNOS mRNA levels were not elevated significantly in all tissues examined at 24 h. In the plasma, TNF-alpha and IL-1beta culminated within 1 h, and reduced gradually to baseline levels in a relatively short period (within 9 h). The results suggest that local NO production by activation of iNOS mRNA expression and cytokine release may contribute to LPS-induced organ dysfunction at various time points.     

Upregulation of Slc39a10 gene expression in response to thyroid hormones in intestine and kidney

A novel zinc transporter has been purified and cloned from rat renal brush border membrane. This transporter was designated as Zip10 encoded by Slc39a10 gene and characterized as zinc importer. Present study documents the impact of thyroid hormones on the expression of Zip10 encoded by Slc39a10 gene in rat model of hypo and hyperthyroidism. Serum T(3) and T(4) levels were reduced significantly in hypothyroid rats whereas these levels were significantly elevated in hyperthyroid rats as compared to euthyroid rats thereby confirming the validity of the model. Kinetic studies revealed a significant increase in the initial and equilibrium uptake of Zn(++) in both intestinal and renal BBMV of hyperthyroid rats in comparison to hypothyroid and euthyroid rats. By RT-PCR, Slc39a10 mRNA expression was found to be significantly decreased in hypothyroid and increased in hyperthyroid as compared to euthyroid rats. These findings are in conformity with the immunofluorescence studies that revealed markedly higher fluorescence intensity at periphery of both intestinal and renal cells isolated from hyperthyroid rats as compared to hypothyroid and euthyroid rats. Higher expression of Zip10 protein in hyperthyroid group was also confirmed by western blot. These findings suggest that expression of zinc transporter protein Zip10 (Slc39a10) in intestine and kidney is positively regulated by thyroid hormones.     

Tumor necrosis factor-alpha reduces argininosuccinate synthase expression and nitric oxide production in aortic endothelial cells

Endothelial dysfunction associated with elevated serum levels of TNF-alpha observed in diabetes, obesity, and congenital heart disease results, in part, from the impaired production of endothelial nitric oxide (NO). Cellular NO production depends absolutely on the availability of arginine, substrate of endothelial nitric oxide synthase (eNOS). In this report, evidence is provided demonstrating that treatment with TNF-alpha (10 ng/ml) suppresses not only eNOS expression but also the availability of arginine via the coordinate suppression of argininosuccinate synthase (AS) expression in aortic endothelial cells. Western blot and real-time RT-PCR demonstrated a significant and dose-dependent reduction of AS protein and mRNA when treated with TNF-alpha with a corresponding decrease in NO production. Reporter gene analysis demonstrated that TNF-alpha suppresses the AS proximal promoter, and EMSA analysis showed reduced binding to three essential Sp1 elements. Inhibitor studies suggested that the repression of AS expression by TNF-alpha may be mediated, in part, via the NF-kappaB signaling pathway. These findings demonstrate that TNF-alpha coordinately downregulates eNOS and AS expression, resulting in a severely impaired citrulline-NO cycle. The downregulation of AS by TNF-alpha is an added insult to endothelial function because of its important role in NO production and in endothelial viability.     

Synergistic cytokine-induced nitric oxide production in human alveolar epithelial cells.

Nitric oxide (NO) is an important mediator molecule in regulating normal airway function, as well as in the pathophysiology of inflammatory airway diseases. In addition, cytokines are potent messenger molecules at sites of inflammation. The specific relationship among IL-1beta, TNF-alpha, and IFN-gamma on iNOS induction and NO synthesis in human alveolar epithelial cells has not been determined. In addition, rigorous methods to determine potential synergistic action between the cytokines have not been employed. We exposed monolayer cultures of A549 cells to a factorial combination of three cytokines (IL-1beta, TNF-alpha, and IFN-gamma) and three concentrations (0, 5, and 100 ng/mL). TNF-alpha alone does not induce NO production directly; however, it does have a stimulatory effect on IL-1beta-induced NO production. IL-1beta and INF-gamma both induce NO production alone, yet at different concentration thresholds, and act synergistically when present together. In the presence of all three cytokines, the net effect of NO production exceeds the predicted additive effect of each individual cytokine and the two-way interactions. Several plausible mechanisms of synergy among IL-1beta, TNF-alpha, and IFN-gamma in NO production from human alveolar epithelial cells (A549) are proposed. In order to verify the proposed mechanisms of synergy, future experimental and theoretical studies must address several molecular steps through which the iNOS gene is expressed and regulated, as well as the expression and regulation of enzyme cofactors and substrates.     

Increased nitric oxide production following chronic hypoxia contributes to attenuated systemic vasoconstriction

Attenuated vasoconstrictor reactivity following chronic hypoxia (CH) is associated with endothelium-dependent vascular smooth muscle (VSM) cell hyperpolarization and diminished intracellular [Ca(2+)]. We tested the hypothesis that increased production of nitric oxide (NO) after CH contributes to blunted vasoconstrictor responsiveness. We found that basal NO production of mesenteric arteries from CH rats (barometric pressure = 380 Torr; 48 h) was greater than that of controls (barometric pressure = 630 Torr). In addition, studies employing pressurized mesenteric arteries (100-200 microM ID) abluminally loaded with the Ca(2+) indicator fura 2-AM demonstrated that although NO synthase (NOS) inhibition normalized agonist-induced vasoconstrictor responses between groups, VSM cell [Ca(2+)] in vessels from CH rats remained diminished compared with controls. To determine whether elevated NO production following CH results from increased NOS protein levels, we performed Western blots for NOS isoforms by using mesenteric arteries from control and CH rats. Endothelial NOS levels did not differ between groups, and other NOS isoforms were not detected in these samples. Selective endothelial loading of fura 2-AM was employed to test the hypothesis that elevated endothelial cell [Ca(2+)] following CH accounts for enhanced NOS activity. These experiments demonstrated greater endothelial cell [Ca(2+)] in mesenteric arteries isolated from CH rats compared with controls. We conclude that enhanced production of NO resulting from elevated endothelial cell [Ca(2+)] contributes to attenuated reactivity following CH by decreasing VSM cell Ca(2+) sensitivity.     

Cytotoxicity of IFN-gamma and TNF-alpha for vascular endothelial cell is mediated by nitric oxide

Endothelial cell injury is a critical event in tissue damage accompanying inflammation, in which both inflammatory cytokines and reactive oxygen species may play pivotal roles, although the exact mechanism has not yet been clarified. We found that combined stimulation with interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) induced both cytotoxicity to murine vascular endothelial cell line F-2 and an increase in nitric oxide (NO). Therefore, in the present study, the implication of NO in cytotoxicity was examined. A potent iNOS-specific inhibitor ONO-1714 completely blocked both cytokine-induced cytotoxicity and NO production. NO scavengers such as carboxy-PTIO and hemoglobin blocked cytotoxicity. Moreover, exogenous NO from NOC 18 also caused cytotoxicity. These results together demonstrated that cytotoxicity of IFN-gamma and TNF-alpha for endothelial cell F-2 was mediated by NO, suggesting a pathogenic role of cytokine-induced NO production in endothelial damage under inflammatory conditions.     

Viral hemorrhagic septicemia virus alters turbot Scophthalmus maximus macrophage nitric oxide production.

The effect of viral hemorrhagic septicemia virus (VHSV) in vitro infection on the nitric oxide (NO) production by turbot Scophthalmus maximus kidney macrophages has been addressed in the past. Previously, we had determined that only a small fraction of turbot possess head kidney macrophages that respond to a single exposure of lipopolysaccharide (LPS) with NO production (LPS-responsive macrophages), whereas macrophage cultures from other individuals were not activated by LPS alone and needed a combination of stimuli to respond (LPS-non-responsive macrophages). In the current work, we examined the effect of VHSV on NO production by macrophages characterized as LPS-responsive macrophages or LPS-non-responsive macrophages. Combinations of LPS and tumor necrosis factor alpha (TNF-alpha) and macrophage-activating factor (MAF) were also used to stimulate the cells for NO production. The effect of VHSV on NO production depends on the response to LPS alone. When a low multiplicity of infection was used (1.78 x 10(-3)), the NO production in response to LPS in LPS-responsive macrophages was significantly decreased. However, LPS-non-responsive macrophage cultures produced NO when a combination of LPS and VHSV was used. In the case of a higher VHSV multiplicity of infection (1.78), no significant change was observed in LPS-non-responsive animals. Combinations of LPS with TNF-alpha, LPS with MAF, and TNF-alpha with MAF were used to induce NO production in LPS-non-responsive macrophages. In all these cases, VHSV suppressed NO production, although at a significant level only when a combination of TNF-alpha and MAF was used for the induction of NO.     

Incidence of congenital hypothyroidism: retrospective study of neonatal laboratory screening versus clinical symptoms as indicators leading to diagnosis.

Filter paper blood samples taken routinely from 100 239 newborn infants were radioimmunoassayed five years later for plasma thyrotrophin concentrations. In 32 cases (0.03%) these were found to be raised. Thirty one of these children were traced and subjected to follow up examination by a paediatrician and a psychologist. Of the 31 children, 15 were found to have been receiving treatment for congenital hypothyroidism since a median of 5 months of age (diagnosed group). Of the 16 others, seven children were found to have raised serum thyrotrophin concentrations and were classified as hypothyroid (undiagnosed group). The remaining nine children were euthyroid. Children in the diagnosed group had a mean Griffiths developmental quotient of 87 (control value 103; p less than 0.01), and five out of 13 showed impaired neurological development. Of the remainder, those in the undiagnosed group had a mean developmental quotient of 100 and those in the euthyroid group a mean developmental quotient of 107. In this study achieving a detection rate of congenital hypothyroidism of one in 3000 in a neonatal screening programme resulted in overdiagnosis of about a quarter of patients considered to have true positive findings. This was outweighed, however, by the early identification of all infants with the disease.     

Nitric oxide production in newborns under phototherapy.

Nitrogen monoxide (NO) is a potent endogenous vasodilator and is involved in cytotoxicity, neurotransmission, and immunological defense mechanisms. Phototherapy has long been known to change the distribution of blood flow throughout the body in newborn infants. The objective of this study was to investigate the effect of phototherapy on NO production in otherwise healthy newborns. Urinary NO levels were measured before and 6 h after phototherapy by a chemiluminescence method using Sievers NOA. Ten newborns (gestational age, 36.4 +/- 3.9 weeks; birth weight, 2863 +/- 677.44 g; postnatal age, 5.1 +/- 2.72 days) were started on phototherapy according to AAP guidelines and urine for NO measurement was collected prior to therapy and 6 h after the commencement of treatment. Urinary NO levels measured during phototherapy were significantly higher (108.8+/-50.69 micromol/mmol creatinine) than the levels measured before phototherapy (73.13+/-34.15 micromol/mmol creatinine; P < 0.05). These results suggest that newborns receiving phototherapy might have increased NO production, which might result in hemodynamic changes. However, further studies on the effects of phototherapy on NO and photorelaxation are needed before reaching firm conclusions.     

Time course of nitric oxide production after endotoxin challenge in mice

Nitric oxide (NO) regulates numerous processes during endotoxemia and inflammation. However, the sequential changes in whole body (Wb) nitric oxide (NO) production during endotoxemia in vivo remain to be clarified. Male Swiss mice were injected intraperitoneally with saline (control group) or lipopolysaccharide (LPS group). After 0, 2, 4, 6, 9, 12, and 24 h, animals received a primed constant infusion of L-[guanidino-(15)N(2)-(2)H(2)]arginine, L-[ureido-(15)N]citrulline, L-[5-(15)N]glutamine, and L-[ring-(2)H(5)]phenylalanine in the jugular vein. Arterial blood was collected for plasma arginine (Arg), citrulline (Cit), glutamine (Gln), and phenylalanine (Phe) concentrations and tracer-to-tracee ratios. NO production was calculated as plasma Arg-to-Cit flux, Wb de novo Arg synthesis as plasma Cit-to-Arg flux, and Wb protein breakdown as plasma Phe flux. LPS reduced plasma Arg and Cit and increased Gln and Phe concentrations. Two peaks of NO production were observed at 4 and 12 h after LPS. Although LPS did not affect total Arg production, de novo Arg production decreased after 12 h. The second peak of NO production coincided with increased Wb Cit, Gln, and Phe production. In conclusion, the curve of NO production in both early and late phases of endotoxemia is not related to plasma Arg kinetics. However, because Wb Cit, Gln, and Phe fluxes increased concomitantly with the second peak of NO production, NO production is probably related to the catabolic phase of endotoxemia.     

N-acetylcysteine inhibits in vivo nitric oxide production by inducible nitric oxide synthase.

This in vivo study evaluates the effect of N-acetylcysteine (NAC) administration on nitric oxide (NO) production by the inducible form of nitric oxide synthase (iNOS). NO production was induced in the rat by the ip administration of 2 mg/100 g lipopolysaccharide (LPS). This treatment caused: (1) a decrease in body temperature within 90 min, followed by a slow return to normal levels; (2) an increase in plasma levels of urea, nitrite/nitrate, and citrulline; (3) the appearance in blood of nitrosyl-hemoglobin (NO-Hb) and in liver of dinitrosyl-iron-dithiolate complexes (DNIC); and (4) increased expression of iNOS mRNA in peripheral blood mononuclear cells (PBMC). Rat treatment with 15 mg/100 g NAC ip, 30 min before LPS, resulted in a significant decrease in blood NO-Hb levels, plasma nitrite/nitrate and citrulline concentrations, and liver DNIC complexes. PBMC also showed a decreased expression of iNOS mRNA. NAC pretreatment did not modify the increased levels of plasma urea or the hypothermic effect induced by the endotoxin. The administration of NAC following LPS intoxication (15 min prior to sacrifice) did not affect NO-Hb levels. These results demonstrate that NAC administration can modulate the massive NO production induced by LPS. This can be attributed mostly to the inhibitory effect of NAC on one of the events leading to iNOS protein expression. This hypothesis is also supported by the lack of effect of late NAC administration.     

Augmented nitric oxide production and up-regulation of endothelial nitric oxide synthase during cecal ligation and perforation

Nitric oxide (NO) has been pointed out as being the main mediator involved in the hypotension and tissue injury taking place during sepsis. This study aimed to investigate the cellular mechanisms implicated in the acetylcholine (ACh)-induced relaxation detected in aortic rings isolated from rats submitted to cecal ligation and perforation (CLP group), 6h post-CLP. The mean arterial pressure was recorded, and the concentration-effect curves for ACh were constructed for endothelium-intact aortic rings in the absence (control) or after incubation with one of the following NO synthase inhibitors: L-NAME (non-selective), L-NNA (more selective for eNOS), 7-nitroindazole (more selective for nNOS), or 1400W (selective for iNOS). The NO concentration was determined by using confocal microscopy. The protein expression of the NOS isoforms was quantified by Western blot analysis. The prostacyclin concentration was indirectly analyzed on the basis of 6-keto-prostaglandin F(1α) (6-keto-PGF(1α)) levels measured by enzyme immunoassay. There were no differences between Sham- and CLP-operated rats in terms of the relaxation induced by acetylcholine. However, the NOS inhibitors reduced this relaxation in both groups, but this effect remained more pronounced in the CLP group as compared to the Sham group. The acetylcholine-induced NO production was higher in the rat aortic endothelial cells of the CLP group than in those of the Sham group. eNOS protein expression was larger in the CLP group, but the iNOS protein was not verified in any of the groups. The basal 6-keto-PGF(1α) levels were higher in the CLP group, but the acetylcholine-stimulated levels did not increase in CLP as much as they did in the Sham group. Taken together, our results show that the augmented NO production in sepsis syndrome elicited by cecal ligation and perforation is due to eNOS up-regulation and not to iNOS.     

Lipoteichoic acid-induced nitric oxide production depends on the activation of platelet-activating factor receptor and Jak2

NO production by macrophages in response to lipoteichoic acid (LTA) and a synthetic lipopeptide (Pam3CSK4) was investigated. LTA and Pam3CSK4 induced the production of both TNF-alpha and NO. Inhibitors of platelet-activating factor receptor (PAFR) blocked LTA- or Pam3CSK4-induced production of NO but not TNF-alpha. Jak2 tyrosine kinase inhibition blocked LTA-induced production of NO but not TNF-alpha. PAFR inhibition blocked phosphorylation of Jak2 and STAT1, a key factor for expressing inducible NO synthase. In addition, LTA did not induce IFN-beta expression, and p38 mitogen-activated protein serine kinase was necessary for LTA-induced NO production but not for TNF-alpha production. These findings suggest that Gram-positive bacteria induce NO production using a PAFR signaling pathway to activate STAT1 via Jak2. This PAFR/Jak2/STAT1 signaling pathway resembles the IFN-beta, type I IFNR/Jak/STAT1 pathway described for LPS. Consequently, Gram-positive and Gram-negative bacteria appear to have different but analogous mechanisms for NO production.