Induction Of iNOS mRNA Research Articles

Time course of expression of mRNA of inducible nitric oxide synthase and generation of nitric oxide by ultraviolet B in keratinocyte cell lines.

Nitric oxide (NO), the ubiquitous free radical, has been implicated in the pathogenesis of various inflammatory diseases, including sunburn and ultraviolet (UV) radiation-induced pigmentation, and it also seems to play an important part in host defence against bacterial infection. The purpose of this study was to determine the time course of production of NO and time course of expression of inducible NO synthase (iNOS) by UVB irradiation and lipopolysaccharide (LPS) stimulation in keratinocyte cell lines. Furthermore, we intended to elucidate the relationship between iNOS and NO in various stimulated conditions. Normal human keratinocytes (NHK), HaCaT cells and PAM212 cells were irradiated with UVB at a dose of 50 mJ cm-2 and 100 mJ cm-2. Separately, the cell lines were stimulated with 20 micro g of LPS. NO was measured by the Griess assay and iNOS mRNA was isolated by reverse transcriptase-polymerase chain reaction at 12, 24, 48 and 72 h after stimulation. The generation of NO was induced by UVB irradiation and LPS stimulation. NO production was significantly increased at 72 h after irradiation of UVB 100 mJ cm-2 in NHK, and at 48 and 72 h in HaCaT cells. In PAM212 cells, NO production was significantly increased at 12, 24, 48 and 72 h by UVB 100 mJ cm-2 and at 72 h by LPS. Induction of iNOS mRNA peaked at 48 h and then decreased to basal level at 72 h when treated with UVB irradiation. The time course of production of NO was approximately correlated with the timing of induction of iNOS mRNA. These results suggest that the expression of iNOS mRNA is upregulated by UVB irradiation, and that NO produced by this inducible enzyme may play a part as a mediator or an immunomodulator in UV-induced skin reactions such as sunburn reaction and photo-induced immune alterations.

Expression of inducible nitric oxide synthase (iNOS) mRNA in target organs of lethal and non-lethal strains of murine malaria.

Nitric oxide (NO) is a putative mediator of the immunological and/or pathological responses to malaria, consequently it is a potential target for novel drug therapy. Numerous cell types increase expression of inducible nitric oxide synthase (iNOS) under inflammatory conditions, the most relevant stimuli being cytokines and endotoxins. In this study the expression of iNOS mRNA in several target organs (brain, liver, spleen) of malaria have been investigated in MF1 mice during lethal Plasmodium (P.) berghei and non-lethal P. c. chabaudi infection. In P. berghei malaria, iNOS mRNA decreased in liver and was unchanged in spleen during the period of rising parasitaemia, but increased in both organs late in the infection, when parasitaemia was high and death imminent. In mice infected with P. c. chabaudi, spleen iNOS mRNA increased progressively throughout the early, peak and recovery periods of parasitaemia, but decreased in liver. Brain iNOS mRNA decreased in samples collected throughout the time courses of both infections. Hence it is evident that changes in iNOS mRNA in murine malaria depend upon the tissue, day of infection, degree of parasitaemia and strain of Plasmodium. These data indicate induction of iNOS mRNA in the spleen has a role in combating these strains of Plasmodium in MF1 mice. Failure to clear lethal P. berghei parasitaemia was associated with increased iNOS mRNA expression in the liver, which may contribute to the pathology of this malaria.

Lovastatin induced cytokine‐mediated inducible nitric oxide synthase (INOS) expression in brain transformed cell lines

Nitric oxide (NO) produced by inducible nitric‐oxide synthase (iNOS) in different cells including brain cells in response to proinflammatory cytokines plays an important role in the pathophysiology of neurodegenerative diseases. The present study underlines the involvement of RhoA protein in the expression of iNOS via NF‐κB in transformed brain cell lines [rat C6 glioma, human astrocytoma (T98G and A172) and immortal rat astrocytes]. Treatment of C6 glioma cells with lovastatin resulted in the induction of iNOS mRNA and increased NO production in a dose and time dependent manner. Addition of mevalonate and geranylgeranylpyrophosphate reversed the lovastatin‐mediated effect. A selective inhibitor of geranylgeranyltransferase inhibitor (GGTI 298) further enhanced the lovastatin as well as cytokine‐mediated iNOS expression suggesting the involvement of geranylgeranylated proteins in the down regulation of iNOS expression. Inactivators of Rho A family (bacterial toxin B), Rho A protein (C3 toxin from C. botulinum) and Rho A associated kinases (Y‐27632) increased the iNOS expression. Lovastatin induced NO production mediated by increasing IkB? degradation and NF‐κB translocation via inactivation of Rho A protein. Co‐transfection studies using dominant negative form of RhoA along with iNOS luciferase and NF‐κB luciferase reporter constructs further supported these observations. Taken together, these studies document a role for Rho A in lovastatin mediated induction of iNOS expression in brain glial cell lines.Acknowledgements: Supported by NIH grants NS‐22576, NS‐34741 and NS‐37766.

Induction of nitric oxide synthase in cytokine‐stimulated human astrocytes by interleukin‐12 P40 monomer

Excessive production of IL‐12 participates in the neuroinflammatory disease process of Multiple Sclerosis (MS). IL‐12 is composed of two different subunits – p40 and p35. However, it has been found that p40 but not that p35 is expressed in excessive amount in the CNS of MS patients. However, the biological significance of this overexpression of p40 in the CNS of patients with MS is not known. The present study underlines the importance of human IL‐12 (p70, the heterodimeric form) and p40 monomer in inducing the expression of iNOS in IFN‐g‐stimulated human astrocytes. IL‐12, p40 or IFN‐γ alone was poor inducer of NO production in human U373MG astrocytoma cells. However, the combination of either IL‐12 and IFN‐γ or p40 and IFN‐γ but not that of the IL‐12 and p40 markedly induced the production of NO. This induction of NO production was accompanied by an induction of iNOS protein and mRNA. Similar to U373MG astrocytoma cells, IL‐12 or p40 also induced the production of NO in human primary astrocytes. Although IFN‐γ alone was a weak inducer of NF‐κB activation, it efficiently induced the activation of CCAAT/enhancer‐binding protein (C/EBP)b and g‐activation site (GAS). On the other hand, p40 alone induced the activation of NF‐κB and C/EBPb but not that of GAS suggesting that p40 alone cannot induce the expression of iNOS probably due to its inability to induce the activation of GAS. This study delineates a novel role of IL‐12 p70 or IL‐12 p40 monomer in inducing the expression of iNOS in human astrocytes which may participate in the pathogenesis of neuroinflammatory diseases.Acknowledgements: This study was supported by NIH grants (NS39940 and AG19487).

Pharmacological preconditioning with resveratrol: role of nitric oxide.

Resveratrol (trans-3,4',5-trihydroxystilbene), a recently described grape-derived polyphenolic antioxidant, has been found to protect the heart from ischemic-reperfusion injury. The present study sought to determine the mechanism of cardioprotection by investigating the ability of resveratrol to precondition the heart. Isolated perfused rat hearts were randomly divided into six groups: group I was perfused for 15 min with Kreb-Henseleit buffer (KHB) only; group II was perfused with 10 microM resveratrol; group III was perfused with 10 microM resveratrol plus 100 microM N(G)-nitro-L-arginine methyl ester (L-NAME), a nonselective nitric oxide (NO) synthase (NOS) inhibitor; group IV was perfused with 10 microM resveratrol plus 100 microM aminoguanidine (AG), an inducible NOS (iNOS) blocker; and groups V and VI consisted of hearts perfused with L-NAME and AG, respectively. The perfusion was then switched to working mode, and all hearts were made globally ischemic for 30 min followed by 2 h of reperfusion. Preconditioning of the hearts with resveratrol provided cardioprotection as evidenced by improved postischemic ventricular functional recovery (developed pressure and aortic flow) and reduced myocardial infarct size and cardiomyocyte apoptosis. Resveratrol-mediated cardioprotection was completely abolished by both L-NAME and AG. In a separate study, hearts were examined for iNOS mRNA induction. Resveratrol caused an induction of the expression of iNOS mRNA beginning at 30 min after reperfusion, increasing steadily up to 60 min of reperfusion, and then decreasing progressively up to 2 h after reperfusion. Preperfusion of the hearts with AG almost completely blocked the induction of iNOS. The results of our study demonstrate that resveratrol can pharmacologically precondition the heart in a NO-dependent manner.

Induction of hepatic inducible nitric oxide synthase by cholesterol in vivo and in vitro.

Cholesterol-rich diet impairs endothelial NO synthase (eNOS) and enhances inducible NOS (iNOS) expression. In this study, we investigated effects of cholesterol on iNOS expression in high-fat-fed rat models, HepG2 and RAW264.7 cells. The high-fat diet increased the plasma total cholesterol level 6-7 fold and low-density lipoprotein cholesterol level (LDL-C) approximately 70 fold and slightly increased the level of lipid peroxidation as determined by thiobarbituric acid-reactive substance assay. The high-fat diet also increased plasma nitric oxide (NO) concentrations up to 5 fold, and induced iNOS mRNA expression in liver. The contractile responses of the endothelium-denuded thoracic aortic rings to phenylephrine were significantly damaged in high-fat-fed rats when assessed by organ chamber study. Treatment with estrogen for 4 days failed to reduce iNOS expressions as well as aortic contractility, although it improved lipid profiles. In cultured HepG2 or murine macrophage RAW264.7 cells, 3 days treatment with either 25-hydroxycholesterol or 7-ketocholesterol induced iNOS mRNA expression, as determined by RT-PCR. Our data suggested that the chronic exposure of hepatocytes and macrophage cells to high concentration of cholesterol or oxysterols may induce iNOS expression and subsequent synthesis of NO, which may be important in the pathogenesis of atherosclerosis.

Host systemic and local nitric oxide levels do not correlate with rejection of pig proislet xenografts in mice.

The rejection of pig proislet xenografts in mice is a CD4 T cell-dependent process in which macrophages play an important role. To assess the potential for activated macrophages to act as effector cells in xenograft destruction, we have examined the relationship between proislet xenograft rejection, two principal markers of macrophage activation, transcription of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO), and their temporal relationship to intragraft cytokine gene expression. Xenograft rejection in CBA/H mice correlated with early induction of intragraft host iNOS mRNA and marked intragraft production of NO (reactive nitrogen intermediates, RNI). Intragraft mRNA expression for IFN-gamma, IL-1beta and TNF, cytokines associated with macrophage activation, was also found. These findings suggested that activated macrophages could be contributing to xenograft destruction via local NO-mediated toxicity at the graft site. To test the role of NO in this model: (1) Q-fever antigen (QFA) was administered to recipient mice in order to induce high systemic RNI levels and (2) in another experiment, pig proislets were transplanted into iNOS-/- mice. Treatment with QFA correlated with prolonged xenograft survival at 7 days post-transplant. Splenocytes from QFA-treated, but not control mice at 7 and 22 days post-transplant, exhibited inhibition of secondary xenogeneic mouse antiporcine mixed lymphocyte reaction (MLR) that was reversed by culture with the NOS inhibitor N-methylarginine (NMA). Despite continued elevated NO production, xenograft protection was temporary with complete rejection by day 22. Evidence that locally produced NO was not contributing to rejection was seen when pig proislets transplanted into iNOS-/- mice were rejected with normal kinetics; in these animals intragraft NO production was not detected (despite porcine iNOS gene expression). Failure of activated macrophages to achieve indefinite xenograft survival suggests that other factors are also required. Macrophage potential to effect either destructive or protective roles after pig proislet xenotransplantation suggests that such functions may depend on the site and magnitude of macrophage activation. Together these findings clearly demonstrate that high NO levels in the periphery are not damaging to xenogeneic islet tissue, neither host nor donor NO production is essential for islet xenograft rejection and consequently elevated plasma RNI levels do not represent a direct marker for rejection.

P38 mitogen-activated protein kinase mediates lipopolysaccharide, not interferon-γ, -induced inducible nitric oxide synthase expression in mouse BV2 microglial cells

The present study examined the role of mitogen-activated protein kinases (MAPKs) in inducible nitric oxide synthase (iNOS) induction by lipopolysaccharide (LPS) or interferon-γ (IFN-γ) in the murine microglial cell line BV2 cells. LPS rapidly (<2 h) induced iNOS mRNA expression whereas IFN-γ did not within 8 h after stimulation. LPS activated three MAPK subtypes, extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) as early as 15 min after stimulation. In contrast, IFN-γ activated only ERK after 6 h of stimulation and did not alter the activity of p38 and JNK. LPS-induced iNOS expression was markedly decreased by the p38 inhibitor SB203580, but not by the MAPK or ERK kinase inhibitor PD98059. IFN-γ-induced nitric oxide (NO) generation was partially inhibited by PD98059 and SB203580 only in combination. The results demonstrate that MAPKs differentially mediate NO production by LPS- and IFN-γ in BV2 cells.

Induction of secretory and tumoricidal activities in peritoneal macrophages by ginsan

The immunomodulatory effect of ginsan based on the production of cytokines and the activation of macrophage was studied. Murine peritoneal macrophages (PM) on in vitro treatment with ginsan isolated from Panax ginseng induced mRNA of cytokines such as tumor necrosis factor (TNF)-α, interleukin-1 (IL-1)β, interleukin-6 (IL-6) and interleukin-12 (IL-12); TNF-α mRNA induction was maximum within 3 h, IL-6 mRNA was gradually induced up to 24 h, and IL-1β and IL-12 mRNA were highly induced at 24 h. IL-1β and IL-6 protein levels also increased within 24 h in a dose-dependent manner and reached a maximum with 100 μg/ml ginsan. IL-12 was induced after 3 days and a high level of induction was detected after 4 days post treatment. Ginsan enhanced the lytic death of L929 cells through TNF-α activation. The mRNA expression of nitric oxide synthase (iNOS) was highly induced after 24 h treatment of ginsan, and then NO production was maximum after 48-h treatment with a low dose of 1 μg/ml. The level of iNOS mRNA induction by ginsan was slightly less than that of macrophages activating agents such as LPS plus IFN-γ. The tumoricidal activity of macrophage cultured with ginsan on Yac-1 cells was enhanced in a dose-dependent manner; growth inhibition increased 1.6-fold with 100 μg/ml ginsan. These results suggest that ginsan exerts as an effective immunomodulator and enhances antitumor activity of macrophages.

Effects of sanguiin H-6, a component of Sanguisorbae Radix, on lipopolysaccharide-stimulated nitric oxide production

The present study was conducted to evaluate the effect of sanguiin H-6, a component of Sanguisorbae Radix, on the production of nitric oxide (NO), using macrophages activated by lipopolysaccharide (LPS). Sanguiin H-6 inhibited nitrite production, taken as an index for NO, in a concentration-dependent fashion. This compound decreased inducible NO synthase (iNOS) activity, with the inhibitory effect at a concentration of 25 μM being equal to that of the known iNOS inhibitor aminoguanidine at 50 μM. However, unlike aminoguanidine, sanguiin H-6 was associated with improved cell viability. Reverse transcription-polymerase chain reaction analysis revealed that the expression of iNOS mRNA in activated macrophages was suppressed by sanguiin H-6 in a concentration-dependent manner. In addition, sanguiin H-6 even at a low concentration showed a clear scavenging effect on the NO generated from sodium nitroprusside (an NO donor). These findings indicate that not only does sanguiin H-6 act directly as an NO scavenger, but it also inhibits NO production in LPS-activated macrophages by the concomitant inhibition of iNOS mRNA induction and enzyme activity.

Expression and regulation of inducible nitric oxide synthase from human primary airway epithelial cells.

Elevated levels of exhaled nitric oxide are seen in inflammatory airway diseases such as asthma, but the cellular source remains unknown. This study investigated whether human airway epithelial cells express inducible nitric oxide synthase (iNOS). Human bronchial epithelial cells stimulated with 50 ng/ml interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma express iNOS mRNA, protein and increased nitrite in the cell culture media, which was inhibited by the selective iNOS inhibitor 1400W. Cells derived from subjects with asthma produced less nitrite than cells from normal subjects (6.59 +/- 0.99 microM nitrite, n = 15 versus 3.89 +/- 0.42 microM nitrite, n = 20; P < 0.05). This was not attributed to steroid treatment of subjects with asthma because there was no difference in the amount of nitrite released from steroid-naive and steroid-treated cells (3.51 +/- 0.46 versus 4.27 +/- 0.7 microM nitrite, n = 10). Neither dexamethasone nor budesonide inhibited iNOS mRNA induction, protein expression, or nitrite accumulation. The cells were not steroid insensitive because steroids inhibited GM-CSF release. Therefore, although these cells express iNOS under inflammatory conditions, they do not appear to be regulated directly by glucocorticosteroids.

Cytokine responses to group B streptococci induce nitric oxide production in respiratory epithelial cells.

Streptococcus agalactiae (group B streptococcus [GBS]) is a leading cause of neonatal pneumonia, sepsis, and meningitis. Early-onset GBS pneumonia is characterized by marked pulmonary epithelial and endothelial cell injury. Innate proinflammatory responses to GBS infection that may contribute to the respiratory pathology include the synthesis and release of cytokines, prostaglandins, and nitric oxide (NO). The hypothesis that NO is directly induced in lung epithelial cells by invading GBS or indirectly induced by cytokines released by GBS-infected mononuclear cells was tested. A549 transformed human respiratory epithelial cells were directly cultured with GBS, cocultured with GBS-infected human mononuclear cells or purified macrophages, or exposed to conditioned culture medium from human mononuclear cells infected by GBS. The culture medium of A549 cultures was assayed for NO secretion, and the cell lysates were tested for presence of inducible nitric oxide synthase (iNOS) mRNA by reverse transcriptase PCR (RT-PCR). GBS-treated A549 cells neither secreted detectable NO nor expressed iNOS mRNA. GBS interaction with human mononuclear cells, however, stimulated release of soluble factors that readily induced iNOS mRNA expression and NO secretion by A549 cells. Inflammatory mediator-induced nitric oxide (NO) production by alveolar epithelium may exceed that of other lung cell types such as macrophages, and induction during GBS infection may play a significant role in pulmonary defense or free-radical-mediated lung injury.

Brain expression of inducible cyclooxygenase 2 messenger RNA in rats undergoing cardiopulmonary bypass.

We hypothesized that systemic proinflammatory cytokines or endotoxemia, or both, associated with cardiopulmonary bypass (CPB) would increase expression of inducible cyclooxygenase (COX-2) or inducible nitric oxide synthase (iNOS) messenger RNA (mRNA), or both, in brain. Isoflurane-anesthetized Sprague-Dawley rats were randomly selected for CPB (n = 6) or sham surgery (n = 6). All animals underwent tracheotomy and controlled ventilation, arterial and venous pressure monitoring, insertion of a jugular venous outflow catheter, insertion of a subclavian arterial inflow catheter, systemic anticoagulation (500 U/kg heparin) and, except during CPB, servoregulation of pericranial temperature at 37.5 degrees C. Animals selected for CPB underwent 1 h of CPB at 165 ml x kg(-1) x min(-1) (31.8 +/- 0.2 degrees C), whereas animals having sham surgery underwent no intervention during this interval. Thereafter, all animals were given protamine and remained anesthetized for 4 more h. Brain and liver COX-2 and iNOS mRNA expression were determined by a ribonuclease protection assay with ribosomal L32 mRNA as a loading control. Arterial blood was analyzed for interleukin 1beta, interleukin 6, and endotoxin concentrations. Endotoxin concentrations did not increase above baseline values in either group. At 4 h after the CPB interval, interleukin 6 concentrations were significantly greater in CPB animals (101 +/- 45 pg/ml) versus sham animals (44 +/- 17 pg/ml) (P = 0.025). Brain COX-2 expression was significantly greater in CPB animals (0.36 +/- 0.11) versus shams (0.19 +/- 0.08) (P = 0.013). Brain COX-2 expression correlated with interleukin 6 concentration 4 h after CPB (r = 0.91; P = 5 x 10(-5)). In brain, iNOS mRNA was not detected in any animal. Cardiopulmonary bypass animals had only trace COX-2 and iNOS mRNA induction in liver. Cardiopulmonary bypass was associated with increased systemic interleukin 6 concentrations and increased brain COX-2 expression.

Interleukin-17 stimulates inducible nitric oxide synthase activation in rodent astrocytes

The effect of interleukin-17 (IL-17) on production of nitric oxide (NO) in rodent astrocytes was investigated. While IL-17 by itself did not induce NO production, it caused a dose-dependent enhancement of IFN-γ-triggered NO synthesis in both mouse and rat primary astrocytes. In contrast, IL-17 was unable to stimulate NO synthesis in either murine or rat macrophages. IFN-γ-triggered expression of mRNA for iNOS, but not for its transcription factor interferon regulatory factor-1 (IRF-1), was markedly elevated in IL-17-treated astrocytes. The induction of iNOS mRNA by IL-17 in IFN-γ-pretreated astrocytes was abolished by antagonists of nuclear factor-κB (NF-κB) activation—a proteasome inhibitor MG132 and an antioxidant agent PDTC, as well as with specific p38 MAP kinase inhibitor SB203580. While IL-17 stimulated both IL-1β and IL-6 production in astrocytes, only IL-1 was partly responsible for IL-17-induced NO release. Finally, IL-17 synergized with exogenous IL-1β and TNF-α for astrocyte NO production. Having in mind a well-known neurotoxic action of NO, these results suggest a possible role for IL-17 in the inflammatory diseases of the CNS.

Beneficial effect of HCL-31D in murine models of endotoxaemia.

We evaluated the effect of HCL-31D, a novel cAMP-specific phosphodiesterase inhibitor, on the induction of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma)-treated rat aortic smooth muscle cells (RASMC) and on survival in a murine model of severe endotoxaemia. Treatment of cultured RASMC with LPS and IFN-gamma resulted in an increase of nitrite, tumour necrosis factor (TNF-alpha) production and induction of iNOS mRNA. However, incubation with HCL-31D (1 approximately 50 microM) for 24 h caused significant attenuation of nitrite and TNF-alpha formation as well as iNOS mRNA induction in a dose-dependent manner but no effect on iNOS activity in RASMC. In addition, administration of HCL-31D (5 mg/kg, i.p.) resulted in that the increase of both plasma nitrate and TNF-alpha levels induced by LPS in vivo was significantly reduced in LPS-treated rats. Treatment of conscious mice with a high dose of LPS (60 mg/kg, i.p.) to ICR mice resulted in a 24-h survival rate of only 10%. However, administration of HCL-31D (5 mg/kg, i.p. at 0 h and 6 h after LPS) improved the 24-h survival to 50%, indicating that HCL-31D has a beneficial effect in murine model endotoxaemia. These effects may be mainly due to inhibition of TNF-alpha formation and of the induction of iNOS. We proposed that the elevation of cAMP levels by HCL-31D may be involved in the prevention of TNF-alpha formation and iNOS induction.

Dexamethasone suppresses iNOS gene expression by inhibiting NF-κB in vascular smooth muscle cells

Bacterial lipopolysaccharide (LPS) and other immunostimulants induce an isoform of nitric oxide synthase (iNOS) gene expression in vascular smooth muscle cells (VSMC). This process is dependent on nuclear factor-kappa B (NF-κB) activation and is suppressed by glucocorticoids. The aim of this study was to investigate the molecular mechanisms of inhibition of iNOS expression by the synthetic glucocorticoid, dexamethasone (DEX), in rat VSMC. Treatment of VSMC with LPS plus interferon-γ (LPS/IFN) caused activation of NF-κB and the iNOS promoter. LPS/IFN induced iNOS mRNA and NO synthesis. DEX markedly depressed LPS/IFN-stimulated iNOS mRNA expression and NO production. DEX also suppressed LPS/IFN-stimulated activity of a 1.7-kb iNOS promoter, indicating that the inhibition of iNOS expression by DEX occurs at the level of transcription. NF-κB activation by LPS/IFN was repressed by DEX. The inhibition of NF-κB by DEX exhibited dose-dependent kinetics, which corresponded to DEX suppression of iNOS promoter activation, iNOS mRNA expression, and NO production. However, activation of activator protein-1 (AP-1), which is also contained in the iNOS promoter, was not enhanced by LPS/IFN or inhibited by DEX. Thus, glucocorticoids appear to block iNOS expression, at least in part, through inhibition of NF-κB activation, which results in decreased NO production.

Serum after Partial Hepatectomy Stimulates iNOS Gene Transcription via Downstream NF-κB Site

It has been known that the expression of inducible nitric oxide synthase (iNOS) is up-regulated during hepatic regeneration. The present study characterized the molecular mechanisms involved in the transcriptional activation of iNOS gene by using the serum after partial hepatectomy (post-PH serum) in vitro. The post-PH serum rapidly induced iNOS mRNA expression, which was blocked by anti-tumor necrosis factor-α (TNF-α) antibody in BNL CL.2 cells, murine embryonic liver cell line. In addition, EMSAs using a NF-κB-specific oligomer showed that the up-regulated iNOS mRNA expression in cells treated with post-PH serum correlated with transient activation of NF-κB complex (p50/p65 heterodimer). Transient transfection of BNL CL.2 cells with iNOS promoter linked to a CAT reporter gene showed the transcriptional activation of iNOS promoter by post-PH serum. Furthermore, site-directed mutational analysis of the two NF-κB sites individually or in combination revealed that iNOS expression by post-PH serum is regulated by the downstream NF-κB site, but not by upstream NF-κB site. Taken together, these results suggest that the downstream NF-κB site acts as an essential component for the iNOS expression by post-PH serum during hepatic regeneration.

Group B streptococci and inducible nitric oxide synthase: Modulation by nuclear factor kappa B and ibuprofen

Group B Streptococci (GBS) neonatal infections cause a complex inflammatory process involving numerous biochemical mediators. Nitric Oxide (NO) is generated by many cell types in response to different inflammatory signals. Nuclear factor kappa B (NFkappaB) plays an important role in the inflammatory process and may induce a number of biochemical mediator genes, including the inducible nitric oxide synthase (iNOS). We tested the hypothesis that GBS induces iNOS gene expression through activation of NFkappaB. We also tested whether ibuprofen (IBU) will suppress iNOS expression by blocking NFkappaB activation. Cerebral microvascular endothelial cells isolated from newborn piglets were harvested for the determination of iNOS gene expression and activation of NFkappaB. GBS significantly induced iNOS mRNA expression (5- to 6-fold, P < .005) and iNOS protein (3- to 4-fold, P < .01) at 24 hours. DNA-NFkappaB binding activity was detected within 15 minutes of GBS treatment and reached a maximal effect at 3 hours. Treatment with IBU significantly suppressed GBS-induced iNOS mRNA expression at 24 hours, and NFkappaB activity at 3 hours, suggesting that suppression of GBS-induced iNOS mRNA expression by IBU occurs by blocking of NFkappaB activation. These data show that NFkappaB activation is an early step in the induction of iNOS gene expression by GBS and that this interaction may play a vital role in the pathogenesis of GBS neonatal infections.

Induction of Nitric-oxide Synthase and Activation of NF-κB by Interleukin-12 p40 in Microglial Cells

Interleukin-12 (IL-12) is composed of two different subunits, p40 and p35. Expression of p40 mRNA but not that of p35 mRNA in excessive amount in the central nervous system of patients with multiple sclerosis (MS) suggests that IL-12 p40 may have a role in the pathogenesis of the disease. However, the mode of action of p40 is completely unknown. Because nitric oxide produced from the induction of nitric-oxide synthase (iNOS) also plays a vital role in the pathophysiology of MS, the present study was undertaken to explore the role of p40 in the induction of NO production and the expression of iNOS in microglia. Both IL-12 and p40(2), the p40 homodimer, dose-dependently induced the production of NO in BV-2 microglial cells. This induction of NO production was accompanied by an induction of iNOS protein and mRNA. Induction of NO production by the expression of mouse p40 cDNA but not that of the mouse p35 cDNA suggests that the p40 but not the p35 subunit of IL-12 is involved in the expression of iNOS. In addition to BV-2 glial cells, p40(2) also induced the production of NO in mouse primary microglia and peritoneal macrophages. However, both IL-12 and p40(2) were unable to induce the production of NO in mouse primary astrocytes. Because activation of NF-kappaB is important for the expression of iNOS, we investigated the effect of p40(2) on the activation of NF-kappaB. Induction of the DNA binding as well as the transcriptional activity of NF-kappaB by p40(2) and inhibition of p40(2)-induced expression of iNOS by SN50, a cell-permeable peptide carrying the nuclear localization sequence of p50 NF-kappaB, but not by SN50M, a nonfunctional peptide mutant, suggests that p40(2) induces the expression of iNOS through the activation of NF-kappaB. This study delineates a novel role of IL-12 p40 in inducing the expression of iNOS in microglial cells, which may participate in the pathogenesis of neuroinflammatory diseases.

Differential expression and regulation of inducible nitric oxide synthase (iNOS) mRNA in human trophoblasts in vitro.

To determine the expression of inducible nitric oxide synthase (iNOS) in human trophoblast and to examine the possible regulation of iNOS gene by cytokines. Total RNA was isolated from: 1) homogenized placental tissue; from 2) isolated and purified cytotrophoblast cells; and 3) cytotrophoblast and syncytiotrophoblast cells treated with cytokines in vitro. RNA was reverse transcribed and amplified by polymerase chain reaction, using specific primers for iNOS. Trophoblast cells were treated in vitro by interferon-gamma (IFN-gamma) in a dose of 10 ng/mL, Interleukin 1beta (IL-1beta) (4 ng/mL) and leukemia inhibitory factor (LIF) (1 ng/mL). Trophoblasts were also subjected to immunocytochemistry using iNOS-specific antibody to detect iNOS protein expression in these cells. The expression of iNOS mRNA was found both in placental tissue and isolated cytotrophoblast cells. In culture, the highly differentiated syncytiotrophoblast expressed more mRNA than cytotrophoblast cells. IFN-gamma and LIF, but not IL-1beta, induced iNOS mRNA expression in trophoblast cells in vitro. The effects of these cytokines on iNOS mRNA were only observed in syncytiotrophoblast cells, but not in cytotrophoblast cells. Immunocytochemical staining confirmed the trophoblast cells as a major source of the iNOS synthase production. 1) Human trophoblast cells are able to express the iNOS mRNA, hence suggesting a role for NO in placental growth and function. 2) LIF and IFN-gamma, but not IL-1beta, induce the iNOS mRNA expression in syncytiotrophoblast cells in vitro, suggesting possible similar regulatory mechanisms in vivo. 3) This study, for the first time, demonstrates the stimulating effect of LIF on iNOS gene expression in human tissue.