Protein kinase C-eta (PKC-eta) is required for the development of inducible nitric oxide synthase (iNOS) positive phenotype in human monocytic cells.

Several murine and human monocytic cell lines and monocyte-derived macrophages (MDM) from healthy volunteers were studied to compare their production of nitric oxide (NO) and induction of iNOS following endotoxin treatment. Although the human cells were sensitive to endotoxin and responded well by producing TNF-alpha and matrix metalloproteases (MMP), there was no induction of iNOS expression or NO production by any of these cells. Murine cells, however, produced large amounts of NO and expressed iNOS following similar endotoxin stimulation. We investigated the expression of PKC isotypes in all human and murine cell lines as well as in MDM, and found that the human cells lacked PKC-eta while the murine counterparts lacked PKC-beta1. Subsequently, human cells that were transfected with PKC-eta were found to make large quantities of NO following endotoxin exposure, an observation not seen in untransfected cells. We propose that PKC-eta is essential for the development of the iNOS positive phenotype in human monocytic cells, and may be responsible for the development of a number of inflammatory related conditions. As such it may be a suitable target for therapeutic intervention.     

Expression of inducible nitric oxide synthase in tumoral and non-tumoral epithelia from bladder cancer patients.

We have previously demonstrated that nitric oxide (NO) is elevated in the urine from bladder cancer patients. As the inducible nitric oxide synthase (iNOS) produces high NO output, the aim of this study was to examine iNOS expression and activity in tumoral (BT) and non-tumoral bladder tissue (NT). iNOS expression was determined by Western blot in 42 BT, 22 NT, and 4 normal bladders (normal B). iNOS activity was evaluated by conversion of [(14)C]l-arginine to [(14)C]l-citrulline plus NO, in additional 15 BT, 8 NT, and 1 normal B. iNOS tissue localization was studied by immunohistochemistry. iNOS expression and activity were found in almost 50% of bladder cancer patients, in both BT and in NT. A similar positive or negative iNOS expression in each pair of NT and BT tissue compared was observed, suggesting that high urine NO levels could be generated by an active iNOS present not only in the tumor but also in the non-tumoral bladder tissue. By immunohistochemistry, heterogeneous iNOS staining was detected in tumor cells from superficial and invasive tumors, while it was not evident in the normal bladder epithelium. A follow-up of 21 patients during 2 years showed recurrences in 80% with positive iNOS. On the contrary, no recurrences were observed in 73% of iNOS negative patients. Our results suggest that iNOS expression in bladder tissue may predispose to cancer recurrences.     

The major rheumatoid factor cross-reactive idiotype is dominantly expressed by Staphylococcus aureus Cowan strain I-activated CD5+ control B cells.

Some seropositive (RF+) and seronegative (RF-) rheumatoid arthritis (RA) patients selectively express high concentrations of the major RF cross-reactive idiotype (RCRI) in their sera and generate high frequencies of RCRI+ pokeweed mitogen (PWM)-induced plasma cells from their peripheral blood mononuclear cells (PBM). To determine if normal individuals can express RCRI in vitro, B cells from controls were activated with Staphylococcus aureus Cowan strain I (SAC) bacteria to identify RCRI and RF production. In addition, we studied the relationship of RCRI expression with the subset of B cells bearing CD5. Control CD5+ B cells are responsible for RCRI expression following SAC activation. We also observed that RCRI is dominantly expressed by control SAC-induced B cells in frequencies comparable to that expressed by some RA and juvenile rheumatoid arthritis patients' PBM activated by PWM. Therefore, the frequency of RCRI+ B cells in control and arthritis patients' PBL may be similar, or the selection and/or regulation of RCRI+ B-cell expression in vitro and in vivo may be different in arthritis patients compared to normal individuals.     

Nitric oxide and the Th2 response combine to prevent severe hepatic damage during Schistosoma mansoni infection.

During infection with Schistosoma mansoni, NO production increases following the deposition of parasite eggs in the liver. In wild-type C57BL/6 mice, NO levels peak during the sixth week of infection and are subsequently down-regulated. Inducible NO synthase (iNOS) mRNA was found in diseased liver tissue along with TNF-alpha and IFN-gamma, which are known promoters of iNOS expression. Mice treated with aminoguanidine, a selective inhibitor of iNOS, exhibited cachexia and exacerbated liver pathology, suggesting that NO limits hepatocyte damage when the liver is first exposed to eggs. Hepatic iNOS is up-regulated in SCID mice, indicating that NO production is part of an innate response. Studies with infected highly susceptible IL-4-/- mice revealed that prolonged NO production is in itself deleterious and that a major function of the Th2 response, which is severely compromised in the absence of IL-4, is to regulate NO production. In these animals, plasma NO levels are high compared with those in infected wild-type mice and remain elevated until death. Nevertheless, the underlying importance of NO is illustrated by the finding that aminoguanidine treatment leads to more severe liver disease and reduced time to death in infected IL-4-/- mice.     

Hypoxia induces nitric oxide synthase in rheumatoid synoviocytes: consequences on NADPH oxidase regulation

We investigated the effects of hypoxia on inducible NO synthase (iNOS) activity and expression in rheumatoid arthritis (RA) synoviocytes. We further studied the relationship between nitrosative stress and NADPH oxidase (NOX) in such conditions. Human cultured synoviocytes were treated for 24 hours with IL-1β, TNF-α or neither, and submitted to hypoxia or normoxia for the last 6 hours. Nitrite production and iNOS expression were increased under hypoxia conditions in RA cells in comparison to normoxia. Hypoxia did not potentate the basal and cytokine-induced superoxide productions, while NOXs' subunit expression and p47-phox phosphorylation were increased. Nitrosylation of NOXs and p47-phox was not raised under hypoxia conditions. Finally, peroxynitrite production was significantly increased under hypoxia conditions, in comparison to normoxia. Our results provide evidence for upregulation of iNOS and NOX activities in RA synoviocytes under hypoxia conditions, associated to an increased peroxynitrite production. Synovial cell metabolism under hypoxia conditions might be different from that in normoxia.     

Overexpression of iNOS and down-regulation of BMPs-2, 4 and 7 in retinoic acid induced cleft palate formation

The present work studied the induction of cleft palate formation in embryos developed from pregnant BALB/c mice treated orally with retinoic acid (RA). Previous studies on mature somatic cell types showed that RA exerted inhibitory effects on inducible nitric oxide synthase (iNOS) production. For the first time, our study has shown that RA actually stimulates significant expression of iNOS at specific zones of the affected embryonic palatal tissues at three consecutive stages, from gestation day 13 (GD13) to day 16 (GD16). Enzymatically, iNOS facilitates intracellular nitric oxide (NO) synthesis from L-arginine. When NO reacts with reactive superoxides it may result in irreparable cell injury. NO was also reported to induce apoptosis in some mammalian cell systems. Based on our findings, we propose that such an increase in NO production might be associated with apoptosis in the embryonic palatal tissues in the RA-treated mice. The detrimental effects of NO resulted in a reduction in proliferating palatal cells and therefore disturbed the normal plasticity of the palatal shelves. With iNOS overexpression, our findings also showed that there was significant concomitant down-regulation in the expressions of Bone Morphogenetic Proteins (BMPs) -2, 4, and 7 with regional variations particularly in the palatal mesenchymal cells for those embryos developing cleft palate. Since specific spatial and temporal expressions of BMPs -2, 4, and 7 are critical during normal palatal morphogenesis, any deficiency in the epithelial-mesenchymal interaction may result in retarding growth at the embryonic palatal shelves. Taken together, our study has demonstrated cleft palate formation in the BALB/c embryos involved overexpression of iNOS and down-regulation of BMPs-2, 4 and 7.     

Inducible nitric oxide synthase expression before and after eradication of Helicobacter pylori in different forms of gastritis

An increased expression of inducible nitric oxide synthase (iNOS) has been observed in the inflamed human gastric mucosa as well as in some tumors. This observation suggests a pathobiological role of elevated NO production. The purpose of this study was to compare the immunohistochemical iNOS expression in the different kinds of gastritis before and after the eradication of Helicobacter pylori. We performed iNOS and H. pylori immunohistochemical staining and counted iNOS positive cells. We detected elevated expression of iNOS around sites infected with H. pylori. iNOS expression in chemical gastritis was strongly elevated in mucosal glands. After treatment, we found a significant difference in iNOS expression in patients with classical H. pylori-induced antrum predominant gastritis and in patients with active autoimmune gastritis. In the special case of progressed gastritis with intestinal metaplasia we found persistence of intestinal metaplasia, and we could not find a significant difference in the number of positive iNOS cells before and after treatment. The persistence of IM as a possibly precancerous lesion is probably at least in the antrum a source of continuous iNOS induction even after H. pylori eradication.     

Nitric oxide produced by iNOS is associated with collagen synthesis in keloid scar formation

Nitric oxide (NO) has emerged as an important mediator of many physiological functions. Recent reports have shown that NO participates in the wound healing process, however, its role in keloid formation remains unclear. This study aimed to investigate the effect of NO on keloid fibroblasts (KF) and to determine the levels of inducible nitric oxide synthase (iNOS) expression in clinical specimens of keloid. Scar tissue from seven keloid patients with matched perilesion skin tissue controls was studied for inducible nitric oxide synthase expression and location. In addition, primary keloid and normal scar skin fibroblast cultures were set up to investigate the effects of NO in inducing collagen type I expression. Inducible nitric oxide synthase expression, and NO production were elevated in keloid scar tissues but not in matched perilesion skin tissues. Furthermore, exposure of KF to exogenous NO resulted in increased expression of collagen type I in a dose-dependent manner. NO exposure also induced time-course dependent collagen I expression that peaked at 24h in KF. Taken together, these results indicate that excess collagen formations in keloid lesion may be attributed to iNOS overexpression.     

Troglitazone inhibits the expression of inducible nitric oxide synthase in adipocytes in vitro and in vivo study in 3T3-L1 cells and Otsuka Long-Evans Tokushima Fatty rats

The aim of this study was to determine the mechanism of troglitazone action on nitric oxide (NO) production via inducible NO synthase (iNOS) in adipocytes in vitro and in vivo. The treatment of 3T3-L1 adipocytes with the combination of lipopolysaccharide (LPS), tumor necrosis factor-alpha and interferon-gamma synergistically induced de novo iNOS expression leading to enhanced NO production. The NO production was inhibited by co-treatment with aminoguanidine or N-nitro-L-arginine methylester hydrochloride. Troglitazone inhibited the NO production in a dose dependent manner by the suppression of iNOS expression. In the 24 week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats, the mean weight and the blood glucose were 21% and 30%, respectively, higher than in their lean counterparts. The serum nitrite concentration was increased after injection of LPS (4 mg/kg, i.p.), more markedly in OLETF rats than in the lean rats. The epididymal fats from LPS-injected groups, but not the ones from the non-injected groups, expressed mRNA and protein of iNOS. Troglitazone pre-treatment blocked the LPS-induced expression of iNOS in adipose tissue and the increase in serum nitrite concentration. These results suggest that troglitazone inhibits the cytokine-induced NO production in adipocytes by blocking iNOS expression both in vitro and in vivo.     

Age Related Changes from Youth to Adulthood in Rat Brain Cortex: Nitric Oxide Synthase and Mitochondrial Respiratory Function

Age related changes in brain cortex NO metabolism were investigated in mitochondria and cytosolic extracts from youth to adulthood. Decreases of 19%, 40% and 71% in NO production were observed in mitochondrial fractions from 3, 7, and 14 months old rats, respectively, as compared with 1-month-old rats. Decreased nNOS protein expression in 14 months old rats was also observed in mitochondria as compared with the nNOS protein expression in 1-month-old rats. Low levels of eNOS protein expression close to the detection limits and no iNOS protein expression were significantly detected in mitochondrial fraction for both groups of age. NO production in the cytosolic extracts also showed a marked decreasing tendency, showing higher levels than those observed in mitochondrial fractions for all groups of age. In the cytosolic extracts, however, the levels were stabilized in adult animals from 7 to 14 months. nNOS protein expression showed a similar age-pattern in cytosolic extracts for both groups of age, while the protein expression pattern for eNOS was higher expressed in adult rats (14 months) than in young animals. As well as in mitochondrial extracts iNOS protein expression was not significantly detected in cytosolic extracts at any age. RT-PCR assays indicated increased levels of nNOS mRNA in 1-month-old rats as compared with 14 months old rats, showing a similar pattern to that one observed for protein nNOS expression. A different aged pattern was observed for eNOS mRNA expression, being lower in 1-month-old rats as compared with 14 months old animals. iNOS mRNA was very low expressed in both groups of age, showing a residual iNOS mRNA that was not significantly detected. State 3 respiration rates were 78% and 85% higher when succinate and malate-glutamate were used as substrates, respectively, in 14 months rats as compared with 1-month-old rats. No changes were observed in state 4 respiration rates. These results could indicate 1 that nNOS and eNOS mRNA and protein expression can be age-dependent, and confirmed the nNOS origin for the mitochondrial NOS. During rat growth, the respiratory function seems to be modulated by NO produced by the different NOS enzymes: nNOS, eNOS and mtNOS present in the cytosol and in the mitochondria.     

Hypoxia induces nitric oxide synthase in rheumatoid synoviocytes: consequences on NADPH oxidase regulation

Abstract

We investigated the effects of hypoxia on inducible NO synthase (iNOS) activity and expression in rheumatoid arthritis (RA) synoviocytes. We further studied the relationship between nitrosative stress and NADPH oxidase (NOX) in such conditions. Human cultured synoviocytes were treated for 24 hours with IL-1β, TNF-α or neither, and submitted to hypoxia or normoxia for the last 6 hours. Nitrite production and iNOS expression were increased under hypoxia conditions in RA cells in comparison to normoxia. Hypoxia did not potentate the basal and cytokine-induced superoxide productions, while NOXs’ subunit expression and p47-phox phosphorylation were increased. Nitrosylation of NOXs and p47-phox was not raised under hypoxia conditions. Finally, peroxynitrite production was significantly increased under hypoxia conditions, in comparison to normoxia. Our results provide evidence for upregulation of iNOS and NOX activities in RA synoviocytes under hypoxia conditions, associated to an increased peroxynitrite production. Synovial cell metabolism under hypoxia conditions might be different from that in normoxia.     

Localization and activity of iNOS in normal human lung tissue and lung cancer tissue

Inducible nitric oxide synthase (iNOS) is one of three enzymes generating nitric oxide (NO) from the amino acid L-arginine. iNOS-derived NO plays an important role in several physiological and pathophysiological conditions. NO is a free radical which produces many reactive intermediates that account for its bioactivity. In the human lung, the alveolar macrophage is an important producer of cytokines and this production may be modified by NO. Moreover, high concentrations of NO have been shown to increase nuclear factor kappaB (NF-kB) activation. Recent investigations of NO expression in tumor tissue indicated that, at least for certain tumors, NO may mediate one or more roles during the growth of human cancer. We have studied iNOS in two tissue groups: normal human lung tissue and human lung cancer tissue. We localized iNOS in these tissues by immunohistochemistry and tested the mRNA expression by RT-PCR, the protein level by Western blot, and the protein activity by radiometric analysis. The results demonstrate different expression, localization and activity of iNOS in normal versus tumor tissue. This is suggestive of a role for NO production from iNOS in human lung cancer because high concentrations of this short molecule may transform to highly reactive compounds such as peroxynitrite (ONOO-); moreover, through the upregulator NF-kB, they can induce a chronic inflammatory state representing an elevated risk for cell transformation to cancer.     

Expression of inducible nitric oxide synthase and elevation of tyrosine nitration of a 32-kilodalton cellular protein in brain capillary endothelial cells from rats infected with a neuropathogenic murine leukemia virus

PVC-211 murine leukemia virus (MuLV) is a neuropathogenic variant of Friend MuLV (F-MuLV) which causes a rapidly progressive spongiform neurodegenerative disease in rodents. The primary target of PVC-211 MuLV infection in the brain is the brain capillary endothelial cell (BCEC), which is resistant to F-MuLV infection. Previous studies have shown that changes in the envelope gene of PVC-211 MuLV confer BCEC tropism to the virus. However, little is known about how infection of BCECs by PVC-211 MuLV induces neurological disease. Previous results suggest that nitric oxide (NO), which has been implicated as a potential neurotoxin, is involved in PVC-211 MuLV-induced neurodegeneration. In this study, we show that expression of inducible nitric oxide synthase (iNOS), which produces NO from L-arginine, is induced in BCECs from PVC-211 MuLV-infected rats. Furthermore, elevated levels of a 32-kDa cellular protein modified by 3-nitrotyrosine, which is a hallmark of NO production, were observed in virus-infected BCECs. BCECs from rats infected with BCEC-tropic but nonneuropathogenic PVF-e5 MuLV, which is a chimeric virus between PVC-211 MuLV and F-MuLV, fail to induce either iNOS expression or elevation of tyrosine nitration of a 32-kDa protein. These results suggest that expression of iNOS and nitration of tyrosine residues of a 32-kDa protein in PVC-211 MuLV-infected BCECs may play an important role in neurological disease induction.     

Expression of ID family genes in the synovia from patients with rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by aggressive proliferation of synovial tissue leading to destruction of cartilage and bone. To identify molecules which play a crucial role for the pathogenesis, we compared mRNA expression pattern of RA synovium with that of osteoarthritis (OA), using the differential display. From the panel of differentially expressed genes, ID1 (inhibitor of differentiation 1) was considered to be particularly relevant to the pathogenesis of RA, because Id family genes have been shown to play a role in cell proliferation and angiogenesis. To examine whether the up-regulation of these genes is consistently observed in the patients with RA, mRNA levels of ID1 and ID3 in the synovial tissues from 13 patients with RA and 6 patients with OA were semi-quantitatively analyzed by RT-PCR. Mean mRNA levels of ID1 and ID3 were significantly elevated in RA synovia compared with OA by 8.6-fold (P = 0.0044) and 3.3-fold (P = 0.0085), respectively. Immunohistochemistry revealed striking staining of Id1 and Id3 in the endothelial cells, suggesting a possible role of Id in severe angiogenesis observed in RA. The expression of Id family genes in the synovium constitutes a new finding of particular interest. Their functional role as well as their contribution to the genetic susceptibility to RA requires further investigation.