iNOS KO Research Articles

INOS gene deficiency protects from sepsis induced cognitive long-term deficits

Septic encephalopathy (SE) is caused by systemic inflammation in the absence of direct brain infection. While the acute reactions to SE have been well defined, the long-term consequences of SE on cerebral metabolism, cognition, learning and memory capabilities remain to be elucidated. Using a murine model of sepsis, we demonstrate that a single injection of lipopolysaccharide (LPS), a gramnegative bacterial cell wall component, results in long-term cognitive deficits. Two month after the initial challenge, LPS treated mice committed more working and reference memory errors than controls. These behavioural changes were accompanied by reduced cortical glucose uptake as evidenced by small animal 18F-FDG-PET studies. In contrast, mice deficient for the inducible nitric oxide synthase gene (iNOS KO), did not show any cognitive changes and revealed a normal cerebral glucose uptake when challenged with LPS using the identical protocol. LPS induced SE did not cause neuronal cell death, since serial sectioning of brains and subsequent immunohistological analysis did not detect any changes. However, further analysis of inflammatory genes showed that LPS treated iNOS KO mice had lower mRNA levels of proinflammatory cytokines and chemokines, including tumor necrosis factor alpha and interleukin 1 beta, compared to LPS challenged wild type animals, suggesting that NO is involved in the transcriptional control of these genes. Synaptosome preparations and detection of key synaptic proteins revealed distinct LPS induced changes of protein levels along with sepsis induced and NO mediated nitration and nitrosylation of tyrosine and cysteine residues. Together these data suggest that sepsis is causing long-lasting cognitive deficits, which are influenced by iNOS dependent NO generation and NO mediated alterations at the synaptic level.

Role of inflammation in blood‐retinal barrier breakdown: animal models

Abstract Purpose Animal models are valuable tools to study the pathogenic mechanisms of many diseases. Diabetic retinopathy is a low‐grade chronic inflammatory disease. Nitric oxide (NO) is involved in leukostasis and blood‐retinal barrier (BRB) breakdown in the early stages of the disease. However, the role of the different NO synthase (NOS) isoforms was not elucidated. We aimed to clarify the involvement of constitutive (eNOS, nNOS) and inducible NOS (iNOS) isoforms and the mechanisms underlying NO‐mediated leukostasis and BRB breakdown, by using an experimental model of diabetes and iNOS KO mice. Methods Diabetes was induced by streptozotocin in normal and KO mice (2 weeks of diabetes). Normal mice were treated with L‐NAME (NOS inhibitor). Vessel leakage was assessed with Evans blue. Leukostasis was quantified in flat‐mounted retinas and in vivo. ICAM‐1, occludin, ZO‐1 and nitrotyrosine levels were assessed by Western blotting or immunohistochemistry. Results Diabetes increased the permeability of BRB and leukostasis, which were reduced by L‐NAME. Similar effects were observed in diabetic iNOS KO mice. In diabetic mice, the immunoreactivity of tight junction proteins, occludin and ZO‐1, decreased, whereas ICAM‐1 protein levels increased. Those effects were prevented by L‐NAME and in diabetic iNOS KO mice. Diabetes also upregulated all NOS isoforms and increased nitrotyrosine levels in normal mice, but did not significantly increase eNOS and nNOS, and nitrotyrosine levels, in iNOS KO mice. Conclusion These data demonstrate that iNOS plays a predominant role in leukostasis and BRB breakdown. The mechanism involves ICAM‐1 upregulation and tight junction proteins downregulation. Support: FCT, Portugal

Characterization of the non‐adrenergic/non‐cholinergic response to perivascular nerve stimulation in the double‐perfused mesenteric bed of the mouse

Calcitonin gene-related peptide (CGRP), a capsaicin-sensitive neuromodulator of splanchnic vascular tone in several animal species, remains poorly investigated in mouse models. We therefore assessed whether endogenous CGRP is a non-adrenergic/non-cholinergic (NANC) neuromodulator in the mesenteric vascular bed of the mouse. Arterial and venous changes in perfusion pressure in response to perivascular nerve stimulation (PNS) were monitored in the mouse mesenteric bed under basal conditions or precontracted with KCl (artery) or U46619 (vein) in circuits pretreated with guanethidine, atropine, indomethacin and prazosin. Arterial responses to NANC were also characterized with a CGRP1 antagonist, halphaCGRP8-37. Finally, the PNS-induced release of arterial CGRP was measured by enzyme immunoassay. HalphaCGRP8-37 enhanced PNS-induced arterial increases in perfusion pressure under basal tone. PNS-induced stimulation of NANC triggered an halphaCGRP8-37 or capsaicin- sensitive reduction in perfusion pressure of the pre-contracted arterial bed only. Chemical removal of the endothelium inhibited PNS- and halphaCGRP- induced reduction in perfusion pressure in the arterial mesenteric bed. Responses to NANC nerves were reduced by guanylate and adenylate cyclase inhibitors (1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one (ODQ)) and [9-(tetrahydro-2-furanyl)-9H-purin-6-amine] (SQ 22,536), respectively. A neuronal NOS inhibitor (7-nitroindazole; 7-NI) also enhanced the response to NANC in vessels from wild-type, eNOS KO but not iNOS KO mice. Finally, PNS enhanced the release of immunoreactive CGRP from the perfused arterial mesenteric bed. Our study demonstrates a role for CGRP in the NANC-dependent reduction in perfusion pressure of the arterial but not venous mesenteric bed of the mouse.

Exacerbated Pathology of Viral Encephalitis in Mice with Central Nervous System-Specific Autoantibodies

We examine here the outcome of viral encephalomyelitis [mouse hepatitis virus (MHV) A59, Theiler’s encephalomyelitis virus, and Coxsackievirus B3] in mice with autoantibodies to a central nervous system (CNS)-specific antigen, myelin oligodendrocyte glycoprotein, that usually develop no clinical disease. Morbidity and mortality of the acute viral CNS disease was augmented by the presence of the autoantibodies in all three viral infections. Transfer of serum containing the autoantibodies at the time of infection with MHV was sufficient to reproduce the exacerbated disease. The presence of the autoantibodies was found to result in increased infiltration of mononuclear cells into the brain. Early demyelination was severely augmented in brains and spinal cords of MHV-infected mice with CNS-specific autoantibodies. The antibody-mediated exacerbation was shown to be independent of the complement system but to require expression of Fc receptors, because it was observed in C′-3-deficient but not in Fc receptor-deficient mice. Our study illustrates the possibility that infections can lead to much more profound immunopathology in the presence of an otherwise latent autoimmune condition.

Endothelially Derived Nitric Oxide Affects the Severity of Early Acetaminophen‐induced Hepatic Injury in Mice

The precise mechanism of hepatocellular toxicity following acetaminophen (APAP) poisoning remains unclear. Nitric oxide is implicated in APAP toxicity as an inflammatory signaling molecule and as a precursor to the free radical peroxynitrate. The effects of inducible nitric oxide synthase (iNOS)-derived NO in APAP toxicity are known; however, the role of endothelial nitric oxide synthase (eNOS)-derived NO is unknown. The authors sought to evaluate the effect of eNOS-derived NO during APAP toxicity. C57BL6/J mice deficient in eNOS (eNOS KO) or iNOS (iNOS KO) and wild-type mice (WT) were treated with 300 mg/kg APAP. Alanine aminotransferase levels and plasma nitrate and nitrite levels were measured. Hypoxia inducible factor (HIF)-1alpha and Glucose Transporter 1 (Glut-1) levels were determined by Western blot. Alanine aminotransferase levels were significantly elevated in all treated animals. Alanine aminotransferase levels were significantly lower in eNOS KO and iNOS KO than in treated WT animals. Plasma nitrate/nitrite levels were significantly higher in WT animals than in iNOS KO and eNOS KO animals. HIF-1alpha expression was increased in WT mice and decreased in iNOS KO mice. Glut-1 is a downstream, indirect marker of HIF function. Glut-1 expression was increased in WT and eNOS KO mice. Deficiency of either iNOS or eNOS results in decreased NO production and is associated with reduced hepatocellular injury following APAP poisoning. HIF-1alpha and Glut-1 levels are increased following APAP poisoning, implying that HIF-1alpha is functional during the pathogenic response to APAP poisoning.

Endothelially Derived Nitric Oxide Affects the Severity of Early Acetaminophen-induced Hepatic Injury in Mice

Objectives: The precise mechanism of hepatocellular toxicity following acetaminophen (APAP) poisoning remains unclear. Nitric oxide is implicated in APAP toxicity as an inflammatory signaling molecule and as a precursor to the free radical peroxynitrate. The effects of inducible nitric oxide synthase (iNOS)-derived NO in APAP toxicity are known; however, the role of endothelial nitric oxide synthase (eNOS)-derived NO is unknown. The authors sought to evaluate the effect of eNOS-derived NO during APAP toxicity. Methods: C57BL6/J mice deficient in eNOS (eNOS KO) or iNOS (iNOS KO) and wild-type mice (WT) were treated with 300 mg/kg APAP. Alanine aminotransferase levels and plasma nitrate and nitrite levels were measured. Hypoxia inducible factor (HIF)-1α and Glucose Transporter 1 (Glut-1) levels were determined by Western blot. Results: Alanine aminotransferase levels were significantly elevated in all treated animals. Alanine aminotransferase levels were significantly lower in eNOS KO and iNOS KO than in treated WT animals. Plasma nitrate/nitrite levels were significantly higher in WT animals than in iNOS KO and eNOS KO animals. HIF-1α expression was increased in WT mice and decreased in iNOS KO mice. Glut-1 is a downstream, indirect marker of HIF function. Glut-1 expression was increased in WT and eNOS KO mice. Conclusions: Deficiency of either iNOS or eNOS results in decreased NO production and is associated with reduced hepatocellular injury following APAP poisoning. HIF-1α and Glut-1 levels are increased following APAP poisoning, implying that HIF-1α is functional during the pathogenic response to APAP poisoning.

Thermoregulatory role of inducible nitric oxide synthase in lipopolysaccharide-induced hypothermia

We have tested the hypothesis that nitric oxide (NO) arising from inducible nitric oxide synthase (iNOS) plays a role in hypothermia during endotoxemia by regulating vasopressin (AVP) release. Wild-type (WT) and iNOS knockout mice (KO) were intraperitoneally injected with either saline or Escherichia coli lipopolysaccharide (LPS) 10.0 mg/kg in a final volume of 0.02 mL. Body temperature was measured continuously by biotelemetry during 24 h after injection. Three hours after LPS administration, we observed a significant drop in body temperature (hypothermic response) in WT mice, which remained until the seventh hour, returning then close to the basal level. In iNOS KO mice, we found a significant fall in body temperature after the fourth hour of LPS administration; however, the hypothermic response persisted until the end of the 24 h of the experiment. The pre-treatment with β-mercapto-β,β-cyclopentamethylenepropionyl 1, O-Et-Tyr 2, Val 4, Arg 8-Vasopressin, an AVP V 1 receptor antagonist (10 μg/kg) administered intraperitoneally, abolished the persistent hypothermia induced by LPS in iNOS KO mice, suggesting the regulation of iNOS under the vasopressin release in this experimental model. In conclusion, our data suggest that the iNOS isoform plays a role in LPS-induced hypothermia, apparently through the regulation of AVP release.

Enhanced Oxidative Stress in iNOS-Deficient Mice after Traumatic Brain Injury: Support for a Neuroprotective Role of iNOS

Studies in experimental traumatic brain injury (TBI) suggest both deleterious and protective effects of inducible nitric oxide synthase (iNOS). Early after injury, iNOS may be detrimental via formation of peroxynitrite and iNOS inhibitors are protective. In contrast, we reported impaired long-term functional outcome after TBI in iNOS knockout (ko) versus wild-type (wt) mice. To elucidate potential neuroprotective and neurotoxic mechanisms for iNOS, we studied nitric oxide formation by electron paramagnetic resonance (EPR) spectroscopy using diethyldithiocarbamate-iron (DETC-Fe) as a spin trap and markers of nitrosative (S-nitrosothiol (RSNO, Fluorescent assay); nitrotyrosine (3NT, ELISA)) and oxidative stress (ascorbate, HPLC) at 72 h after controlled cortical impact (CCI) in iNOS ko and wt and in uninjured iNOS ko and wt mice. 3NT immunostaining with macrophage and myeloperoxidase (MPO) dual labeling was also assessed in brain sections. Brain DETC-Fe-NO low-temperature EPR signal intensity was approximately 2-fold greater in wt versus iNOS ko at 72 h after CCI. Ascorbate levels decreased in injured hemisphere in wt and iNOS ko versus uninjured -this decrease was more pronounced in iNOS ko. In wt mice, RSNO and 3NT levels were increased after CCI versus uninjured (50% and 400%, respectively, P < 0.05). RSNO levels were not increased in iNOS ko after CCI. Nitrotyrosine levels increased after CCI in wt and ko versus respective uninjured -this increase was more pronounced in wt (2.34 +/- 0.95 versus 1.27 +/- 0.49 pmol/mg protein, P < 0.05). Increased 3NT immunoreactivity was detected in wt versus iNOS ko at 72 h after CCI, and colocalized with macrophage marker and MPO. Our data support a role for iNOS-derived NO as an endogenous antioxidant after CCI. iNOS also contributes protein nitrosylation and nitration. Colocalization of 3NT with macrophages and MPO suggests generation of nitrating agents by macrophages and/or phagocytosis of nitrated proteins.

Inducible Nitric Oxide Synthase Has Divergent Effects on Vascular and Metabolic Function in Obesity

Previous studies have suggested an involvement of inducible nitric oxide synthase (iNOS) in obesity, but the relation, if any, between this and mechanisms underlying endothelial dysfunction in obesity is unknown. We studied mice fed an obesogenic high-fat or standard diet for up to 8 weeks. Obesity was associated with elevated blood pressure; resistance to the glucoregulatory actions of insulin; resistance to the vascular actions of insulin, assessed as the reduction in phenylephrine constrictor response of aortic rings after insulin preincubation (lean -21.7 +/- 11.5 vs. obese 18.2 +/- 15.5%; P < 0.05); and evidence of reactive oxygen species (ROS)-dependent vasodilatation in response to acetylcholine in aortic rings (change in maximal relaxation to acetylcholine after exposure to catalase: lean -2.1 +/- 6.0 vs. obese -15.0 +/- 3.8%; P = 0.04). Obese mice had increased expression of iNOS in aorta, with evidence of increased vascular NO production, assessed as the increase in maximal constriction to phenylephrine after iNOS inhibition with 1400W (lean -3.5 +/- 9.1 vs. obese 42.1 +/- 11.2%; P < 0.001). To further address the role of iNOS in obesity-induced vascular and metabolic dysfunction, we studied the effect of a high-fat diet in iNOS knockout mice (iNOS KO). Obese iNOS KO mice were protected against the development of resistance to insulin's glucoregulatory and vascular effects (insulin-dependent reduction in maximal phenylephrine response: obese wild-type 11.2 +/- 15.0 vs. obese iNOS KO -20.0 +/- 7.7%; P = 0.02). However, obese iNOS KO mice remained hypertensive (124.0 +/- 0.7 vs. 114.9 +/- 0.5 mmHg; P < 0.01) and had evidence of increased vascular ROS production. Although these data support iNOS as a target to protect against the adverse effects of obesity on glucoregulation and vascular insulin resistance, iNOS inhibition does not prevent the development of raised blood pressure or oxidative stress.

Impact of interleukin-12, oxidative burst, and iNOS on the survival of murine fecal peritonitis

Abdominal sepsis due to secondary fecal peritonitis following anastomosis insufficiency is a rare but life threatening complication of colorectal surgery. The induction of IFN-gamma by IL-12 is believed to play a key role in sepsis as it promotes antibacterial effector mechanisms such as oxidative burst or nitric oxide induction. The impact of gene deficiency for IL-12 (IL-12p40 KO), oxidative burst (p47(phox) KO), or NO induction (iNOS KO) on the outcome of fecal peritonitis was characterized using the murine Colon Ascendens Stent Peritonitis model (CASP). In the IL-12p40 KO model, 3 and 12 h after surgery, serum cytokine levels of IL-1beta, TNF, IL-18, and IL-10 were analyzed. Expression of IL-1beta, IL-10, IP-10, and MIP-1alpha was measured in lung and liver by RNAse Protection Assay. IL-12p40 and iNOS-deficient mice exhibited a significantly higher susceptibility to CASP as compared to the controls, whereas no significant difference was observed in p47(phox) KO mice. Absence of IL-12 resulted in delayed expression of proinflammatory cytokines and chemokines in both the liver and the lung, and was associated with significant reduction of IL-1beta levels in the serum 12 h after CASP. IL-12 and iNOS possess protective functions in fecal murine peritonitis. Surprisingly, no significant contribution of oxidative burst to the immune response was observed. Overall, these findings suggest that IL-12 deficiency causes a profound delay of the immune response after polymicrobial challenge resulting in significantly increased susceptibility in the CASP model.

Inducible nitric oxide synthase mediates bone development and P. gingivalis-induced alveolar bone loss

The role of inducible nitric oxide synthase (iNOS) in bone development and bacterially induced periodontal bone loss was examined using mice with targeted mutation of the iNOS gene. Femurs of iNOS KO mice showed 30% and 9% higher bone mineral density compared to wild type (WT) at 4 and 9 weeks of age, respectively. Micro-computed tomography revealed that cortical thickness and cortical bone density is increased in the absence of iNOS, while trabecular bone thickness and bone density remains unchanged. Histochemical analysis using TRAP staining showed that osteoclast numbers are lower by 25% in iNOS KO femurs compared to WT femurs. When bone marrow cells were stimulated with M-CSF and RANKL in vitro, iNOS KO cultures developed 51% fewer TRAP-positive multinuclear cells compared to WT cultures. When similar cultures were grown on dentine discs, resorption pit area was decreased by 54% in iNOS KO cultures. Gene expression studies showed that iNOS expression is induced by M-CSF and RANKL in WT bone marrow cultures, while no iNOS transcript was detected in iNOS KO. No compensatory change was detected in the expression of neuronal or endothelial NOS isoforms. There was no difference in RANK and osteoprotegerin expression between iNOS KO and WT bone marrow cultures after M-CSF and RANKL-treatment, while Traf6 expression was significantly lower in the absence of iNOS. In the alveolar bone of the maxilla, the distance between the cementoenamel junction and the alveolar bone crest was larger in iNOS KO compared to WT mice from 6 to 14 weeks of age, indicating a developmental effect of iNOS in oral tissues. Oral administration of the periodontal pathogen Porphyromonas gingivalis caused alveolar bone loss in the maxilla of WT mice, but failed to do so in iNOS KO mice. Expression of the osteoclast marker cathepsin K was 25% lower in iNOS KO alveolar bone. These data indicate that iNOS promotes bone resorption during bone development as well as after bacterial infection, and that iNOS is an important signal for normal osteoclast differentiation.

Vasopressin release during endotoxemic shock in mice lacking inducible nitric oxide synthase

Background We have tested the hypothesis that nitric oxide (NO) arising from inducible nitric oxide synthase (iNOS) is responsible for the deficiency in vasopressin release and consequent hypotension during endotoxemic shock. Methods and results Wild-type (WT) and iNOS knockout mice (KO) were intravenously injected with either saline or Escherichia coli lipopolysaccharide (LPS) 1.0 mg/kg in a final volume of 0.03 ml. Mean arterial blood pressure (MAP) was measured and vasopressin (AVP) plasma levels determined before and after LPS injection. In WT mice we found a significant drop in MAP after LPS administration, which lasted low until the end of the experiment. We also observed an increase in AVP plasma levels at the second hour of experiment, returning thereafter to basal levels. Conversely LPS injection in iNOS KO mice elicited sustained increase in plasma AVP concentration and maintenance of the blood pressure. Conclusion These data indicate that NO arising from the iNOS plays an important inhibitory role in AVP release during endotoxemia and may be responsible for the hypotension occurring during this vasodilatory shock. Clinical Pharmacology & Therapeutics (2005) 77, P9–P9; doi: 10.1016/j.clpt.2004.11.038

Thermoregulation during endotoxemia in mice lacking inducible nitric oxide synthase: Involvment of the arginine-vasopressin

Background We have tested the hypothesis that nitric oxide (NO) arising from inducible nitric oxide synthase (iNOS) plays a role in hypothermia during endotoxemic shock, by regulating vasopressin (AVP) release. Methods and results Wild-type (WT) and iNOS knockout mice (KO) were intraperitoneally injected with either saline or Escherichia coli lipopolysaccharide (LPS) 10.0 mg/kg in a final volume of 0.02 ml. Body temperature was measured continuously by biotelemetry during 24 hours after LPS administration. One hour after LPS administration we observed a significant drop in body temperature (hypothermic response) in WT, which remained until the eighth hour after LPS injection, returning the close to the basal level. In iNOS KO mice, we found a significant fall in body temperature after the first hour of LPS administration, however the hypothermic response persisted until the end of the twenty-four hour of experiment. The pre-treatment with (b-mercapto-b,b-cyclopentamethylenepropionyl1, O-Et-Tyr2, Val4, Arg8-Vasopressin), an AVP V1 receptor blocker (10 μg/kg) administered i.p., abolished the persistent hypothermia induced by LPS in iNOS KO mice, suggesting the regulation of iNOS under the vasopressin release in this patophysiological model. Conclusion In conclusion, our data indicate that the inducible NOS isoform plays an inhibitory role in AVP release during endotoxemia, which seems to be critical for the thermoregulation during this pathophysiological state. Clinical Pharmacology & Therapeutics (2005) 77, P9–P9; doi: 10.1016/j.clpt.2004.11.037

Nitric-oxide-dependent and independent mechanisms of protection from CNS inflammation during Th1-mediated autoimmunity: evidence from EAE in iNOS KO mice

Experimental autoimmune encephalomyelitis (EAE) disease was accelerated iNOS-deficient (KO) mice: coinciding with greatly increased numbers of Ag-specific Th1 cells in the periphery that appeared to rapidly shift from the spleen to the CNS during onset of disease symptoms. iNOS KO mice had significantly increased Th1 cells in the CNS versus wild-type mice. Apoptosis of CNS-infiltrating CD4 + T cells was impaired in iNOS KO mice at peak of disease; consequently, these mice had more CNS-infiltrating CD4 + T cells. Subsequently, iNOS KO mice up-regulated apoptosis of CNS-CD4 + T cells. During chronic EAE, CNS macrophages were greatly decreased, suggesting elimination of CNS-infiltrating CD4 + T cells and activated macrophages by iNOS-independent mechanisms. INOS is not only required for apoptosis of CNS-CD4 + T cells but also prevents overexpansion of autoreactive Th1 cells in the periphery and the CNS.

Role of nitric oxide in tolerance to lipopolysaccharide in mice

The injection of repeated doses of lipopolysaccharide (LPS) results in attenuation of the febrile response, which is called endotoxin tolerance. We tested the hypothesis that nitric oxide (NO) arising from inducible NO synthase (iNOS) plays a role in endotoxin tolerance, using not only pharmacological trials but also genetically engineered mice. Body core temperature was measured by biotelemetry in mice treated with NG-monomethyl-L-arginine (L-NMMA, 40 mg/kg; a nonselective NO synthase inhibitor) or aminoguanidine (AG, 10 mg/kg; a selective iNOS inhibitor) and in mice deficient in the iNOS gene (iNOS KO) mice. Tolerance to LPS was induced by means of three consecutive LPS (100 microg/kg) intraperitoneal injections at 24-h intervals. In wild-type mice, we observed a significant reduction of the febrile response to repeated administration of LPS. Injection of L-NMMA and AG markedly enhanced the febrile response to LPS in tolerant animals. Conversely, iNOS-KO mice repeatedly injected with LPS did not become tolerant to the pyrogenic effect of LPS. These data are consistent with the notion that NO modulates LPS tolerance in mice and that iNOS isoform is involved in NO synthesis during LPS tolerance.

Genetic ablation of iNOS modulates pro-inflammatory cytokine levels after trauma and confers a survival advantage following infectious challenge

Introduction: Significant immune changes occur after trauma. Prostaglandin-E2 (PGE2) and nitric oxide (NO ) are key inflammatory mediators involved in trauma-related immune dysfunction. The relative importance of the early response genes nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) after trauma is controversial. This study investigated macrophage cytokine production after trauma and followed survival from subsequent sepsis in COX-2 and iNOS knockout (−/−) and wild-type mice. Methods: Female C57/Bl6 COX-2 and iNOS KO and WT mice were randomized to control or trauma (femur fracture plus 40% blood volume hemorrhage) groups. Expt. 1. Seven days after injury, splenic macrophages were isolated, stimulated with LPS (10ng/mL) for 24 hours and pro- (IL-6 and TNFa) and anti-inflammatory (IL-10 and IL-12) cytokine levels measured by cytometric bead array. Expt. 2. At 7 days after trauma the mice were subjected to cecal ligation and puncture and followed for survival. Results: Macrophages produced significantly decreased TNFa levels in iNOS (−/−) mice compared with WT mice after trauma. iNOS (−/−) mice has a significant survival advantage compared with WT mice following sepsis (10/13 vs 4/12 p < 0.005). IL-10 levels were significantly elevated in COX-2 (−/−) but IL-6, IL-10 and IL-12 levels were unchanged compared with WT mice. Survival was similar between the COX-2 (−/−) and WT mice following septic challenge. Conclusions: These data suggest that inhibition of iNOS is more important than COX-2 in protecting the host from sepsis after trauma. Significantly decreased TNFa production in iNOS (−/−) mice correlated with improved outcome suggesting that NO regulates TNFa production in trauma.

Altered AP-1/Ref-1 redox pathway and reduced proliferative response in iNOS-deficient vascular smooth muscle cells

We previously reported that injury-induced medial vascular smooth muscle cell (VSMC) proliferation and neointima formation in carotid arteries of inducible nitric oxide synthase knockout (iNOS KO) mice were significantly reduced compared with wild type (WT). However, the molecular pathway underlying such differences is not known. In this in vitro study, we discovered that the AP-1/Ref-1/thioredoxin signaling pathway is altered in aortic VSMC from iNOS KO mice, which leads to reduced growth response when compared with aortic VSMC from WT mice. After equal initial seeding, the cell number after 7 days in serum medium was less in iNOS KO cells compared with WT VSMC (1.2 +/- 0.6 x 10(5) vs 3.2 +/- 1.1 x 10(5); p < 0.05). Significantly more iNOS KO cells remained in the G0/G1 phase compared with WT cells after 24-h serum treatment (82.6 +/- 13.7% vs 62.3 +/- 14.6%; p < 0.05) by cell-cycle analysis. Nuclear PCNA expression was also less in the iNOS KO cells, which was not affected by exogenous NO or superoxide. Superoxide generation after 24-h serum stimulation was less in the iNOS KO cells compared with WT cells. After 30-min serum stimulation, AP-1 DNA binding was reduced and a lack of increase in nuclear c-Jun protein was observed in iNOS KO VSMC. RT-PCR analysis confirmed a lack of inducible c-Jun mRNA after serum stimulation in the KO cells. In addition, KO cells had less nuclear reducing factor-1 (Ref-1) and serum-inducible thioredoxin protein expression. Reduced proliferative response of iNOS KO VSMC to serum treatment is associated with altered AP-1 /Ref-1 /thioredoxin pathway activation.

Role of inducible nitric oxide synthase-derived nitric oxide in lipopolysaccharide plus interferon-γ-induced pulmonary inflammation

Exposure of mice to lipopolysaccharide (LPS) plus interferon-γ (IFN-γ) increases nitric oxide (NO) production, which is proposed to play a role in the resulting pulmonary damage and inflammation. To determine the role of inducible nitric oxide synthase (iNOS)-induced NO in this lung reaction, the responses of inducible nitric oxide synthase knockout (iNOS KO) versus C57BL/6J wild-type (WT) mice to aspirated LPS + IFN-γ were compared. Male mice (8–10 weeks) were exposed to LPS (1.2 mg/kg) + IFN-γ (5000 U/mouse) or saline. At 24 or 72 h postexposure, lungs were lavaged with saline and the acellular fluid from the first bronchoalveolar lavage (BAL) was analyzed for total antioxidant capacity (TAC), lactate dehydrogenase (LDH) activity, albumin, tumor necrosis factor-α (TNF-α), and macrophage inflammatory protein-2 (MIP-2). The cellular fraction of the total BAL was used to determine alveolar macrophage (AM) and polymorphonuclear leukocyte (PMN) counts, and AM zymosan-stimulated chemiluminescence (AM-CL). Pulmonary responses 24 h postexposure to LPS + IFN-γ were characterized by significantly decreased TAC, increased BAL AMs and PMNs, LDH, albumin, TNF-α, and MIP-2, and enhanced AM-CL to the same extent in both WT and iNOS KO mice. Responses 72 h postexposure were similar; however, significant differences were found between WT and iNOS KO mice. iNOS KO mice demonstrated a greater decline in total antioxidant capacity, greater BAL PMNs, LDH, albumin, TNF-α, and MIP-2, and an enhanced AM-CL compared to the WT. These data suggest that the role of iNOS-derived NO in the pulmonary response to LPS + IFN-γ is anti-inflammatory, and this becomes evident over time.

Spontaneous sleep in mice with targeted disruptions of neuronal or inducible nitric oxide synthase genes

Nitric oxide (NO) affects almost every physiological process, including the regulation of sleep. There is strong evidence that NO plays an important role in rapid eye movement sleep (REMS) regulation. To further investigate the role of NO in sleep, we characterized spontaneous sleep in mice with targeted disruptions (knockout; KO) in the neuronal nitric oxide synthase (nNOS) or inducible (i)NOS genes. REMS in nNOS KO mice was substantially lower than that of their control mice. In contrast, the iNOS KO mice had significantly more REMS than their controls. Inducible NOS KO mice also had less non-REMS (NREMS) during the dark period. Results suggest that nNOS and iNOS play opposite roles in REMS regulation.

Response of Alveolar Macrophages from Inducible Nitric Oxide Synthase Knockout or Wild-Type Mice to an in Vitro Lipopolysaccharide or Silica Exposure

The role of nitric oxide (NO) in pulmonary disease has been controversial with both antiinflammatory (scavenging radicals and inhibiting NF-êB activation) and proinflammatory (forming highly reactive peroxynitrite and augmenting NF-êB activation by inflammatory agents) actions reported. Therefore, a study has been initiated to determine whether deletion of the inducible nitric oxide synthase (iNOS) gene in the C57BL/6J mouse alters the pulmonary macrophage response to lipopolysaccharide (LPS) or silica. The objective of the initial phase of this study was to determine the difference in responsiveness of alveolar macrophages (AMs), harvested from naive wild-type (WT) or iNOS knockout (iNOS KO) mice, to an in vitro LPS or silica exposure. Primary AMs were obtained by bronchoalveolar lavage (BAL) from age- and weightmatched iNOS KO and WT mice. The cells were treated with interferon-gamma (IFN-ã) (50 U/ml), IFN-ã (50 U/ml) + LPS (1 g/ml), LPS (0.01-100 g/ml), or silica (25-250 g/ml). The following parameters were measured: nitrate and nitrite (NOx), tumor necrosis factor-á (TNF-á), macrophage inflammatory protein-2 (MIP-2), intracellular generation of the reactive oxygen species (ROS) hydrogen peroxide (H2O2) and superoxide (O ” . 2 ), and basal (unstimulated) total antioxidant capacity. Data show a significant increase in NOx production upon exposure to IFN-ã - LPS in the WT but not iNOS KO AMs. NOx production by iNOS KO or WT AMs was not altered by in vitro exposure to LPS or silica alone. LPS, but not silica, induced TNF-á and MIP-2 production in both iNOS KO and WT AMs. Statistical analysis of concentrationresponse curves found a significant tendency for greater mediator production in the iNOS KO versus WT AMs. Basal intracellular production of H2O2 and O ” . 2 was significantly greater in the iNOS KO compared to WT AMs. In contrast, LPS- (10 g/ml) or silica- (100 g/ml) stimulated intracellular oxidant production was lower in iNOS KO AMs, but overall (basal + stimulated) inflammatory capacity was similar between the cell types. The basal total antioxidant production of the iNOS KO AMs was approximately twofold higher than the WT AMs. In conclusion, certain compensatory changes appear to occur in AMs from iNOS KO mice. In response to the inability to induce NO production, iNOS KO AMs exhibit significantly higher basal generation of H2O2 and O ” . 2 as well as higher total antioxidant levels. In addition, LPSinduced TNF-á and MIP-2 production tend to be higher in AMs from iNOS KO mice. Such compensatory changes in the AM response may affect the response of iNOS KO mice to inflammatory exposures.