Neutrophil iNOS Expression Research Articles

E3 Ubiquitin Ligase Smurf1 Regulates the Inflammatory Response in Macrophages and Attenuates Hepatic Damage during Betacoronavirus Infection.

The E3 ubiquitin ligase Smurf1 catalyzes the ubiquitination and proteasomal degradation of several protein substrates related to inflammatory responses and antiviral signaling. This study investigated the role of Smurf1 in modulating inflammation induced by Betacoronavirus infection. Bone marrow-derived macrophages (BMDMs) from C57BL/6 (wild-type) or Smurf1-deficient (Smurf1-/-) mice were infected with MHV-A59 to evaluate the inflammatory response in vitro. Smurf1 was found to be required to downregulate the macrophage production of pro-inflammatory mediators, including TNF, and CXCL1; to control viral release from infected cells; and to increase cell viability. To assess the impact of Smurf 1 in vivo, we evaluated the infection of mice with MHV-A59 through the intranasal route. Smurf1-/- mice infected with a lethal inoculum of MHV-A59 succumbed earlier to infection. Intranasal inoculation with a 10-fold lower dose of MHV-A59 resulted in hematological parameter alterations in Smurf1-/- mice suggestive of exacerbated systemic inflammation. In the lung parenchyma, Smurf1 expression was essential to promote viral clearance, downregulating IFN-β mRNA and controlling the inflammatory profile of macrophages and neutrophils. Conversely, Smurf1 did not affect IFN-β mRNA regulation in the liver, but it was required to increase TNF and iNOS expression in neutrophils and decrease TNF expression in macrophages. In addition, Smurf1-/- mice exhibited augmented liver injuries, accompanied by high serum levels of alanine aminotransferase (ALT). These findings suggest that Smurf1 plays a critical role in regulating the inflammatory response in macrophages and attenuating systemic inflammation during Betacoronavirus infection.

Nitrosative stress and cytokines are linked with the severity of sepsis and organ dysfunction

ABSTRACTObjective: An imbalance in oxidant-antioxidant status may impact the severity of sepsis. We hypothesised links between nitrosative stress and pro-inflammatory cytokines and their correlation with the severity of sepsis and associated organ dysfunction.Methods: The hypothesis was tested in 110 patients with sepsis (in whom a disease severity score (APACHE II) and assessment of organ failure score (SOFA) were determined) and 55 healthy volunteers. Neutrophil inducible nitric oxide synthase (iNOS) expressions at mRNA and protein levels were estimated by real-time PCR and immuno-precipitation followed by Western blotting, respectively. Nitric oxide (NO) content was assessed in neutrophils by confocal microscopy, plasma nitrite by the Griess reaction and inflammatory cytokines (TNF-α, IFN-γ and IL-8) by ELISA (in plasma) and real-time PCR (in neutrophils). Serum bilirubin and creatinine were determined by routine methods and lung function by the PaO2/FiO2 ratio.Results: Increased neutrophil iNOS expression and NO content, plasma total nitrite content and pro-inflammatory cytokines were present in sepsis patients (all P < 0.001). Plasma nitrite correlated with cytokines, APACHE II, SOFA, PaO2/FiO2 ratio, serum bilirubin and creatinine clearance (all r2 0.63–0.85, P < 0.001). Cytokines correlated with nitrite, APACHE II, SOFA, PaO2/FiO2 ratio, serum bilirubin and creatinine clearance (all r2 0.35–0.85, P < 0.001).Conclusion: Neutrophils iNOS expression, NO content, plasma nitrite and cytokines have a role in the assessment of the severity of sepsis and organ toxicity.

The role of MAP kinases in the induction of iNOS expression in neutrophils exposed to NDMA: the involvement transcription factors

The role of MAP kinases in the activation of AP-1 (c-Jun, c-Fos) and NF-κB p65 engaged in the regulation of iNOS expression in human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA) was analyzed in the study. The study included a group of 20 healthy individuals. Isolated human PMN were incubated in the presence of NDMA. Selective MAP kinases inhibitors were used. The expression of proteins in the cytoplasmic and nuclear fractions was assessed using Western blot method. The results show that NDMA intensifies iNOS, c-Jun, NF-κB p65 and IκB-α expression in the analyzed PMNs. The blocking of the p38 pathway led to lower iNOS expression, and higher expression of c-Jun and c-Fos in the cytoplasmic fraction, and also lower c-Jun expression in the nuclear fraction of PMNs exposed to NDMA. A decrease in iNOS expression in the cytoplasmic fraction, and also c-Jun in both fractions of the examined cells, was observed as a result of JNK pathway inhibition. The blocking of the ERK5 pathway led to higher iNOS, c-Jun and c-Fos expression in the cytoplasmic fraction, and higher c-Jun expression in the nuclear fraction of PMNs exposed to NDMA. The study also demonstrated that blocking of the p38 and JNK pathways resulted in higher expression of NF-κB p65 and IκB-α in the cytoplasmic fraction and their lower expression in the nuclear fraction of these cells. Our data indicate the role of MAP kinases p38 and JNK in the activation of c-Jun and NF-κB p65 transcription factors engaged in the regulation of iNOS expression in human neutrophils exposed to NDMA. However ERK5 kinase is not involved in the regulation of iNOS and NO production by those cells.

Abstract 37: Inducible Nitric Oxide Synthase In Infiltrating Neutrophils Mediates Neurotoxicity After Ischemic Brain Injury In Mice

Nitric oxide produced by inducible nitric oxide synthase (iNOS) contributes to brain damage after cerebral ischemia and iNOS-deficient mice have smaller infarcts after middle cerebral artery occlusion (MCAO) (J Neurosci, 17:9157, 1997). However, the cell type(s) expressing iNOS and mediating tissue damage remain to be defined. We tested the hypothesis that iNOS expressed in neutrophils infiltrating the ischemic brain contributes to tissue injury. To this end, we examined the outcome of cerebral ischemia in iNOS -/- mice reconstituted with iNOS+/+ leukocytes. Lethally irradiated iNOS -/- mice were transplanted with iNOS+/+ bone marrow (BM) to produce chimeric mice expressing iNOS in peripheral leukocytes. Control chimeras were also obtained by transplanting iNOS+/+ or -/- BM in iNOS+/+ or -/- mice, respectively. Five weeks later, mice underwent transient MCAO (n=10/group) and injury volume was assessed after 3 days in cresyl violet-stained brain sections. Chimerism was 89%, indicating successful BM engraftment. iNOS -/- mice transplanted with iNOS+/+ BM had larger infarcts (40±3 mm3; Mean±SE) than iNOS -/- mice receiving iNOS -/- BM (25±3 mm3; p&lt;0.05; ANOVA), and not different from those of iNOS+/+ mice transplanted with iNOS+/+ BM (36±5 mm3; p&gt;0.05). Cell sorting demonstrated iNOS expression in neutrophils infiltrating the infarct of iNOS -/- mice transplanted with iNOS+/+ BM. To confirm that iNOS in neutrophils contributes to tissue damage, adoptive transfer experiments were performed. Neutrophils (Ly6G positive) from iNOS+/+ or -/- mice were isolated by negative selection and 5x105 cells were administered i.v. to iNOS+/+ or -/- mice 24h after MCAO (n=10/group). Consistent with the result in chimeric mice, transfer of iNOS+/+ neutrophils into iNOS -/- mice increased the infarct 3 days after MCAO (vehicle: 35±6; iNOS+/+ neutrophils: 47±6 mm3; p&gt;0.05 from iNOS+/+ mice receiving vehicle), but not when iNOS -/- neutrophils were transferred (38±8 mm3; p&gt;0.05 from iNOS -/- mice). The findings identify iNOS in infiltrating leukocytes, particularly neutrophils, as a mediator of neurotoxicity in the post-ischemic brain and establish iNOS in circulating leukocytes as a therapeutic target for ischemic brain injury. Supported by NIH grant NS34179

Role of p38 MAPK Pathway in Induction of iNOS Expression in Neutrophils and Peripheral Blood Mononuclear Cells in Patients With Squamous Cell Carcinoma of the Oral Cavity

The aim of the present study was to assess the role of the p38 mitogen-activated protein kinase (MAPK) pathway in the induction of inducible nitric oxide synthase (iNOS) expression and the production of NO by neutrophils (polymorphonuclear neutrophils [PMNs]) and peripheral blood mononuclear cells (PBMCs) in patients with squamous cell carcinoma (SCC) of the oral cavity. PMNs and PBMCs were isolated from 24 patients with SCC. The expression of iNOS and phospho-p38 MAPK was estimated by Western blotting. Total NO was measured in the cell supernatants and serum using the Griess method. The generation of superoxide anion radicals by the cells was estimated using the cytochrome-c reduction test. The cyclic guanosine monophosphate level in the cell supernatants and plasma was assessed using an enzyme-linked immunosorbent assay kit, and the concentrations of malonyldialdehyde in serum were assessed using a thiobarbituric acid method. The results of the present study of patients with stage II and III disease showed lowered expression of iNOS and phospho-p38 MAPK in PMNs and PBMCs. Moreover, in these patients, a lower production of NO by PMNs and PBMCs was observed. However, the opposite relationship was observed between the expression of phospho-p38 MAPK and iNOS in the leukocytes of patients with stage IV disease. The concentration of total NO in the PMN and PBMC supernatants of patients with advanced disease stages did not differ from that of the control group. In all the patients with SCC, a lowered ability of neutrophils to generate superoxide anion radicals and an increased production of cyclic guanosine monophosphate by PMNs and PBMCs was confirmed. Furthermore, a greater concentration of cyclic guanosine monophosphate was found in the plasma and total NO in the serum of patients with stage IV disease compared with the levels in the control group. A greater concentration of malonyldialdehyde in the serum of all patients compared with that in the control group was also observed. Our results indicate that in the leukocytes of patients with stage II and III SCC, the p38 MAPK pathway performs an essential role in the induction of iNOS expression, and the process of lipid peroxidation is not dependent on NO. In contrast, in patients with advanced-stage SCC, iNOS expression did not seem to be linked with the p38 MAPK pathway, and NO directly influenced the process of lipid peroxidation.

Exhaled NO and iNOS expression in sputum cells of healthy, obese and OSA subjects

Obstructive sleep apnoea (OSA) is associated with airways inflammation; a key role in this regard seems to be played by nitric oxide (NO). The aim of this study was to measure exhaled NO and expression of its enzyme, the inducible nitric oxide synthase (iNOS) in cells of induced sputum in OSA patients and in obese subjects without sleep apnoea and to correlate these inflammatory markers with severity of OSA. We enrolled 18 obese patients with OSA (10 men, age 48.2 +/- 8.4 years), 15 obese patients without OSA (eight men, age 52.8 +/- 11 years) and 10 healthy subjects (five men, age 42 +/- 4 years). Exhaled NO was measured using a chemiluminescence analyser; iNOS expression was measured in the sputum cells by immunocytochemistry. Exhaled NO resulted significantly increased in OSA and in obese patients (23.1 +/- 2.1 and 17.9 +/- 2.1 p.p.b.) than in healthy subjects (7.2 +/- 0.6 p.p.b.; P < 0.001). OSA and obese patients showed a higher percentage of neutrophils and a lower percentage of macrophages in the induced sputum compared to healthy subjects. In addition, OSA and obese patients showed higher iNOS expression in neutrophils and in macrophages with respect to healthy subjects. A positive correlation between exhaled NO, iNOS expression and AHI was observed. These data confirm the presence of airway inflammation in OSA and in obese patients, and suggest the possible role for NO and iNOS expression in neutrophils of the induced sputum as noninvasive markers to identify and monitor the airway inflammation in these subjects.

Enhanced iNOS expression in leukocytes and circulating endothelial cells is associated with the progression of coronary artery lesions in acute Kawasaki disease.

Nitric oxide (NO) serves many vasoprotective roles, but the massive release of NO causes arterial wall degeneration. We investigated whether enhanced nitric oxide synthase (iNOS) expression in peripheral blood leukocytes and circulating endothelial cells mirrors the progression of coronary arterial lesions in 55 children with acute Kawasaki disease (KD), including 24 with and 31 without coronary artery lesions (CAL). Patients were treated with i.v. gamma-globulin at the time of diagnosis and blood samples were collected before and after treatment. The cellular origin of NO synthesis was determined by flow cytometric analysis of iNOS expression in peripheral blood, and by immunohistochemical analysis of circulating endothelial cells and coronary arteries. iNOS expression in neutrophils peaked at the time of diagnosis, but did not peak in monocytes until 2 wk post onset of disease. Levels were significantly higher in both cell types in patients with CAL (p = 0.001 and p = 0.035, respectively). In addition, the number of circulating endothelial cells and levels of iNOS expression were higher in patients with CAL (p = 0.011 and p = 0.012, respectively). Immunohistochemical analysis of the coronary arteries from three patients with acute KD revealed iNOS immunoreactivity in endothelial cells, as well as infiltrating monocytes/macrophages in the aneurysms. We conclude that the expression of iNOS in peripheral blood leukocytes, as well as circulating endothelial cells, correlates with the severity of coronary arterial wall injury and the progression of CAL in patients with acute KD.