Neutrophil Nicotinamide Adenine Dinucleotide Phosphate Oxidase Research Articles

Enhanced effect of limb remote ischemic postconditioning combined with paeoniflorin on alleviating cerebral ischemic injury via neutrophil NADPH pathway

BackgroundLimb remote ischemic postconditioning (LRIP) and paeoniflorin (PF) both can ameliorate cerebral ischemia reperfusion (I/R) injury. At present, whether LRIP combined with PF can achieve better therapeutic effect is unknown. PurposeThis study explored the alleviating effect and mechanism of LRIP in combination with PF on cerebral I/R injury in rats. MethodsMiddle cerebral artery occlusion (MCAO) surgery was performed on rats except Sham group. Then PF (2.5 mg/kg, 5 mg/kg, 10 mg/kg) was administrated by intraperitoneal injection 10 min before the start of reperfusion. LRIP was operated on the left femoral artery at 0 h of reperfusion. Behavioral testing was used to assess neurological impairment, while TTC staining was used to examine infarct volume. Protein expression of MyD88, TRAF6, p38-MAPK and phosphorylation of p47phox in neutrophils from rat peripheral blood were tested by Western blot. Rat bone marrow neutrophils were extracted and incubated for 24 h with serum from rats after LRIP combined with PF. p38 MAPK inhibitor group was administrated SB203580 while the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor group was administrated Apocynin. Neutrophils were stimulated by fMLP (10 μM). Reactive oxygen species (ROS) production and protein expression of MyD88, TRAF6, p38 MAPK, and p47phox (ser 304 and ser 345) were detected. ResultsLRIP combined with PF (5 mg/kg) reduced cerebral infarct volume, ameliorated neurological deficit score (NDS), decreased fMLP-stimulated ROS release and downregulated the protein expression of MyD88, TRAF6, p38-MAPK and phosphorylation of p47phox (ser 304 and ser 345) in neutrophils. ConclusionThe protective effect of LRIP combined with PF on cerebral I/R injury was better than either alone. Taken together, we provided solid evidence to demonstrate that the combination of LRIP and PF had potential to alleviate cerebral I/R injury, which was regulated by MyD88-TRAF6-p38 MAPK pathway and neutrophil NADPH oxidase pathway.

Role of oxidase and antioxidant enzymes in neutrophils and blood circulation in patients with acute coronary syndrome

Acute coronary syndrome (ACS), a subcategory of cardiovascular diseases, has become a major cause of mortality and morbidity worldwide. Oxidative stress resulting from increased production of reactive oxygen species and decreased antioxidants plays a major role in the pathophysiology of ACS. This study evaluated the activities of certain oxidase and antioxidant enzymes in circulation and neutrophils to determine their roles in increased oxidative stress in patients with ACS. A total of 52 patients with ACS admitted in the coronary care unit of two tertiary hospitals and 52 healthy controls were enrolled. Blood samples were collected from all subjects, and various oxidase and antioxidant enzymes in neutrophils and circulation were assayed. The patients had significantly higher white blood cell and neutrophil counts than the controls. In patients, the mean (±SD) serum activities of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (12.83±6.6 U/L) and superoxidase dismutase (4.34±1.41 U/mL) were significantly higher than in the control group (4.88±3.03 U/L and 3.02±1.7 U/mL, respectively) while the catalase had significantly lower activities (33.36±13.16 U/mL vs. 63.98±31.86 U/mL). In neutrophils, the activities of myeloperoxidase, NADPH oxidase and catalase were significantly higher in ACS patients, while superoxidase dismutase was significantly lower. Further, significant positive correlations were found between activities of myeloperoxidase and catalase, and NADPH oxidase and superoxidase dismutase in neutrophils of ACS patients. These findings revealed that higher activities of myeloperoxidase and NADPH oxidase, both in serum and neutrophils, lead to increased oxidative stress and form the inflammatory basis of ACS, and the antioxidant enzymes combat the events.

Limb Remote Ischemic Postconditioning Reduces Ischemia-Reperfusion Injury by Inhibiting NADPH Oxidase Activation and MyD88-TRAF6-P38MAP-Kinase Pathway of Neutrophils.

Limb remote ischemic postconditioning (LRIP) has been confirmed to reduce the ischemia-reperfusion injury but its mechanisms are still not clear. This study clarified the mechanism of LRIP based on the nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase and Myeloid differentiation factor 88 (MyD88)-Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6)-P38 pathway of neutrophils. Rat middle cerebral artery occlusion (MCAO) model was used in this study. Ischemia-reperfusion injury was carried out by MCAO 1.5 h followed by 24 h reperfusion. LRIP operation was performed to the left femoral artery at 0, 1 or 3 h after reperfusion. Behavioral testing, including postural reflex test, vibrissae-elicited forelimb placing test and tail hang test, showed that LRIP operated at 0 h of reperfusion could significantly ameliorate these behavioral scores. Pathological examinations, infarct size, Myeloperoxidase (MPO) activity showed that LRIP operated at 0 h of reperfusion could significantly ameliorate the pathological scores, reduce the infarct size and MPO activity in the brain and increase the MPO activity in the left leg. By using Neutrophil counting, immunofluorescence and real-time PCR techniques, we found that LRIP operated at 0 h of reperfusion could reduce neutrophil counts in the peripheral blood and downregulate the activation of neutrophil in the peripheral blood and rat brain. Western blots revealed that MyD88, TRAF6, p38 mitogen-activated protein kinase (p38-MAPK) in neutrophils and the phosphorylation of p47phox (Ser 304 and Ser 345) in neutrophil could be downregulated by LRIP. Our study suggests that LRIP inhibits the number and activation of neutrophils in the rat brain and peripheral blood linked to down-regulating the activation of NADPH oxidase in neutrophils by MyD88/TRAF6/p38-MAPK pathway.

Role of Granulocyte-Macrophage Colony-Stimulating Factor Signaling in Regulating Neutrophil Antifungal Activity and the Oxidative Burst During Respiratory Fungal Challenge.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that plays a critical role in regulating myeloid cell host defense. In this study, we demonstrated that GM-CSF signaling plays an essential role in antifungal defense against Aspergillus fumigatus. Mice that lack the GM-CSF receptor β chain (GM-CSFRβ) developed invasive hyphal growth and exhibited impaired survival after pulmonary challenge with A. fumigatus conidia. GM-CSFRβ signaling regulated the recruitment of inflammatory monocytes to infected lungs, but not the recruitment of effector neutrophils. Cell-intrinsic GM-CSFRβ signaling mediated neutrophil and inflammatory monocyte antifungal activity, because lung GM-CSFRβ(-/-) leukocytes exhibited impaired conidial killing compared with GM-CSFRβ(+/+) counterparts in mixed bone marrow chimeric mice. GM-CSFRβ(-/-) neutrophils exhibited reduced (hydrogenated) nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in vivo. Conversely, administration of recombinant GM-CSF enhanced neutrophil NADPH oxidase function, conidiacidal activity, and lung fungal clearance in A. fumigatus-challenged mice. Thus, our study illustrates the functional role of GM-CSFRβ signaling on lung myeloid cell responses against inhaled A. fumigatus conidia and demonstrates a benefit for systemic GM-CSF administration.

Newly diagnosed chronic granulomatous disease in a 44 year old male presenting with recurrent groin cellulitis and colitis

Background Chronic Granulomatous Disease (CGD) is an inherited deficiency which results from the absence or dysfunction of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits in phagocytic cells. This condition is usually diagnosed in early childhood and presents with recurrent infections at epithelial surfaces such as the skin, lungs, and gut. We present a case of CGD which was diagnosed late in adulthood. Case presentation A forty-four year old male was admitted to hospital with fever, testicular pain and an acute onset of swelling of his penis and scrotum. His past history was significant for Crohn’s disease diagnosed at age sixteen; stable on treatment with mesalamine (Asacol W ). He also had recurrent soft tissue infections of his penis and/or scrotum since childhood, approximately 2 or 3 in total, to his recollection. There was no history of abscesses or sexually transmitted infections. However, the patient did complain of frequent upper respiratory tract infections each winter. He had no family history of primary immunodeficiency disease. During admission, he was assessed by urology and general surgery. Both confirmed the absence of any anatomic abnormalities. An immunologist suggested work up for immunodeficiency. Basic laboratory investigations were normal, with a white blood cell count of 7.8 × 10 9 L. C-reactive protein was elevated at 69.7 mg/L. Both his rheumatoid factor and HIV testing were negative. Serum levels of IgA, IgM and IgG levels were all within normal limits (Table 1 reports full details of his immunology investigations) Blood cultures were negative. A CT scan of the chest was normal. CT scan of the abdomen and pelvis revealed no subcutaneous air to suggest a necrotizing infection. Quantitative dihydrorhodamine (DHR)123 flow cytometry assay revealed the complete absence of an oxidative burst response; this was confirmed three times. This was suggestive of a neutrophil NADPH oxidase deficiency associated with CGD. The patient was treated with intravenous cefazolin and metronidazole and discharged home on clindamycin. His cellulitis resolved within weeks, but he experienced a recurrence approximately 6 months later. He was started on trimethoprim-sulfamethoxazole (TMP-SMX), amoxicillin, and itraconazole as prophylactic therapy to prevent future infections. He was assessed by the Infectious Disease service, who simplified his prophylactic regimen to TMP-SMX 160/800 q Mon/Wed/Fri and Penicillin V K 300mg OD based on risk assessment and previous infective patterns. Interferon-gamma was considered, but based on restricted availability at the time and his relatively stable clinical course to date, we elected to pursue antibiotic prophylaxis and active surveillance alone. Genetic testing, performed by the Hospital for Sick Children in Toronto, confirmed a diagnosis of CGD and revealed a homozygous mutation in the NCFI gene (P47). It was recommended that directly family members receive DHR flow cytometry screening but this was declined.

Gutkha chewing induces oxidative disturbances and cellular DNA damage in humans

Chewing of gutkha is a risk factor for several diseases. The purpose of this study was to determine the effects of gutkha on oxidative damage and antioxidant enzyme status in serum, lymphocyte, and neutrophils. Male subjects were randomly selected from university students (21–24 years) in a rural community in the District of Midnapore, West Bengal, India. In order to examine oxidative damage and antioxidant enzyme status, superoxide radical generation (), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was measured in lymphocytes and neutrophils; and in serum, neutrophils, and lymphocytes the levels of lipid peroxidation (MDA), reduced glutathione (GSH), and oxidized glutathione (GSSG) as well as activities of lactate dehydrogenase (LDH), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) were measured. Deoxyribonucleic acid (DNA) damage in lymphocytes and neutrophils was determined by diphenylamine (DPA) assay and 1.2% gel electrophoresis. There was a significant increase in generation, NADPH oxidase, LDH activity, and DPA in lymphocytes and neutrophils, and a rise in the levels of MDA and GSSG in serum, lymphocytes, and neutrophils of gutkha chewers. GSH levels, redox ratio, and the activity of SOD, CAT, GPx, and GR of serum, lymphocyte, and neutrophils in gutkha chewers were significantly decreased. Agarose gel electrophoresis showed a smeary pattern of DNA. These findings suggest that chewing of gutkha produced oxidative stress and impaired antioxidant defense system.

Differential activation of RAGE by HMGB1 modulates neutrophil-associated NADPH oxidase activity and bacterial killing

The receptor for advanced glycation end products (RAGE) plays an important role in host defense against bacterial infection. In the present experiments, we investigated the mechanisms by which RAGE contributes to the ability of neutrophils to eradicate bacteria. Wild-type (RAGE(+/+)) neutrophils demonstrated significantly greater ability to kill Escherichia coli compared with RAGE(-/-) neutrophils. After intraperitoneal injection of E. coli, increased numbers of bacteria were found in the peritoneal fluid from RAGE(-/-) as compared with RAGE(+/+) mice. Exposure of neutrophils to the protypical RAGE ligand AGE resulted in activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and enhanced killing of E. coli, and intraperitoneal injection of AGE enhanced bacterial clearance during peritonitis. However, incubation of neutrophils with high mobility group box 1 protein (HMGB1), which also binds to RAGE, diminished E. coli-induced activation of NADPH oxidase in neutrophils and bacterial killing both in vitro and in vivo. Deletion of the COOH-terminal tail of HMGB1, a region necessary for binding to RAGE, abrogated the ability of HMGB1 to inhibit bacterial killing. Incubation of neutrophils with HMGB1 diminished bacterial or AGE-dependent activation of NADPH oxidase. The increase in phosphorylation of the p40(phox) subunit of NADPH oxidase that occurred after culture of neutrophils with E. coli was inhibited by exposure of the cells to HMGB1. These results showing that HMGB1, through RAGE-dependent mechanisms, diminishes bacterial killing by neutrophils as well as NADPH oxidase activation provide a novel mechanism by which HMGB1 can potentiate sepsis-associated organ dysfunction and mortality.

G6PC3 mutations are associated with a major defect of glycosylation: a novel mechanism for neutrophil dysfunction.

Glucose-6-phosphatase, an enzyme localized in the endoplasmic reticulum (ER), catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate. In humans, there are three differentially expressed glucose-6-phosphatase catabolic genes (G6PC1-3). Recently, it has been shown that mutations in the G6PC3 gene result in a syndrome associating congenital neutropenia and various organ malformations. The enzymatic function of G6PC3 is dependent on G6P transport into the ER, mediated by G6P translocase (G6PT). Mutations in the gene encoding G6PT result in glycogen storage disease type-1b (GSD-1b). Interestingly, GSD-1b patients exhibit a similar neutrophil dysfunction to that observed in G6PC3-deficient patients. To better understand the causes of neutrophil dysfunction in both diseases, we have studied the neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of patients with G6PC3 and G6PT syndromes. Unexpectedly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments indicated hypo-glycosylation of gp91(phox), the electron-transporting component of the NADPH oxidase, in all of these patients. Rigorous mass spectrometric glycomic profiling showed that most of the complex-type antennae which characterize the neutrophil N-glycome of healthy individuals were severely truncated in the patients' neutrophils. A comparable truncation of the core 2 antenna of the O-glycans was also observed. This aberrant neutrophil glycosylation is predicted to have profound effects on the neutrophil function and merit designation of both syndromes as a new class of congenital disorders of glycosylation.

The prolyl isomerase Pin1 acts as a novel molecular switch for TNF-α–induced priming of the NADPH oxidase in human neutrophils

AbstractNeutrophils play a key role in host defense by releasing reactive oxygen species (ROS). However, excessive ROS production by neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can damage bystander tissues, thereby contributing to inflammatory diseases. Tumor necrosis factor-α (TNF-α), a major mediator of inflammation, does not activate NADPH oxidase but induces a state of hyperresponsiveness to subsequent stimuli, an action known as priming. The molecular mechanisms by which TNF-α primes the NADPH oxidase are unknown. Here we show that Pin1, a unique cis-trans prolyl isomerase, is a previously unrecognized regulator of TNF-α–induced NADPH oxidase hyperactivation. We first showed that Pin1 is expressed in neutrophil cytosol and that its activity is markedly enhanced by TNF-α. Inhibition of Pin1 activity with juglone or with a specific peptide inhibitor abrogated TNF-α–induced priming of neutrophil ROS production induced by N-formyl-methionyl-leucyl-phenylalanine peptide (fMLF). TNF-α enhanced fMLF-induced Pin1 and p47phox translocation to the membranes and juglone inhibited this process. Pin1 binds to p47phox via phosphorylated Ser345, thereby inducing conformational changes that facilitate p47phox phosphorylation on other sites by protein kinase C. These findings indicate that Pin1 is critical for TNF-α–induced priming of NADPH oxidase and for excessive ROS production. Pin1 inhibition could potentially represent a novel anti-inflammatory strategy.

Phosphorylation of threonine 154 in p40phox is an important physiological signal for activation of the neutrophil NADPH oxidase

AbstractThe neutrophil nicotinamide adenine dinucleotide phosphate-oxidase is a multisubunit enzyme (comprising gp91phox, p22phox, p67phox, p40phox, p47phox, and Rac) that plays a vital role in microbial killing. The recent discovery of a chronic granulomatous disease patient who expresses a mutant p40phox subunit, together with the development of mouse models of p40phox function, indicate phosphatidylinositol 3-phosphate binding to the PX domain of p40phox is an important signal for oxidase activation. However, the presence of other conserved residues and domains in p40phox suggest further regulatory roles for this protein. To test this, we introduced wild-type and mutated versions of p40phox into fully differentiated mouse neutrophils by retroviral transduction of p40phox−/− bone marrow progenitors and repopulation of the bone marrow compartment in radiation chimaeras. Phosphorylation of p40phox on threonine 154, but not serine 315, was required for full oxidase activation in response to formylated bacterial peptide fMLP, serum-opsonized S aureus, and immunoglobulin-opsonized sheep red blood cells. A functional SH3 domain was not required for oxidase activation, and deletion of the entire domain resulted in enhanced oxidase responses. Phosphorylation of threonine 154 in response to S aureus was mediated by protein kinase Cδ and was required for full translocation of p47phox to phagosomes. These results define an important new element in the physiological activation of the oxidase.

Hemorrhagic Shock Activates Lung Endothelial Reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase Via Neutrophil NADPH Oxidase

The vascular endothelium plays an important role in the regulation of inflammatory responses after trauma and hemorrhage. Interactions of neutrophils with endothelial cells (ECs) contribute to the activation of specific EC responses involved in innate immunity. We have previously reported that oxidants derived from the neutrophil reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a critical regulator to EC activation. Our objective was to test the role of neutrophil NADPH oxidase-derived oxidants in mediating and enhancing hemorrhagic shock (HS)-induced activation of lung endothelial NADPH oxidase. Mice were subjected to HS and neutrophil depletion. The mice were also replenished with the neutrophil from NADPH oxidase-deficient mice. The resultant activation of lung NADPH oxidase was analyzed. The in vivo studies were also recapitulated with in vitro neutrophil-EC coculture system. HS induces NADPH oxidase activation in neutrophils and lung through high-mobility group box 1/Toll-like receptor 4-dependent signaling. In neutropenic mice, shock-induced NADPH oxidase activation in the lung was reduced significantly, but was restored upon repletion with neutrophils obtained from wild-type mice subjected to shock, but not with neutrophils from shock mice lacking the gp91(phox) subunit of NADPH oxidase. The findings were recapitulated in mouse lung vascular ECs cocultured with neutrophils. The data further demonstrate that neutrophil-derived oxidants are key factors mediating augmented High mobility group box 1 (HMGB1)-induced endothelial NADPH oxidase activation through a Rac1-dependent, but p38 mitogen-activated protein kinase-independent, pathway. Oxidant signaling by neutrophil NADPH oxidase is an important determinant of activation of endothelial NADPH oxidase after HS.

Rab27a and Rab27b Regulate Neutrophil Azurophilic Granule Exocytosis and NADPH oxidase Activity by Independent Mechanisms

Neutrophils rely on exocytosis to mobilize receptors and adhesion molecules and to release microbicidal factors. This process should be strictly regulated because uncontrolled release of toxic proteins would be injurious to the host. In vivo studies showed that the small GTPase Rab27a regulates azurophilic granule exocytosis. Using mouse neutrophils deficient in Rab27a (Rab27a(ash/ash)), Rab27b [Rab27b knockout (KO)] or both [Rab27a/b double KO (DoKo)], we investigated the role of the Rab27 isoforms in neutrophils. We found that both Rab27a and Rab27b deficiencies impaired azurophilic granule exocytosis. Rab27a(ash/ash) neutrophils showed upregulation of Rab27b expression which did not compensate for the secretory defects observed in Rab27a-deficient cells, suggesting that Rab27 isoforms play independent roles in neutrophil exocytosis. Total internal reflection fluorescence microscopy analysis showed that Rab27a(ash/ash) and Rab27b KO neutrophils have a decreased number of azurophilic granules near the plasma membrane. The effect was exacerbated in Rab27a/b DoKo neutrophils. Rab27-deficient neutrophils showed impaired activation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase at the plasma membrane although intraphagosomal reactive oxygen species (ROS) production was not affected. Exocytosis of secretory vesicles in Rab27-deficient neutrophils was functional, suggesting that Rab27 GTPases selectively control the exocytosis of neutrophil granules.

Involvement of a functional NADPH oxidase in neutrophils and macrophages during programmed cell clearance: implications for chronic granulomatous disease

Resolution of inflammation requires clearance of activated neutrophils by macrophages in a manner that prevents injury to adjacent tissues. Surface changes, including phosphatidylserine (PS) exposure, may target neutrophils for phagocytosis. In this study, we show that externalization of PS is defective in phorbol myristate acetate (PMA)-activated neutrophils obtained from chronic granulomatous disease (CGD) patients with mutations in components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Moreover, activated neutrophils from CGD patients failed to undergo clearance upon cocultivation with macrophages from normal donors. In line with these results, treatment of donor neutrophils with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, blocked PMA-induced PS oxidation and externalization and prevented their engulfment by macrophages. Furthermore, primary macrophages from CGD patients or human gp91(phox)-deficient PLB-985 cells differentiated into macrophage-like cells were defective for engulfment of apoptotic target cells. Pretreatment of normal macrophages with DPI also suppressed the subsequent ingestion of PS-positive target cells. Together, these data demonstrate that NADPH oxidase plays an important role in the process of macrophage disposal of target cells (programmed cell clearance). Thus we speculate that the lack of a functional NADPH oxidase results in impaired neutrophil clearance and the exaggerated inflammation that is characteristic for CGD.

The role of respiratory burst oxidase homologues in elicitor-induced stomatal closure and hypersensitive response in Nicotiana benthamiana

Active oxygen species (AOS) are central components of the defence reactions of plants against pathogens. Plant respiratory burst oxidase homologues (RBOH) of gp91phox, a plasma membrane protein of the neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, play a prominent role in AOS production. The role of two RBOH from Nicotiana benthamiana, NbrbohA and NbrbohB that encode plant NADPH oxidase in the process of elicitor-induced stomatal closure and hypersensitive cell death is described here. NbrbohA was constitutively expressed at a low level, whereas NbrbohB was induced when protein elicitors exist (such as boehmerin, harpin, or INF1). The virus-induced gene-silencing (VIGS) method was used to produce single-silenced (NbrbohA or NbrbohB) and double-silenced (NbrbohA and NbrbohB) N. benthamiana plants. The hypersensitive response (HR) of cell death and pathogenesis-related (PR) gene expression of these gene-silenced N. benthamiana plants, induced by various elicitors, are examined. The HR cell death and transcript accumulation of genes related to the defence response (PR1) were slightly affected, suggesting that RBOH are not essential for elicitor-induced HR and activation of these genes. Interestingly, gene-silenced plants impaired elicitor-induced stomatal closure and elicitor-promoted nitric oxide (NO) production, but not elicitor-induced cytosolic calcium ion accumulation and elicitor-triggered AOS production in guard cells. These results indicate that RBOH from N. benthamiana function in elicitor-induced stomatal closure, but not in elicitor-induced HR.

Downregulation of p22phox in Retinal Pigment Epithelial Cells Inhibits Choroidal Neovascularization in Mice

Choroidal neovascularization (CNV) occurs in a variety of chorioretinal diseases including age-related macular degeneration (AMD), and is the major cause of severe visual loss in patients with AMD. Oxidative stress has been thought to play an important role in the development of CNV. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is one of the major intracellular sources of reactive oxygen species (ROS) in the vascular system. In this study, we examined the expression of p22phox, an integral subunit in the NADPH oxidase complex, in the mouse eye. We determined that p22phox is expressed in the retinal pigment epithelial (RPE) cells and inner retinal neurons. A small-interfering RNA (siRNA) designed against p22phox efficiently reduced the expression of the protein in the eye when delivered by means of recombinant adeno-associated virus (AAV) vector. Vector treatment inhibited CNV in the mouse when delivered into the subretinal space where RPE cells were transduced. These results suggest that NADPH oxidase-mediated ROS production in RPE cells may play an important role in the pathogenesis of neovascular AMD, and that this pathway may represent a new target for therapeutic intervention in AMD.

Priming of the neutrophil NADPH oxidase activation: role of p47phox phosphorylation and NOX2 mobilization to the plasma membrane

Neutrophils play an essential role in host defense against microbial pathogens and in the inflammatory reaction. Upon activation, neutrophils produce superoxide anion (O*2), which generates other reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical (OH*) and hypochlorous acid (HOCl), together with microbicidal peptides and proteases. The enzyme responsible for O2* production is called the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of two trans-membrane proteins (p22phox and gp91phox/NOX2, which form the cytochrome b558), three cytosolic proteins (p47phox, p67phox, p40phox) and a GTPase (Rac1 or Rac2), which assemble at membrane sites upon cell activation. NADPH oxidase activation in phagocytes can be induced by a large number of soluble and particulate factors. Three major events accompany NAPDH oxidase activation: (1) protein phosphorylation, (2) GTPase activation, and (3) translocation of cytosolic components to the plasma membrane to form the active enzyme. Actually, the neutrophil NADPH oxidase exists in different states: resting, primed, activated, or inactivated. The resting state is found in circulating blood neutrophils. The primed state can be induced by neutrophil adhesion, pro-inflammatory cytokines, lipopolysaccharide, and other agents and has been characterized as a "ready to go" state, which results in a faster and higher response upon exposure to a second stimulus. The active state is found at the inflammatory or infection site. Activation is induced by the pathogen itself or by pathogen-derived formylated peptides and other agents. Finally, inactivation of NADPH oxidase is induced by anti-inflammatory agents to limit inflammation. Priming is a "double-edged sword" process as it contributes to a rapid and efficient elimination of the pathogens but can also induce the generation of large quantities of toxic ROS by hyperactivation of the NADPH oxidase, which can damage surrounding tissues and participate to inflammation. In order to avoid extensive damage to host tissues, NADPH oxidase priming and activation must be tightly regulated. In this review, we will discuss some of the mechanisms of NADPH oxidase priming in neutrophils and the relevance of this process to physiology and pathology.

Apocynin, NADPH Oxidase, and Vascular Cells

Apocynin (4-hydroxy-3-methoxyacetophenone), isolated from the traditional medicinal plant Picrorhiza kurroa , is a naturally occurring methoxy-substituted catechol, experimentally used as an inhibitor of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase.1 Activated neutrophil NADPH oxidase, a multisubunit complex composed of membrane-associated gp91phox (Nox2) and p22phox and cytosolic subunits, p47phox, p67phox, and p40phox, produces superoxide anion, a precursor of microbicidal reactive oxygen species (ROS), thereby playing a crucial role in host defense. Apocynin inhibits the release of superoxide anion by NADPH oxidase by blocking migration of p47phox to the membrane, critically involved in initiating assembly of the functional NADPH oxidase complex.1 The underlying chemistry of apocynin’s actions has been elucidated in phagocytic cells.2 Apocynin needs to be activated (oxidized) for it to inhibit the oxidase. In the presence of H2O2 and myeloperoxidase (MPO), abundant in neutrophils, apocynin is oxidized and has as products dimer and trimer derivatives resulting in diapocynin formation, the metabolically active compound of apocynin. The reactivity of apocynin radical with thiol compounds is possibly the mechanism involved in the inhibitory effect of apocynin on the NADPH oxidase complex.2,3 In the early 1990s, with the discovery that cardiovascular cells possess functionally active NADPH oxidase critically involved in ROS generation and oxidative stress in the vascular wall, many investigators extended neutrophil findings and used apocynin as a pharmacological agent to specifically inhibit the vascular cell oxidase. Convincing data from numerous in vitro studies demonstrated that, in endothelial cells, vascular smooth muscle cells, and adventitial fibroblasts, apocynin blocks NADPH oxidase activity and superoxide anion generation.4,5 In vivo studies, in which mice and …

Dysregulation of innate immune receptors on neutrophils in chronic granulomatous disease

Chronic granulomatous disease (CGD) is the most common inherited disorder of neutrophil function, is caused by mutations in the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and results in recurrent bacterial infections. We sought to investigate the expression and function of innate immune receptors on neutrophils in patients with CGD. We quantified mRNA and protein expression of Toll-like receptors (TLRs), complement receptors, and chemokine receptors on neutrophils from 15 patients with CGD compared with that seen in healthy control subjects (n = 15) and control patients with bacterial pneumonia (n = 15). Phagocytosis, chemotaxis, and TLR function of isolated neutrophils were analyzed. The effect of NADPH oxidase inhibition on receptor expression and function was analyzed in control neutrophils. Neutrophils from patients with CGD had lower expression levels of TLR5, TLR9, CD11b, CD18, CD35, and CXCR1 compared with those from healthy control subjects, whereas similar or increased receptor expressions were found in patients without CGD but with bacterial pneumonia. Reduced TLR5 expression resulted in impaired neutrophil activation by bacterial flagella, reduced CD11b/CD18 expression was associated with impaired phagocytosis of Staphylococcus aureus, and reduced CXCR1 expression was associated with decreased chemotaxis. TLR5 and CD18 expression levels correlated with disease severity in patients with CGD. TLR5 and TLR9 expression were greater in patients with residual NADPH oxidase activity. Inhibition of the NADPH oxidase in control neutrophils in vitro decreased TLR5 and TLR9 expression and impaired TLR5 function. These results provide the first evidence that innate immune receptors are dysregulated in patients with CGD.

Combination of Wu Lin San and Shan Zha ameliorates substance P-induced hyperactive bladder via the inhibition of neutrophil NADPH oxidase activity

Substance P (SP) via neurokinin type 1 receptor activates leukocytes to produce burst release of reactive oxygen species (ROS) and increases leukocytes adhesion to the vessels in the inflamed bladder. Activation of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity may contribute to the neutrophil ROS production. We explored the therapeutic effect of traditional Chinese formula for urinary dysfunction, Wu Lin San (WLS), and a modified formula WLS plus Shan Zha (WLSSZ) on SP-induced bladder hyperactivity. We evaluated WLS, Shan Zha, and WLSSZ effect on neutrophils NADPH oxidase activity in SP-stimulated neutrophils in vitro, and isovolumetric cystometrogram and ROS activity in vivo in anesthetized rat bladder with SP stimulation. Our results showed that WLS, Shan Zha, and WLSSZ inhibited SP-induced NADPH oxidase activity in an order WLSSZ > Shan Zha > WLS. Exogenous SP enhanced systemic vasodilation, bladder hyperactivity and bladder ROS. One week of oral administration of WLS or WLSSZ significantly reduced SP-induced bladder ROS amount and leukocyte accumulation and ameliorated the hyperactive bladder response. The therapeutic action was better in WLSSZ than in WLS. Our results indicate that a modified formula Wu Lin San plus Shan Zha can potentially ameliorate SP-induced neurogenic inflammation possibly via the inhibition of leukocyte NADPH oxidase activity.

Structural organization of the neutrophil NADPH oxidase: phosphorylation and translocation during priming and activation

The reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is part of the microbicidal arsenal used by human polymorphonuclear neutrophils (PMNs) to eradicate invading pathogens. The production of a superoxide anion (O2-) into the phagolysosome is the precursor for the generation of more potent products, such as hydrogen peroxide and hypochlorite. However, this production of O2- is dependent on translocation of the oxidase subunits, including gp91phox, p22phox, p47phox, p67phox, p40phox, and Rac2 from the cytosol or specific granules to the plasma membrane. In response to an external stimuli, PMNs change from a resting, nonadhesive state to a primed, adherent phenotype, which allows for margination from the vasculature into the tissue and chemotaxis to the site of infection upon activation. Depending on the stimuli, primed PMNs display altered structural organization of the NADPH oxidase, in that there is phosphorylation of the oxidase subunits and/or translocation from the cytosol to the plasma or granular membrane, but there is not the complete assembly required for O2- generation. Activation of PMNs is the complete assembly of the membrane-linked and cytosolic NADPH oxidase components on a PMN membrane, the plasma or granular membrane. This review will discuss the individual components associated with the NADPH oxidase complex and the function of each of these units in each physiologic stage of the PMN: rested, primed, and activated.