C242T CYBA polymorphism of the NADPH oxidase is associated with reduced respiratory burst in human neutrophils

Oxidative stress contributes to the pathogenesis of atherosclerosis. p22phox-based NAD(P)H oxidases exist in the vessel wall, acting as important superoxide-generating systems in the vasculature. Some studies have identified reduced atherosclerosis in the presence of the C242T CYBA polymorphism, whereas others have not. Because vascular p22phox is identical to neutrophil p22phox, we studied the association between the C242T, A640G, and -930A/G CYBA polymorphisms and the quantity of superoxide produced from neutrophils isolated from healthy adults to determine if these polymorphisms had any functional impact on NADPH oxidase function. Neutrophils were isolated from 90 subjects by Percoll density gradient centrifugation. Genotypes were determined by polymerase chain reaction (PCR) and restriction mapping, as well as real-time PCR. The oxidative burst was stimulated with phorbol 12-myristate 13-acetate. Superoxide was quantified using the superoxide dismutase inhibitable oxidation of the spin probe hydroxylamine 1-hydroxy-3-carboxy-pyrrolidine, detected by electron paramagnetic resonance. Superoxide production was significantly affected by the C242T polymorphism, being 8.7+/-0.7, 7.9+/-0.6, and 5.9+/-1.2 micromol/L per minute per 10(6) neutrophils for the C242T CC, CT, and TT genotypes, respectively (P<0.05). In contrast, the A640G and the -930A/G polymorphisms did not alter the neutrophil respiratory burst. Phagocytic respiratory burst activity in homozygous individuals with the T allele of the C242T CYBA polymorphism is significantly lower than of wild-type carriers and heterozygous individuals. Because p22phox exists in both the neutrophil and vessel wall, vascular oxidative stress is likely diminished in individuals with this polymorphism.     

Stimulation of the respiratory burst in human neutrophils by crystal phagocytosis

Exposure to monosodium urate crystals (MSU) stimulated the respiratory burst of human neutrophils as measured by increased O2 consumption and the generation of superoxide radicals (O(2)). From the comparison of data derived from nitroblue tetrazolium and cytochrome C reduction (two methods of detecting O(2) release), it appears tht O(2) production in response to MSU may be compartmentalized, i.e., occur predominantly in the intracellular space. After exposure to MSU, neutrophils from patients with chronic granulomatous disease lost viability at the normal rate; thus products of the respiratory burst are not likely to be responsible for cell death.     

Activation of the neutrophil respiratory burst oxidase

The neutrophil respiratory burst oxidase is a multicomponent activatable enzyme comprising one of the major phagocyte antimicrobial systems. In the genetic disorder chronic granulomatous disease, absent oxidase function is associated with recurrent, severe, and often life-threatening infections. The components of the oxidase system include both membrane-bound and soluble cytosolic proteins. A primary feature of stimulus-dependent activation is the translocation of a complex of cytosolic factors to the membrane, where they associate with a flavocytochrome enzyme. Interactions among the various oxidase components occur through a number of specific regions, including SH3 domains and proline-rich motifs. The fully assembled complex functions as an electron transport system, moving electrons from cytosolic NADPH to molecular oxygen to form superoxide, which, along with subsequent reactive products, exerts microbicidal and cytotoxic activities.     

Role of myeloperoxidase in the respiratory burst of human neutrophils

Myeloperoxidase (MPO), a heme enzyme present in the primary granules of polymorphonuclear leukocytes (PMNs), has been demonstrated to participate in the oxygen-dependent microbicidal activity of these cells. Evidence for the importance of MPO in this role comes in part from studies of normal PMNs treated with the heme enzyme inhibitor, sodium azide. MPO has also been suggested to regulate the respiratory activity of PMNs during phagocytosis. The role of MPO in PMN oxygen metabolism was examined by studying parameters of the respiratory burst of PMNs from a number of unrelated MPO-deficient subjects; in addition, the ability of heme enzyme inhibitors to duplicate the MPO-deficient state was studied by treating normal and MPO-deficient cells with these compounds. MPO-deficient PMNs were found to have a time-dependent hypermetabolic response as assessed by measurement of oxygen consumption, superoxide generation, hydrogen peroxide release, and hexose monophosphate shunt activity. Catabolic pathways for hydrogen peroxide were normal, suggesting the increased recovery of oxygen metabolites reflects increased production rather than decreased catabolism of H2O2. These observations support the concept that MPO may play an important role in terminating the respiratory burst of normal PMNs. The three heme enzyme inhibitors studied--sodium azide, potassium cyanide, and 3-aminotriazole--differed greatly in the degree to which they inhibited various enzymatic systems in the PMN. Nonetheless, as a group, they exerted qualitatively similar effects on oxygen metabolism of normal and of MPO-deficient PMNs. This indicates that many of the mechanisms by which heme enzyme inhibitors influence PMN metabolism are independent of the inhibition of MPO. Conclusions from studies using such treatment of PMNs should be interpreted with caution.     

Creation of a genetic system for analysis of the phagocyte respiratory burst: high-level reconstitution of the NADPH oxidase in a nonhematopoietic system

The phagocyte nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) oxidase was functionally reconstituted in monkey kidney COS-7 cells by transfection of essential subunits, gp91(phox), p22(phox), p47(phox), and p67(phox). COS-7 cells express the essential small guanosine 5'-triphosphatase, Rac1. Transgenic COS-phox cells were capable of arachidonic acid-induced NADPH oxidase activity up to 80% of that of human neutrophils, and of phorbol myristate acetate (PMA)-induced activity up to 20% of that of neutrophils. Expression of all 4 phox components was required for enzyme activity, and enzyme activation was associated with membrane translocation of p47(phox), p67(phox), and Rac1. Expression of p47(phox) Ser303Ala/Ser304Ala or Ser379Ala phosphorylation-deficient mutants resulted in significantly impaired NAPDH oxidase activity, compared with expression of wild-type p47(phox) or the p47(phox) Ser303Glu/Ser304Glu phosphorylation mimic, suggesting that p47(phox) phosphorylation contributes to enzyme activity in the COS system, as is the case in neutrophils. Hence, COS-phox cells should be useful as a new whole-cell model that is both capable of high-level superoxide production and readily amenable to genetic manipulation for investigation of NADPH oxidase function. PMA-elicited superoxide production in COS-phox cells was regulated by activation of protein kinase C (PKC) and Rac. Although COS-7 cells differ from human neutrophils in PKC isoform expression, transient expression of major neutrophil isoforms in COS-phox cells did not increase PMA-induced superoxide production, suggesting that endogenous isoforms were not rate limiting. Val204 in p67(phox), previously shown to be required for NADPH oxidase activity under cell-free conditions, was found to be essential for superoxide production by intact COS-phox cells, on the basis of transfection studies using a p67(phox) (Val204Ala) mutant.     

Tumor necrosis factor primes neutrophils by shortening the lag period of the respiratory burst

Pretreatment of neutrophils (PMNs) with low-dose tumor necrosis factor (TNF) enhances their capacity to produce oxidant radicals after stimulation with a variety of agents ('priming'). We used a continuous cytochrome C assay to investigate the superoxide production by human PMNs primed with TNF and subsequently stimulated with phorbol myristate acetate (PMA). There was no difference in the maximum rate of superoxide production by primed and unprimed PMNs stimulated with either high (2 x 10(-6) M) or low levels of PMA (2 x 10(-8) M). Following stimulation with high levels of PMA, primed PMNs demonstrated a significantly shorter lag period than unprimed cells (103.3 +/- 14.4 vs. 142.1 +/- 21.7 seconds) and larger amounts of superoxide generated in the intervals between 100 seconds (3.1 +/- 0.5 vs. 1.7 +/- 0.3 nmol/10(6) PMNs) and 300 seconds (14.9 +/- 1.2 vs. 12.2 +/- 1.1). Primed cells stimulated with low levels of PMA (2 x 10(-8) M) displayed a significantly shorter lag period (1225.8 +/- 96.8 vs. 1573.8 +/- 74.3 seconds) and a greater production of superoxide between 1300 seconds (5.4 +/- 0.9 vs. 3.0 +/- 0.4 nmol/10(6) PMNs) and 1900 seconds (25.7 +/- 4.3 vs. 14.9 +/- 2.4) than unprimed cells. These results indicate that priming of PMNs with TNF increases superoxide production during the early phase of the respiratory burst through a shortening of the post-stimulation lag period.     

Fc gamma R-stimulated activation of the NADPH oxidase: phosphoinositide-binding protein p40phox regulates NADPH oxidase activity after enzyme assembly on the phagosome

The phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b(558) and cytosolic p67(phox), p47(phox), and p40(phox) subunits that undergo membrane translocation upon cellular activation. The function of p40(phox), which binds p67(phox) in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40(phox) and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized phagosomes. To better define the role of p40(phox) in FcgammaR-induced oxidase activation, we used immunofluorescence and real-time imaging of FcgammaR-induced phagocytosis. YFP-tagged p67(phox) and p40(phox) translocated to granulocyte phagosomes before phagosome internalization and accumulation of a probe for PI3P. p67(phox) and p47(phox) accumulation on nascent and internalized phagosomes did not require p40(phox) or PI3 kinase activity, although superoxide production before and after phagosome sealing was decreased by mutation of the p40(phox) PI3P-binding domain or wortmannin. Translocation of p40(phox) to nascent phagosomes required binding to p67(phox) but not PI3P, although the loss of PI3P binding reduced p40(phox) retention after phagosome internalization. We conclude that p40(phox) functions primarily to regulate FcgammaR-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after phagosome internalization.     

Effect of Cysticercus cellulosae fractions on the respiratory burst of pig neutrophils

Neutrophils, eosinophils and macrophages are cells that interact with invading parasites and naive hosts have been shown to have anti-parasitic activity. The initial reaction of these leukocytes is the generation of reactive oxygen species (ROS) to play in parasite expulsion. The present work was carried out to study the effect of total extract, scolex and membrane fractions from Cysticercus cellulosae on respiratory burst by pig neutrophils. Hydrogen peroxide (H2O2) production by neutrophils incubated with metacestode fractions from C. cellulosae showed an increase of: 190% (total extract), 120% (scolex) and 44% (membrane). High antioxidant catalatic activity (33%, 28%, 28% by total extract, scolex and membrane, respectively) was observed in neutrophils incubated with metacestode fractions, which could be an attempt at self-protection. Scolex and membrane fractions increased the phagocytic capacity of neutrophils (44% and 28%, respectively). On the other hand, total cysticerci did not alter the phagocytosis, possibly due to modifications in membrane function, caused by high ROS production from neutrophils in the presence of total cysticerci. Total fraction from C. cellulosae is toxic for neutrophils as shown by the decrease in phagocytic capacity, probably caused by high levels of ROS formation. The difference in toxicity of total extract, scolex and membrane fractions on neutrophils can be explained by the presence of an antigenic effect of the vesicular fluid in the total extract of C. cellulosae.     

Dual granule localization of the dormant NADPH oxidase and cytochrome b559 in human neutrophils

The subcellular localization of the microbicidal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and associated b-cytochrome was investigated in human neutrophils. In unperturbed neutrophils 85% of b-cytochrome and the major part of membrane-bound components of the NADPH oxidase co-sedimented with markers for specific granules and gelatinase. Using cytochrome b559 as a marker for membrane-bound components of the NADPH oxidase in quantitative studies we observed that, of the remaining 15%, the vast majority co-sedimented with latent alkaline phosphatase, a marker for a newly identified mobilizable intracellular compartment. Only a small fraction co-localized with the plasma membranes. Azurophil granules contained a protease activity which rapidly inactivated the NADPH oxidase components present in other membranes. Stimulation of the neutrophils with formyl-methionyl-leucyl-phenyl-alanine and leukotriene B4 which caused minimal degranulation of specific granules, resulted in translocation of b-cytochrome to the plasma membrane, concomitant with incorporation of alkaline phosphatase into the plasma membrane.     

Different effects of glucose on extracellular and intracellular respiratory burst response in normal human neutrophils activated with the soluble agonist fMet-Leu-Phe.

AIMS: The study evaluated the effects of glucose concentration on the extracellular and intracellular activation of the respiratory burst in fMet-Leu-Phe-stimulated normal human neutrophils. METHODS: Specific extracellular respiratory burst activity was measured as superoxide dismutase (SOD)-inhibitable cytochrome c reduction. Intracellular respiratory burst activity was studied using luminol-enhanced chemiluminescence in the presence of SOD and catalase, to quench extracellular chemiluminescence activity. Myeloperoxidase (MPO) release from activated neutrophils was studied by using the guaiacol technique. RESULTS: The extracellular respiratory burst following activation with 1 microM fMet-Leu-Phe was significantly reduced at 15 and 25 mM D-glucose (9.5 +/- 1.0 and 8.5 +/- 0.8 nmol/10(6) cells and 10 min; P < 0.01 and P < 0.001, respectively) as compared with that at 5 mM glucose (10.3 +/- 1.0 nmol/10(6) cells and 10 min). When specifically studying the intracellular respiratory burst, no difference was found between the responses at 5, 15 or 25 mM glucose. Increasing glucose concentrations also reduced the secretion of MPO from fMet-Leu-Phe-activated neutrophils. CONCLUSIONS: Elevated glucose concentrations inhibit the generation of extracellularly released reactive oxygen metabolites but have no effects on the intracellular respiratory burst in fMet-Leu-Phe-stimulated normal human neutrophils.     

Regulation of the superoxide-forming NADPH oxidase of human neutrophils is not altered in essential hypertension.

Neutrophils possess a plasma-membrane-bound reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase which catalyzes superoxide (O2-) formation and is activated by a variety of stimuli. Recently, neutrophils of patients with essential hypertension (EHT) have been reported to generate O2- at rates up to fourfold higher than those of normotensive (NT) subjects upon exposure to the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe). We studied regulation of O2- formation in neutrophils of 25 EHT subjects and 25 age- and sex-matched NT subjects. The intercellular signal molecules fMet-Leu-Phe, platelet-activating factor and leukotriene B4 activated O2- formation in neutrophils, but the latter two receptor agonists were less effective than the former. fMet-Leu-Phe activated O2- formation with a 50% effective concentration (EC50) of about 30 nmol/l, the effect of the chemotactic peptide being maximal at 0.1-1 mumol/l. fMet-Leu-Phe-induced O2- formation was potentiated by platelet-activating factor, adenosine 5'-[gamma-thio]triphosphate and cytochalasin B and was inhibited by the activators of adenylyl cyclase, isoproterenol, prostaglandin E1 and histamine. 4 beta-Phorbol 12-myristate 13-acetate, 1,2-dioctanoyl-sn-glycerol, gamma-hexachlorocyclohexane and arachidonic acid, which circumvent receptor stimulation, also activated O2- formation. Significant differences between NT and EHT subjects were not evident in respect of any of the parameters studied. Our data suggest that regulation of the neutrophil NADPH oxidase is not disturbed in EHT and that altered O2- formation does not represent a genetic marker for abnormalities in plasma-membrane signal transduction in EHT.     

Decrease of respiratory burst in neutrophils of patients with ankylosing spondylitis by combined radon-hyperthermia treatment.

OBJECTIVE: To define the respiratory burst activity of neutrophils, the total anti-oxidative status of plasma, and the parameters of systemic inflammation in patients with ankylosing spondylitis (AS) before and after a combined radon-hyperthermia treatment in the thermal tunnels of B?ckstein-Bad Gastein in Austria. METHODS: In 20 patients with AS the effects of a total of 15 hours of radon-hyperthermia-treatment spread over a period of three weeks were studied. The respiratory burst activity of neutrophils was measured fluorometrically using dichlorofluorescein diacetate, the total anti-oxidant status was measured using azinodiethyl-benzthiazoline-sulphonate, and inflammation parameters were determined by routine laboratory assays. RESULTS: Before treatment, the basal neutrophil respiratory burst in patients (n = 20) was 409 +/- 62 fluorescence arbitrary units (AU; mean +/- SEM) and 359 +/- 37 AU in controls (n = 9; p > 0.5); the stimulated respiratory burst (fMet-Leu-Phe, 10(-6) M) was 1,027 +/- 133 AU in patients and 1,152 +/- 218 AU in controls (p > 0.5). After treatment, the basal neutrophil respiratory burst in patients (n = 19) was 137 +/- 16 and in controls it was 174 +/- 35 AU (n = 8; p > 0.1); the stimulated respiratory burst was 670 +/- 66 and 1,305 +/- 82 AU, in patients and controls respectively (p < 0.001). No effects of treatment on the total anti-oxidant status of the plasma or on the parameters of inflammation were detected. CONCLUSION: Combined radon-hyperthermia treatment reduces the respiratory burst activity of the blood circulating neutrophils in patients with AS. If respiratory burst activity from the neutrophils plays a role in the pathophysiology of ankylosing spondylitis, the observed reduction may be related to the beneficial effects of radon-hyperthermia treatment.     

Chronic oxidative stress reduces the respiratory burst response of neutrophils from beta-thalassaemia patients

Beta-thalassaemia patients are susceptible to infections by mechanisms that are not fully understood. Polymorphonuclear neutrophils (PMN) destroy microbes by producing a burst of reactive oxygen species (ROS) (respiratory burst) in response to bacterial components, as well as to phorbol-myristate-acetate (PMA). In the present study, we compared ROS generation by normal and beta-thalassaemia PMN and assessed their response to PMA. Blood cells were subjected to gelatin separation, staining with dichlorofluorescin-diacetate and flow cytometry. At basal level, the fluorescence (mean fluorescence channel) of normal and thalassaemia PMN were 12.7 +/- 4.5 and 95.6 +/- 19.8 respectively; it changed to 283.4 +/- 72.5 and 39.5 +/- 14.3, respectively, upon PMA stimulation, indicating that thalassaemia PMN have a higher basal ROS but a reduced response to PMA. When normal PMN were treated with the oxidants hydrogen peroxide and butyl-hydroxyperoxide, as well as iron and haemin, which are elevated in thalassaemia, their basal ROS increased 5-22-fold, but the PMA response was abolished. Treating thalassaemic PMN with antioxidants (N-acetyl-L-cysteine or vitamins C and E) reduced their basal ROS but enhanced their PMA response. Our findings indicate that chronically stressed PMN, e.g. in thalassaemia, have reduced capacity to elicit a respiratory burst, which may compromise their antibacterial capacity, and imply prophylactic treatment with antioxidants for recurrent infections.     

Effect of glucose on the respiratory burst of neutrophils from normal and diabetic subjects

Summary Superoxide production was enhanced in stimulated circulating neutrophils from both diabetic and normal subjects by adding glucose, but concentrations above 4 mmol/l caused a relative decline. Cells from a population of diabetics under a variety of treatment regimes had a respiratory burst equivalent to that of control cells in the presence of glucose from 0–50 mmol/l. No correlations were found between indices of hyperglycaemia (fasting plasma glucose and haemoglobin Ale, HbA1c) and the magnitude of the respiratory burst in cells from diabetics. When the isolated cells from diabetics were restored to a medium containing glucose in the original concentration of the patients' serum at phlebotomy, the rate of superoxide production was approximately doubled in every case. Incubation of cells for 1 h in the presence of 0–50 mmol/l glucose had no effect on the respiratory burst. Thus circulating neutrophils from the diabetic population under the conditions studied are just as competent as control cells in their ability to sustain superoxide production.     

Effects of oxygen tension and pH on the respiratory burst of human neutrophils.

The respiratory burst of human neutrophils was measured under conditions of hypoxia and low pH. O2 -- production by neutrophils activated with opsonized zymosan fell slowly as the oxygen concentration declined to 1%, then dropped more sharply, reaching negligible levels at oxygen concentrations less than 0.25%. Production was half maximal at an oxygen concentration of 0.35% (equivalent to approximately 10-microM dissolved oxygen). O2- production by the cell-free O2- -forming system prepared from zymosan-activated neutrophils showed a similar dependence on oxygen concentration. A drop in pH caused decreases in both oxygen consumption and O2-- production by zymosan-treated neutrophils, values at PH 6.0 being 10%--20% of those observed at pH 7.5. Experiments with the cell-free O2-- -forming system suggested that this decline in respiratory burst activity at low pH was due to inefficient activation of the O2-- -forming enzyme under acidic conditions.     

Superoxide inhibition following different stimuli of respiratory burst and metabolism of aminosalicylates in neutrophils

Reactive oxygen species such as superoxide radicals have been proposed to play an important role in the pathogenesis of inflammatory bowel disease. Some of the antiinflammatory actions of aminosalicylates have been ascribed to their capability to scavenge superoxide radicals directly or to inhibit its production in stimulated neutrophils. However, as a controversy still exists with regard to the precise mechanisms of inhibition and the metabolism within inflammatory cells, we compared scavenger properties of 5-aminosalicylic acid, 4-aminosalicylic acid,N-acetyl aminosalicylic acid, olsalazine, and benzalazine in systems with defined superoxide radical generation such as the dimethyl sulfoxide-NaOH and the potassium superoxide system. We also studied possible inhibition of the superoxide production following different stimuli of the respiratory burst in neutrophils and investigated the uptake and potential metabolism (N-acetylation) of 5-aminosalicylic acid in lipopolysaccharide-primed and resting neutrophils. We found that 5-aminosalicylic acid and 4-aminosalicylic acid had defined scavenger properties in the dimethyl sulfoxide-NaOH or potassium superoxide systems, respectively, whereas compounds with a modified aminophenolic structure had no effects. At the cellular level, 5-aminosalicylic acid inhibited phorbol myristate acetate (100 ng/ml)-activated superoxide generation to 82.3±9.3%, the formylmethionyl leucyl peptide (10^–5 M) to 61.0±6.8%, and the NaF (20 mM) -stimulated production to 32.3±3.2% (X±sd,P<0.01). the=" actions=" of=" the=" other=" drugs=" were=" less=" pronounced.=" almost=" identical=" retention=" times=">R _t=11.2 min) of³H-labeled phorbol myristate acetate in the presence and absence of 5-aminosalicylic acid revealed noin vitro interactions. 5-Aminosalicylic acid permeates cells in a dose- and time-dependent manner; there was, however, no acetylation of 5-aminosalicylic acid regardless whether the cells had been stimulated or not with lipopolysaccharide. From our results we suggest that (1) the extra- (scavenger) and intracellular inhibition of superoxide radicals by 5-aminosalicylic acid may be an important mechanism of action, (2) an intact aminophenolic structure may be necessary for such actions, and (3) the inability of inflammatory neutrophils to acetylate and, therefore, inactivate 5-aminosalicylic acid could be an important determinant for its local actions.This work was supported by a grant from Kabi-Pharmacia GmbH, Erlangen, Germany.     

Evidence for participation of vicinal dithiols in the activation sequence of the respiratory burst of human neutrophils

Phenylarsine oxide (PAO) specifically forms a stable ring complex with vicinal dithiols that can be reversed with 2,3-dimercaptopropanol (DMP). Pretreatment of human neutrophils with micromolar concentrations of PAO inhibited release of superoxide anion (O2-) stimulated by N- formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol 12-myristate 13- acetate (PMA); the inhibition was reversed with DMP, but not with 2- mercaptoethanol. PAO did not affect O2- release in previously stimulated cells. PAO did not affect the FMLP-induced Ca2+ response, suggesting that PAO affects a postreceptor event that does not modulate the Ca2+ transient. Treatment of isolated membrane or cytosolic fractions with PAO did not change the rates of arachidonate-stimulated O2- production in a cell-free system. Pretreatment of unstimulated neutrophils with PAO inactivated cytosolic protein kinase C (PKC); the inactivation was reversed with DMP. However, PAO did not affect PMA- induced translocation of beta-PKC protein or reduce the PKC activity translocated to the membrane. PAO had no effect on tyrosine kinase activity but inactivated phosphotyrosine phosphatase; stimulus-induced tyrosine phosphorylation of several proteins was markedly enhanced. These results suggest that vicinal dithiols play an essential role in activation of the respiratory burst oxidase. Possible sites for the activity of these essential vicinal dithiols include PKC and the regulatory balance of tyrosine phosphatase activity and tyrosine phosphorylation.