The neutrophil NADPH oxidase.

The NADPH oxidase of phagocytes catalyzes the conversion of oxygen to O2(-). This multicomponent enzyme complex contains five essential protein components, two in the membrane and three in the cytosol. Unassembled and inactive in resting phagocytes, the oxidase becomes active after translocation of cytosolic components to the membrane to assemble a functional oxidase. Multiple factors regulate its assembly and activity, thus serving to maintain this highly reactive system under spatial and temporal control until recruited for antimicrobial or proinflammatory events. The recent identification of homologs of one of the membrane components in nonphagocytic cells will expand understanding of the biological contexts in which this system may function.     

RhoG regulates the neutrophil NADPH oxidase

RhoG is a Rho family small GTPase implicated in cytoskeletal regulation, acting either upstream of or in parallel to Rac1. The precise function(s) of RhoG in vivo has not yet been defined. We have identified a novel role for RhoG in signaling the neutrophil respiratory burst stimulated by G protein-coupled receptor agonists. Bone marrow-derived neutrophils from RhoG knockout (RhoG(-/-)) mice exhibited a marked impairment of oxidant generation in response to C5a or fMLP, but normal responses to PMA or opsonized zymosan and normal bacterial killing. Activation of Rac1 and Rac2 by fMLP was diminished in RhoG(-/-) neutrophils only at very early (5 s) time points (by 25 and 32%, respectively), whereas chemotaxis in response to soluble agonists was unaffected by lack of RhoG. Additionally, fMLP-stimulated phosphorylation of protein kinase B and p38MAPK, activation of phospholipase D, and calcium fluxes were equivalent in wild-type and RhoG(-/-) neutrophils. Our results define RhoG as a critical component of G protein-coupled receptor-stimulated signaling cascades in murine neutrophils, acting either via a subset of total cellular Rac relevant to oxidase activation and/or by a novel and as yet undefined interaction with the neutrophil NADPH oxidase.     

Bacillus anthracis toxins inhibit human neutrophil NADPH oxidase activity

Bacillus anthracis, the causative agent of anthrax, is a Gram-positive, spore-forming bacterium. B. anthracis virulence is ascribed mainly to a secreted tripartite AB-type toxin composed of three proteins designated protective Ag (PA), lethal factor, and edema factor. PA assembles with the enzymatic portions of the toxin, the metalloprotease lethal factor, and/or the adenylate cyclase edema factor, to generate lethal toxin (LTx) and edema toxin (ETx), respectively. These toxins enter cells through the interaction of PA with specific cell surface receptors. The anthrax toxins act to suppress innate immune responses and, given the importance of human neutrophils in innate immunity, they are likely relevant targets of the anthrax toxin. We have investigated in detail the effects of B. anthracis toxin on superoxide production by primary human neutrophils. Both LTx and ETx exhibit distinct inhibitory effects on fMLP (and C5a) receptor-mediated superoxide production, but have no effect on PMA nonreceptor-dependent superoxide production. These inhibitory effects cannot be accounted for by induction of neutrophil death, or by changes in stimulatory receptor levels. Analysis of NADPH oxidase regulation using whole cell and cell-free systems suggests that the toxins do not exert direct effects on NADPH oxidase components, but rather act via their respective effects, inhibition of MAPK signaling (LTx), and elevation of intracellular cAMP (ETx), to inhibit upstream signaling components mediating NADPH oxidase assembly and/or activation. Our results demonstrate that anthrax toxins effectively suppress human neutrophil-mediated innate immunity by inhibiting their ability to generate superoxide for bacterial killing.     

Characterisation of electron currents generated by the human neutrophil NADPH oxidase

Electron transport by the human neutrophil NADPH oxidase is an important microbicidal weapon for phagocytes. The electron current (I_e) generated by the neutrophil NADPH oxidase is poorly characterised due to the lack of appropriate electrophysiological data. In this study, I fully characterise the neutrophil generated I_e when the NADPH oxidase is activated by NADPH and GTPγS. The neutrophil I_e was markedly voltage-dependent in the entire voltage range in comparison to those electron currents measured after chloride was removed from the external bath solution. The difference in I_e measured in chloride free conditions was not due to a change in the activation kinetics of voltage-gated proton channels. The I_e depolarises the neutrophil plasma membrane at a rate of 2.3 V s⁻¹ and this depolarisation was opposed when voltage-gated proton channels are activated. 3 mM ZnCl₂ depolarised the membrane potential to +97.8 ± 2.5 mV (n = 4), and this depolarisation was abolished after NADPH oxidase inhibition.     

Exploration of the diaphorase activity of neutrophil NADPH oxidase.

In the O2- generating flavocytochrome b, the membrane-bound component of the neutrophil NADPH oxidase, electrons are transported from NADPH to O2 in the following sequence: NADPH --> FAD --> heme b -->O2. Although p-iodonitrotetrazolium (INT) has frequently been used as a probe of the diaphorase activity of the neutrophil flavocytochrome b, the propensity of its radical to interact reversibly with O2 led us to question its specificity. This study was undertaken to reexamine the interaction of INT with the redox components of the neutrophil flavocytochrome b. Two series of inhibitors were used, namely the flavin analog 5-deaza FAD and the heme inhibitors bipyridyl and benzylimidazole. The following results indicate that INT reacts preferentially with the hemes rather than with the FAD redox center of flavocytochrome b and is not therefore a specific probe of the diaphorase activity of flavocytochrome b. First, in anaerobiosis, reduced heme b in activated membranes was reoxidized by INT as efficiently as by O2 even in the presence of concentrations of 5-deaza FAD which fully inhibited the NADPH oxidase activity. Second, the titration curve of dithionite-reduced heme b in neutrophil membranes obtained by oxidation with increasing amounts of INT was strictly superimposable on that of dithionite-reduced hemin. Third, INT competitively inhibited the O2 uptake by the activated NADPH oxidase in a cell-free system. Finally, the heme inhibitor bipyridyl competitively inhibited the reduction of INT in anaerobiosis, and the oxygen uptake in aerobiosis.     

NADPH binding component of neutrophil superoxide-generating oxidase

The 2',3'-dialdehyde derivative of NADPH was used as an affinity labeling reagent of a solubilized NADPH-dependent superoxide-generating oxidase preparation of pig neutrophils. The analogue served as both an electron donor and a competitive inhibitor of the NADPH oxidase against NADPH. The apparent Michaelis constant (Km) for the derivative (31 microM) was essentially the same as that for NADPH (33 microM). The activity of the superoxide formation in the presence of 2',3'-dialdehyde NADPH was about a half of that in the presence of NADPH. Incubation of the enzyme with the derivative inactivated the superoxide-generating activity and the inactivation was prevented by the addition of NADPH. We performed the labeling of the oxidase preparation with 2',3'-dialdehyde NADPH and sodium cyanoboro[3H]hydride, based on the above results. A protein of 66,000 daltons was selectively labeled among more than 20 bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis which were visualized with Coomassie Brilliant Blue. The protein was not labeled when the oxidase preparation was pretreated with p-chloromercuribenzoate or it was labeled in the presence of excess NADPH. The protein is suggested to be the NADPH binding component of the neutrophil superoxide-generating oxidase system.     

Activity of neutrophil NADPH oxidase in iron-deficient anemia

This study was designed to measure the effects of iron supplementation on respiratory burst in iron-deficient anemia. The performance of neutrophils was evaluated by measuring the activity of NADPH oxidase in 18 patients with iron-deficient anemia before and after body iron stores are saturated. The activity of NADPH oxidase was significantly lower in pretreatment patients relative to controls (p<0.05). The activity increased after iron supplementation to levels that had no significant differences relative to controls.     

A menadione-stimulated pyridine nucleotide oxidase from resting bovine neutrophil membranes. Purification, properties, and immunochemical cross-reactivity with the human neutrophil NADPH oxidase

A menadione-stimulated, superoxide-generating enzyme was purified 127-fold from resting bovine polymorphonuclear leukocyte (neutrophil) membranes with a yield of 34%. The enzyme was extracted with Triton X-100 and purified by chromatography on DEAE-Sepharose CL-6B, NAD-agarose, and Sephacryl S-200. The purified enzyme contained FAD and had an apparent molecular mass of 93 kDa by sodium dodecyl sulfate gel electrophoresis. In a nondenaturing gel electrophoresis system, the enzyme was multimeric (Mr greater than 400,000). The oxidase showed 3-4-fold higher activity (Vm) with NADH compared with NADPH, but the Km for both pyridine nucleotides was similar (39 and 47 microM, respectively). The enzyme transferred electrons to cytochrome c, dichlorophenolindophenol, and nitro blue tetrazolium. Cytochrome c reduction was stimulated 4-fold by menadione and was inhibited 70% by superoxide dismutase. Cytochrome c reduction was not inhibited by several mitochondrial respiratory chain inhibitors (azide, cyanide, and rotenone) but was sensitive to thiol-reactive agents (p-chloromercuribenzoate and monoiodo acetate). The catalytic properties of this enzyme distinguish it from the NADPH-dependent superoxide-generating respiratory burst oxidase (NADPH-oxidase) of human neutrophils. Nevertheless, antibodies to this enzyme inhibited not only the purified menadione-stimulated oxidase, but also the respiratory burst oxidase in membranes isolated from activated human neutrophils, indicating similar antigenic determinants are shared by these enzymes. Western blots of human neutrophil membranes visualized a plasma membrane protein of molecular mass 67 kDa, corresponding in size to a protein previously reported in preparations of the human respiratory burst oxidase.     

Requirement for posttranslational processing of Rac GTP-binding proteins for activation of human neutrophil NADPH oxidase.

Rac1 and Rac2 are closely related, low molecular weight GTP-binding proteins that have both been implicated in regulation of phagocyte NADPH oxidase. This enzyme system is composed of multiple membrane-bound and cytosolic subunits and when activated catalyzes the one-electron reduction of oxygen to superoxide. Superoxide and its highly reactive derivatives are essential for killing microorganisms. Rac proteins undergo posttranslational processing, primarily the addition of an isoprenyl group to a carboxyl-terminal cysteine residue. We directly compared recombinant Rac1 and Rac2 in a human neutrophil cell-free NADPH oxidase system in which cytosol was replaced by purified recombinant cytosolic components (p47-phox and p67-phox). Processed Rac1 and Rac2 were both highly active in this system and supported comparable rates of superoxide production. Under different cell-free conditions, however, in which suboptimal amounts of cytosol were present in the assay mixture, processed Rac2 worked much better than Rac1 at all but the lowest concentrations. This suggests that a factor in the cytosol may suppress the activity of Rac1 but not of Rac2. Unprocessed Rac proteins were only weakly able to support superoxide generation in either system, but preloading of Rac1 or Rac2 with guanosine 5'-O-(3-thio-triphosphate) (GTP gamma S) restored activity. These results indicate that processing is required for nucleotide exchange but not for interaction with oxidase components.     

Sequential phospholipase activation in the stimulation of the neutrophil NADPH oxidase

Abstract Stimulation of human neutrophils with the chemotactic peptide fMet-Leu-Phe results in activation of a rapid, transient burst of oxidant secretion, which reaches a maximal rate by about 1 min after stimulation. This phase of oxidant secretion is then followed by intracellular oxidant production, which is detected by luminol chemiluminescence but not by assays such as cytochrome c reduction or scopoletin oxidation. The rapid phase of oxidant secretion requires increases in intracellular free Ca²⁺ and phospholipase A₂ activity, but not the activities of phospholipase D or D or protein kinase C. In contrast, intracellular oxidant production requires the activities of phospholipase D and protein kinase C. A model is thus proposed suggesting the sequential activation of different phospholipases which activate oxidase molecules on the plasma membrane or else from the membranes of specific granules.     

Purification and characterization of Rac 2. A cytosolic GTP-binding protein that regulates human neutrophil NADPH oxidase.

Human neutrophils and other phagocytes generate superoxide anion (O2-) as a means of destroying ingested microorganisms. O2- is produced by an NADPH-consuming oxidase composed of membrane and cytosolic components. Activation of the NADPH oxidase is absolutely dependent upon GTP, indicating the requirement for a GTP-binding protein in this process. We have utilized a five-step chromatographic procedure to isolate a GTP-binding protein from human neutrophil cytosol which can stimulate NADPH oxidase activity in a cell-free assay. Oxidase enhancing activity was shown to coisolate with this GTP-binding component, which was purified to apparent homogeneity. The GTP-binding protein was identified as Rac 2 by immunological reactivity and amino acid sequencing. Thus, Rac 2 appears to be a third cytosolic component required for human neutrophil NADPH oxidase activation. Recombinant Rac 2 was shown to bind guanine nucleotides in a Mg(2+)-dependent fashion. GDP dissociation rates were determined and shown to be regulated by the free Mg2+ concentration. Rac 2 was found to possess the highest rate of intrinsic GTP hydrolysis of any of the characterized members of the Ras superfamily. The biochemical properties of Rac 2 indicate it is likely to be subject to regulatory cofactors in vivo.     

Human neutrophil cytosolic activation factor of the NADPH oxidase. Characterization of activation kinetics

The kinetics of sodium dodecyl sulfate-induced activation of respiratory burst oxidase (NADPH oxidase) in a fully soluble cell-free system from resting (control) or phorbol myristate acetate (PMA)-stimulated human neutrophils were investigated. In a cell-free system containing solubilized membranes and cytosol fractions (cytosol) derived from control neutrophils (control cell-free system), the values of Km and Vmax for NADPH of the NADPH oxidase from control neutrophils continuously increased with increasing concentrations of cytosol, but with increasing concentrations of solubilized membranes from the control neutrophils, Km values continuously decreased, suggesting cytosolic activation factor-dependent continuous changes in the affinity of NADPH oxidase to NADPH. In a cell-free system containing solubilized membranes and cytosol prepared from PMA-stimulated neutrophils, NADPH oxidase was not activated after the addition of NADPH. However, cytosol from control neutrophils activated the NADPH oxidase of PMA-stimulated neutrophils in a cell-free system. Cytosol from PMA-stimulated neutrophils did not activate the control neutrophil oxidase, although it contained no inhibitors of NADPH oxidase activation. The results suggest that, in PMA-stimulated neutrophils, cytosolic activation factors may be consumed or exhausted with an increasing period of time after the stimulation of neutrophils, and that the affinity of PMA-stimulated neutrophil NADPH oxidase to NADPH may almost be the same as that of control neutrophil oxidase. It was concluded that the affinity of NADPH oxidase to NADPH was closely associated with interaction between solubilized membranes and cytosolic activation factors, as indicated by the concentration ratio.     

Bilirubin inhibits the activation of superoxide-producing NADPH oxidase in a neutrophil cell-free system.

We studied the effect of bilirubin on the NADPH-dependent superoxide production induced by sodium dodecyl sulfate in a cell-free system consisting of the membrane and cytosolic fractions of pig neutrophils. Preincubation of the cytosolic fraction with bilirubin before the addition of sodium dodecyl sulfate resulted in the time- and dose-dependent inhibition of the superoxide production while the preincubation of the membrane fraction with the tetrapyrrole did not result in the inhibition. When the pigment was added after the initiation of the reaction, the ongoing production was not affected by the addition. Other tetrapyrroles, such as hemin, protoporphyrin and biliverdin, also inhibited the production. The results indicate that bilirubin inhibits the activation process of the superoxide producing NADPH oxidase by decreasing the potency of the cytosolic fraction and its inhibitory effect seems to be due to the hydrophobic nature of the tetrapyrrole.     

Dialdehyde-GDP blocks activity of cytosolic components of neutrophil NADPH oxidase

Superoxide production by neutrophil NADPH oxidase activated in a cell-free system consisting of plasma membranes, cytosol and arachidonate is enhanced by nonhydrolyzable analogs of GTP and reduced by GDP. To characterize the interaction of guanine nucleotides with the system, dialdehyde analogs of GTP and GDP (oGTP and oGDP) were employed. oGDP or oGTP caused an irreversible and dose dependent inactivation of NADPH oxidase-supporting cytosolic activity. Cytosol was fractionated on S and Q Sepharose ion exchange columns into three fractions, combinations of which synergistically supported activation of NADPH oxidase. Two fractions shown by immunoblotting to contain the oxidase-linked p47 and p67 proteins were inactivated by oGDP. Labeling with [alpha-32P]-oGTP lead to incorporation of the label into several proteins.     

Anti-inflammatory action of gentamycin through inhibitory effect on neutrophil NADPH oxidase activity

The effects of gentamycin on the NADPH oxidase (EC 1.6.99.6) from human neutrophils in both whole-cell and fully soluble (cell-free) systems were investigated. Gentamycin was found to inhibit, concentration-dependently, the superoxide generation of neutrophils exposed to phorbol myristate acetate in a whole-cell system and the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate in a cell-free system. The concentrations of the drug required for 50% inhibition of the oxidase (IC₅₀) were 150 μM in the whole-cell system and 10 μM in the cell-free system. In addition, in the cell-free system, the drug did not change the K_m value for NADPH of the oxidase. However, gentamycin did not the superoxide generation of NADPH oxidase after its activation in the cell-free system, suggesting that the drug do not have superoxide-scavenger action. These results suggest that gentamycin, an aminoglycoside antibiotic, may exhibit an anti-inflammatory action due to inhibition of neutrophil NADPH oxidase activation.     

Role of NADPH oxidase versus neutrophil proteases in antimicrobial host defense

NADPH oxidase is a crucial enzyme in mediating antimicrobial host defense and in regulating inflammation. Patients with chronic granulomatous disease, an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates (ROIs), suffer from life-threatening bacterial and fungal infections. The mechanisms by which NADPH oxidase mediate host defense are unclear. In addition to ROI generation, neutrophil NADPH oxidase activation is linked to the release of sequestered proteases that are posited to be critical effectors of host defense. To definitively determine the contribution of NADPH oxidase versus neutrophil serine proteases, we evaluated susceptibility to fungal and bacterial infection in mice with engineered disruptions of these pathways. NADPH oxidase-deficient mice (p47(phox-/-)) were highly susceptible to pulmonary infection with Aspergillus fumigatus. In contrast, double knockout neutrophil elastase (NE)(-/-)×cathepsin G (CG)(-/-) mice and lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI)-deficient mice that are defective in neutrophil serine protease activation demonstrated no impairment in antifungal host defense. In separate studies of systemic Burkholderia cepacia infection, uniform fatality occurred in p47(phox-/-) mice, whereas NE(-/-)×CG(-/-) mice cleared infection. Together, these results show a critical role for NADPH oxidase in antimicrobial host defense against A. fumigatus and B. cepacia, whereas the proteases we evaluated were dispensable. Our results indicate that NADPH oxidase dependent pathways separate from neutrophil serine protease activation are required for host defense against specific pathogens.     

NADPH oxidase in human lung fibroblasts

Reactive oxygen species produced by NADPH oxidase appear to play a role in the response of human lung fibroblast cells to rhinovirus infection. The purpose of the following studies was to characterize the NADPH oxidase components in these cells, to examine the effect of rhinovirus challenge on the expression of these proteins, and to confirm previous studies suggesting a role for p47-phox in the oxidant response to rhinovirus challenge. The results revealed that the NADPH oxidase components p47-phox, p67-phox, p22-phox, and NOX4 were expressed in lung fibroblast cells. In contrast, gp91-phox was not expressed in this cell line. Expression of p67-phox was upregulated by rhinovirus challenge. The functional role of NADPH oxidase in the rhinovirus-induced oxidant stress and elaboration of IL-8 was confirmed by detection of significant reductions in oxidant stress and IL-8 elaboration following transfection of the cells with antisense nucleotides to p47-phox. The lack of gp91-phox in cultured lung fibroblast cells, the induction of p67-phox by rhinovirus, and the confirmation of participation of p47-phox in rhinovirus-induced oxidant stress are significant findings of this study and form a basis for future investigations into understanding the mechanisms of the NADPH oxidase response to rhinovirus infection.     

Purification and some properties of the 45 kDa diphenylene iodonium-binding flavoprotein of neutrophil NADPH oxidase.

The 45 kDa diphenylene iodonium-binding flavoprotein of the human neutrophil superoxide-generating oxidase has been purified by affinity chromatography. The polypeptide was eluted from Blue Memsep or 2',5'-ADP-agarose columns with either NADP or low concentrations of the specific inhibitor diphenylene iodonium. The purified protein was shown to bind FAD at a ratio of 1.09 mol of FAD/mol of protein. The reconstituted flavoprotein had a fluorescence spectrum similar, but not identical, to that of free FAD. It had an isoelectric point of approx. 4.0. The reconstituted flavoprotein displayed no diaphorase activity towards a range of artificial electron acceptors. Polyclonal antibodies raised against the pure protein inhibited superoxide generation by solubilized oxidase in a dose-dependent manner, and inhibited superoxide generation when incubated with either cytosol or membrane fractions in a reconstituted system. These antibodies precipitated the 45 kDa polypeptide together with a haem-containing 23 kDa protein thought to be the small subunit of cytochrome b-245. Antibodies raised against cytochrome P-450 reductase also precipitated these two polypeptides. These results are consistent with the 45 kDa polypeptide being the flavoprotein of the neutrophil superoxide-generating oxidase.