Phospholipase D-dependent and-independent activation of the neutrophil NADPH oxidase

Stimulation of the respiratory burst of human neutrophils by fMet-Leu-Phe (in the absence of cytochalasin B) is largely unaffected when the activities of protein kinase C and phospholipase D are inhibited. This has been confirmed using three separate assays to measure the respiratory burst. However, whilst these enzymes are not required for the initiation or maximal rate of oxidant generation, they are required to sustain oxidase activity. In contrast, in the presence of cytochalasin B, fMet-Leu-Phe stimulated oxidase activity is much more dependent on phospholipase D activity. It is proposed that (in the absence of cytochalasin B) activation of the NADPH oxidase utilises cytochrome b molecules that are already present on the plasma membrane and activation occurs independently of phospholipase D and protein kinase C. Once these complexes are inactivated, then new cytochrome b molecules must be recruited from sub-cellular stores. This translocation and/or activation of these molecules is phospholipase D dependent. Some support for this model comes from the finding that the translocation of CD11b (which co-localises with cytochrome b) onto the cell surface is phospholipase D dependent.Abbreviations GM-CSF granulocyte-macrophage colony-stimulating factor - fMet-Leu-Phe N-formylmethionyl-leucyl-phenylalanine luminol 5-amino-2,3-dihydro-1,4-phthalazinedione, O₂,-superoxide radical     

Assembly of the phagocyte NADPH oxidase

Stimulated phagocytes undergo a burst in respiration whereby molecular oxygen is converted to superoxide anion through the action of an NADPH-dependent oxidase. The multicomponent phagocyte oxidase is unassembled and inactive in resting cells but assembles at the plasma or phagosomal membrane upon phagocyte activation. Oxidase components include flavocytochrome b₅₅₈, an integral membrane heterodimer comprised of gp91phox and p22phox, and three cytosolic proteins, p47phox, p67phox, and Rac1 or Rac2, depending on the species and phagocytic cell. In a sense, the phagocyte oxidase is spatially regulated, with critical elements segregated in the membrane and cytosol but ready to undergo nearly immediate assembly and activation in response to stimulation. To achieve such spatial regulation, the individual components in the resting phagocyte adopt conformations that mask potentially interactive structural domains that might mediate productive intermolecular associations and oxidase assembly. In response to stimulation, post-translational modifications of the oxidase components release these constraints and thereby render potential interfaces accessible and interactive, resulting in translocation of the cytosolic elements to the membrane where the functional oxidase is assembled and active. This review summarizes data on the structural features of the phagocyte oxidase components and on the agonist-dependent conformational rearrangements that contribute to oxidase assembly and activation.     

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.     

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.     

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.     

Activation of guinea-pig and bovine neutrophil NADPH oxidase by N,N′-dicyclohexylcarbodiimide

Dicyclohexylcarbodiimide (DCCD) is a potent stimulant of superoxide generation in guinea-pig peritoneal and bovine blood neutrophils. The dependence of DCCD-elicited respiratory burst on the compositon of the medium was investigated. At 37°C, the superoxide generation was short-lived and a rapid losses of enzymatic activity was observed; at 0°C, the activity could be preserved for hours. Superoxide generation by whole cells was accompanied by exocytic degranulation. Prolonged incubation with DCCD at 37°C resulted also in a progressive loss of cellular integrity evidenced by the release of a fraction of lactate dehydrogenase. K_m values of the particulate NADPH oxidase isolated from DCCD-triggered guinea-pig and bovine cells were 31.7 and 50.0 μM, respectively. Cells pre-equilibrated with the potential sensitive dye Di-S-C₃-(5) exhibited changes in the transmembrane potential upon stimulation. Stimulation with DCCD resulted also in the release of membrane-associated calcium, indicated by quenching of the fluorescence of chlortetracyclineloaded neutrophils. Both effects were observed also in human neutrophils which did not generate superoxide upon exposure to DCCD. The mechanism of DCCD-induced responses is discussed.     

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.     

The oxidation of apocynin catalyzed by myeloperoxidase: proposal for NADPH oxidase inhibition

Apocynin has been used as an efficient inhibitor of the NADPH oxidase complex and its mechanism of inhibition is linked to prior activation through the action of peroxidases. Here we studied the oxidation of apocynin catalyzed by myeloperoxidase (MPO) and activated neutrophils. We found that apocynin is easily oxidized by MPO/H2O2 or activated neutrophils and has as products dimer and trimer derivatives. Since apocynin impedes the migration of the cytosolic component p47phox to the membrane and this effect could be related to its conjugation with essential thiol groups, we studied the reactivity of apocynin and its MPO-catalyzed oxidation products with glutathione (GSH). We found that apocynin and its oxidation products do not react with GSH. However, this thiol compound was efficiently oxidized by the apocynin radical during the MPO-catalyzed oxidation. We suggest that the reactivity of apocynin radical with thiol compounds could be involved in the inhibitory effect of this methoxy-catechol on NADPH oxidase complex.     

Inositol lipids and phosphatidic acid inhibit cell-free activation of neutrophil NADPH oxidase

The effect of inositol lipids on the SDS-initiated cell-free activation of NADPH oxidase in membranes of human neutrophils was investigated. In a system consisting of low density membranes, cytosol and SDS, low doses of phosphatidylinositol, phosphatidylinositol mono- and biphosphates and phosphatidic acid interfered with activation of the oxidase. The inhibition was relieved by increasing concentrations of the cytosol. Conversely, preincubation of multilamellar phosphoinositide vesicles with cytosol reduced its ability to support activation of the oxidase.     

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.     

Arachidonate supports hydrolysis of phosphatidylinositol by neutrophil cytosolic phospholipase C: relation to NADPH oxidase

NADPH oxidase is a superoxide-generating, membrane-bound complex activated in stimulated phagocytes or in a reconstituted system consisting of membranes, cytosolic components and arachidonate or SDS. To delineate mechanism of oxidase activation in the cell-free system, hydrolysis of phosphoinositides in the combined membrane-cytosol oxidase mixture was investigated. Arachidonate promoted hydrolysis of membrane-[3H]-phosphatidylinositol by cytosolic phospholipase C. PI hydrolysis was similarly supported by other unsaturated fatty acids and by SDS. Unlike activation of the NADPH oxidase, PI hydrolysis required the presence of calcium ions. Implications of these findings to the mechanism of NADPH oxidase activation are discussed.     

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.     

GTP-dependent and -independent activation of superoxide producing NADPH oxidase in a neutrophil cell-free system

GTP and GTP-gamma-S enhanced several-fold the NADPH-dependent superoxide production induced by sodium dodecyl sulfate in a cell-free system of pig neutrophils consisting of the membrane fraction and two cytosolic fractions separated by gel filtration. The enhanced activity was decreased by the addition of GDP in a dose-dependent manner, but 70% of the activity in the absence of GTP remained even at 1 mM GDP. Only one cytosol fraction besides the membrane fraction was required for the activation in the presence of GTP. The cytosol fraction was analyzed by chromatography on 2',5'-ADP agarose and two components responsible for the GTP-dependent and independent activation were separated. These results suggest that at least two pathways are available for the activation of superoxide production in the cell-free system of pig neutrophils.     

Distinct ligand-dependent roles for p38 MAPK in priming and activation of the neutrophil NADPH oxidase

In response to certain cytokines and inflammatory mediators, the activity of the neutrophil NADPH oxidase enzyme is primed for enhanced superoxide production when the cells receive a subsequent oxidase-activating stimulus. The relative role of p38 MAPK in the priming and activation processes is incompletely understood. We have developed a 2-step assay that allows the relative contributions of p38 MAPK activity in priming to be distinguished from those involved in oxidase activation. Using this assay, together with in vitro kinase assays and immunochemical studies, we report that p38 MAPK plays a critical role in TNFalpha priming of the human and porcine NADPH oxidase for superoxide production in response to complement-opsonized zymosan (OpZ), but little, if any, role in neutrophil priming by platelet-activating factor (PAF) for OpZ-dependent responses. The OpZ-mediated activation process per se is independent of p38 MAPK activity, in contrast to oxidase activation by fMLP, where 70% of the response is eliminated by p38 MAPK inhibitors regardless of the priming agent. We further report that incubation of neutrophils with TNFalpha results in the p38 MAPK-dependent phosphorylation of a subpopulation of p47(phox) and p67(phox) molecules, whereas PAF priming results in phosphorylation only of p67(phox). Despite these phosphorylations, TNFalpha priming does not result in significant association of either of these oxidase subunits with neutrophil membranes, demonstrating that the molecular basis for priming does not appear to involve preassembly of the NADPH oxidase holoenzyme/cytochrome complex prior to oxidase activation.     

A protein of neutrophil granules interferes with activation of NADPH oxidase in a cell-free system

A soluble extract of neutrophil granules interfered with activation of the NADPH oxidase in a cell-free system. The extract had no effect on superoxide production by preactivated enzyme. The inhibitory activity was retained during dialysis and was lost upon exposure to proteinase K indicating that the active substance was a protein. The inhibitor exhibited a high stability at elevated temperatures. Chromatography of granules extract on ion exchangers implied that the inhibitor was a positively charged protein eluting from S Sepharose cation exchanger above 0.4M concentration of NaCl.     

Interactions between NADPH oxidase and voltage-gated proton channels: why electron transport depends on proton transport

Leukocytes kill microbes by producing reactive oxygen species, using a multi-component enzyme complex, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Electrons pass from intracellular NADPH through a redox chain within the enzyme, to reduce extracellular O2 to O2-. Electron flux is electrogenic, and rapidly depolarizes the membrane potential. Excessive depolarization can turn off electron transport by self-inhibition, but this is prevented by proton flux that balances the electron flux. Although the membrane potential depolarizes by approximately 100 mV during the respiratory burst (NADPH oxidase activity), NADPH oxidase activity is independent of voltage in this range, which permits optimal function and prevents self-inhibition.     

Characterization of FcalphaR-triggered Ca(2+) signals: role in neutrophil NADPH oxidase activation

Human neutrophil IgA receptors (FcalphaR) trigger phagocytosis of IgA-opsonized particles and activate the NADPH oxidase complex ultimately leading to pathogen destruction. Signal transduction events triggered by FcalphaR have not been investigated in the context of NADPH oxidase activation. In this study, we show that crosslinking FcalphaR triggers the release of Ca(2+) from an intracellular store that was unchanged by the addition of extracellular EGTA. This was in contrast to the thapsigargin-triggered Ca(2+) signal, which activates store-operated Ca(2+) entry pathways (SOCP) and is sensitive to extracellular EGTA. Buffering extracellular Ca(2+) with EGTA had no effect on FcalphaR-triggered NADPH oxidase activation, suggesting that SOCP was not required for activation by FcalphaR. EGTA inhibited thapsigargin-triggered NADPH oxidase activation but had no effect on PMA-triggered responses. The intracellular Ca(2+) chelator BAPTA caused dose-dependent inhibition of both FcalphaR-triggered and thapsigargin-triggered NADPH oxidase activation but had no effect on PMA-triggered responses. Our data demonstrate that FcalphaR-triggered NADPH oxidase activation is dependent on the release of Ca(2+) from an intracellular store, but is independent of SOCP.     

A new role of Pro-73 of p47phox in the activation of neutrophil NADPH oxidase

The PX domain of p47phox is thought to be involved in autoinhibition. However, when the domain was deleted, the ability to activate the phagocyte NADPH oxidase was markedly diminished. We have mutated the proline-rich region of the PX domain and examined the mutants for the ability to activate. Substitution of Gln for Pro-73 of p47phox(1-286) (P73Q) resulted in a considerably lower activity than the wild type and P73Q had a much lower affinity for the oxidase complex. Whereas, Gln substitution for Pro-76 (P76Q) showed a slightly enhanced activation and the mutant had a slightly higher affinity for the complex than the wild type. Affinity for p67phox(1-210) was slightly decreased either by P73Q or P76Q. Optimal SDS concentration for the activation was lowered by these mutations. Binding of PX domain with phosphatidylinositol-3,4-bisphosphate was diminished by P73Q mutation. The results in this study suggest that Pro-73 has a role in interaction with the catalytic component cytochrome b558.     

Phosphatidylinositol 3-phosphate-dependent and -independent functions of p40phox in activation of the neutrophil NADPH oxidase

In response to bacterial infection, the neutrophil NADPH oxidase assembles on phagolysosomes to catalyze the transfer of electrons from NADPH to oxygen, forming superoxide and downstream reactive oxygen species (ROS). The active oxidase is composed of a membrane-bound cytochrome together with three cytosolic phox proteins, p40(phox), p47(phox), and p67(phox), and the small GTPase Rac2, and is regulated through a process involving protein kinase C, MAPK, and phosphatidylinositol 3-kinase. The role of p40(phox) remains less well defined than those of p47(phox) and p67(phox). We investigated the biological role of p40(phox) in differentiated PLB-985 neutrophils, and we show that depletion of endogenous p40(phox) using lentiviral short hairpin RNA reduces ROS production and impairs bacterial killing under conditions where p67(phox) levels remain constant. Biochemical studies using a cytosol-reconstituted permeabilized human neutrophil cores system that recapitulates intracellular oxidase activation revealed that depletion of p40(phox) reduces both the maximal rate and total amount of ROS produced without altering the K(M) value of the oxidase for NADPH. Using a series of mutants, p47PX-p40(phox) chimeras, and deletion constructs, we found that the p40(phox) PX domain has phosphatidylinositol 3-phosphate (PtdIns(3)P)-dependent and -independent functions. Translocation of p67(phox) requires the PX domain but not 3-phosphoinositide binding. Activation of the oxidase by p40(phox), however, requires both PtdIns(3)P binding and an Src homology 3 (SH3) domain competent to bind to poly-Pro ligands. Mutations that disrupt the closed auto-inhibited form of full-length p40(phox) can increase oxidase activity approximately 2.5-fold above that of wild-type p40(phox) but maintain the requirement for PX and SH3 domain function. We present a model where p40(phox) translocates p67(phox) to the region of the cytochrome and subsequently switches the oxidase to an activated state dependent upon PtdIns(3)P and SH3 domain engagement.     

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.