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.     

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.     

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.     

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.     

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.     

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.     

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.     

Activation of human neutrophil NADPH oxidase and lateral mobility of membrane proteins. A study with crosslinkers

Fluorescence photobleaching recovery was employed to investigate the relationship between the activation of neutrophil NADPH oxidase and lateral mobility of membrane proteins. Treatment of neutrophils with the crosslinking reagent disuccinimidyl suberate (DSS) blocked activation of the respiratory burst without affecting the lateral motion of concanavalin A receptors. Neutrophils treated with DSS after prestimulation with concanavalin A generated superoxide in response to another stimulator, phorbol myristate acetate, in spite of the lateral immobilization of concanavalin A receptors. The apparent lack of correlation between the activation of NADPH oxidase and the lateral motion of membrane proteins is discussed.     

Activation of human neutrophil NADPH oxidase and lateral mobility of membrane proteins: A study with crosslinkers

Fluorescence photobleaching recovery was employed to investigate the relationship between the activation of neutrophil NADPH oxidase and lateral mobility of membrane proteins. Treatment of neutrophils with the crosslinking reagent disuccinimidyl suberate (DSS) blocked activation of the respiratory burst without affecting the lateral motion of concanavalin A receptors. Neutrophils treated with DSS after prestimulation with concanavalin A generated superoxide in response to another stimulator, phorbol myristate acetate, in spite of the lateral immobilization of concanavalin A receptors. The apparent lack of correlation between the activation of NADPH oxidase and the lateral motion of membrane proteins is discussed.     

Activation of the neutrophil NADPH oxidase is inhibited by SB 203580, a specific inhibitor of SAPK2/p38.

Activation of the neutrophil NADPH oxidase by either the bacterial peptide fMLP or phorbol myristate acetate (PMA) is partially suppressed by SB 203580, a specific inhibitor of the MAP kinase family member, SAPK2/p38. The concentration of SB 203580 that suppresses activation of NADPH oxidase is similar to that which inhibits SAPK2/p38 in vitro, and both fMLP and PMA induce an extremely rapid and potent activation of SAPK2/p38 in neutrophils. SB 203580 does not exert its effect by preventing the neutrophil priming reaction, by suppressing the phosphorylation of p47phax, or by preventing the translocation of p47phax/p67phax to the plasma membrane.     

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     

The generation of hydroxyl radicals following superoxide production by neutrophil NADPH oxidase

Stimulated neutrophils generate superoxide and hydroxyl radicals. A membrane-bound NADPH oxidase, inactive in the resting state, is responsible for superoxide production. The production of hydroxyl radicals is through a secondary reaction. A Fenton-catalysed Haber—Weiss reaction is proposed. Transferrin was used as the catalyst in this investigation.     

Activation of NADPH oxidase in Alzheimer's disease brains

The present study is the first to show that superoxide (O(-)(2)) forming NADPH oxidase is activated in Alzheimer's disease (AD) brains by demonstrating the marked translocation of the cytosolic factors p47-phox and p67-phox to the membrane. In conjunction with a recent in vitro study showing that amyloid beta activates O(-)(2) forming NADPH oxidase in microglia, where these phox proteins are localized in this study, the present results suggest that, in AD, NADPH oxidase is activated in microglia, resulting in the formation of reactive oxygen species which can be toxic to neighboring neurons in AD.     

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.     

Charge-dependent regulation of NADPH oxidase activity in guinea-pig polymorphonuclear leukocytes

The mechanism of respiratory burst was studied by modulating membrane surfaces with lipophilic ions in guinea-pig polymorphonuclear leukocytes and their subcellular membranes. Positively charged alkylamines in concentration ranges of 0.5 to 15 microM (ED50 values) inhibited the O2- generation with phorbol 12-myristate 13-acetate, N-formylmethionylleucylphenylalanine, A23187, myristate and arachidonate in intact cells, and the inhibition was relieved by negatively charged agents. A similar molecular size of alkylalcohols had no effects. A similar charge-dependent O2- generation was also observed with fatty acids in subcellular membrane fractions prepared from unstimulated control cells, and this was insensitive to H-7 and W-7. These results suggest that triggering of NADPH oxidase activation involves a reaction(s) that is regulated by membrane charges.     

Activation of NADPH oxidase in human neutrophils. Synergism between fMLP and the neutrophil products PAF and LTB4

The induction of the respiratory burst in human neutrophils by combinations of fMLP and either PAF or LTB4 was studied. Pretreatment with PAF (0.0001 to 10 uM), which by itself did not elicit the burst, greatly enhanced the rate and extent of fMLP-induced superoxide production. A synergism of a different kind was observed with the reversed stimulus sequence: Pretreatment with fMLP made the neutrophils capable to respond to PAF with superoxide production. A moderate enhancement of the fMLP response was also obtained following pretreatment with LTB4. The response of the cells to LTB4, however, was not influenced by fMLP, and no synergism was observed between the two neutrophil products PAF and LTB4. The results of this study demonstrate a marked synergism between fMLP and PAF and suggest that PAF may function as an amplifier of the respiratory burst response of stimulated neutrophils.     

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.     

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.     

Inactivation of rice bran thiamine-binding protein by N,N'-dicyclohexylcarbodiimide

The addition of a carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) to thiamine-binding protein isolated from rice bran resulted in a remarkable loss of its binding activity with [14C]thiamine. Thiamine and chloroethylthiamine substantially protected the protein against inactivation by DCCD, whereas thiamine phosphates did not. Another carboxyl reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) also inactivated rice bran thiamine-binding protein. Inactivation of the thiamine-binding protein was accompanied by covalent binding of DCCD to the protein as shown by the use of [14C]DCCD. The binding of [14C]DCCD to the thiamine-binding protein was specific, and significantly inhibited by the addition of thiamine. The loss of thiamine-binding activity was proportional to the specific binding of [14C]DCCD. For complete inactivation of the thiamine-binding activity, the binding of 2.46 mol of [14C]DCCD per mol of thiamine-binding protein was required. Furthermore, limited proteolysis of the binding protein by trypsin yielded two polypeptides with molecular weights of 35,000 (large polypeptide) and 12,500 (small polypeptide) which were separated by SDS-polyacrylamide gel electrophoresis. The binding sites of [14C]DCCD were found to be located on the large polypeptide. These results suggest that a specific carboxyl residue in the large polypeptide releasable from rice bran thiamine-binding protein by trypsin digestion when modified by DCCD is involved in the binding of thiamine.