Role of Rac GTPase activating proteins in regulation of NADPH oxidase in human neutrophils

Precise spatiotemporal regulation of O₂⁻-generating NADPH oxidases (Nox) is a vital requirement. In the case of Nox1–3, which depend on the small GTPase Rac, acceleration of GTP hydrolysis by GTPase activating protein (GAP) could represent a feasible temporal control mechanism. Our goal was to investigate the molecular interactions between RacGAPs and phagocytic Nox2 in neutrophilic granulocytes. In structural studies we revealed that simultaneous interaction of Rac with its effector protein p67^phox and regulatory protein RacGAP was sterically possible. The effect of RacGAPs was experimentally investigated in a cell-free O₂⁻-generating system consisting of isolated membranes and recombinant p47^phox and p67^phox proteins. Addition of soluble RacGAPs decreased O₂⁻ production and there was no difference in the effect of four RacGAPs previously identified in neutrophils. Depletion of membrane-associated RacGAPs had a selective effect: a decrease in ARHGAP1 or ARHGAP25 level increased O₂⁻ production but a depletion of ARHGAP35 had no effect. Only membrane-localized RacGAPs seem to be able to interact with Rac when it is assembled in the Nox2 complex. Thus, in neutrophils multiple RacGAPs are involved in the control of O₂⁻ production by Nox2, allowing selective regulation via different signaling pathways.     

Blockade of class IA phosphoinositide 3-kinase in neutrophils prevents NADPH oxidase activation- and adhesion-dependent inflammation

We examined the role of class IA phosphoinositide 3-kinase (PI3K) in the regulation of activation of NADPH oxidase in PMNs and the mechanism of PMN-dependent lung inflammation and microvessel injury induced by the pro-inflammatory cytokine TNF-alpha. TNF-alpha stimulation of PMNs resulted in superoxide production that was dependent on CD11b/CD18-mediated PMN adhesion. Additionally, TNF-alpha induced the association of CD11b/CD18 with the NADPH oxidase subunit Nox2 (gp91(phox)) and phosphorylation of p47(phox), indicating the CD11b/CD18 dependence of NADPH oxidase activation. Transduction of wild-type PMNs with Deltap85 protein, a dominant-negative form of the class IA PI3K regulatory subunit, p85alpha, fused to HIV-TAT (TAT-Deltap85) prevented (i) CD11b/CD18-dependent PMN adhesion, (ii) interaction of CD11b/CD18 with Nox2 and phosphorylation of p47(phox), and (iii) PMN oxidant production. Furthermore, studies in mice showed that i.v. infusion of TAT-Deltap85 significantly reduced the recruitment of PMNs in lungs and increase in lung microvascular permeability induced by TNF-alpha. We conclude that class IA PI3K serves as a nodal point regulating CD11b/CD18-integrin-dependent PMN adhesion and activation of NADPH oxidase, and leads to oxidant production at sites of PMN adhesion, and the resultant lung microvascular injury in mice.     

Composition of partially purified NADPH oxidase from pig neutrophils.

The superoxide (O2.-)-forming enzyme NADPH oxidase from pig neutrophils was solubilized and partially purified by gel-filtration chromatography. The purification procedure allowed the separation of NADPH oxidase activity from NADH-dependent cytochrome c reductase and 2,6-dichlorophenol-indophenol reductase activities. O2.-forming activity was co-purified with cytochrome b-245 and was associated with phospholipids. However, active fractions endowed with cytochrome b were devoid of ubiquinone and contained only little FAD. The cytochrome b/FAD ratio was 1.13:1 in the crude solubilized extract and increased to 18.95:1 in the partially purified preparations. Most of FAD was associated with fractions containing NADH-dependent oxidoreductases. These results are consistent with the postulated role of cytochrome b in O2.-formation by neutrophil NADPH oxidase, but raise doubts about the participation of flavoproteins in this enzyme activity.     

Heat shock inhibits NADPH oxidase in human neutrophils

The heat shock response is a conserved, physiological, transient cellular response to injury. Several studies have suggested a link between the heat shock response and oxidative injury. We have investigated the effects of heat shock on superoxide anion generation by human neutrophils stimulated with opsonized zymosan or phorbol myristate acetate. Human neutrophils exposed to elevated temperatures or to the heavy metal cadmium synthetized a variety of heat shock proteins. In parallel to this protein synthesis, we observed a selective, reversible and temperature-dependent inhibition of NADPH oxidase activation, which was independent from variations of cytosolic pH or thiol group oxidation. Inhibition of NADPH oxidase by heat shock appeared related to the synthesis of heat shock proteins and may represent an intrinsic cellular mechanism to down regulate superoxide production.     

Calmodulin-dependent stimulation of the NADPH oxidase of human neutrophils

The NADPH oxidase of human neutrophils is highly sensitive to calcium concentration and is inhibited in intact cells and cell-free preparations by various phenothiazine drugs. Addition of calmodulin to preparations of NADPH oxidase stimulates enzymatic rates from 1.4-2.5-fold. Addition of calmodulin and calcium, but not calcium alone, to NADPH oxidase preparations which have been inactivated by EDTA results in the restoration of activity. No activation is observed when membrane preparations containing latent NADPH oxidase are exposed to calcium and calmodulin. These studies suggest a role for calmodulin in the control of NADPH oxidase but that calmodulin alone is not sufficient for activation.     

Calmodulin-dependents stimulation of the NADPH oxidase of human neutrophils

The NADPH oxidase of human neutrophils is highly sensitive to calcium concentration and is inhibited in intact cells and cell-free preparations by various phenothiazine drugs. Addition of calmodulin to preparations of NADPH oxidase stimulates enzymatic rates from 1.4–2.5-fold. Addition of calmodulin and calcium, but not calcium alone, to NADPH oxidase preparations which have been inactivated by EDTA results in the restoration of activity. No activation is observed when membrane preparations containing latent NADPH oxidase are exposed to calcium and calmodulin. These studies suggest a role for calmodulin in the control of NADPH oxidase but that calmodium alone is not sufficient for activation.     

Guanine nucleotides stimulate NADPH oxidase in membranes of human neutrophils

In the chain of events by which chemotactic peptides stimulate NADPH oxidase-catalyzed superoxide formation in human neutrophils, the involvements of a pertussis toxin-sensitive guanine nucleotide-binding protein (N-protein), mobilization of intracellular calcium and protein kinase C stimulation have been proposed. Superoxide formation was studied in membranes from human neutrophils; NADPH oxidase was stimulated by arachidonic acid in the presence of neutrophil cytosol. Fluoride and stable GTP analogues, such as GTP gamma S and GppNHp, which all activate N-proteins, enhanced NADPH oxidase activity up to 4-fold. GDP beta S inhibited the effect of GTP gamma S. These data suggest that NADPH oxidase is regulated by an N-protein, independent of an elevation of the cytoplasmic calcium concentration.     

Relationship between calcium release and NADPH oxidase inhibition in human neutrophils

The aim of this study was to investigate the possible relationship between NADPH oxidase activity and changes in cytosolic Ca²⁺ in response to different agonists. Treatment of neutrophils with leukotriene B4 (LTB₄) demonstrated characteristic changes to cytoslic Ca²⁺ yielding an EC₅₀ of 4 nM. The pA₂ values for the specific LTB₄ receptor (BLT) antagonists, U-75302 and LY-255283 were 6.32 and 6.38, respectively. Similarly, neutrophils treated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and platelet activating factor (PAF) exhibited changes in cytoslic Ca²⁺ in a dose dependant manner with pD₂ values of 9.0 and 9.9, respectively. The phorbol ester PMA prevented elevations in cytosolic Ca²⁺ in response to LTB₄, FMLP and PAF with IC₅₀ values of 5.88, 1.44 and 5.71 nM, respectively. In addition, potent NADPH oxidase inhibitors apocynin and diphenyleneiodonium (DPI) inhibited FMLP mediated cytosolic Ca²⁺ release. These results demonstrate that inhibition of the NADPH oxidase suppresses cytosolic Ca²⁺ release in FMLP activated human neutrophils.     

Reconstitution of GTPgammaS-induced NADPH oxidase activity in streptolysin-O-permeabilized neutrophils by specific cytosol fractions.

GTPgammaS activates the NADPH oxidase and this activity declines rapidly with time after preexposure to streptolysin O. This was not due to loss of p47(phox), p67(phox), or Rac. To identify the component(s) leaking out of the permeabilized cell responsible for loss of activity, a GTPgammaS-dependent reconstitution assay was established. Neutrophil cytosol was subjected to chromatographic fractionation steps for purification of the minimum fraction required to restore activity. The reconstitution of the GTPgammaS-stimulated activity was dependent on ATP. The inhibitors staurosporine and calphostin C greatly reduced the activity in the reconstitution assay, implicating the involvement of a protein kinase C (PKC) pathway. PKC isoforms beta and delta were eliminated as the active factors in the most pure reconstitution fraction. With this novel cell-based reconstitution assay, we have identified the requirement for a protein kinase, or its substrate, for the restoration of GTPgammaS activation of the NADPH oxidase.     

Cytosolic factors involved in the activation of NADPH oxidase from guinea pig neutrophils.

Partial purification of the cytosolic factors which are required for the activation of O2- producing enzyme (NADPH oxidase) was performed using guinea pig neutrophils. Three active cytosolic factors were obtained by using the combination of IEC-SP (cation-exchange) and IEC-QA (anion-exchange) HPLC. One factor (termed SP-1e which was adsorbed on IEC-SP column, somewhat activated the NADPH oxidase by itself. The molecular weight of SP-1 was estimated to be approximately 260 kDa. In contrast, the other two factors (termed QA-1 and QA-2, respectively), which were adsorbed on IEC-QA column, did not activate the NADPH oxidase by themselves but activated the enzyme only in the presence of SP-1. When three factors were combined, they activated the oxidase synergistically, and the activity recovered was almost the same as that observed with the unfractionated cytosol. These results suggest that at least three different cytosolic factors are required for the full activation of NADPH oxidase in guinea pig neutrophils.     

Activation of NADPH oxidase and phospholipase D in permeabilized human neutrophils. Correlation between oxidase activation and phosphatidic acid production.

A major function of human neutrophils (PMN) during inflammation is formation of oxygen radicals through activation of the respiratory burst enzyme, NADPH oxidase. Stimulus-induced production of both phosphatidic acid (PA) and diglyceride (DG) has been suggested to mediate oxidase activity; however, transductional mechanisms and cofactor requirements necessary for activation are poorly defined. We have utilized PMN permeabilized with Staphylococcus aureus alpha-toxin to elucidate the signal pathway involved in eliciting oxidase activity and to investigate whether PA or DG act as second messengers. PMN were permeabilized in cytoplasmic buffer supplemented with ATP and EGTA for 15 min before addition of NADPH and various cofactors. Oxidase activation was assessed by superoxide dismutase inhibitable reduction of ferricytochrome C; PA and DG levels were measured by radiolabeled product formation or by metabolite mass formation. Both superoxide (O2-) and PA formation were initiated by 10 microM GTP gamma S; addition of cytosolic levels of calcium ions (Ca2+, 120 nM) enhanced O2- and PA formation 1.5-2 fold. DG levels showed little change during these treatments. PA formation preceded O2- production and varying GTP gamma S levels had parallel effects on O2- and PA formation. However, while PA formation and oxidase activation occurred in tandem at Ca2+ levels of < 1 microM, higher calcium enhanced PA formation but inhibited O2- production. Removal of ATP completely blocked O2- production but had little effect on PA formation; in contrast, if ATP was replaced with ATP gamma S, parallel production of PA and O2- occurred in the absence of other cofactors. Finally, while inhibition of PA production by ethanol pretreatment led to inhibition of O2- formation in PMN treated with GTP gamma S alone, in cells stimulated with a combination of GTP gamma S and Ca2+, ethanol continued to inhibit PA formation but had no effect on O2- production. Our results do not support a role for DG in the signal transduction path leading to oxidase activation and, while we show a close correlation between oxidase activation and PA production under many physiologic conditions, we also demonstrate that PA is not sufficient to induce oxidase activation and O2- formation can occur when PA production is inhibited.     

Lectin-induced activation of plasma membrane NADPH oxidase in cholesterol-depleted human neutrophils

The gp91phox subunit of flavocytochrome b₅₅₈ is the catalytic core of the phagocyte plasma membrane NADPH oxidase. Its activation occurs within lipid rafts and requires translocation of four subunits to flavocytochrome b₅₅₈. gp91phox is the only glycosylated subunit of NADPH oxidase and no data exist about the structure or function of its glycans. Glycans, however, bind to lectins and this can stimulate NADPH oxidase activity. Given this information, we hypothesized that lectin–gp91phox interactions would facilitate the assembly of a functionally active NADPH oxidase in the absence of lipid rafts. To test this, we used lectins with different carbohydrate-binding specificity to examine the effects on H₂O₂ generation by human neutrophils treated with the lipid raft disrupting agent methyl-β-cyclodextrin (MβCD). MβCD treatment removed membrane cholesterol, caused changes in cell morphology, inhibited lectin-induced cell aggregation, and delayed lectin-induced assembly of the NADPH oxidase complex. More importantly, MβCD treatment either stimulated or inhibited H₂O₂ production in a lectin-dependent manner. Together, these results show selectivity in lectin binding to gp91phox, and provide evidence for the biochemical structures of the gp91phox glycans. Furthermore, the data also indicate that in the absence of lipid rafts, neutrophil NADPH oxidase activity can be altered by these select lectins.     

Activation of neutrophils NADPH oxidase by PMA: Cytosol activity is translocated in phorbol-primed neutrophils

• 1.1. Translocation of cytosol activity in phorbol-primed neutrophils was studied.
• 2.2. Prior exposure of PMA or FMLP could potentiate the oxidative response by subsequent heterogeneous stimulus, FMLP or PMA.
• 3.3. In FMLP-primed neutrophils, the cytosol had almost the same activity as resting one and cytosol activity was not eluted from the membrane.
• 4.4. In PMA-primed neutrophils, however, the cytosol had less activity and cytosol activity was correspondingly eluted from the membrane.
• 5.5. These observations suggested that cytosol activity was translocated in PMA-primed cells.

     

NADPH oxidase of human neutrophils. Subcellular localization and characterization of an arachidonate-activatable superoxide-generating system

The superoxide-forming NADPH oxidase of human neutrophils was studied in subcellular fractions of unstimulated cells. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions: alpha, azurophil granules; beta, mostly specific granules; gamma, plasma membrane, and cytosol. NADPH-dependent O2-. formation by these fractions was quantitated as the rate of superoxide dismutase-inhibitable reduction of ferricytochrome c. In the presence of cytosol, NADPH, and either arachidonic acid (optimum 90 microM) or sodium dodecyl sulfate (optimum 160 microM), 70-75% of the oxidase was in the beta fraction and about 25% was in the gamma fraction. A similar distribution was found for cytochrome b559 and FAD, two putative components of the oxidase. The reaction rates observed with arachidonic acid activation were sufficient to account for 25-75% of the O2-. generated by intact neutrophils. The properties of the beta and gamma enzymes were similar and closely resembled those of the oxidase in intact neutrophils or disrupted prestimulated cells. These included resistance to azide and cyanide, a pH optimum of 7.4, and a preference for NADPH (Km approximately 40-45 microM) rather than NADH (Km approximately 2.5 mM) as the electron donor. The combination of beta and gamma fractions displayed additive activity. The activatable oxidase required Mg2+ but not Ca2+. ATP was required for maximum reaction rates. When beta and gamma membranes were preincubated with cytosol and arachidonic acid in the presence of millimolar Mg2+ and then ultracentrifuged membrane-bound O2-. -forming activity was recovered in the pellet and the enzyme required only NADPH (i.e. no cytosol, arachidonic acid, or Mg2+) for expression of activity. These data suggest that cytosol contains a Mg2+-dependent oxidase-activating factor. Molecular sieve chromatography of cytosol indicated a single peak of activity (i.e. ability to activate O2-. generation by beta and/or gamma fraction) eluting with molecules of about 10,000 daltons.     

Presence of cytochrome b-245 in NADPH oxidase preparations from human neutrophils

The composition of NADPH oxidase purified by Red Sepharose chromatography of extracts from human neutrophil membranes was investigated. In contrast to that was recently reported by others, the enzyme isolated according to this procedure contained a high concentration of cytochrome b-245 and little FAD. The results reinforce the belief that cytochrome b-245 is a major component of the NADPH oxidase and plays a fundamental role in the formation of O2-by neutrophils.     

Protein kinase C phosphorylates a component of NADPH oxidase of neutrophils

A protein of 31.5 kDa belonging to the NADPH oxidase of neutrophils was phosphorylated following stimulation of the cells with phorbol myristate acetate. The same protein was phosphorylated in vitro in the presence ofcytosol and of Ca²⁺ and phosphatidylserine. The phosphorylation in vitro of the 31.5 kDa protein was increased by phorbol myristate acetate and was inhibited by trifluoperazine. The data are compatible with an involvement of protein kinase C in the activation of NADPH oxidase.

NADPH oxidase Cytochrome b₋₂₄₅ Phosphorylation Protein kinase Neutrophil activation Respiratory burst     

(-)-Epicatechin elevates nitric oxide in endothelial cells via inhibition of NADPH oxidase

Dietary (-)-epicatechin is known to improve bioactivity of (*)NO in arterial endothelium of humans, but the mode of action is unclear. We used the fluorophore 4,5-diaminofluorescein diacetate to visualize the (*)NO level in living human umbilical vein endothelial cells (HUVEC). Untreated cells showed only a weak signal, whereas pretreatment with (-)-epicatechin (10 microM) or apocynin (100 microM) elevated the (*)NO level. The effects were more pronounced when the cells were treated with angiotensin II with or without preloading of the cells with (*)NO via PAPA-NONOate. While (-)-epicatechin scavenged O2(*-), its O-methylated metabolites prevented O2(*-) generation through inhibition of endothelial NADPH oxidase activity, even more strongly than apocynin. From the effect of 3,5-dinitrocatechol, an inhibitor of catechol-O-methyltransferase (COMT), on HUVEC it is concluded that (-)-epicatechin serves as 'prodrug' for conversion to apocynin-like NADPH oxidase inhibitors. These data indicate an (*)NO-preserving effect of (-)-epicatechin via suppression of O2(*-)-mediated loss of (*)NO.     

NADPH oxidase activation and assembly during phagocytosis

Generation of superoxide (O2-) by the NADPH-dependent oxidase of polymorphonuclear leukocytes is an essential component of the innate immune response to invading microorganisms. To examine NADPH oxidase function during phagocytosis, we evaluated its activation and assembly following ingestion of serum-opsonized Neisseria meningitidis, serogroup B (NMB), and compared it with that elicited by serum-opsonized zymosan (OPZ). Opsonized N. meningitidis- and OPZ-dependent generation of reactive oxygen species by polymorphonuclear leukocytes peaked early and then terminated. Phosphorylation of p47phox coincided with peak generation of reactive oxygen species by either stimulus, consistent with a role for p47phox phosphorylation during NADPH oxidase activation, and correlated with phagosomal colocalization of flavocytochrome b558 (flavocytochrome b) and p47phox and p67phox (p47/67phox). Termination of respiratory burst activity did not reflect dephosphorylation of plasma membrane- and/or phagosome-associated p47phox; in contrast, the specific activity of phosphorylated p47phox at the phagosomal membrane increased. Most significantly, termination of oxidase activity paralleled the loss of p47/67phox from both NMB and OPZ phagosomes despite the continued presence of flavocytochrome b. These data suggest that 1) the onset of respiratory burst activity during phagocytosis is linked to the phosphorylation of p47phox and its translocation to the phagosome; and 2) termination of oxidase activity correlates with loss of p47/67phox from flavocytochrome b-enriched phagosomes and additional phosphorylation of membrane-associated p47phox.     

Effect of 2',3'-dialdehyde NADPH on activation of superoxide-producing NADPH oxidase in a cell-free system of pig neutrophils

We studied the effect of the 2',3'-dialdehyde derivative of NADPH on the activation of superoxide-producing oxidase in a cell-free system of pig neutrophils. The system consisted of a membrane fraction, two cytosolic fractions prepared by gel filtration, and arachidonic acid. Preincubation of one of the cytosolic fractions with the derivatives of NADPH and NADP+ caused the loss of its ability to activate the enzyme. The inactivation was effectively prevented by the addition of NADPH and NADP+. Neither the membrane fraction nor the other cytosolic fraction was affected by the derivatives. The results indicate that the NADPH binding component of the oxidase is present in the cytosolic fraction and may be translocated to the membrane fraction during the activation process in the cell-free system.