Restraint of spreading-dependent activation of polymorphonuclear leukocyte NADPH oxidase in an acidified environment

Elucidation of the mechanisms by which environmental pH affects or regulates the functions of polymorphonuclear leukocytes (PMNs) is important because severe acidification of the microenvironment often prevails at sites of inflammation where they act in host defense. In the present study, we investigated the effect of an acidic environment on spreading-dependent activation of O2- -producing NADPH oxidase in PMNs. We found that PMNs underwent spreading spontaneously over type I collagen and plastic surfaces at both neutral and acidic pH, although spreading over fibrinogen surfaces, for which cellular stimulation with H2O2 is required, was inhibited by acidic pH. At acidic pH, however, PMNs were unable to undergo spreading-dependent production of O2-. Pharmacological experiments showed that p38 mitogen-activated protein kinase (MAPK) was involved in the signaling pathways mediating the spreading-dependent activation of NADPH oxidase, and that its spreading-dependent phosphorylation of Thr-180 and Tyr-182, a hallmark of activation, was impaired at acidic pH. Furthermore, the inhibition by acidic pH of O2- production as well as p38 MAPK phosphorylation subsequent to spreading induction was reversible; environmental neutralization and acidification after induction of spreading at acidic and neutral pH, respectively, up- and down-regulated the two phenomena. Acidic pH did not affect the O2- production activity of NADPH oxidase pre-activated by phorbol 12-myristate 13-acetate (PMA). These results suggest that, in PMNs, the p38 MAPK-mediated signaling pathway functions as a pH-sensing regulator of spreading-dependent NADPH oxidase activation.     

Electrophoretic isolation of a membrane-bound NADPH oxidase from guinea-pig polymorphonuclear leukocytes

Electrophoretic isolation of a membrane-bound NADPH oxidase of guinea-pig polymorphonuclear leukocytes was attempted with the O2- -generating membranes of cells unstimulated or stimulated with C3b-zymosan or sodium dodecyl sulfate, and also with the phagosomes isolated from the phorbol myristate acetate-coated latex particle-phagocytosing cells. When these vesicles were subjected to discontinuous polyacrylamide gel electrophoresis in the presence of Triton X-100 and then assayed for NADPH-Nitroblue tetrazolium reducing activity, the activity was detected by the appearance of a single, blue band of the reduced dye on the gel, independent of the source of vesicles. In addition, the enzyme was able to generate O2- and its activity was significantly augmented with the homologous liver microsomal cytochrome b5. Its activity was heat-labile and inactivated by N-ethylmaleimide and p-chloromercuribenzene sulfonate. The enzyme, with an apparent molecular weight of 150 000, in the phagosomes was easily susceptible to limited proteolysis by trypsin and formed an active fragment with a molecular weight of 70 000, accompanying the loss of O2- -generating activity of the vesicles.     

The NADPH oxidase of guinea pig polymorphonuclear leucocytes

Summary NADPH oxidase from stimulated guinea pig granulocytes was extracted with deoxycholate. The solubilized enzyme was stable in 20% glycerol. Solubilized enzyme was free of myeloperoxidase activity. The properties of the deoxycholate solubilized enzyme indicated that it is a high molecular weight complex with a flavoprotein, calmodulin and cytochrome b possibly forming part of the complex. Maximum activity was between pH 7.0 and 7.5. The K_m value was 15.8 µM for NADPH and 434 µM for NADH indicating that NADPH is the preferential substrate.     

Superoxide-forming NADPH oxidase preparation of pig polymorphonuclear leucocyte.

The effect of corticosterone on myofibrillar protein breakdown in diabetic rats was investigated in order to assess the possible counteracting effects of the secondary rise in plasma insulin concentrations which normally accompanies such treatment. N^τ-Methylhistidine excretion, an index of myofibrillar protein breakdown, was compared before and after corticosterone treatment (4.0 mg/100 g body wt. per day) of normal control, adrenalectomized, 10-day-streptozotocin-diabetic and adrenalectomized diabetic rats. Diabetic rats received 1.5 units of insulin/100 g body wt. per day throughout the experiment and showed marked hyperglycaemia and glucosuria during corticosterone treatment, whereas non-diabetic rats had only mild hyperglycaemia but elevated insulin concentrations. Corticosterone treatment increased the average rate of myofibrillar protein breakdown by 68% and 95% respectively in non-diabetic and diabetic rats. Net loss of muscle non-collagen protein for the same 7-day period was greater in diabetic than in non-diabetic animals (4.15 versus 2.84% per day), and the calculated average synthesis rates were lowest in diabetic rats. Adrenalectomy had little effect except to decrease slightly the rate of muscle protein breakdown. These results show that the rise in plasma insulin concentrations that accompanies exogenous corticosterone administration to non-diabetic rats diminishes the catabolic effect of this glucocorticoid on muscle. Insulin appears to antagonize the effects of the glucocorticoid by attenuating the increased rates of myofibrillar protein breakdown and, to a lesser extent, by limiting the decrease in synthesis rates.     

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.     

Lipoxin A augments release of thromboxane from human polymorphonuclear leukocyte suspensions

Lipoxin A (LXA) is a novel eicosanoid, generated by the interactions of lipoxygenases, which has a variety of biological actions. When added to human polymorphonuclear leukocytes, LXA stimulated thromboxane formation which was monitored as TxB2 by radioimmunoassay. The compound augmented the formation of TxA2 stimulated by the ionophore of divalent cations (A23187). Formation of thromboxane was inhibited by two non-steroidal anti-inflammatory drugs (i.e. indomethacin and proglumetacin). Results of the present study indicate that LXA can provoke the release and transformation of endogenous arachidonic acid to thromboxane. Moreover, they suggest a relationship between lipoxin A and the formation of cyclooxygenase pathway products.     

Reconstituted high-density lipoprotein suppresses leukocyte NADPH oxidase activation by disrupting lipid rafts

Reconstituted discoidal high-density lipoprotein (rHDL) has potent vascular protective actions. Native HDL suppresses cellular generation of reactive oxygen species, whereas this antioxidant effect of rHDL is less clear. This study examined the effects of rHDL on NADPH oxidase, a major source of cellular superoxide generation, in both leukocytes and human umbilical vein endothelial cells. Superoxide was measured with lucigenin-enhanced chemiluminescence. Expression of NADPH oxidase sub-units was determined by real-time PCR. Pre-treatment of HL-60 cells with rHDL (10 and 25 µM) for 1 h significantly reduced phorbol 12-myristate 13-acetate-stimulated superoxide production. Treatment with rHDL for up to 24 h did not change the mRNA expression of NADPH oxidase sub-units. In HL-60 cells, depletion of cholesterol from the plasma membrane by methyl-β-cyclodextrin mimicked the effect of rHDL, whereas cholesterol repletion blunted the effects of rHDL. Treatment with rHDL induced disruption of the lipid raft structures and blunted PMA-induced redistribution of p47phox into lipid rafts. In contrast, treatment of endothelial cells with rHDL for up to 18 h had no effect on either basal or tumour necrosis factor-α-stimulated NADPH oxidase activity, but markedly suppressed the cytokine-induced expression of proinflammatory adhesion molecules. The results suggest that rHDL inhibits NADPH oxidase activation in leukocytes, probably by interrupting the assembly of NADPH oxidase sub-units at the lipid rafts. This effect may contribute to the vascular protective actions of rHDL against inflammation-mediated oxidative damage.     

Involvement of membrane charges in constituting the active form of NADPH oxidase in guinea pig polymorphonuclear leukocytes

NADPH oxidase activity in a membrane fraction prepared from phorbol 12-myristate 13-acetate (PMA)-stimulated guinea pig polymorphonuclear leukocytes (PMNL) was inhibited by positively charged myristylamine. The inhibitory effect of myristylamine was significantly suppressed by simultaneous addition of a negatively charged fatty acid, such as myristic acid. However, the suppression by myristylamine was not sufficiently restored when myristic acid was added later. On the other hand, pretreatment of PMA-stimulated PMNL with glutaraldehyde, a protein crosslinking reagent, stabilized NADPH oxidase activity against inhibition by myristylamine, but not against that by p-chloromercuribenzenesulfonic acid. In a cell-free system of reconstituted plasma membrane and cytosolic fractions prepared from unstimulated PMNL, arachidonic acid-stimulated NADPH oxidase activity was also inhibited by myristylamine. During the activation of NADPH oxidase by PMA in intact PMNL and by arachidonic acid in the cell-free system, cytosolic activation factor(s) translocated to plasma membranes. The bound cytosolic activation factor(s) was released from the membranes by myristylamine, accompanied by a loss of NADPH oxidase activity. It is plausible from these results that the inhibitory effect of alkylamine on NADPH oxidase is due to induction of the decoupling and/or dissociation of the cytosolic activation component(s) from the activated NADPH oxidase complex by increments of positive charges in the membranes, and that the glutaraldehyde treatment prevents the dissociation of component(s).     

The NADPH oxidase of human polymorphonuclear leukocytes. Evidence for regulation by multiple signals

Activation of the membrane-bound NADPH oxidase in human polymorphonuclear leukocytes can be triggered by chemoattractants, the tumor promoter phorbol myristate acetate or the calcium ionophore A23187. We have shown previously that these stimuli have markedly different temporal patterns of oxidase activation (McPhail, L. C., and Snyderman, R. (1983) J. Clin. Invest. 72, 192-200), suggesting that each follows, at least in part, a unique transductional pathway. We now report that if leukocytes were sequentially exposed to any of several combinations of heterologous stimuli, the pattern of activation by the second stimulus was strikingly altered, resulting in a more rapid rate and enhanced level of oxidase activation by the second stimulus. This suggests that exposure of cells to the first stimulus (priming) had influenced an intermediate also used by the second stimulus. The signal for priming could be clearly distinguished from the signal causing oxidase activation by the dose-response curves for each, as well as by the use of several pharmacologic agents. In addition, if leukocytes were given sequential doses of homologous stimuli, either partial (phorbol myristate acetate) or full (N-formyl-methionyl-leucyl -phenylalanine and A23187) desensitization of oxidase activation was observed. These results demonstrate that these stimuli share a common intermediate in the pathway of oxidase activation. Moreover, the data indicate that NADPH oxidase activation is regulated by at least three distinct signals: signal 1 (priming), signal 2 (activation), and signal 3 (inactivation). It is likely that more than one intracellular messenger exerts a modulating influence on NADPH oxidase activity and that its regulation involves the interplay between several cellular control proteins.     

Effect of glutaraldehyde on NADPH oxidase system of guinea pig polymorphonuclear leukocytes

In an attempt to elucidate properties and activation mechanisms of the NADPH oxidase system, which is known to be responsible for the production of superoxide anion (O2-) in cell membranes of polymorphonuclear leukocytes (PMNL), intact guinea pig PMNL were treated with glutaraldehyde, a protein crosslinking reagent, before or after stimulation with phorbol 12-myristate 13-acetate (PMA). Then, PMNL were disrupted and NADPH oxidase activity was measured. After the treatment of resting PMNL with glutaraldehyde, NADPH oxidase was no longer activated by PMA. On the other hand, the NADPH oxidase activity enhanced by PMA in advance was markedly retained by the glutaraldehyde treatment of such PMA-stimulated PMNL as compared to that in untreated cells. Similar retention by glutaraldehyde of the stimulated NADPH oxidase activity was observed in PMNL stimulated by formyl-methionyl-leucyl-phenylalanine (FMLP) and cytochalasin D. Furthermore, the oxidase activity of glutaraldehyde-treated PMNL was stable during incubation at 37 degrees C, the half life of the oxidase activity of the treated PMNL being more than 90 min whereas that of the untreated PMNL is about 15 min. This ability of the glutaraldehyde treatment to retain the activity was also observed against inactivation by high concentrations of NaCl and by positively charged alkylamine.     

Effect of nedocromil sodium on polymorphonuclear leukocyte plasma membrane

The effect of nedocromil sodium on the plasma membrane fluidity of polymorphonuclear leukocytes (PMNs) was investigated by measuring steady-state fluorescence anisotropy of 1-[4-trimethylammonium-phenyl]-6-phenyl- 1,3,5-hexatriene (TMA-DPH) incorporated in the membrane. Our results show that nedocromil sodium 300 muM significantly decreased membrane fluidity of PMNs. The decrease in membrane fluidity of PMNs induced by fMLP was abolished in the presence of nedocromil sodium. These data suggest that nedocromil sodium interferes with the plasma membranes of PMNs and modulates their activities.     

NADPH dehydrogenase activity of p67PHOX, a cytosolic subunit of the leukocyte NADPH oxidase

The leukocyte NADPH oxidase catalyzes the one-electron reduction of oxygen to O2- at the expense of NADPH. It is a multicomponent enzyme comprising a membrane-bound flavocytochrome (cytochrome b558) and at least four cytosolic components: p47PHOX, p67PHOX, p40PHOX, and Rac, a small GTPase. All the oxidase components except p40PHOX are required for enzyme activity. Many aspects of their function, however, are unclear. Using the electron acceptor ferricyanide, we found that recombinant p67PHOX from baculovirus-infected Sf9 cells could mediate the dehydrogenation of NADPH. NADPH dehydrogenation was not dependent on FAD and was insensitive to superoxide dismutase. Several control experiments showed that NADPH dehydrogenation was accomplished by p67PHOX, not by a trace contaminant in the p67PHOX preparation. The NADPH dehydrogenase activity of p67PHOX was proportional to enzyme concentration, and showed saturation kinetics with NADPH (Km 92 +/- 5 microM), but was inhibited at high concentrations of ferricyanide. NADH was also used as a substrate by p67PHOX (Km 123 +/- 38 microM). Taken together, these results show that p67PHOX is able to mediate pyridine nucleotide dehydrogenation. These findings raise the possibility that p67PHOX might participate directly in electron transfer between NADPH and the oxidase flavin.     

Activation of guinea pig polymorphonuclear leukocytes with soluble stimulators leads to nonrandom distribution of NADPH oxidase in the plasma membrane

Guinea pig polymorphonuclear leukocytes (PMN) were briefly activated with soluble stimulators such as sodium myristate (SM) or phorbol myristate acetate (PMA) and then disrupted by the nitrogen cavitation method to study the subcellular distribution of NADPH oxidase, which is responsible for O2 - generation. Fc-receptor and 5'-nucleotidase activities were measured as plasma membrane markers. 1) The homogenate was first fractionated by differential centrifugation. The O2- -generating activity of PMN activated either by SM or PMA was recovered in a 2 X 10(4) g pellet which contained a large amount of granules and about 50% of the plasma membrane markers, but not in a 1 X 10(5) g pellet which consisted of plasma membranes and few granules. 2) Further separation of the 2 X 10(4) g pellet from PMA-activated PMN was attempted by an iso-osmotic Percoll density gradient centrifugation. The O2- -generating activity was recovered in light fractions in which plasma membrane markers were found, but neither in specific nor in azurophil granules. The 1 X 10(5) g pellet showed a similar distribution of the plasma membrane markers to that of the 2 X 10(4) g pellet, except that the peak of the O2- -generating activity was much smaller on an identical density gradient. The results showed that NADPH oxidase is located in the plasma membranes precipitated by centrifugation at 2 X 10(4) X g but not in the ones precipitated at 1 X 10(5) X g. The results suggest that the plasma membrane of activated PMN has a mosaic distribution of NADPH oxidase.     

Mechanism of hydrogen peroxide formation catalyzed by NADPH oxidase in thyroid plasma membrane

The thyroid plasma membrane contains a Ca2(+)-regulated NADPH-dependent H2O2 generating system which provides H2O2 for the thyroid peroxidase-catalyzed biosynthesis of thyroid hormones. The plasma membrane fraction contains a Ca2(+)-independent cytochrome c reductase activity which is not inhibited by superoxide dismutase. But it is not known whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of super-oxide anion (O2-). Indirect evidence from electron scavenger studies indicate that the H2O2 generating system does not liberate O2-, but studies using the modified peroxidase, diacetyldeuteroheme horseradish peroxidase, to detect O2- indicate that H2O2 is provided via the dismutation of O2-. The present results provide indirect evidence that the cytochrome c reductase activity is not a component of the NADPH-dependent H2O2 generator, since it was removed by washing the plasma membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid without affecting H2O2 generation. Spectral studies with diacetyldeuteroheme-substituted horseradish peroxidase showed that the thyroid NADPH-dependent H2O2 generator does not catalyze superoxide anion formation. The O2- adduct compound (compound III) was formed but was completely inhibited by catalase, indicating that the initial product was H2O2. The rate of NADPH oxidation also increased in the presence of diacetylheme peroxidase. This increase was blocked by catalase and was greatly enhanced by superoxide dismutase. The O2- adduct compound (compound III) was produced in the presence of NADPH when glucose-glucose oxidase (which does not produce O2-) was used as the H2O2 generator. NADPH oxidation occurred simultaneously and was enhanced by superoxide dismutase. We conclude that O2- formation occurs in the presence of an H2O2 generator, diacetylheme peroxidase and NADPH, but that it is not the primary product of the H2O2 generator. We suggest that O2- formation results from oxidation of NADPH, catalyzed by the diacetylheme peroxidase compound I, producing NADP degree, which in turn reacts with O2 to give O2-.     

Platelet release products modulate some aspects of polymorphonuclear leukocyte activation.

The aim of this research was to evaluate in vitro interactions between platelets and polymorphonuclear leukocytes. The effects of supernatant from thrombin-activated platelets and two platelet release products (adenosine triphosphate and beta-thromboglobulin) were tested on the following features of polymorphonuclear leukocytes activation: opsonized zymosan and phorbol myristate acetate stimulated chemiluminescence, release of membrane bound calcium, NADPH-oxidase activity, and membrane fluidity (fluorescent polarization). The results showed that the addition of platelet supernatant to polymorphonuclear leukocytes induces a significant activation of cells. On the other hand, after three hours of preincubation of polymorphonuclear leukocytes with platelet supernatant, a decreased response of polymorphonuclear leukocytes to stimulation with phorbol myristate acetate, a significant decrease in NADPH-oxidase activity, and a lowered membrane fluidity were observed. Adenosine triphosphate modulated only opsonized zymosan stimulated chemiluminescence, with and without preincubation with polymorphonuclear leukocytes. Beta-thromboglobulin caused a decrease of the chemiluminescent response of polymorphonuclear leukocytes, using both agonists, with and without preincubation with polymorphonuclear leukocytes. Moreover beta-thromboglobulin only caused a decrease of the polymorphonuclear leukocytes membrane fluidity without preincubation with the cells. These results support the thesis that platelets have a "time-related" modulating activity on polymorphonuclear leukocytes.     

Charge-dependent regulation of NADPH oxidase activities in intact and subcellular systems of polymorphonuclear leukocytes

It has been reported that respiratory bursts with N-formylmethionylleucylphenylalanine, A23187, phorbol ester and fatty acids are switched off and on by modulating the net charges of plasma membranes in guinea-pig neutrophils (Miyahara, M. et al. (1987), Biochim. Biophys. Acta, 929, 253-262). In the present study, this was further extended in cells treated with protein kinase C inhibitors which completely suppressed the phorbol ester-dependent respiratory burst. This suggested that the initiation of the respiratory burst, which is generally accepted as linked to protein kinase C activation, might also be implicated in the net charge changes of plasma membranes. The above results were also supported by data obtained with a cell-free system reconstituted with plasma membranes and cytosolic fractions from unstimulated neutrophils, guanosine 5'-[gamma-thio]triphosphate and NADPH. Arachidonate stimulated NADPH oxidase activity accompanied by a marked phosphorylation of membrane proteins. The phosphorylation was sensitive to H-7, but it did not appear to be essential for the respiratory burst, because the oxidase activation was insensitive to H-7. Pretreating the plasma membranes with positively charged cetylamine inhibited the oxidase activation by arachidonate. These results suggest that a charge-dependent process, which does not use protein kinase C, may play an important role in the reaction leading to NADPH oxidase activation, and this may be related to the interaction of plasma membranes with the cytosolic activation factor.     

Protein kinase C-independent pathway for NADPH oxidase activation in guinea pig peritoneal polymorphonuclear leukocytes by cytochalasin D

Cytochalasin D (CD) induced production of the superoxide radical (O(2)(-)) in guinea pig polymorphonuclear leukocytes (PMNs). The protein kinase C (PKC) inhibitor GF109203X (GFX) was rarely without effect on CD-induced O(2)(-) production. CD as well as PMA induced the translocation of p47(phox) to the membrane fraction, and this translocation was slightly decreased by GFX. Moreover, the inhibitory effect of a PKCzeta antagonist with sequences based on the endogenous PKCzeta pseudosubstrate region was weaker than the inhibitory effect on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced O(2)(-) production. On the other hand, the production of O(2)(-) induced by CD was more strongly suppressed by the PLD inhibitor ethanol and phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin than that induced by fMLP, and the activation of phospholipase D (PLD) by CD was restrained by wortmannin. These findings suggest that NADPH oxidase is activated by CD through a PKC-independent signaling pathway in PMNs, and this pathway involves the activation of PLD through PI3-K.     

Microglia proliferation is regulated by hydrogen peroxide from NADPH oxidase

Microglia are resident brain macrophages that become activated and proliferate following brain damage or stimulation by immune mediators, such as IL-1beta or TNF-alpha. We investigated the mechanisms by which microglial proliferation is regulated in primary cultures of rat glia. We found that basal proliferation of microglia was stimulated by proinflammatory cytokines IL-1beta or TNF-alpha, and this proliferation was completely inhibited by catalase, implicating hydrogen peroxide as a mediator of proliferation. In addition, inhibitors of NADPH oxidase (diphenylene iodonium or apocynin) also prevented microglia proliferation, suggesting that this may be the source of hydrogen peroxide. IL-1beta and TNF-alpha rapidly stimulated the rate of hydrogen peroxide produced by isolated microglia, and this was inhibited by diphenylene iodonium, implying that the cytokines were acting directly on microglia to stimulate the NADPH oxidase. Low concentrations of PMA or arachidonic acid (known activators of NADPH oxidase) or xanthine/xanthine oxidase or glucose oxidase (generating hydrogen peroxide) also increased microglia proliferation and this was blocked by catalase, showing that NADPH oxidase activation or hydrogen peroxide was sufficient to stimulate microglia proliferation. In contrast to microglia, the proliferation of astrocytes was unaffected by the presence of catalase. In conclusion, these findings indicate that microglial proliferation in response to IL-1beta or TNF-alpha is mediated by hydrogen peroxide from NADPH oxidase.