Superoxide Generation In Human Neutrophils Research Articles

Effect of timosaponin E1 and E2 on superoxide generation induced by various stimuli in human neutrophils and on platelet aggregation in human blood

We have reported that six steroidal saponins isolated from Anemarrhenae rhizoma had various effects on stimulus-induced superoxide generation in human neutrophils. In this paper, two novel steroidal saponins, timosaponins E1 and E2 were isolated from Anemarrhenae rhizoma, and the effects of these steroidal saponins on superoxide generation in human neutrophils were investigated. Timosaponins E1 and E2 significantly inhibited N-formyl–methionyl–leucyl–phenylalanine (fMLP)-induced superoxide generation in a concentration-dependent manner, but not that induced by arachidonic acid (AA). On the other hand, both compounds enhanced superoxide generation induced by phorbol 12-myristate 13-acetate (PMA) in a concentration-dependent manner. The superoxide generation induced by PMA with timosaponins E1 and E2 was suppressed by staurosporine, an inhibitor of protein kinase C, but was not suppressed by genistein, an inhibitor of protein tyrosine kinase. Tyrosyl phosphorylation of a 58 kDa protein, which was increased by fMLP, was inhibited by timosaponins E1 and E2. Timosaponins E1 and E2 also inhibited the generation of a 47 kDa protein and platelet aggregation in human blood. The results suggest that protein tyrosine kinase participates in fMLP-mediated superoxide generation by timosaponin E1- and E2-treated human neutrophils.

1060 Fenoterol inhibits the β receptor-independent signaling pathways of superoxide anion generation inhuman neutrophils

1060 Fenoterol inhibits the β receptor-independent signaling pathways of superoxide anion generation inhuman neutrophils

Trp-Lys-Tyr-Met-Val-Met activates mitogen-activated protein kinase via a PI-3 kinase-mediated pathway independent of PKC

Trp-Lys-Tyr-Met-Val-Met (WKYMVM) is a novel potent peptide which can stimulate phosphoinositide hydrolysis in U937 as well as U266 and HL-60 cells (Baek et al., J. Biol. Chem. 271, 8170 (1996)). The peptide also induces Superoxide generation in human neutrophils (Seo et al., J. Immunol. 158, 1896 (1997)). However, the signaling pathway down-stream of PLC set in motion by the peptide is not yet completely understood. We studied the signaling pathway of the peptide with the goal of elucidating the mechanism of the peptide's action. WKYMVM induced a rapid and transient activation of the ERKs in human histiocytic lymphoma cells, U937. The ERK1 activation peaked at 5 min and returned to the basal level after 30 min. The ERK1 stimulation by the peptide was partially inhibited by pretreatment of the cells with pertussis toxin (PTX), implicating G-protein involvement in the peptide's action. Pretreatment of staurosporine, protein kinase C (PKC) inhibitor, or PKC down-regulating PMA had no impact on the ERK1 activation by the peptide, indicating that the signaling pathway is independent of PKC activation. Pretreatment of the cells with neomycin and intracellular Ca 2+ mobilizing reagents had also no effect on the ERK1 activation by the peptide. However, pretreatment with wortmannin or LY294002, the inhibitor of phosphatidylinositol 3 kinase (PI-3K), strongly inhibited peptide-stimulated ERK1 activation. Our results suggest that PI-3K may be an important participant in the ERK cascade induced by the peptide. Furthermore, the treatment of U937 cells with the peptide activated p74 raf-1, an upstream kinase of ERK. Taken together, our results suggest that the peptide activate ERK via a G-protein/PI-3K/Ras/Raf-1 mediated signaling pathway in U937 cells.

Effect of Steroidal Saponins of Anemarrhenae Rhizoma on Superoxide Generation in Human Neutrophils

Effect of six steroidal saponins isolated from Anemarrhenae rhizoma on superoxide generation in human neutrophils was investigated. The steroidal saponins examined were anemarrhenasaponin-I (An-I), anemarrhenasaponin-Ia (An-Ia), timosaponin B-I (TB-I), timosaponin B-II (TB-II), timosaponin B-III (TB-III) and timosaponin A-III (TA-III). An-I, An-Ia, and TB-III suppressed the superoxide generations induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and arachidonic acid (AA) in a concentration-dependent manner, but enhanced that induced by phorbol 12-myristate 13-acetate (PMA). While TB-II also suppressed and enhanced the superoxide generations induced by fMLP and PMA, respectively, the compound significantly enhanced the AA-induced superoxide generation. TB-I enhanced the fMLP-induced superoxide generation in a low concentration range (peak at 40 μM), gave no effect on the PMA-induced superoxide generation and weakly enhanced the AA-induced superoxide generation. TA-III enhanced the fMLP-induced superoxide generation more than twice as much as that by TB-I in the same concentration range. However, TA-III enhanced the PMA-induced superoxide generation and most significantly suppressed the AA-induced superoxide generation.

Superoxide Production in Human Neutrophils: Evidence for Signal Redundancy and the Involvement of More Than One PKC Isoenzyme Class

Selective protein kinase C (PKC) activators and inhibitors and a physiological agonist, fMLP, were used to study superoxide production and PKC isoenzyme activation in human neutrophils. The data show that the classical PKC isoenzymes, α and β, were activated by TPA and at a time prior to NADPH oxidase complex assembly. fMLP induced activation of PKC-β over a similar time course. Inhibition of c-PKCs reduced, but did not block, TPA-induced superoxide production completely, suggesting additional PKC isoenzymes were involved beyond NADPH oxidase assembly. PKC inhibitors were unable to inhibit fMLP-induced superoxide generation, indicative of signal redundancy in the induction of superoxide generation in human neutrophils.

PROPIONIC ACID STIMULATES SUPEROXIDE GENERATION IN HUMAN NEUTROPHILS

Short-chain carboxylic acids are the metabolic by-products of pathogenic anaerobic bacteria and are found at sites of infection in millimolar quantities. We previously reported that propionic acid, one of the short-chain carboxylic acids, induces an increase in intracellular Ca2+([Ca2+]i) in human neutrophils. Here we investigate the effect of propionic acid on superoxide generation in human neutrophils. Propionic acid (10mm) induced inositol 1,4,5-trisphosphate (IP3) formation and a rapidly transient increase in [Ca2+]i, but not superoxide generation, whereas 1μmformylmethionyl-leucyl-phenylalanine (fMLP), a widely used neutrophil-stimulating bacterial peptide, stimulated not only IP3formation and Ca2+mobilization but also superoxide generation. The IP3level induced by propionic acid was slightly lower than that induced by fMLP. The transient increase in [Ca2+]iinduced by propionic acid immediately returned to the basal level, whereas a sustained increase in [Ca2+]i, which was higher than the basal level, following a transient increase in [Ca2+]iwas induced by fMLP. The peak level induced by propionic acid was lower than that with fMLP. In the absence of extracellular Ca2+, thapsigargin, a potent inhibitor of endoplasmic reticulum Ca2+-ATPase, induced an increase in [Ca2+]ieven after propionic acid stimulation, but not after fMLP. The Ca2+ionophore A23187 and thapsigargin induced superoxide generation by themselves. Propionic acid enhanced the superoxide generating effect of A23187 and thapsigargin. These results suggest that Ca2+mobilization induced by propionic acid is much weaker than that with fMLP, and propionic acid is able to generate superoxide in the presence of a Ca2+ionophore and a Ca2+influx activator.

Distribution of the receptor for a novel peptide stimulating phosphoinositide hydrolysis in human leukocytes.

Previously we reported that a synthetic peptide, WKYMVm, stimulates phosphoinositide (PI) hydrolysis in several hematopoietic cell lines and human neutrophils. In addition, the peptide induces superoxide generation in human neutrophils. The biochemical stimulation appeared to be mediated by specific receptors on leukocytes. In this report we try to find whether the specific receptor(s) exists on specific types of cells in human leukocytes. To study distribution of the peptide-specific receptors, we prepared a probe, biotin-labeled WKYMVm, and analyzed distribution of the peptide receptor using a biochemical study, cytochemical staining, and flow cytometric analysis. The peptide-specific receptors are expressed in several human leukocytes including granulocytes, monocytes, and B-lymphocytes but not T-lymphocytes and erythrocytes. These data suggest that the receptors for the peptide restricted to certain types of leukocytes and this specificity is probably related to the specific functions of the stimulated leukocytes.

P38 Mitogen-Activated Protein Kinase Activation Is Required for Human Neutrophil Function Triggered by TNF-α or FMLP Stimulation

AbstractMitogen-activated protein (MAP) kinase-mediated signal-transduction pathways convert extracellular stimulation into a variety of cellular functions. However, the roles of MAP kinases in neutrophils are not well understood yet. Protein phosphorylation analysis of cellular MAP kinases indicates that exposure of human neutrophils to chemotactic factor FMLP as well as granulocyte-macrophage CSF, PMA, or ionomycin rapidly induced the activation of p38 and p44/42 MAP kinases, but stimulation with inflammatory cytokine TNF-α triggered the activation of p38 MAP kinase only. To study the cellular functions of these MAP kinases, the inhibitor SB20358, which specifically inhibited enzymatic activity of cellular p38 MAP kinase, and the inhibitor PD98059, which specifically blocked the induced protein phosphorylation and activation of p44/42 MAP kinase in intact neutrophils, were utilized. Inhibition of the cellular p38 MAP kinase activation almost completely abolished the TNF-α-stimulated IL-8 production and superoxide generation of human neutrophils. In addition, the FMLP-induced neutrophil chemotaxis as well as superoxide generation were suppressed markedly by inhibiting the activation of cellular p38 MAP kinase, but not p44/42 MAP kinase. Moreover, RIA indicates that the activation of cellular p38 MAP kinase was required for the neutrophil IL-8 production stimulated by granulocyte-macrophage CSF or LPS as well as TNF-α, but not for that induced by PMA or ionomycin. These results demonstrate that the activation of cellular p38 MAP kinase is indispensable for the TNF-α- or FMLP-mediated cellular functions in human neutrophils, and suggest that p38 MAP kinase may play a different role in response to distinct stimulation.

A peptide with unique receptor specificity: stimulation of phosphoinositide hydrolysis and induction of superoxide generation in human neutrophils.

Abstract Previously, we identified peptides that stimulate phosphoinositide hydrolysis in several leukocyte cell lines from mixtures of random hexapeptide sequences. Moreover, the peptides activate phospholipase C via a pertussis toxin-sensitive G protein-coupled receptor. We now investigate the structure-activity relationship of the peptides with the goal of improving the activity of the peptides, as well as the biologic function of the peptides. Substitution of the L-methionine at the C terminus of peptides with D-methionine markedly increased the effectiveness of the peptides. The half-maximal effective concentrations of MKYMPm-NH2 and WKYMVm-NH2 for stimulation of phosphoinositide hydrolysis in U266 cells were 30 and 0.5 nM, respectively. By BIAcore analysis we confirmed the existence of a receptor for WKYMVm-NH2. Furthermore, the intracellular calcium concentration increase induced by WKYMVm-NH2 was not inhibited by several chemoattractants (FMLP, IL-8, platelet-activating factor, C5a, granulocyte-macrophage CSF, and granulocyte CSF) suggests that WKYMVm-NH2 has a unique cell surface receptor on leukocytes. WKYMVm-NH2 stimulated the phosphoinositide hydrolysis in U937, HL60, and U266 cells, as well as in human neutrophils. Moreover, WKYMVm-NH2 is more effective than FMLP in the production of superoxide in human neutrophils. The data suggest that WKYMVm-NH2 may have the ability to activate the microbicidal functions of human neutrophils.

Modulation of degranulation and superoxide generation in human neutrophils by unsaturated fatty acids of odd carbon numbers

Unsaturated fatty acids of odd carbons, 13:1(12), 17:1(10trans), 19:1(7) and 19:1(10) inhibited release of myeloperoxidase (MPO) from fMet-Leu-Phe-cytochalasin B-treated neutrophils. The inhibitory effect was smaller than that of aseanostatins which have been isolated as microbial-derived free fatty acids with a methyl blanch (i-14:0 and ai-15:0) (Journal of Antibiotics (1991) 44, 524–532). These unsaturated fatty acids also inhibited lactoferrin release by the same treatment. On the other hand, 13:1(12), 15;1(10) and 19:1(10) inhibited fMet-Leu-Phe-stimulated superoxide generation of neutrophils, and the fatty acids 15:1(10), 17:(10) and 19:2(10,13) induced superoxide generation in both unstimulated cells and the cell-free system. However, none of unsaturated fatty acids of odd carbons tested inhibited β-glucuronidase release, whereas 15:1(10), 17:1(10), 19:1(7), 19:1(10) and 19:2(10,13) rather enhanced an increase in β-glucuronidase activity liberated from cells at high concentrations over 35 μM, indicating cellular damages by these fatty acids. These observations suggest that unsaturated free fatty acids having odd carbons such as 13, 15, 17 and 19 may act as modulators of neutrophil functions including degranulation and superoxide generation.

Involvement of protein phosphatase 2A in PKC-independent pathway of neutrophil superoxide generation by fMLP

We examined the effects of okadaic acid, a protein phosphatase 1 and 2A inhibitor, on superoxide generation in human neutrophils. Superoxide generation induced by fMLP was inhibited by low-dose okadaic acid (10-100 nM), but it had no effect on superoxide synthesis by PMA, and the fMLP-induced rise of the intracellular Ca2+ concentration was not affected by low-dose okadaic acid. These findings suggested that the inhibitory mechanism of okadaic acid might involve PKC-independent and Ca(2+)-independent pathways in fMLP induced NADPH oxidase activation. Both fMLP-stimulated phosphorylation of serine residues in p47phox and its translocation to the plasma membrane were suppressed by low-dose okadaic acid. On the other hand, PMA-induced phosphorylation and translocation of p47phox were not affected by such a low dose of okadaic acid. These findings suggested that fMLP induced phosphorylation of serine residues in p47phox was regulated by protein phosphatase 2A, and its phosphorylation was necessary for translocation and superoxide generation in fMLP-activated human neutrophils.

Mechanism of vasodilating action of nebivolol

A decrease in endothelium-derived relaxing factor or nitric oxide has been proposed as a potential mechanism of increased vascular resistance in hypertension. An increase in the generation of superoxide anions, which degrade nitric oxide and induce platelet aggregation, may also compromise regional blood flow in hypertension. Recent studies show that human neutrophils generate nitric oxide, which has a similar biologic profile to the endothelium-derived relaxing factor. This study measured nitric oxide synthase activity and superoxide generation in human neutrophils and platelet aggregation in patients with essential hypertension. Nitric oxide synthase activity, measured as conversion of 3H-L-arginine to 3H-L-citrulline, in peripheral blood neutrophils was decreased in hypertensive subjects (percent conversion of 3H-L-arginine: 4.2 ± 0.5 vs 9.0 ± 3.0 in control subjects; p < 0.01). Neirtrophil superoxide anion generation, measured as conversion of ferricytochrome C to ferrocytochrome C, in response to phorbol-12-myristate 13-acetate (100 ng/ml) was higher in hypertensive subjects (17.5 ± 8.1 vs 13.2 ± 3.0 nmoles/106 cells/10 minutes in control subjects; p < 0.05). Patients were treated with a selective β blocker, celiprolol, for 8 weeks. Supine blood pressure decreased from 177/103 mm Hg (mean ± SD 18/7) to 160/92 mm Hg (mean ± 10/5; p < 0.02), while heart rate was unchanged (73 ± 11 vs 69 ± 10 beats/min). Epinephrine and adenosine diphosphate-induced platelet aggregation was also increased in hypertensive subjects. Nitric oxide synthase activity increased after therapy with celiprolol (4.2 ± 0.5 to 8.9 ± 0.4%; p < 0.01), and superoxide anion generation decreased (17.5 ± 8.1 to 3.0 ± 1.0 nmoles/106 cells/10 minutes; p < 0.01). Platelet aggregation also decreased during celiprolol therapy. The increase in superoxide radical generation, decrease in nitric oxide synthase activity, and increased platelet aggregation may relate to the pathophysiology of hypertension. The novel effects of celiprolol described in this study may be the basis of cardioprotective effects of β blockers.

PGI2 Analogue, sodium beraprost, suppresses superoxide generation in human neutrophils by inhibiting p47PHOX phosphorylation

Sodium beraprost, a newly synthesized PGI2 analogue inhibited in a dose-dependent manner formyl-methionyl-leucyl-phenylalanine (fMLP) induced superoxide generation of human neutrophils, but it had no effect on the superoxide synthesis by phorbol myristate acetate (PMA) or A23187. Sodium beraprost inhibited Ca 2+ influx in fMLP stimulated neutrophils employing fluorometry and confocal microscopy. These findings suggested that the inhibitory effect of sodium beraprost on fMLP induced speroxide generation was due to suppression of Ca 2+ influx. To examine the relationship between the effect of sodium beraprost and phosphorylation of p47phox (the 47kDa cytosolic phagocyte oxidase factor), immuno-precipitation of p47phox and western blotting for phospho-amino acids were performed. Phosphorylation of serine residues of p47phox induced by fMLP was reduced in the presence of sodium beraprost in a dose-dependent manner. The reduction in phosphorylation was accompanied by a reduction in p47phox and p67phox translocation to the plasma membrane and superoxide generation. These findings suggested that p47phox phosphorylation was necessary for translocation and superoxide generation in fMLP activated neutrophils, and that p47phox phosphorylation was regulated by a Ca 2+ dependent mechanism. These observations suggested that sodium beraprost inhibited fMLP induced superoxide generation of human neutrophils by the inhibition of p47phox phosphorylation and translocation by aCa 2+dependent mechanism.

Ibudilast, an anti-allergic and cerebral vasodilator, modulates superoxide production in human neutrophils.

We evaluated the effect of ibudilast on superoxide generation in human neutrophils by chemiluminescence development using luciferine analog, FCLA. By incubating neutrophils with ibudilast (2-200 microM) for more than 10 minutes, fMLP- or PMA-induced chemiluminescence was enhanced. However, the fMLP-induced chemiluminescence was suppressed by incubation for less than 10 minutes. This suppressed effect was missing with PMA-induced chemiluminescence. On the both fMLP- and PMA-induced chemiluminescence, the priming effect of ibudilast was further enhanced by the treatment with H-7, a protein kinase C inhibitor. In contrast, the priming effect of ibudilast on the fMLP-induced chemiluminescence was abolished by the treatment with ST-638, a selective inhibitor of tyrosine kinase. Ibudilast showed a transient stimulatory effect on cyclic AMP accumulation which continued for only a few minutes. Ibudilast showed no significant effect on phospholipase D dependent chemiluminescence, 1,4,5 trisphosphate level, or protein kinase C activity. Ibudilast inhibited extracellular calcium influx. These results suggest that ibudilast acts on or through tyrosine kinase to achieved its priming effect on the fMLP-induced chemiluminescence. The early and transient increase in cyclic AMP level may explain the inhibitory effect of ibudilast on the fMLP-induced chemiluminescence after short time of incubation.

Ibudilast, an anti-allergic and cerebral vasodilator, modulates superoxide production in human neutrophils

We evaluated the effect of ibudilast on superoxide generation in human neutrophils by chemiluminescence development using luciferine analog, FCLA. By incubating neutrophils with ibudilast (2–200 μM) for more than 10 minutes, fMLP- or PMA-induced chemiluminescence was enhanced. However, the fMLP-induced chemiluminescence was suppressed by incubation for less than 10 minutes. This suppressed effect was missing with PMA-induced chemiluminescence. On the both fMLP- and PMA-induced chemiluminescence, the priming effect of ibudilast was further enhanced by the treatment with H-7, a protein kinase C inhibitor. In contrast, the priming effect of ibudilast on the fMLP-induced chemiluminescence was abolished by the treatment with ST-638, a selective inhibitor of tyrosine kinase. Ibudilast showed a transient stimulatory effect on cyclic AMP accumulation which continued for only a few minutes. Ibudilast showed no significant effect on phospholipase D dependent chemiluminescence, 1,4,5 triphosphate level, or protein kinase C activity. Ibudilast inhibited extracellular calcium influx. These results suggest that ibudilast acts on or through tyrosine kinase to achieved its priming effect on the fMLP-induced chemiluminescence. The early and transient increase in cyclic AMP level may explain the inhibitory effect of ibudilast on the fMLP-induced chemiluminescence after short time of incubation.

Alterations in nitric oxide synthase activity, superoxide anion generation, and platelet aggregation in systemic hypertension, and effects of celiprolol

A decrease in endothelium-derived relaxing factor or nitric oxide has been proposed as a potential mechanism of increased vascular resistance in hypertension. An increase in the generation of superoxide anions, which degrade nitric oxide and induce platelet aggregation, may also compromise regional blood flow in hypertension. Recent studies show that human neutrophils generate nitric oxide, which has a similar biologic profile to the endothelium-derived relaxing factor. This study measured nitric oxide synthase activity and superoxide generation in human neutrophils and platelet aggregation in patients with essential hypertension. Nitric oxide synthase activity, measured as conversion of 3H-L-arginine to 3H-L-citrulline, in peripheral blood neutrophils was decreased in hypertensive subjects (percent conversion of 3H-L-arginine: 4.2 ± 0.5 vs 9.0 ± 3.0 in control subjects; p < 0.01). Neirtrophil superoxide anion generation, measured as conversion of ferricytochrome C to ferrocytochrome C, in response to phorbol-12-myristate 13-acetate (100 ng/ml) was higher in hypertensive subjects (17.5 ± 8.1 vs 13.2 ± 3.0 nmoles/10 6 cells/10 minutes in control subjects; p < 0.05). Patients were treated with a selective β blocker, celiprolol, for 8 weeks. Supine blood pressure decreased from 177/103 mm Hg (mean ± SD 18/7) to 160/92 mm Hg (mean ± 10/5; p < 0.02), while heart rate was unchanged (73 ± 11 vs 69 ± 10 beats/min). Epinephrine and adenosine diphosphate-induced platelet aggregation was also increased in hypertensive subjects. Nitric oxide synthase activity increased after therapy with celiprolol (4.2 ± 0.5 to 8.9 ± 0.4%; p < 0.01), and superoxide anion generation decreased (17.5 ± 8.1 to 3.0 ± 1.0 nmoles/10 6 cells/10 minutes; p < 0.01). Platelet aggregation also decreased during celiprolol therapy. The increase in superoxide radical generation, decrease in nitric oxide synthase activity, and increased platelet aggregation may relate to the pathophysiology of hypertension. The novel effects of celiprolol described in this study may be the basis of cardioprotective effects of β blockers.

Ceramide does not mediate the effect of tumour necrosis factor alpha on superoxide generation in human neutrophils.

The effect of tumour necrosis factor alpha (TNF alpha) on superoxide generation in human neutrophils was investigated using the Nitro Blue Tetrazolium reduction assay. TNF alpha stimulated superoxide generation in a time- and concentration-dependent fashion. The maximally effective concentration of TNF alpha for superoxide generation was 10 nM and maximal response was obtained after 15-20 min. The monoclonal antibody (mAb), utr-1, which was raised against the 75 kDa receptor and behaves as an antagonist, had no effect on superoxide generation, but partially inhibited the response to TNF alpha. mAb htr-9, which was raised against the 55 kDa receptor and behaves as an agonist, mimicked the effect of TNF alpha, but with a lower maximal response. As it has been reported that ceramide might act as a second messenger to mediate many of the effects of TNF alpha, the effects of exogenous sphingomyelinase and the cell-permeable ceramide analogue, C2- ceramide, on production of superoxide anions, induction of priming in response to formylmethionyl-leucyl-phenylalanine, and cell-shape change were examined. Neither sphingomyelinase nor C2-ceramide mimicked the effect of TNF alpha. Ceramide is converted into ceramide 1-phosphate by ceramide kinase and we have measured levels of this metabolite to clarify the effect of TNF alpha on sphingomyelinase activity in neutrophils. Although exogenous sphingomyelinase increased the amount of ceramide 1-phosphate in a time-dependent manner, and C2-ceramide was rapidly converted into C2-ceramide phosphate, TNF alpha had no effect on the level of ceramide 1-phosphate. These results suggest that TNF alpha stimulates superoxide generation through both the 55 kDa and 75 kDa receptors, but that ceramide does not act as an intracellular mediator for TNF alpha in human neutrophils.

P-581 - Mechanisms for substance P-induced superoxide generation in human neutrophils.

P-581 - Mechanisms for substance P-induced superoxide generation in human neutrophils.

Modulation of TNF-α-priming and stimulation-dependent superoxide generation in human neutrophils by protein kinase inhibitors

Human peripheral blood polymorphonuclear leukocytes (HPPMN) from healthy individuals are not primed and, hence, weak stimulation-dependent responses are induced by certain stimuli which bind to membrane receptors. When HPPMN were exposed to recombinant human tumor necrosis factor α (rHuTNF-α) or recombinant human granulocyte colony stimulating factor (rG-CSF), they underwent priming and the rate of superoxide anion O 2 ∸ generation was increased by subsequent exposure to formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ). However, the degree of enhancement was very small upon exposure to phorbol myristate acetate (PMA) or dioctanoyl glycerol (DOG). The oxygen burst induced by FMLP or OZ was inhibited by genistein and α-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamid (ST638), which are inhibitors of tyrosine kinase (TK), and was enhanced by 1-(5-isoquinoline-sulfonyl)-3-methyl-piperazine (H-7) and staurosporine, which are inhibitors of protein kinase C (PKC). Without priming, however, O 2 ∸ generation from HPPMN by high concentrations of FMLP was not inhibited strongly by genistein or ST638. On the contrary, the oxygen burst induced by PMA or DOG was stimulated by genistein or ST638 and was inhibited by H-7 or staurosporine. Furthermore, O 2 ∸ generation by guinea pig peritoneal neutrophils, which are already primed in vivo, was induced markedly by FMLP by a mechanism which was stimulated by a low concentration of genistein or ST638. Thus, FMLP-mediated O 2 ∸-generation of HPPMN is coupled with rHuTNF-α- or rG-CSF-priming and is inhibited by TK inhibitors, whereas PMA- or DOG-induced O 2 ∸ generation is not coupled with TNF-α or G-CSF-priming and is inhibited by PKC inhibitors. These results suggest that both PKC and TK play critical roles in the regulatory mechanism of priming and NADPH-oxidase activation in neutrophils.

Effects of anti-allergic drugs on human neutrophil superoxide-generating NADPH oxidase

The effects of anti-allergic drugs with or without H 1-receptor antagonism on the NADPH oxidase (EC 1.6.99.6) from human neutrophils in both whole-cell and fully soluble (cell-free) systems were investigated. Three anti-allergic drugs with H 1-receptor antagonism, azelastine, ketotifen and oxatomide, were found to inhibit the Superoxide generation of human neutrophils exposed to phorbol myristate acetate in a whole-cell system and the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate in a cell-free system. The concentrations of these drugs required for 50% inhibition of the oxidase (Ic 50) were: azelastine—0.7 μM in the whole-cell system and 0.5 μM in the cell-free system; ketotifen—60 μM in the whole-cell system and 6.8 μM in the cell-free system; and oxatomide—25 μM in the whole-cell system and 9.7 μM in th cell-free system. In addition, in the cell-free system, these drugs did not change the K m values for the NADPH of the oxidase. However, these drugs did not inhibit the Superoxide generation of NADPH oxidase after its activation in whole-cell and cell-free systems, suggesting that these drugs do not have superoxide-scavenger actions. Concentrations of less than 200 μM of anti-allergic drugs without H 1receptor antagonism, tranilast, repirinast and ibudilast, did not inhibit neutrophil NADPH oxidase in whole-cell and cell-free systems. The Ic 50 of hydrocortisone in the cell-free system was 60 μM. These results suggest that anti-allergic drugs with H 1-receptor antagonism inhibit activation of the solubilized membrane-bound enzyme by sodium dodecyl sulfate in cell-free systems and that they have much stronger anti-inflammatory action than hydrocortisone.