Priming Effect of Pseudomonal Leukocidin on Chemiluminescence Response of Rabbit Polymorphonuclear Leukocytes

To clarify effects of pseudomonal leukocidin (42.5 kd) on chemiluminescence (CL) production of polymorphonuclear leukocytes (PMNs), rabbit PMNs were stimulated by zymosan or phorbol myristate acetate (PMA) after pretreatment with the leukocidin, which by itself stimulated little chemiluminescence response. The extent of CL responses stimulated by zymosan or PMA was respectively 5.3- or 3.5-fold greater in leukocidin (1.5 μg/ml)-pretreated PMNs than in non-pretreated ones. The priming effect of the leukocidin was greater than that of G-CSF and related to some steps before NADPH oxidase activation. The increased CL productions might be related to tissue damages caused by pseudomonal infections in vivo.     

Suppressive effect of rebamipide, an antiulcer agent, against activation of human neutrophils exposed to formyl-methionyl-leucyl-phenylalanine

Rebamipide, an antiulcer agent, has been shown to be able to prevent gastric mucosal injury resulting in part from activation of neutrophils. The mechanism of its suppressive action, however, remains to be established. The present study aimed to determine the effect of rebamipide on activation of isolated human neutrophils and to identify the signal transduction pathway involved in its regulation. In unstimulated cells, alkaline phosphatase activity was found residing in short rod-shaped intracellular granules. Upon stimulation with a chemotactic peptide formyl-methionyl-leucyl-phenylalanine, the granules fused to form elongated tubular structures and spherical vacuoles. Rebamipide inhibited reorganization of alkaline phosphatase-containing granules along with upregulation of alkaline phosphatase activity and CD16, a marker of the granules. It also suppressed chemotaxis, an increase in intracellular calcium ion concentration, and NADPH oxidase activation in cells stimulated with formyl-methionyl-leucyl-phenylalanine. In contrast, the drug showed no inhibitory action toward upregulation of alkaline phosphatase activity and CD16, and activation of NADPH oxidase in cells stimulated with phorbol myristate acetate, an activator of protein kinase C. These findings demonstrate that rebamipide exerts a broad spectrum of suppressive actions toward biological functions of human neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine, but not with phorbol myristate acetate, and suggest that the upstream point of protein kinase C is the signal transduction pathway involved in its regulation.     

Modulation by phorbol myristate acetate of arachidonic acid release and leukotriene synthesis by human polymorphonuclear leukocytes stimulated with A23187

Phorbol myristate acetate augmented the release of 3H-AA and the synthesis of leukotriene B4 and 5-hydroxyeicosatetraenoic acid by human polymorphonuclear leukocytes stimulated by A23187. PMA alone had no effect. Enhancement of the response to A23187 was not seen when the inactive phorbol ester 4-alpha phorbol didecanoate was added with A23187. These data are consistent with the hypothesis that activation of protein kinase C enhances AA release and metabolism in stimulated polymorphonuclear leukocytes.     

Actin polymerization modifies stimulus-oxidase coupling in rat neutrophils

Oxidase activity in rat neutrophils was monitored by oxygen consumption rate and luminol-dependent chemiluminescence. Two agents which inhibit actin polymerization, cytochalasin B and dihydrocytochalasin B, produced a marked enhancement (up to 10-fold) of oxidase activation induced by two Ca2+-dependent stimuli, chemotactic peptide and ionophore A23187. In contrast, activation by the calcium-independent stimulus, phorbol myristate acetate, was unaffected by these agents. Other agents that interact with the cytoskeleton, phalloidin and colchicine have no effect on activation by any stimulus tested. The effect of cytochalasin B, when added after stimulation by chemotactic peptide, was transient with t0.5 approx. 10 s. Similarly, the degree of actin polymerization following stimulation by chemotactic peptide was transient, decaying with a t0.5 of approx. 10 s. The half-maximal concentration of cytochalasin B for inhibition of actin polymerization was similar to that for enhancement of oxidase activation. It was concluded, therefore, that the intracellular Ca2+ rise in rat neutrophils that accompanies stimulation by chemotactic peptide affects actin polymerization in a manner that modifies oxidase activation.     

NADPH oxidase-dependent oxidation and externalization of phosphatidylserine during apoptosis in Me2SO-differentiated HL-60 cells. Role in phagocytic clearance

Resolution of inflammation requires clearance of activated neutrophils by phagocytes in a manner that protects adjacent tissues from injury. Mechanisms governing apoptosis and clearance of activated neutrophils from inflamed areas are still poorly understood. We used dimethylsulfoxide-differentiated HL-60 cells showing inducible oxidase activity to study NADPH oxidase-induced apoptosis pathways typical of neutrophils. Activation of the NADPH oxidase by phorbol myristate acetate caused oxidative stress as shown by production of superoxide and hydrogen peroxide, depletion of intracellular glutathione, and peroxidation of all three major classes of membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In addition, phorbol myristate acetate stimulation of the NADPH oxidase caused apoptosis, as evidenced by apoptosis-specific phosphatidylserine externalization, increased caspase-3 activity, chromatin condensation, and nuclear fragmentation. Furthermore, phorbol myristate acetate stimulation of the NADPH oxidase caused recognition and ingestion of dimethylsulfoxide-differentiated HL-60 cells by J774A.1 macrophages. To reveal the apoptosis-related component of oxidative stress in the phorbol myristate acetate-induced response, we pretreated cells with a pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), and found that it caused partial inhibition of hydrogen peroxide formation as well as selective protection of only phosphatidylserine, whereas more abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, were oxidized to the same extent in the absence or presence of z-VAD-fmk. In contrast, inhibitors of NADPH oxidase activity, diphenylene iodonium and staurosporine, as well as antioxidant enzymes, superoxide dismutase/catalase, completely protected all phospholipids against peroxidation, inhibited expression of apoptotic biomarkers and externalization of phosphatidylserine, and reduced phagocytosis of differentiated HL-60 cells by J774A.1 macrophages. Similarly, zymosan-induced activation of the NADPH oxidase resulted in the production of superoxide and oxidation of different classes of phospholipids of which only phosphatidylserine was protected by z-VAD-fmk. Accordingly, zymosan caused apoptosis in differentiated HL-60 cells, as evidenced by caspase-3 activation and phosphatidylserine externalization. Finally, zymosan triggered caspase-3 activation and extensive SOD/catalase-inhibitable phosphatidylserine exposure in human neutrophils. Overall, our results indicate that NADPH oxidase-induced oxidative stress in neutrophil-like cells triggers apoptosis and subsequent recognition and removal of these cells through pathways dependent on oxidation and externalization of phosphatidylserine.     

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     

Phorbol myristate acetate mediates redistribution of protein kinase C in human neutrophils: potential role in the activation of the respiratory burst enzyme

Protein kinase C may be important in leukocyte function, because it is activated by phorbol myristate acetate (PMA), a potent stimulus of the respiratory burst in neutrophils. The localization of protein kinase C was compared in unstimulated and PMA-stimulated human neutrophils. Protein kinase C was primarily cytosolic in unstimulated cells but became associated with the particulate fraction after treatment of cells with PMA. The particulate-associated kinase activity did not require added calcium and lipids, but when extracted by Triton X-100 (greater than or equal to 0.2%), calcium and phospholipid dependence could be demonstrated. The EC50 of PMA for stimulating kinase redistribution and activation of NADPH oxidase, the respiratory burst enzyme, were similar (30 to 40 nM). Redistribution of protein kinase C occurred rapidly (no lag) and preceded NADPH oxidase activation (30 sec lag). These results suggest that redistribution of protein kinase C is linked to activation of the respiratory burst in human neutrophils.     

Prostaglandins E1 and E2 enhance the stimulation of superoxide release by 1-oleoyl-2-acetylglycerol from human neutrophils

Superoxide release from human neutrophils was stimulated either by receptor activation (using fMet-Leu-Phe) or by activating, independently, each of the two pathways considered to be involved in signal transduction--calcium mobilization (using the ionophore, A23187) and protein kinase C activation (using phorbol myristate acetate or 1-oleoyl-2-acetylglycerol). Prostaglandin E1 (3 X 10(-5) M) decreased fMet-Leu-Phe-stimulated superoxide release, had no effect on superoxide release stimulated by A23187, or by phorbol myristate acetate, and markedly enhanced the superoxide release stimulated by 1-oleoyl-2-acetylglycerol. Similar enhancement was obtained with prostaglandin E2.     

Chemiluminescence of mononuclear cells is enhanced during antigen recognition

Stimulation of phagocytes by several cytokines causes superoxide generation and consequently chemiluminescence. Since antigen-activated lymphocytes generate cytokines, we investigated whether antigen recognition by mononuclear cells, which contain both lymphocytes and monocytes, is accompanied by changes in lucigenin-dependent chemiluminescence. Mononulcear cells which underwent antigen-induced proliferation showed a delayed rise in lucigenin-dependent chemiluminescence in the absence of other stimuli. The common recall antigen Candida albicans increased spontaneous chemiluminescence of mononuclear cells from unselected donors up to 20-fold over control values after 48–72h of culture. With Rabies virus vaccine as specific antigenic stimulus, only mononuclear cells from rabies immunized individuals responded with enhanced delayed chemiluminescence. In contrast to opsonized zymosan and phorbol myristate acetate, antigens induced no oxidative burst within one hour after addition. Delayed mononuclear cel chemiluminescence was inhibited by the superoxide scavenger superoxide dismutase and by di-phenylene iodonium, a selective inhibitor of the phagocyte NADPH oxidase. A neutralizing monoclonal antibody against interferon-gamma completely abrogated antigen-induced chemiluminescence. Recombinant interferon-gamma by itself induced delayed mononuclear cell chemiluminescence. Thus, antigen-induced delayed mononuclear cell chemiluminescence represents activation of phagocyte NADPH oxidase by interferon-gamma generated by activated lymphocytes.     

Phorbol myristate acetate potentiates superoxide release and membrane depolarization without affecting an increase in cytoplasmic free calcium in human granulocytes stimulated by the chemotactic peptide, lectins and the calcium ionophore

We investigated the inter-relationships of superoxide (O2-) release, membrane depolarization and an increase in cytoplasmic free Ca2+, [Ca2+]i, in human granulocytes stimulated by various agonists. When concanavalin A or the Ca2+ ionophore ionomycin was used as stimulus, an increase in [Ca2+]i clearly preceded the onset of membrane depolarization, which was followed by O2- release. On the other hand, when N-formylmethionylleucylphenylalanine or wheat-germ agglutinin was used as stimulus, no demonstrable lag was seen in any of the responses. O2- release and membrane depolarization stimulated by all these agonists were markedly potentiated in parallel by pretreatment of cells with a low concentration of phorbol myristate acetate (0.25 ng/ml), whereas an increase in [Ca2+]i was not affected or minimally potentiated. The lag time between addition of the stimulus (concanavalin A or ionomycin) and onset of membrane depolarization or O2- release was significantly reduced by pretreatment of cells with phorbol myristate acetate, whereas the lag time between addition of concanavalin A and onset of the increase in [Ca2+]i was not affected. The dose-response curves for triggering of O2- release and membrane depolarization by each of receptor-mediated agonists in phorbol myristate acetate-pretreated or control cells were identical. These findings suggest that; (a) an increase in [Ca2+]i stimulates membrane depolarization indirectly; (b) a low concentration of phorbol myristate acetate potentiates membrane depolarization and O2- release by acting primarily at the post-receptor level, in particular, at the level distal to an increase in [Ca2+]i, but not by augmenting an increase in [Ca2+]i; and (c) the system provoking membrane depolarization and the system activating NADPH oxidase share a common pathway, which may be susceptible to a low concentration of phorbol myristate acetate.     

Response of superoxide anion production by guinea pig eosinophils to various soluble stimuli: comparison to neutrophils

The effect of various soluble stimuli on the superoxide production by guinea pig eosinophils was studied in comparison to neutrophils. Phorbol myristate acetate, A23187, digitonin, NaF, concanavalin A (Con A), and cytochalasin E stimulated eosinophils and neutrophils to release O2-. The O2- production by these active agents, excluding Con A and cytochalasin E, was much greater in eosinophils than in neutrophils. Formyl-Met-Leu-Phe stimulated the O2- production in neutrophils but not in eosinophils. Neither histamine nor Val/Ala-Gly-Ser-Glu stimulated the O2- production in both types of leukocytes. A23187- or Con A-stimulated O2- production was greatly enhanced by cytochalasin B pretreatment in neutrophils but not in eosinophils. Lineweaver-Burk analysis of NADPH oxidase in particulate fractions showed that eosinophils possessed the same Km values as neutrophils and greater Vmax values than neutrophils, suggesting that eosinophils have a similar, but more active, O2- -generating enzyme system than neutrophils.     

Comparative study on effect of N-ethylmaleimide on the superoxide-generating system of guinea-pig eosinophils stimulated by soluble stimuli

1. The effect of N-ethylmaleimide (MalNEt) modification on O2- production by guinea-pig eosinophils mediated by different soluble stimuli was studied. 2. MalNEt pretreatment inhibited the O2- production stimulated by concanavalin A (Con A), cytochalasin E or digitonin, but not A23187 or sodium fluoride. 3. Particulate fractions from MalNEt-pretreated eosinophils before exposure to the stimulus showed the inhibition of the enhancement of NADPH-dependent O2- production induced by Con A, cytochalasin E or digitonin, but not A23187. 4. Treatment of eosinophils with MalNEt after stimulation had no effect on the NADPH oxidase activity. 5. These findings suggest that at least two pathways exist for the activation of the O2(-)-generating enzyme system, probably the NADPH oxidase system, in guinea-pig eosinophils.     

Phorbol 12, myristate 13, acetate potentiates the respiratory burst while inhibits phosphoinositide hydrolysis and calcium mobilization by formyl-methionyl-leucyl-phenylalanine in human neutrophils

It is widely believed that the transduction pathway in the activation of the NADPH oxidase by formyl-methionyl-leucyl-phenylalanine (FMLP) in neutrophils involves the stimulation of phosphoinositide hydrolysis, the increase in [Ca2+]i and the activity of the Ca2+ and phospholipid dependent protein kinase C. The results presented here show that the activation of the respiratory burst by FMLP can be dissociated by the stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate and Ca2+ changes. In fact, in neutrophils pretreated (primed) with non stimulatory doses of phorbol myristate acetate the respiratory burst by chemotactic peptide is greatly potentiated while the increase in [3H] inositol phosphates formation and in [Ca2+]i are depressed due to the inhibition of phospholipase C. This finding indicates that FMLP can trigger also a sequence of transduction reactions for the activation of the NADPH oxidase different from that involving the formation of the second messengers diacylglycerol and inositol phosphates and the increase in free Ca2+ concentration.     

Effect of maleimide derivatives on superoxide-generating system of guinea-pig neutrophils stimulated by different soluble stimuli

The effect of modification of maleimide derivatives on superoxide production by guinea-pig neutrophils induced by a variety of different soluble stimuli was studied. Pretreatment of neutrophils by showdomycin, a very slowly penetrating-SH reagent, did not affect superoxide production by all of the stimuli used, suggesting no exposure of sulfhydryl groups involved in superoxide-generating system on the cell surface. Pretreatment with N-ethylmaleimide (MalNEt), a considerably penetrating-SH reagent, markedly inhibited superoxide production stimulated by formyl-methionyl-leucyl-phenylalanine (HCO-Met-Leu-Phe), cytochalasin E or digitonin, but not superoxide production stimulated by the ionophore A23187 or sodium fluoride. The oxygen consumption stimulated by HCO-Met-Leu-Phe or cytochalasin E was inhibited by MalNEt pretreatment, whereas the oxygen consumption stimulated by A23187 was not inhibited by MalNEt. The inhibition by MalNEt of superoxide production did not appear to be due to the interference with binding of the affected stimuli, since MalNEt pretreatment did not inhibit the release of lysozyme, granule enzyme, induced by HCO-Met-Leu-Phe, cytochalasin E or digitonin. Particulate fractions from MalNEt-pretreated neutrophils before exposure to the stimulus exhibited the inhibition of the enhancement of NADPH-dependent superoxide production induced by HCO-Met-Leu-Phe, cytochalasin E or digitonin, but not A23187, whereas treatment of neutrophils with MalNEt after activation by these stimuli had no effect on the NADPH oxidase activity in particulate fractions. Direct exposure of particulate fractions from A23187-stimulated neutrophils to MalNEt showed no actual susceptibility of NADPH oxidase to MalNEt inhibition. These findings suggest that the inhibitory effect of MalNEt is caused by the modification of the process of the activation by the affected stimuli of the superoxide system, probably NADPH oxidase and that at least two mechanisms exist for activation of superoxide-generating system in guinea-pig neutrophils on the basis of the susceptibility to MalNEt inhibition.     

Activation of the NADPH oxidase in a cell-free system from human neutrophils stimulated by phorbol myristate acetate

Kinetics of activation of the NADPH oxidase in a fully soluble cell-free system from phorbol myristate acetate (PMA)-stimulated human neutrophils were investigated. In a cell-free system in which Mg2+ and sodium dodecyl sulfate, an anionic detergent required for the activation of NADPH oxidase are contained, cytosol prepared from PMA-stimulated neutrophils failed to activate PMA-stimulated neutrophil oxidase. However, cytosol prepared from resting (control) neutrophils was capable of activating PMA-stimulated neutrophil oxidase in a cell-free system in which its Km for NADPH was almost similar to that of control neutrophil oxidase. Cytosol from PMA-stimulated neutrophils could not activate control neutrophil oxidase, although it did not contain any inhibitors of NADPH oxidase activation. These results suggest that, in PMA-stimulated neutrophils, cytosolic activation factors may be consumed or exhausted, and that the affinity for NADPH of PMA-stimulated neutrophil oxidase may be the same as that of control neutrophil oxidase.     

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.     

A role for protein kinase C activity in interleukin-1 (IL-1) induction of IL-2 gene expression but not in IL-1 signal transduction.

Interleukin-1 (IL-1) is known to synergize with phorbol esters in the induction of interleukin-2 (IL-2) expression in T-lymphoid leukemia cells and proliferation of mouse thymocytes. We used a plasmid construct containing the bacterial gene for chloramphenicol acetyltransferase under the control of the human IL-2 promoter to study the nature of this synergism in the murine thymoma cell line EL4. Although IL-1 induction of the IL-2 promoter in these cells required costimulus with phorbol myristate acetate, the signal induced by IL-1 was qualitatively different. We provide evidence to support the hypothesis that the phorbol ester signal is mediated by protein kinase C, and we show that the IL-1 signal is not. That IL-1 and phorbol myristate acetate represent different stimuli was shown by their response to protein kinase C inhibitors, capacity to synergize with increased intracellular free calcium, and requirement for protein synthesis. In addition we show that pretreatment with IL-1 can prime EL4 cells to subsequent activation by concentrations of phorbol esters not normally sufficient to induce IL-2 expression. Pretreated cells remained primed for at least 40 h after removal of the IL-1. Neither phorbol myristate acetate nor a calcium ionophore was capable of preactivating EL4 cells.     

Synergism between phorbol ester and A23187 in superoxide production by neutrophils

Concentrations of phorbol myristate acetate and the calcium ionophore, A23187, which by themselves are minimally effective in stimulating superoxide generation in human neutrophils show marked mutual potentiation when given together. This supports the hypothesis that synergism between cytosolic calcium and protein kinase C is involved in the stimulus/activation coupling of the respiratory burst in the neutrophil.     

Stabilizing effect of glutaraldehyde on the respiratory burst NADPH oxidase of guinea pig polymorphonuclear leukocytes

The membrane fraction of guinea pig polymorphonuclear leukocytes stimulated with phorbol myristate acetate exhibits the respiratory burst NADPH oxidase activity. This activity is markedly unstable at 37 degrees C, disappearing with a half-life of 11.0 min. When the membrane fraction was pretreated with 0.1% glutaraldehyde, the NADPH oxidase was found to become more stable; its half-life increased about sixfold without any enhancement of the initial activity. The glutaraldehyde treatment of the membrane fraction also protected the NADPH oxidase against inactivation with 0.1-0.2% Triton X-100. These stabilizing effects of glutaraldehyde on the NADPH oxidase seem to be due to its protein cross-linking ability, since its monovalent analogue, butyraldehyde, did not show any effect on the NADPH oxidase activity. In fact, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that glutaraldehyde cross-linked many proteins constituting the membrane.     

Human neutrophil cytosolic activation factor of the NADPH oxidase. Characterization of activation kinetics

The kinetics of sodium dodecyl sulfate-induced activation of respiratory burst oxidase (NADPH oxidase) in a fully soluble cell-free system from resting (control) or phorbol myristate acetate (PMA)-stimulated human neutrophils were investigated. In a cell-free system containing solubilized membranes and cytosol fractions (cytosol) derived from control neutrophils (control cell-free system), the values of Km and Vmax for NADPH of the NADPH oxidase from control neutrophils continuously increased with increasing concentrations of cytosol, but with increasing concentrations of solubilized membranes from the control neutrophils, Km values continuously decreased, suggesting cytosolic activation factor-dependent continuous changes in the affinity of NADPH oxidase to NADPH. In a cell-free system containing solubilized membranes and cytosol prepared from PMA-stimulated neutrophils, NADPH oxidase was not activated after the addition of NADPH. However, cytosol from control neutrophils activated the NADPH oxidase of PMA-stimulated neutrophils in a cell-free system. Cytosol from PMA-stimulated neutrophils did not activate the control neutrophil oxidase, although it contained no inhibitors of NADPH oxidase activation. The results suggest that, in PMA-stimulated neutrophils, cytosolic activation factors may be consumed or exhausted with an increasing period of time after the stimulation of neutrophils, and that the affinity of PMA-stimulated neutrophil NADPH oxidase to NADPH may almost be the same as that of control neutrophil oxidase. It was concluded that the affinity of NADPH oxidase to NADPH was closely associated with interaction between solubilized membranes and cytosolic activation factors, as indicated by the concentration ratio.