The chymotrypsin inhibitor carbobenzyloxy-leucine-tyrosine-chloromethylketone interferes with the neutrophil respiratory burst mediated by a signaling pathway independent of PtdInsP2 breakdown and cytosolic free calcium.

The effects of carbobenzyloxy-leucine-tyrosine-chloromethylketone (zLYCK), an inhibitor of chymotrypsin, were investigated on the activation pathways of the human neutrophil respiratory burst. At 10 microM zLYCK, a parallel inhibition was observed of superoxide production stimulated with the chemo-attractant FMLP and of chymotrypsin-like activity of human neutrophils. By contrast, superoxide production induced by PMA was minimally affected by zLYCK. The known transduction pathways triggered by FMLP were analyzed. zLYCK did not affect either the FMLP-induced cytosolic free calcium transient, inositol 1,4,5 trisphosphate formation, nor the PMA-induced phosphorylation of the 47-kDa substrate of protein kinase C. zLYCK did not affect the activity of protein kinase C extracted from neutrophils. In Ca(2+)-depleted cells, in which phosphatidylinositol 4,5-biphosphate breakdown does not occur, zLYCK inhibited the FMLP-induced respiratory burst in cells primed by low doses of PMA. The activity of the NADPH oxidase tested with active membranes from stimulated neutrophils or in a cell-free system was not inhibited by zLYCK. We conclude that: 1) zLYCK inhibits superoxide production through the inhibition of a chymotrypsin-like protease of the neutrophil, 2) zLYCK inhibits FMLP-induced activation of NADPH oxidase through a pathway independent of PtdInsP2 breakdown and cytosolic free calcium, and 3) zLYCK may prove a useful probe for the characterization of its target protease in neutrophil activation.     

Cytotoxicity testing of wound-dressing materials

A method was developed for testing the cytotoxicity of various bandage-like wound dressings and gel wound dressings. In this method, the ability of human polymorphonuclear neutrophils (PMNs) to initiate a respiratory burst after exposure to the various wound dressings is used as a marker of cytotoxicity. Luminol-amplified chemiluminescence stimulated with opsonised zymosan or phorbol 12-myristate 13-acetate (PMA) is used to measure the degree of activation of the respiratory burst, i.e. the NADPH oxidase activity, after exposure to wound dressings. Opsonised zymosan (material from yeast cell walls) is a phagocytic stimulus that activates the NADPH oxidase by binding to FC-receptors and complement receptors, and functions as an artificial bacterium, whereas PMA activates the NADPH oxidase by direct activation of protein kinase C. NADPH oxidase activity was inhibited by several wound dressings. The down-regulation of the respiratory burst is detrimental to the bactericial effect of PMNs, and can be used as a marker for the cytotoxicity of wound dressing materials.     

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.     

Fluoride-mediated activation of guinea pig neutrophils

In guinea pig periotoneal neutrophils NaF at a concentration of above 5 mM elicited a dose-dependent, delayed and sustained activation of NADPH oxidase. Unlike in human neutrophils, in guinea pig cells, this response was independent of extracellular calcium. Fura2 fluorescence measurements indicated also a fluoride-mediated moderate elevation in the level of cytosolic calcium concentration. Pretreatment of neutrophils with pertussis toxin, blocked fluoride-promoted activation of NADPH oxidase, indicating that NaF stimulation was mediated by a G protein which is a pertussis toxin substrate. NaF-elicited calcium elevation was insensitive to the toxin. Upon transfer of NaF-stimulated cells to a fluoride-free medium, superoxide release declined and calcium levels diminished. The response of the deactivated, fluoride-prestimulated guinea pig neutrophils to a secondary stimulation with phorbol myristate acetate (PMA) or fMet-Leu-Phe, was either unaffected by the previous challenge with NaF (PMA) or augmented by it (the chemotactic peptide). In parallel to the activation of NADPH oxidase, NaF also induced translocation of protein kinase C to cell membranes. This effect was also abolished by a pretreatment with pertussis toxin.     

Fluoride-mediated activation of guinea pig neutrophils

In guinea pig peritoneal neutrophils NaF at a concentration of above 5 mM elicited a dose-dependent, delayed and sustained activation of NADPH oxidase. Unlike in human neutrophils, in guinea pig cells, this response was independent of extracellular calcium. Fura2 fluorescence measurements indicated also a fluoride-mediated moderate elevation in the level of cytosolic calcium concentration. Pretreatment of neutrophils with pertussis toxin, blocked fluoride-promoted activation of NADPH oxidase, indicating that NaF stimulation was mediated by a G protein which is a pertussis toxin substrate. NaF-elicited calcium elevation was insensitive to the toxin. Upon transfer of NaF-stimulated cells to a fluoride-free medium, superoxide release declined and calcium levels diminished. The response of the deactivated, fluoride-prestimulated guinea pig neutrophils to a secondary stimulation with phorbol myristate acetate (PMA) or fMet-Leu-Phe, was either unaffected by the previous challenge with NaF (PMA) or augmented by it (the chemotactic peptide). In parallel to the activation of NADPH oxidase, NaF also induced translocation of protein kinase C to cell membranes. This effect was also abolished by a pretreatment with pertussis toxin.     

Activation of NADPH oxidase of human neutrophils. Potentiation of chemotactic peptide by a diacylglycerol

Formyl-methionyl-leucyl-phenylalanine (fMLP) and 1-oleoyl-2-acetyl-glycerol (OAG) are synergistic stimuli of the respiratory burst of neutrophils. Simultaneous exposure to both agents greatly enhanced superoxide production, both in rate and extent. OAG potentiated the response to fMLP also in Ca++ -free medium. Pretreatment of the neutrophils with fMLP drastically shortened the lag of superoxide production in response to OAG. Our findings lead to the following conclusions: (i) Protein kinase C is likely to be involved in the activation of the NADPH oxidase by fMLP; (ii) OAG appears to be utilized as an intermediate in the activation process; (iii) prestimulation of the cells with fMLP facilitates the response to OAG.     

Comparison of the roles of calmodulin and protein kinase C in activation of the human neutrophil respiratory burst

The roles of calmodulin and protein kinase C in the activation of the human neutrophil respiratory burst were characterized pharmacologically. The protein kinase C inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) did not inhibit superoxide anion generation by neutrophils stimulated for 30 minutes with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) or 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). However, H-7 did depress superoxide production during the first 5 minutes following stimulation. In contrast, the specific calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and the dual calmodulin antagonist/protein kinase C inhibitor trifluoperazine (TFP) were potent inhibitors of the response throughout the 30 minute incubation. Stimulation of neutrophils with submaximal doses of FMLP or PMA failed to promote inhibition of the respiratory burst by H-7 or H-9, but did stimulate a respiratory burst response which was not inhibited by TFP or W-7. These results suggest that while protein kinase C may play a role in the initiation of the respiratory burst response, propagation of the response is dependent on calmodulin-dependent processes. The inability of TFP and W-7 to inhibit superoxide anion generation in response to submaximal stimulatory doses of FMLP or PMA suggests that calmodulin-independent processes may also be involved in activation of the respiratory burst.     

Further evidence for the involvement of a phosphoprotein in the respiratory burst oxidase of human neutrophils.

Phosphorylation of a 47 kDa protein in human neutrophils is induced by phorbol 12-myristate 13-acetate (PMA), opsonized latex beads, fMet-Leu-Phe, calcium ionophore A23187 and fluoride. All of these stimuli activate the specialized microbicidal respiratory burst of neutrophils, and in each case the kinetics of activation correspond with the kinetics of phosphorylation of the 47 kDa protein. Trifluoperazine (50 microM) and chlorpromazine (100 microM), inhibitors of calmodulin and protein kinase C, abolish the increase in oxygen consumption and selectively prevent phosphorylation of the 47 kDa protein after PMA stimulation. Treatment of neutrophils with pertussis toxin totally inhibits both superoxide production and phosphorylation of this protein in response to fMet-Leu-Phe, but not in response to PMA, indicating that a GTP-binding protein modulates the fMet-Leu-Phe receptor signal. Phosphorylation of the 47 kDa protein, a phenomenon absent from the neutrophils of subjects with autosomal recessive chronic granulomatous disease, which lack the respiratory burst, appears to be the common trigger for activation of the burst in normal neutrophils.     

Roles of phospholipase D in phorbol myristate acetate‐stimulated neutrophil respiratory burst

The phorbol myristate acetate (PMA) stimulated nutrophil respiratory burst has been considered to simply involve the activation of protein kinase C (PKC). However, the PLD activity was also increased by 10‐fold in human neutrophils stimulated with 100 nM PMA. Unexpectedly, U73122, an inhibitor of phospholipase C, was found to significantly inhibit PMA‐stimulated respiratory burst in human neutrophils. U73122 at the concentrations, which were sufficient to inhibit the respiratory burst completely, caused partial inhibition of the PLD activity but no inhibition on PKC translocation and activation, suggesting that PLD activity is also required in PMA‐stimulated respiratory burst. Using 1‐butanol, a PLD substrate, to block phosphatidic acid (PA) generation, the PMA‐stimulated neutrophil respiratory burst was also partially inhibited, further indicating that PLD activation, possibly its hydrolytic product PA and diacylglycerol (DAG), is involved in PMA‐stimulated respiratory burst. Since GF109203X, an inhibitor of PKC that could completely inhibit the respiratory burst in PMA‐stimulated neutrophils, also caused certain suppression of PLD activation, it may suggest that PLD activation in PMA‐stimulated neutrophils might be, to some extent, PKC dependent. To further study whether PLD contributes to the PMA stimulated respiratory burst through itself or its hydrolytic product, 1,2‐dioctanoyl‐sn‐glycerol, an analogue of DAG , was used to prime cells at low concentration, and it reversed the inhibition of PMA‐stimulated respiratory burst by U73122. The results indicate that U73122 may act as an inhibitor of PLD, and PLD activation is required in PMA‐stimulated respiratory burst.     

Studies on molecular regulation of phagocytosis in neutrophils. Con A-mediated ingestion and associated respiratory burst independent of phosphoinositide turnover, rise in [Ca2+]i, and arachidonic acid release

The role of the activation of phosphoinositide turnover and of the increase in cytosolic free calcium, [Ca2+]i, in the phagocytosis and associated activation of the respiratory burst was investigated. We report the results obtained on the phagocytosis of yeast cells mediated by Con A in normal and in Ca2+-depleted human neutrophils. In normal neutrophils the phagocytosis was associated with a respiratory burst, a stimulation in the formation of [3H] inositol phosphates and [32P]phosphatidic acid, the release of [3H]arachidonic acid, and a rise in [Ca2+]i. Ca2+-depleted neutrophils are able to perform the phagocytosis of yeast cells mediated by Con A and to activate the respiratory burst without stimulation of [3H]inositol phosphates and [32P]phosphatidic acid formation, [3H]arachidonic acid release, and rise in [Ca2+]i. In both normal and Ca2+-depleted neutrophils the phagocytosis and the associated respiratory burst, 1) were inhibited by cytochalasin B; 2) were insensitive to H-7, an inhibitor of protein kinase C; and 3) did not involve GTP-binding protein sensitive to pertussis toxin. These findings indicate that the activation of phosphoinositide turnover, the liberation of arachidonic acid, the rise in [Ca2+]i, and the activity of protein kinase C are not necessarily required for ingestion of Con A-opsonized particles and for associated activation of the NADPH oxidase, the enzyme responsible for the respiratory burst. The molecular mechanisms of these phosphoinositide and Ca2+-independent responses are discussed.     

Endotoxin priming of neutrophils requires NADPH oxidase-generated oxidants and is regulated by the anion transporter ClC-3

Several soluble mediators, including endotoxin, prime neutrophils for an enhanced respiratory burst in response to subsequent stimulation. Priming of neutrophils occurs in vitro, and primed neutrophils are found in vivo. We previously localized the anion transporter ClC-3 to polymorphonuclear leukocytes (PMN) secretory vesicles and demonstrated that it is required for normal NADPH oxidase activation in response to both particulate and soluble stimuli. We now explore the contribution of the NADPH oxidase and ClC-3 to endotoxin-mediated priming. Lipooligosaccharide (LOS) from Neisseria meningitidis enhances the respiratory burst in response to formyl-Met-Leu-Phe, an effect that was impaired in PMNs lacking functional ClC-3 and under anaerobic conditions. Mobilization of receptors to the cell surface and phosphorylation of p38 MAPK by LOS were both impaired in PMN with the NADPH oxidase chemically inhibited or genetically absent and in cells lacking functional ClC-3. Furthermore, inhibition of the NADPH oxidase or ClC-3 in otherwise unstimulated cells elicited a phenotype similar to that seen after endotoxin priming, suggesting that basal oxidant production helps to maintain cellular quiescence. In summary, NADPH oxidase activation was required for LOS-mediated priming, but basal oxidants kept unstimulated cells from becoming primed. ClC-3 contributes to both of these processes.     

Fluoride can activate the respiratory burst independently of Ca2+, stimulation of phosphoinositide turnover and protein kinase C translocation in primed human neutrophils

Evidences have been provided in our laboratory that in neutrophils different signal transduction sequences for the activation of O2(-)-forming NADPH oxidase can be triggered by the same stimulus (Biochem. Biophys. Res. Commun. 1986, 135, 556-565; 1986, 135, 785-794; 1986, 140, 1-11). The results presented here show that the transduction sequence triggered by fluoride via dissociation of G-proteins and involving messengers produced by stimulation of phosphoinositide turnover, Ca2+ changes and translocation of protein kinase C from the cytosol to the plasmamembrane, can be bypassed when a primed state of neutrophils is previously induced. In fact: i) fluoride causes a pertussis toxin insensitive and H-7 sensitive respiratory burst in human neutrophils, which is linked to the activation of hydrolysis of PIP2, rise in [Ca2+]1 and translocation of PKC. In Ca2+-depleted neutrophils these responses to fluoride do not occur and are restored by addition of CaCl2. ii) The pretreatment of Ca2+-depleted unresponsive neutrophils with non stimulatory doses of PMA restores the activation of the NADPH oxidase by fluoride but not the turnover of phosphoinositides and PKC translocation. The nature of the alternative transduction sequence, the reactions different from phospholipase C activated by G-protein for the alternative sequence and the role of these discrete pathways for NADPH oxidase activation are discussed.     

Cutting edge: infection by the agent of human granulocytic ehrlichiosis prevents the respiratory burst by down-regulating gp91phox

The agent of human granulocytic ehrlichiosis (HGE) is an emerging tick-borne pathogen that resides in neutrophils and can be cultured in a promyelocytic (HL-60) cell line. In response to microbes, polymorphonuclear leukocytes normally activate the NADPH oxidase enzyme complex and generate superoxide anion (O2-). However, HL-60 cells infected with HGE bacteria did not produce O2- upon activation with PMA. RT-PCR demonstrated that HGE organisms inhibited mRNA expression of a single component of NADPH oxidase, gp91phox, and FACS analysis showed that plasma membrane-associated gp91phox protein was reduced on the infected cells. Infection with HGE organisms also decreased gp91phox mRNA levels in splenic neutrophils in a murine model of HGE, demonstrating this phenomenon in vivo. Therefore, HGE bacteria repress the respiratory burst by down-regulating gp91phox, the first direct inhibition of NADPH oxidase by a pathogen.     

Evidence for the involvement of the NADPH oxidase enzyme complex in the optimal accumulation of Platelet-activating factor in the human cell line PLB-985.

Platelet-activating factor (PAF) is an early product of the inflammatory environment, influencing development and resolution of inflammation. Its production is greater in neutrophils and macrophages, which predominantly synthesize 1-alkyl sn-2 acetyl glycerophosphocholine (GPC) than in nongranulocytes (B cells and endothelial cells), which lack a respiratory burst and synthesize 1-acyl sn-2 acetyl GPC as their major PAF species. This study investigated whether the respiratory burst was responsible for the quantitative and qualitative differences in sn-2 acetyl GPC species generation by neutrophils and macrophages versus those cells lacking the NADPH oxidase complex. The myeloid cell line PLB-985 (capable of differentiation into neutrophils) was used to test this hypothesis, since these cells had previously been generated with a non-functional respiratory burst (X-CGD PLB-985). Differentiated PLB-985 cells underwent a large respiratory burst in response to PMA (phorbol ester), and smaller respiratory bursts in response to A23187 (calcium ionophore), and the bacterial polypeptide fMLP (receptor mediated activation). Concurrently, treated cells were assessed for production of 1-hexadecyl and 1-palmitoyl sn-2 acetyl GPC species by gas chromatography/mass spectrometry. Neither cell type generated these lipid species in response to PMA, but both cell types generated equal levels of sn-2 acetyl GPC in response to A23187, with five times more 1-hexadecyl than 1-palmitoyl species. Upon fMLP activation, X-CGD PLB-985 cells produced significantly less 1-hexadecyl and 1-palmitoyl sn-2 acetyl GPC in comparison to the wild-type PLB-985 cells. These findings suggest phagocytic oxidant production by NADPH oxidase is not essential for sn-2 acetyl GPC generation, but appears important for optimal production of PAF in response to some stimuli.     

Tamoxifen does not inhibit the swell activated chloride channel in human neutrophils during the respiratory burst

Effective functioning of neutrophils relies upon electron translocation through the NADPH oxidase (NOX). The electron current generated (I_e) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential in activated human neutrophils. Swelling activated chloride channels have been demonstrated in part to counteract the depolarisation generated by the NADPH oxidase I_e. In the present study, the effects of inhibitors of swell activated chloride channels on ROS production and on the swelling activated chloride conductance was investigated in activated human neutrophils. Tamoxifen (10 μM), a specific inhibitor for swell activated chloride channels in neutrophils, completely inhibited both the PMA and FMLP stimulated respiratory burst. This inhibition of the neutrophil respiratory burst was not due to the blocking effect of tamoxifen on the swelling activated chloride conductance in these cells. These results demonstrate that a tamoxifen insensitive swell activated chloride channel has important significance during the neutrophil respiratory burst.     

Redistribution of protein kinase C activity in human monocytes: correlation with activation of the respiratory burst

Protein kinase C (PKC) was found to be present in purified human monocytes and lymphocytes isolated by countercurrent centrifugal elutriation. In unstimulated monocytes and lymphocytes, approximately 90% of the PKC activity was cytosolic when the cells were disrupted in the presence of EGTA. The role of this kinase in the stimulation of the respiratory burst in monocytes was investigated. Phorbol esters capable of triggering the release of O2- caused a loss of PKC activity from the cytosol and the appearance of the kinase activity in the particulate cell fraction. Kinase activity was partially extractable from the particulate fraction by 0.1% Triton X-100, whereupon it demonstrated calcium and lipid dependence. The EC50 for the phorbols in initiating the respiratory burst correlated well with their EC50 for stimulating the appearance of PKC activity in the particulate fraction (R = 0.998). Redistribution of PKC activity in monocytes by phorbol myristate acetate (PMA) was rapid and appeared to precede the release of O2-. PMA also shifted PKC activity from the cytosol to the extractable particulate fraction of lymphocytes. We conclude that redistribution of PKC activity by active phorbols or other cell stimulants could provide substrate specificity for phosphorylation reactions. By shifting PKC activity to the monocyte particulate fraction, active phorbols may initiate the phosphorylation of a substrate required for stimulation of the respiratory burst.     

Effect of the calcium ionophore A23187 on superoxide generation in phorbol ester stimulated human neutrophils

The calcium ionophore, A23187, and the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), interacted synergistically to elicit an accelerated superoxide production response in human neutrophils. The lag period preceding PMA-induced superoxide generation was decreased in a dose-dependent manner by A23187 at a concentration range from 1.0 X 10(-8) to 1.0 X 10(-5) M. Superoxide production rate, however, was subject to biphasic effects. While the rate was potentiated in a dose-dependent manner at A23187 concentrations below 1.0 X 10(-6) M, inhibitory influences became manifest at higher concentrations. Total superoxide production was subject to inhibitory effects, characterized by a mean inhibitory dose of 1.3 X 10(-6) M. The synergistic interaction of A23187 with PMA is consistent with a role for protein kinase C in neutrophil activation. Inhibition at high A23187 concentrations appeared to result from the effects of elevated intracellular Ca2+ levels on either NADPH oxidase itself, or some step in the transduction process linking protein kinase C to the oxidase complex.     

Activation of neutrophil NADPH oxidase in a cell-free system. Partial purification of components and characterization of the activation process

The superoxide-generating enzyme of human neutrophils, NADPH oxidase, is converted from an inactive to an active form upon stimulation of the neutrophil. This activation process was examined using a recently developed cell-free system in which dormant oxidase is activated by arachidonic acid in the presence of a soluble factor from the neutrophil (Curnutte, J. T. (1985) J. Clin. Invest. 75, 1740-1743). NADPH oxidase from unstimulated human neutrophils was detected only in the membrane fraction. The soluble activation factor was localized entirely to the cytosolic fraction and exhibited two peaks of activity when partially purified under nondenaturing conditions: a major peak with a molecular mass of approximately 250 kDa and a variable minor peak with a mass of approximately 40 kDa. Both forms activated NADPH oxidase in a similar manner and did not exhibit synergy when combined. The cytosolic factor is not protein kinase C (or another kinase) as both peaks of factor activity could be resolved from the protein kinase C peak and neither required calcium or ATP to activate the oxidase. Activation of NADPH oxidase did require the simultaneous presence of the membrane fraction, the cytosolic factor, arachidonic acid, and magnesium. Following activation, however, only the membrane fraction was then required for O2- production. Cytosolic factor levels were normal in five patients with either X-linked or autosomal recessive cytochrome b-negative chronic granulomatous disease. In contrast, the membrane fractions from each failed to generate O2-, indicating that the defects in these two genetic forms of chronic granulomatous disease reside either in the oxidase itself or in a membrane component required for activation.     

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.     

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.