Protein kinase C mediates human neutrophil cytotoxicity

Human neutrophils stimulated with phorbol myristate acetate were able to damage human erythroleukemic K562 cells, in the absence of specific antibody, as assessed by a two hour 51Cr release assay. Neutrophils treated with formyl-peptide fMet-Leu-Phe did not display tumoricidal response, but the addition of diacylglycerol kinase inhibitor R59022 together with formyl-peptide induced the cytotoxic capacity against tumor target cells. Phorbol ester is a potent activator of certain functions of neutrophils because of its ability to directly and irreversibly stimulate protein kinase C; formyl-peptide, on the contrary, activates protein kinase C by inducing a rapid and transient production of diacylglycerol, that is quickly metabolized. The addition of an inhibitor of diacylglycerol kinase, R59022, however potentiated the action of formyl-peptide. These results indicate that protein kinase C is involved in the tumoricidal activity of neutrophils against K562 cells, and that maximal activation of the enzyme is required to achieve the cytotoxic response.     

Rapid priming of calcium mobilization and superoxide anion production in human neutrophils by substimulatory concentrations of phorbol esters: a novel role for protein kinase C and tyrosine phosphorylation in the up-modulation of signal transduction.

The modulatory influences of phorbol esters on the functional responsiveness of human peripheral blood neutrophils have been investigated. These studies focused on measurements of the levels of cytoplasmic free calcium and of tyrosine phosphorylation as well as on their ability to mount an oxidative response. Short incubation times (< 1 min) with low concentrations of phorbol esters (5-50 nM) were shown to enhance the above indices of neutrophil responsiveness to chemotactic factors such as fMet-Leu-Phe and leukotriene B4. On the other hand, a time- and concentration-dependent inhibition of calcium mobilization and superoxide production was also observed. The effects of the phorbol esters were stereo-specific and were antagonized by a novel protein kinase C inhibitor (RO 318220) but were not affected by the oxidative burst inhibitor diphenyleneiodonium. Pre-incubation of the cells with phorbol 12,13-dibutyrate (PDBu) altered in a concentration-dependent manner the tyrosine phosphorylation pattern stimulated by fMet-Leu-Phe. In addition, the tyrosine kinase inhibitor erbstatin inhibited the priming of the mobilization of calcium induced by PDBu. These data demonstrate the rapidity of the effects of the activation of protein kinase C, their potential to modulate positively the early events of the excitation-response coupling sequence and the complexity of the functional interrelationships among the various cellular activation pathways available to human neutrophils and other non-muscle cells.     

Chemoattractant-induced cytoplasmic pH changes and cytoskeletal reorganization in human neutrophils. Relationship to the stimulated calcium transients and oxidative burst

The relationships between the chemotactic factor-stimulated mobilization of calcium, activation of the NADPH-oxidase, changes in cytosolic pH, and in the level of polymerized actin in human neutrophils have been examined. The approach taken was to use intracellular calcium chelators, and pharmacologic modulators (both positive and negative) of the NADPH-oxidase to measure the aforementioned responses under conditions where the calcium transients were abrogated and/or the generation of superoxide anions was either inhibited or augmented. The decrease in cytosolic pH induced by chemoattractants was inhibited by the calcium chelator BAPTA and by the diglyceride kinase inhibitor 6-[2-(4-[(4-fluorophenyl)phenylmethylene]-1-piperidinylethyl ]-7-methyl-5H-thiazolo[3,2-alpha]pyriimidin-5-one (R59022) (this latter compound enhanced the oxidative response of the cells). Furthermore, a specific inhibitor of the NADPH-oxidase (diphenyleneiodonium) had no significant effect on the cytosolic acidification induced by FMLP or leukotriene B4. These results indicate that the initiation of the cytosolic acidification induced by chemotactic factors is a calcium-dependent event that is not directly linked to the activation of the NADPH-oxidase. In contrast, the stimulated polymerization of actin was insensitive to BAPTA, R59022, and diphenyleneiodonium. Thus, neither the calcium transients nor the oxidative burst play a signaling role in the initiation of actin polymerization elicited by chemoattractants. These data indicate that additional investigations are needed to uncover the biochemical basis of the signals initiated in human neutrophils by chemotactic factors that lead to the polymerization of actin and to the cytosolic acidification.     

Xanthine oxidase-catalysed oxidation of paracetamol.

[3H]Arachidonic acid is released after stimulation of rabbit neutrophils with fMet-Leu-Phe or platelet-activating factor (PAF). The release is rapid and dose-dependent, and is inhibited in phorbol 12-myristate 13-acetate (PMA)-treated rabbit neutrophils. The protein kinase C (PKC) inhibitor 1-(5-isoquinoline-sulphonyl)-2-methylpiperazine (H-7) prevents this inhibition. In addition, PMA increases arachidonic acid release in H-7-treated cells stimulated with fMet-Leu-Phe. [3H]Arachidonic acid release, but not the rise in the concentration of intracellular Ca2+, is inhibited in pertussis-toxin-treated neutrophils stimulated with PAF. The diacylglycerol kinase inhibitor R59022 increases the concentration of diacylglycerol and potentiates [3H]arachidonic acid release in neutrophils stimulated with fMet-Leu-Phe. This potentiation is not inhibited by H-7. These results suggest several points. (1) A rise in the intracellular concentration of free Ca2+ is not sufficient for arachidonic acid release in rabbit neutrophils stimulated by physiological stimuli. (2) A functional pertussis-toxin-sensitive guanine nucleotide regulatory protein and/or one or more of the changes produced by phospholipase C activation are necessary for arachidonic acid release produced by physiological stimuli. (3) Agents that stimulate PKC potentiate arachidonic acid release, and this potentiation is not inhibited by H-7. These agents produce their actions in part by direct membrane perturbation.     

Protein kinase C-dependent and -independent activation of the NADPH oxidase of human neutrophils

The protein kinase C inhibitor, staurosporine, inhibited NADPH oxidase activity of human neutrophils activated by phorbol myristate acetate. However, this inhibitor had no effect on either the initiation or the maximal rate of O2- secretion activated by the chemotactic peptide, fMet-Leu-Phe, but resulted in a more rapid termination of oxidant production. Similarly, staurosporine had no effect on the rapid (1 min) increase in luminol-dependent chemiluminescence activated by fMet-Leu-Phe, but the second (intracellular) phase of oxidant production was inhibited. The initial burst of oxidant production during phagocytosis was similarly protein kinase C-independent, but again the later phases of oxidase activity were staurosporine-sensitive. Neutrophils loaded with Quin-2 at concentrations sufficient to act as a Ca2+ buffer could not secrete O2- in response to fMet-Leu-Phe; although the initial (protein kinase C-independent) burst of luminol chemiluminescence was not observed in fMet-Leu-Phe-stimulated Ca2(+)-buffered cells, the second phase of (protein kinase C-dependent) oxidant production was largely unaffected. Hence, the initial burst of oxidant production activated by fMet-Leu-Phe, opsonized zymosan, and latex beads is independent of the activity of protein kinase C-dependent intracellular activation processes, but the activity of this kinase is required to extend or sustain the duration of oxidant production.     

The diacylglycerol kinase inhibitor, R59022, potentiates cholecystokinin-induced enzyme secretion from rabbit pancreatic acini.

The putative inhibitor of diacylglycerol kinase activity, 6-(2-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl)-ethyl-7-meth yl-5H- thiazolo[3,2-a]pyrimidin-5-one (R59022), markedly potentiated cholecystokinin-C-terminal-octapeptide(CCK-8-)stimulated enzyme secretion from isolated rabbit pancreatic acini. Maximal potentiation occurred when acini were stimulated in the presence of 5-10 microM R59022. Potentiation depended both on the concentration of R59022 and CCK-8. No potentiation was observed when acini were half-maximally stimulated, whereas the secretory response to maximal and supramaximal concentrations of secretagogue was increased by 50-60%. R59022 alone had no effect on basal enzyme secretion and the drug did not potentiate the secretory response to the Ca2+ ionophore A23187 or to the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate. Moreover, no increase in basal secretion was observed when acini were incubated in the presence of both R59022 and forskolin. These observations strongly suggest that receptor-mediated activation of the inositol phospholipid pathway is required for R59022-induced potentiation. R59022 inhibited the CCK-8-stimulated incorporation of 32Pi into phosphatidic acid dose dependently, without affecting the CCK-8-stimulated hydrolysis of 32P-labelled phosphatidylinositol 4,5-bisphosphate. This is consistent with an inhibitory effect of R59022 on acinar cell diacylglycerol kinase activity. The potentiating effect of R59022 was mimicked by 12-O-tetradecanoylphorbol 13-acetate added simultaneously with CCK-8. Therefore, it is concluded that in the presence of 5-10 microM R59022 the receptor-mediated increase in acinar cell diacylglycerol content is enhanced leading to enhanced activation of protein kinase C and to potentiation of the secretory response. The fact that the secretory response to maximal and supramaximal concentrations of CCK-8 is potentiated by R59022 suggests that at these concentrations of secretagogue the diacylglycerol/protein kinase C branch of the signal-transduction route is rate-limiting.     

Diacylglycerol kinase inhibitors R59022 and dioctanoylethylene glycol potentiate the respiratory burst of neutrophils by raising cytosolic Ca2+

The diacylglycerol kinase inhibitors, R59022 and dioctanoylethylene glycol (diC8-eg), potentiate stimulation of the respiratory burst by the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) in human neutrophils. However, in contrast to the potentiation observed in intact cells, neither R59022 nor diC8-eg enhanced the effect of fMLP on O2 consumption in electropermeabilized neutrophils, under conditions where cytosolic [Ca2+] was held constant using EGTA. In unstimulated, intact cells treatment with the diacylglycerol kinase inhibitors elicited an increase in cytosolic Ca2+ ([Ca2+]i). The results suggest that enhancement of the respiratory burst by diC8-eg and R59022 is mediated by a rise in [Ca2+]i, rather than by inhibition of diacylglycerol kinase.     

Cell alkalinization is not necessary and increased sodium influx is not sufficient for stimulated superoxide production

Preincubation of rabbit neutrophils for 5 min with the protein kinase C inhibitor H7 causes inhibition of the rise in intracellular pH but not the increase in Na+ influx or stimulated oxidative burst produced by the chemotactic factor formyl-methionyl-leucyl-phenylalanine. On the other hand, the stimulated superoxide production, but not the increase in Na+ influx produced by phorbol 12-myristate 13-acetate, is inhibited by H7. The effect is more pronounced on the rate than the extent of the stimulated superoxide release. Furthermore, cell acidification produced by the phorbol ester but not by the chemotactic factor is decreased in the presence of H7. These results suggest that most of the stimulated Na+ influx is not coupled to H+ efflux, in the case of the chemoattractant, the rise in intracellular pH is not necessary for stimulated superoxide production, the increase in Na+ influx, in the case of the phorbol ester, is not sufficient for the stimulation of the oxidative burst, and the sources of the H+ responsible for the stimulated pH drop are the various metabolic activities of the cell, including NADPH oxidation and activation of the hexose monophosphate shunt.     

Phospholipase D-dependent and-independent activation of the neutrophil NADPH oxidase

Stimulation of the respiratory burst of human neutrophils by fMet-Leu-Phe (in the absence of cytochalasin B) is largely unaffected when the activities of protein kinase C and phospholipase D are inhibited. This has been confirmed using three separate assays to measure the respiratory burst. However, whilst these enzymes are not required for the initiation or maximal rate of oxidant generation, they are required to sustain oxidase activity. In contrast, in the presence of cytochalasin B, fMet-Leu-Phe stimulated oxidase activity is much more dependent on phospholipase D activity. It is proposed that (in the absence of cytochalasin B) activation of the NADPH oxidase utilises cytochrome b molecules that are already present on the plasma membrane and activation occurs independently of phospholipase D and protein kinase C. Once these complexes are inactivated, then new cytochrome b molecules must be recruited from sub-cellular stores. This translocation and/or activation of these molecules is phospholipase D dependent. Some support for this model comes from the finding that the translocation of CD11b (which co-localises with cytochrome b) onto the cell surface is phospholipase D dependent.Abbreviations GM-CSF granulocyte-macrophage colony-stimulating factor - fMet-Leu-Phe N-formylmethionyl-leucyl-phenylalanine luminol 5-amino-2,3-dihydro-1,4-phthalazinedione, O₂,-superoxide radical     

The diacylglycerol kinase inhibitor R 59022 potentiates bombesin stimulation of protein kinase C activity and DNA synthesis in Swiss 3T3 cells

Addition of the inhibitor of diacylglycerol kinase R 59022 to quiescent Swiss 3T3 cells potentiated protein kinase C activation by the neuropeptide bombesin, a potent mitogen for these cells. This was detected as a marked shift in the dose-response relationship for bombesin-stimulated phosphorylation of a Mr 80,000 protein, which is a major, specific substrate of protein kinase C. R 59022 also promoted the inhibition of [125I]epidermal growth factor binding induced by bombesin, an effect mediated by protein kinase C. A salient feature of our results is that R 59022, at concentrations that enhanced the activation of protein kinase C in intact cells (4-6 microM), potentiated long-term mitogenesis elicited by bombesin. Thus, R 59022 may be a valuable tool for elucidating the contribution of the protein kinase C pathway in mitogenesis.     

Effect of an inhibitor of protein kinase C on human polymorphonuclear leukocyte degranulation

The protein kinase C inhibitor C-I reduced superoxide production by human neutrophils in response to phorbol myristate acetate by greater than 50%. In contrast to its effects in oxidative metabolism, 100 microM C-I caused minimal inhibition (5-18%) of lysozyme release in response to phorbol myristate acetate. Enzyme release produced by the formylated oligopeptide FMLP was enhanced by 23-54% in neutrophils pretreated with 100 microM C-I. These findings suggest that protein kinase C activation is not required for phorbol myristate acetate induced enzyme release. Enhancement of FMLP stimulated degranulation by C-I suggests that protein kinase C activation may have inhibitory effects on the release of granule enzymes by human neutrophils.     

Role of the actin cytoskeleton during respiratory burst in chemoattractant-stimulated neutrophils

The aim of this study was to clarify the role of the actin cytoskeleton during chemotactic peptide fMet-Leu-Phe (fMLF)-stimulated respiratory burst in human neutrophil granulocytes. Reactive oxygen species (ROS) was measured as luminol-amplified chemiluminescence (CL) and F-actin content as bodipy phallacidin fluorescence in neutrophils treated with latrunculin B or jasplakinolide, an inhibitor and activator of actin polymerization, respectively. Latrunculin B markedly decreased, whereas jasplakinolide increased, the F-actin content in neutrophils, unstimulated or stimulated with fMLF. Latrunculin B enhanced the fMLF-triggered ROS-production more than tenfold. Jasplakinolide initially inhibited the fMLF-induced CL-response, however, caused a potent second sustained phase (>400% of control). Both actin drugs triggered a substantial CL-response when added 5-25 min after fMLF. This was also valid for chemotactic doses of fMLF, where latrunculin B and jasplakinolide amplified the ROS-production 5-10 times. By using specific signal transduction inhibitors, we found that the NADPH oxidase activation triggered by destabilization of the actin cytoskeleton occurs downstream of phospholipase C and protein kinase C but is mediated by Rho GTPases and tyrosine phosphorylation. In conclusion, rearrangements of the actin cytoskeleton are a prerequisite in connecting ligand/receptor activation, generation of second messengers and assembly of the NADPH oxidase in neutrophil granulocytes.     

Stimulation of a histone H4 protein kinase in Triton X-100 lysates of rabbit peritoneal neutrophils pretreated with chemotactic factors. Effect of fMet-Leu-Phe and partial characterization of the protein kinase

Rabbit peritoneal neutrophils were stimulated with either the chemotactic factor, fMet-Leu-Phe (10(-8) M, 10 s) or the protein kinase C activator, phorbol-12-myristate-13-acetate (PMA), (0.1 microgram/ml, 3 min) at 37 degrees C, lysed with Triton X-100 at the indicated times and the histone H4 kinase activity of the lysate measured. The histone H4 protein kinase activity was increased severalfold by fMet-Leu-Phe but not PMA. The inclusion of the potent protein kinase C inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (50 microM) inhibited little if any of the histone H4 protein kinase activity. The effect of fMet-Leu-Phe was transient, maximum stimulation occurring within 10 s and decaying thereafter. The soluble fraction (extract) of the Triton X-100 lysates from control and fMet-Leu-Phe-treated cells was found to contain both histone H4 protein kinase and calcium-phospholipid-activated protein kinase (protein kinase C) activities. The histone H4 protein kinase activity obtained after fMet-Leu-Phe treatment was very little affected by calcium, phospholipid, and PMA and preferred histone H4 but not H1 or H2A as its substrate. In contrast, the calcium-phospholipid-activated protein kinase activity of the extract preferred histones H1 or H2A as substrates and was strongly inhibited by 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine. The histone H4 protein kinase was partially separated from kinase C by DEAE-cellulose and phenyl-Sepharose 4B chromatography. It phosphorylated mostly serine in histone H4. The results indicate that the chemotactic factor, fMet-Leu-Phe, stimulates a protein kinase with substrate specificity and biochemical properties distinct from calcium-phospholipid-activated protein kinase C.     

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.     

Differential effects of the diacylglycerol kinase inhibitor R59022 on thrombin versus collagen-induced human platelet secretion

R59022 is an inhibitor of the enzyme 1,2-diacylglycerol (DAG) kinase, which, by inhibiting the conversion of DAG to phosphatidic acid, causes an increase in endogenous DAG levels and the activity of the DAG-dependent enzyme protein kinase C. This property of the drug was utilized in the present study to assess the role of DAG, i.e., its relative importance as a potentiatory versus inhibitory mediator, in agonist-induced platelet activation. The phosphorylation of the 40-47-kDa protein by protein kinase C was monitored as an indicator of endogenous DAG levels and correlated with other agonist-induced platelet responses such as platelet aggregation, 5-hydroxytryptamine (5HT) secretion and arachidonate release, the agonists used being those that induce DAG formation, e.g., thrombin and collagen. Pretreatment of platelets with R59022 before agonist addition resulted in the potentiation of 5HT secretion as well as 45 kDa protein phosphorylation induced by thrombin and the DAG analogue, 1,2-dioctanoylglycerol (DiC8). However, collagen-induced 5HT secretion was significantly inhibited (70%) in the presence of R59022, which also had strong inhibitory effects on aggregation induced by collagen, as well as by thrombin and DiC8. The inhibition of collagen-induced secretion by R59022 was in contrast to the potentiatory effects of DiC8 on the same, suggesting that even although DAG acts as a potentiatory signal in this system, the inhibitory effects of R59022 on collagen-induced aggregation can mask any effects of endogenous DAG. This inhibitory effect of R59022 on agonist-induced platelet aggregation makes it unsuitable as a tool in studying the role of DAG in platelet activation induced by agonists such as collagen as well as the 'weak' agonists (ADP, adrenaline and platelet-activating factor), where aggregation mediates other responses such as arachidonate release and secretion. Furthermore, potentiatory effects of R59022 on 5HT secretion induced by phorbol 12-myristate 13-acetate and ionomycin, which are effects unlikely to be related to inhibition of DAG kinase was observed, and these effects further underline the non-specificity in the actions of R59022 and its limitations as a tool in studying platelet stimulus-response coupling.     

Activation of superoxide formation and lysozyme release in human neutrophils by the synthetic lipopeptide Pam3Cys-Ser-(Lys)4. Involvement of guanine-nucleotide-binding proteins and synergism with chemotactic peptides.

Upon exposure to the bacterial chemotactic peptide fMet-Leu-Phe, human neutrophils release lysozyme and generate superoxide anions (O2.-). The synthetic lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam3Cys), which is derived from the N-terminus of bacterial lipoprotein, when attached to Ser-(Lys)4 [giving Pam3Cys-Ser-(Lys)4], activated O2.- formation and lysozyme release in human neutrophils with an effectiveness amounting to about 15% of that of fMet-Leu-Phe. Palmitic acid, muramyl dipeptide, lipopolysaccharide and the lipopeptides Pam3Cys-Ala-Gly, Pam3Cys-Ser-Gly, Pam3Cys-Ser, Pam3Cys-OMe and Pam3Cys-OH did not activate O2.- formation. Pertussis toxin, which ADP-ribosylates guanine-nucleotide-binding proteins (G-proteins) and functionally uncouples formyl peptide receptors from G-proteins, prevented activation of O2.- formation by fMet-Leu-Phe and inhibited Pam3Cys-Ser-(Lys)4-induced O2.- formation by 85%. Lipopeptide-induced exocytosis was pertussis-toxin-insensitive. O2.- formation induced by Pam3Cys-Ser-(Lys)4 and fMet-Leu-Phe was enhanced by cytochalasin B, by a phorbol ester and by a diacylglycerol kinase inhibitor. Addition of activators of adenylate cyclase and removal of extracellular Ca2+ inhibited O2.- formation by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 to different extents. Pam3Cys-Ser-(Lys)4 synergistically enhanced fMet-Leu-Phe-induced O2.- formation and primed neutrophils to respond to the chemotactic peptide at non-stimulatory concentrations. Our data suggest the following. (1) Pam3Cys-Ser-(Lys)4 activates neutrophils through G-proteins, involving pertussis-toxin-sensitive and -insensitive processes. (2) The signal transduction pathways activated by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 are similar but not identical. (3) In inflammatory processes, bacterial lipoproteins and chemotactic peptides may interact synergistically to activate O2.- formation, leading to enhanced bactericidal activity.     

Diacylglycerol accumulation and superoxide anion production in stimulated human neutrophils

Exogenous diacylglycerols stimulate neutrophil superoxide anion production, suggesting that endogenous diacylglycerols may function as second messengers for this biological response. We have measured the diacylglycerol mass in human neutrophils stimulated by fMet-Leu-Phe, ionomycin, and concanavalin A and have correlated the kinetics and magnitude of the diacylglycerol response with those for superoxide anion production. For each stimulus, no increase in diacylglycerol mass was detected prior to the onset of superoxide anion generation. However, large sustained increases in diacylglycerol concentration (260-2000% of basal levels) occurred in parallel with the rise in superoxide anion. The cessation or continuation of diacylglycerol accumulation and superoxide anion production also correlated. The diacylglycerol response was proportional to the stimulus concentration and correlated with the concentration dependence for superoxide anion. Pretreatment of neutrophils with cytochalasin B enhanced both superoxide anion and diacylglycerol responses with all three stimuli. These data support the hypothesis that diacylglycerol functions as a modulator of superoxide anion generation causing a sustained or augmented respiratory burst.     

Chemotactic factor-induced recruitment and activation of Tec family kinases in human neutrophils. II. Effects of LFM-A13, a specific Btk inhibitor

Tyrosine phosphorylation events play major roles in the initiation and regulation of several functional responses of human neutrophils stimulated by chemotactic factors such as the bacterially derived tripeptide formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). However, the links between the G protein-coupled receptors, the activation of the tyrosine kinases, and the initiation of neutrophil functional responses remain unclear. In the present study we assessed the effects of a Btk inhibitor, leflunomide metabolite analog (LFM-A13), on neutrophils. LFM-A13 decreased the tyrosine phosphorylation induced by fMet-Leu-Phe and inhibited the production of superoxide anions and the stimulation of adhesion, chemotaxis, and phospholipase D activity. We observed a decreased accumulation of phosphatidylinositol-3,4,5-trisphosphate in response to fMet-Leu-Phe in LFM-A13-pretreated cells even though the inhibitor had no direct effect on the lipid kinase activity of the p110 gamma or p85/p110 phosphatidylinositol 3-kinases or on the activation of p110 gamma by fMet-Leu-Phe. The phosphorylation of Akt and of extracellular signal-regulated kinases 1/2 and p38 were similarly inhibited by LFM-A13. LFM-A13 also negatively affected the translocation of Rac-2, RhoA, ADP ribosylation factor-1, Tec, Bmx, and Btk induced by fMet-Leu-Phe. The results of this study provide evidence for an involvement of Btk and possibly other Tec kinase family members in the regulation of the functional responsiveness of human neutrophils and link these events, in part at least, to the modulation of levels of phosphatidylinositol-3,4,5-trisphosphate.     

Intracellular calcium rise produced by platelet-activating factor is deactivated by fMet-Leu-Phe and this requires uninterrupted activation sequence: role of protein kinase C

Stimulation of the neutrophils with fMet-Leu-Phe inhibits the rise in intracellular concentration of free calcium produced by the subsequent addition of platelet-activating factor. This deactivation is not observed in pertussis toxin treated cells. In addition, preincubation of the cells with the protein kinase C activator phorbol 12-myristate 13-acetate for three minutes abolishes completely the rise in calcium produced by platelet-activating factor. This inhibition is prevented by the addition of the protein kinase C inhibitor 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine prior to the addition of the phorbol ester. Phorbol 12-myristate 13-acetate, at a concentration that does not produce significant inhibition, accelerates the rate of calcium removal from the cytoplasm, and this is abolished by the protein kinase C inhibitor. In contrast, the deactivation by fMet-Leu-Phe is not prevented by the protein kinase C inhibitor. The results presented here suggest that the protein kinase C system may regulate the opening by platelet-activating factor of possible plasma membrane associated pertussis toxin independent calcium channels and/or the binding of platelet-activating factor to the receptors. In addition, protein kinase C activation increases the rates of the calcium efflux pump and/or calcium sequestering by intracellular organelles. The most simple and straightforward explanation of the observed deactivation by fMet-Leu-Phe is that the addition of fMet-Leu-Phe to neutrophils stimulates the production of platelet-activating factor which then binds to and deactivates the receptors.