Phorbol myristate acetate (PMA) augments chemoattractant-induced diglyceride generation in human neutrophils but inhibits phosphoinositide hydrolysis. Implications for the mechanism of PMA priming of the respiratory burst

Pretreatment ("priming") of neutrophils with a non-activating concentration (2 nM) of phorbol myristate acetate (PMA) augments superoxide (O2-) production in response to the chemoattractant formylmethionylleucylphenylalanine (fMLP). We initially examined the effect of sphinganine, an inhibitor of protein kinase C (Ca2+/phospholipid-dependent enzyme), on activation of primed neutrophils. In both primed and unprimed cells activation by fMLP was blocked, and inhibition occurred at identical concentrations, supporting a common inhibited site. PMA also augmented (about 2-fold) fMLP-induced generation of sn-1,2-diglyceride (DG), the level of which correlated with O2- generation. In contrast to its effects on DG, PMA diminished by about 50% the magnitude of the fMLP-stimulated rise in cytosolic Ca2+. Thus, PMA priming dissociates the fMLP-stimulated Ca2+ increase from DG and O2- generation. The effect of PMA on Ca2+ levels appeared to be due in part to lowered levels of inositol trisphosphate. Lowering of inositol phosphate levels correlated with inhibition of fMLP-induced hydrolysis of inositol-containing phospholipids, particularly phosphatidylinositol 4,5-bisphosphate. PMA did not inhibit (and in fact augmented at early time points) formation of [32P] phosphatidic acid in response to fMLP, indicating that the increase in DG was not due to inhibition of cellular diglyceride kinase. Thus, the data suggest that PMA enhances fMLP-stimulated DG generation concomitant with switching the source of DG from phosphatidylinositol 4,5-bisphosphate to an alternative lipid(s). Increased DG and inhibition of activation by sphinganine are consistent with a role for protein kinase C in activation of the respiratory burst in PMA-primed neutrophils.     

Priming of the respiratory burst of neutrophils by diacylglycerol. Independence from activation or translocation of protein kinase C

Preincubation of neutrophils with certain agonists may "prime" the cells to cause increased responses to a second stimulus ("primed stimulation"). We used two approaches to examine the role of protein kinase C (Ca2+/phospholipid-dependent enzyme) in priming and stimulation by 1-oleoyl-2-acetylglycerol (OAG), phorbol 12-myristate 13-acetate (PMA), and N-formyl-Met-Leu-Phe (fMLP): inhibition of protein kinase C by 1-(5-isoquinolinesulfonyl)-piperazine (C-I) and measurement of protein kinase C translocation induced by priming and stimulatory concentrations of OAG. C-I had little effect on stimulation or primed stimulation by fMLP, suggesting that fMLP invokes events independent of protein kinase C. C-I equally inhibited stimulation and primed stimulation by PMA. Direct stimulation by OAG was inhibited, but priming and primed stimulation by OAG was unaltered by C-I. OAG concentrations greater than or equal to 100 microM caused translocation of protein kinase C, in correlation with direct stimulation of the respiratory burst. Lower OAG concentrations (10-30 microM) primed to stimulation by fMLP and, conversely, stimulated neutrophils primed with fMLP, yet did not cause translocation of protein kinase C. The data are compatible with previous assumptions that PMA and OAG directly stimulate polymorphonuclear neutrophil leukocytes by translocation and activation of protein kinase C. However, priming and primed stimulation by OAG apparently invoke distinct transduction mechanisms other than protein kinase C translocation.     

Phospholipase D is required in the signaling pathway leading to p38 MAPK activation in neutrophil-like HL-60 cells, stimulated by N-formyl-methionyl-leucyl-phenylalanine.

Human acute myelogenous leukemia cells (HL-60 cells) can be induced to differentiate to neutrophils by exposure to dibutyryl-cyclic AMP. The differentiation of HL-60 cells allowed the mitogen-activated protein kinases p38 and p44/p42 to be rapidly and transiently activated upon stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). Western blot analysis using phosphospecific p38 and p44/p42 mitogen-activated protein kinase antibodies showed that increasing concentrations of ethanol or 1-butanol but not 2-butanol (0.05-0.5%) inhibited fMLP-induced p38 activation but did not inhibit p44/p42 activation. These data indicated that activation of phospholipase D (PLD) was required for activation of p38 but not p44/p42. We compared the effect of fMLP with those of tumor necrosis factor alpha (TNF alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF). We found that ethanol did not inhibit p38 phosphorylation upon stimulation with either GM-CSF or TNF alpha. These results suggested that in cells stimulated with fMLP, PLD was upstream of p38. To further test the involvement of PLD, we used antisense inhibition of human PLD1 expression. Treatment with antisense oligonucleotides inhibited p38 but not p44/p42 phosphorylation. These data supported a role for human PLD1 in fMLP-induced p38 activation in neutrophil-like HL-60 cells. In addition, the results obtained with TNF alpha and GM-CSF demonstrated that p38 activation occurred independently of PLD activation.     

Priming of human polymorphonuclear leukocytes with granulocyte-macrophage colony-stimulating factor involves protein kinase C rather than enhanced calcium mobilisation.

Pretreatment of human polymorphonuclear leukocytes with the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) enhances leukotriene biosynthesis in response to a receptor agonist (e.g. N-formyl-methionyl-leucyl-phenylalanine, fMLP) or a Ca(2+)-ionophore (e.g. ionomycin). This priming effect could be traced back to an elevated release of arachidonic acid from the phospholipid pools and hence an increased leukotriene biosynthesis by 5-lipoxygenase. Preincubation of polymorphonuclear leukocytes with GM-CSF did not influence the basal intracellular Ca2+ level and does not enhance cytosolic free calcium after stimulation with fMLP or ionomycin. Only a small increase in the second Ca2+ phase after receptor agonist stimulation was found. However, the Ca(2+)-threshold level necessary for the liberation of arachidonic acid by phospholipase A2 was decreased from 350-400 nM calcium in untreated cells to about 250 nM calcium in primed cells. This allows phospholipase A2 to be activated by a release of calcium from intracellular stores and by ionomycin concentrations which are ineffective in untreated cells. Protein biosynthesis inhibitors like actinomycin D (10 micrograms/ml) and cycloheximide (50 micrograms/ml) had no effect on the enhanced leukotriene biosynthesis in primed cells after stimulation with ionomycin. However, staurosporine (200 nM), an inhibitor of protein kinase C totally abolished the priming effect of GM-CSF after stimulation with ionomycin. The priming effect of GM-CSF could be mimicked by phorbol myristate acetate (PMA; 1 nM) and no additive or synergistic effect was found on leukotriene biosynthesis by simultaneous pretreatment with PMA and GM-CSF and stimulation with either fMLP or ionomycin. These results provide evidence that the enhanced arachidonic acid release in GM-CSF-primed polymorphonuclear leukocytes after stimulation with either fMLP or ionomycin involves activation of protein kinase C which, by a still unknown mechanism, reduces the Ca2+ requirement of phospholipase A2.     

Priming of human neutrophil respiratory burst by granulocyte/macrophage colony-stimulating factor (GM-CSF) involves partial phosphorylation of p47(phox).

Neutrophil superoxide production can be potentiated by prior exposure to "priming" agents such as granulocyte/macrophage colony stimulating factor (GM-CSF). Because the mechanism underlying GM-CSF-dependent priming is not understood, we investigated the effects of GM-CSF on the phosphorylation of the cytosolic NADPH oxidase components p47(phox) and p67(phox). Preincubation of neutrophils with GM-CSF alone increased the phosphorylation of p47(phox) but not that of p67(phox). Addition of formyl-methionyl-leucyl-phenylalanine (fMLP) to GM-CSF-pretreated neutrophils resulted in more intense phosphorylation of p47(phox) than with GM-CSF alone and fMLP alone. GM-CSF-induced p47(phox) phosphorylation was time- and concentration-dependent and ran parallel to the priming effect of GM-CSF on superoxide production. Two-dimensional tryptic peptide mapping of p47(phox) showed that GM-CSF induced phosphorylation of one major peptide. fMLP alone induced phosphorylation of several peptides, an effect enhanced by GM-CSF pretreatment. In contrast to fMLP and phorbol 12-myristate 13-acetate, GM-CSF-induced phosphorylation of p47(phox) was not inhibited by the protein kinase C inhibitor GF109203X. The protein-tyrosine kinase inhibitor genistein and the phosphatidylinositol 3-kinase inhibitor wortmannin inhibited the phosphorylation of p47(phox) induced by GM-CSF and by fMLP but not that induced by phorbol 12-myristate 13-acetate. GM-CSF alone did not induce p47(phox) or p67(phox) translocation to the membrane, but neutrophils treated consecutively with GM-CSF and fMLP showed an increase (compared with fMLP alone) in membrane translocation of p47(phox) and p67(phox). Taken together, these results show that the priming action of GM-CSF on the neutrophil respiratory burst involves partial phosphorylation of p47(phox) on specific serines and suggest the involvement of a priming pathway regulated by protein-tyrosine kinase and phosphatidylinositol 3-kinase.     

Effects of lysophospholipids on the generation of reactive oxygen species by fMLP‐ and PMA‐stimulated human neutrophils

In this study, the effects of exogenous lysophospholipids—lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidylserine—on the kinetics of reactive oxygen species (ROS) production by human neutrophils are described. The ROS production by human neutrophils was monitored by luminol‐amplified chemiluminescence after cell stimulation with the chemotactic tripeptide, fMLP, or with the phorbol ester, PMA. The interaction of lysophospholipids with the membrane of human neutrophils was additionally tested by mass spectrometry. Lysophosphatidylcholine showed the most pronounced effect on the chemiluminescence pattern, as well as the intensity of the fMLP and PMA‐stimulated cells, whereas lysophosphatidic acid showed a slight priming effect when fMLP was used for stimulation. In the case of fMLP‐stimulated cells, lysophosphatidylcholine inhibited the first phase and enhanced the second phase of chemiluminescence, whereas the chemiluminescence of PMA‐stimulated neutrophils was inhibited in a concentration‐dependent manner. We conclude that lysophosphatidylcholine is able to interact with protein kinase C‐dependent signalling pathways leading to NADPH oxidase activation. Copyright © 2002 John Wiley & Sons, Ltd.     

Differential regulation of neutrophil phospholipase d activity and degranulation

One of the proposed functions of phosphatidic acid (PA) formation from phospholipase D (PLD) activation in neutrophils is to promote degranulation induced by receptor agonists. The present study shows that the time course and dose response of PA formation and degranulation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) differed. PLD activation and degranulation also exhibited different dose response to genistein and epigallocatechin gallate (EGCG), inhibitors of protein tyrosine kinases. Genistein inhibited PLD activity with an IC(50) value of 12.2 microM in fMLP- and 107 microM in phorbol myristate acetate (PMA)-stimulated cells. It required higher concentrations of genistein to inhibit degranulation than to inhibit PLD activity induced by fMLP. EGCG in the range of 40-400 microM had no effect on PLD activity but it inhibited the release of beta-glucuronidase and elastase by fMLP-stimulated cells. These results demonstrate differential regulation of PLD activity and degranulation of primary granules by genistein and EGCG in fMLP-stimulated neutrophils.     

Variations of the effect of insulin on neutrophil respiratory burst. The role of tyrosine kinases and phosphatases

The priming effect of insulin on the fMLP-induced respiratory burst of mouse neutrophils as well as the involvement of tyrosine protein kinases and phosphatases in this process have been studied. Peritoneal evoked neutrophils of NMRI strain mice were incubated with 0.01-100 nM insulin for 1-60 min at 22, 30, or 37°C and activated by 0.1-50 M N-formyl-methionyl-leucyl-phenylalanine (fMLP). The production of reactive oxygen species (ROS) by neutrophils was monitored by luminol-dependent chemiluminescence. We found that ¹²⁵I-labeled insulin binding by mouse neutrophils occurred with saturation and high affinity. Insulin itself did not change the basal level of the ROS production but could modulate fMLP-induced respiratory burst. The effect of insulin depended on temperature and duration of pretreatment of the neutrophils with insulin and the concentration combination of the insulin and fMLP. The tyrosine kinase inhibitor tyrphostin 51 decreased the fMLP-induced respiratory burst significantly. Insulin did not change the fMLP response of neutrophils pretreated with tyrphostin. However, the effect of tyrphostin on the response to 50 M fMLP was considerably decreased in neutrophils treated with insulin. There was no such effect during activation by 5 M fMLP, for which the priming effect of insulin was not observed. Insulin did not increase the fMLP-induced respiratory burst in neutrophils treated with the protein phosphatase inhibitors orthovanadate and pyrophosphate. If the inhibitors were added after insulin, the combined effect was nearly additive. It is possible that priming by insulin of the fMLP-induced respiratory burst is triggered by tyrosine phosphorylation, realized with its participation, and involves the signaling pathways initiated by tyrosine phosphorylation but subsequently is not dependent on the latter. The role of protein phosphatases in priming by insulin is of little importance. The data indirectly confirm the idea that priming of the neutrophil respiratory burst is a result of crosstalk of signaling pathways of the insulin and fMLP receptors with the participation of tyrosine phosphorylation.     

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.     

Protein kinase C inhibition by sphingoid long-chain bases: effects on secretion in human neutrophils

Sphingoid long-chain bases (sphinganine and sphingosine) have recently been shown to inhibit protein kinase C both in vitro [Y. Hannun et al. (1986) J. Biol. Chem. 261, 12604-12609] and in intact human neutrophils, in which they block activation of the superoxide-generating respiratory burst [E. Wilson et al. (1986) J. Biol. Chem. 261, 12616-12623]. In the present study we have used sphingosine to investigate the pathways for agonist-induced secretion of neutrophil granule contents. Induction of secretion of the specific granule component lactoferrin by a variety of agonists [phorbol 12-myristate-13-acetate (PMA), formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore A23187] was completely inhibited by sphingosine with an ED50 of 6 to 10 microM. PMA-induced secretion of lysozyme (present in both the azurophilic and specific granules) was completely blocked with an ED50 of 10 microM, whereas fMLP-induced secretion was only about 50% inhibited. Secretion of the azurophilic granule proteins beta-glucuronidase and myeloperoxidase was activated by fMLP and A23187, but not by PMA, and was not affected by sphingosine. The use of A23187 in the presence of sphingosine allowed differentiation between calcium activation of protein kinase C-dependent versus-independent pathways. The effect of sphingosine was not mediated by neutralizing intracellular acidic compartments, since treatment of neutrophils with inhibitory concentrations of sphingosine did not significantly alter the uptake of labeled methylamine. We conclude that at least two mechanisms participate in the regulation of specific and azurophilic granule secretion, respectively: a protein kinase C-dependent pathway and a calcium-dependent pathway which does not involve protein kinase C.     

Binding of GM-CSF to adherent neutrophils activates phospholipase D.

When the hematopoietic growth factor granulocyte-macrophage colony-stimulating factor was incubated with neutrophils adherent to plastic tissue culture plates or plates coated with extracellular matrix proteins, a rapid (3 min) but transient formation of phosphatidic acid was observed. This stimulation was dependent on the dose of GM-CSF, with an EC50 of 140 pM, and was further enhanced (up to 350%) with the PA phosphatase inhibitor propranolol in a dose-dependent manner. Conversely, GM-CSF was unable to trigger any PA formation in neutrophils maintained in suspension, even in the presence of soluble fibronectin. However, GM-CSF did prime the cells for enhanced PA formation in the presence of a secondary stimulus (fMet-Leu-Phe or PAF). GM-CSF also caused a time-dependent stimulation of diacylglycerol formation in adherent, but not suspended, cells and elicited a time-dependent stimulation of phosphatidylethanol formation, with a concomitant decrease in the formation of PA only at early (< 7 min) times. These observations were consistent with a rapid activation of the enzyme phospholipase D in adherent cells stimulated with GM-CSF. Additional data indicated that the source of DAG was PLD coexisting with PLC, especially at later times ( > 7 min) of stimulation with GM-CSF. Finally, the formation of PA and PEt, and to a minor extent, DAG, were inhibited by the protein tyrosine kinase inhibitor erbstatin in conditions in which tyrosine phosphorylation occurred. Taken together the data indicate that GM-CSF rapidly activates PLD in adherent cells, which is responsible for the generation of PA. Thus, PLD activation is an early event in neutrophil signal transduction following exposure of adherent cells to GM-CSF.     

fMLP-induced arachidonic acid release in db-cAMP-differentiated HL-60 cells is independent of phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activation and cytosolic phospholipase A(2) activation

In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca(2+)-dependent translocation of the enzyme to the membrane. Here we investigated the role of phospholipases in N-formylmethionyl-l-leucyl-l-phenylalanine (fMLP; 1 nM-10 microM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells. U 73122 (1 microM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca(2+)-chelator 1, 2-bis?2-aminophenoxy?thane-N,N,N',N'-tetraacetic acid (10 microM) abolished fMLP-mediated Ca(2+) signaling, but had no effect on fMLP-induced AA release. The protein kinase C-inhibitor Ro 318220 (5 microM) or the inhibitor of cPLA(2) arachidonyl trifluoromethyl ketone (AACOCF(3); 10-30 microM) did not inhibit fMLP-induced AA release. In contrast, AA release was stimulated by the Ca(2+) ionophore A23187 (10 microM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 microM). This effect was inhibited by either Ro 318220 or AACOCF(3). Accordingly, a translocation of cPLA(2) from the cytosol to the membrane fraction was observed with A23187 + PMA, but not with fMLP. fMLP-mediated AA release therefore appeared to be independent of Ca(2+) signaling and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by approximately 45% by Clostridium difficile toxin B (10 ng/ml) or by 1-butanol; both block phospholipase D (PLD) activity. The inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 (100 microM), decreased fMLP-mediated AA release by approximately 35%. The effect of D609 + 1-butanol on fMLP-induced AA release was additive and of a magnitude similar to that of propranolol (0.2 mM), an inhibitor of phosphatidic acid phosphohydrolase. This suggests that the bulk of AA generated by fMLP stimulation of db-cAMP-differentiated HL-60 cells is independent of the cPLA(2) pathway, but may originate from activation of PC-PLC and PLD.     

Contribution of mitogen-activated protein kinase to stimulation of phospholipase D by the chemotactic peptide fMet-Leu-Phe in human neutrophils.

Phospholipase D (PLD) plays an important role in signaling through phosphatidylcholine (PC) and in the production of superoxide (respiratory burst) by polymorphonuclear leukocytes (PMN) stimulated by the chemoattractant fMet-Leu-Phe (fMLP). However, the regulation of PLD activity by protein kinases is not fully understood. In the present study, we have used a mitogen-activated protein (MAP) kinase inhibitor (PD 98059) to investigate a possible connection between extracellular signal-regulated kinase (ERK) and PLD activity and respiratory burst. Using a range of concentrations (3-20 microM) which inhibit ERK activity, PD 98059 inhibited PLD activity induced by fMLP in cytochalasin B-primed PMN, as assessed by production-tritiated phosphatidylethanol (PEt), phosphatidic acid (PA), and hydrolysis of PC. However, the inhibition was partial (approximately 50%), while inhibition of PC hydrolysis was almost complete, suggesting a concomitant inhibition of PLA2 activity. In addition, PD 98059 reduced fMLP-induced respiratory burst by 50%, an effect which was correlated with PLD inhibition of PLD (r = 0.981, P < 0.01), and neither did PD 98059 inhibit the PLD activity and respiratory burst induced by PKC upon its direct activation by phorbol myristate acetate. These data provide the first evidence for implication of the ERK cascade in the stimulation of PLD through Gi signaling. They further indicate that PLD stimulation by fMLP receptors occurs through two pathways, dependent and independent on MAP kinase, the former pathway being linked to superoxide production.     

Primary granule release from human neutrophils is potentiated by soluble fibrinogen through a mechanism depending on multiple intracellular signaling pathways

N-Formyl-methionyl-leucyl-phenylalanine (fMLP) is a potent activator of neutrophil degranulation. The intracellular signaling mechanisms involved in the potentiating effect of fibrinogen on fMLP-induced primary granule release from human neutrophils were investigated. Fibrinogen caused a significant leftward shift of the concentration-response curve of fMLP-induced elastase release. An antibody against Mac-1 (CD11b/CD18) prevented the potentiating effect of fibrinogen, suggesting that soluble fibrinogen potentiates fMLP-induced degranulating effect by a mechanism mediated by the integrin Mac-1. Fibrinogen enhanced fMLP-induced tyrosine phosphorylation in human neutrophils and markedly enhanced the phosphorylation of mitogen-activated protein kinases (MAPK) caused by fMLP. However, U0126, an inhibitor of p44/42 MAPK activation, or SB-203580, an inhibitor of p38 MAPK, did not alter the effect of fibrinogen on fMLP-induced elastase release. Wortmannin, a phosphatidylinositol 3-kinase (PI3K) kinase inhibitor, and genistein, a nonspecific tyrosine kinase inhibitor, strongly inhibited fMLP-induced elastase release both in the presence and in the absence of fibrinogen. An Akt/PKB inhibitor failed to alter the potentiating effect of fibrinogen, suggesting that the effect of fibrinogen is mediated by Akt-independent pathways. Go6976, an inhibitor of classical PKC isoforms, caused a significant inhibition of fMLP-induced elastase release in the presence or absence of fibrinogen, while nonselective inhibitors of PKC, Ro 31-8220, GF-109203X, and staurosporine, caused potentiation of fMLP-induced elastase release. We conclude that fibrinogen potentiation of primary granule release induced by fMLP is mediated by the integrin CD11b/CD18 through pathways dependent on PI3K and tyrosine kinases, but other regulatory mechanisms may be also involved.     

Sphingosine kinase: a point of convergence in the action of diverse neutrophil priming agents

Neutrophils are a vital component of the early acute inflammatory response, but can cause profound tissue damage when activated to excess or prevented from undergoing apoptosis. However, much remains unknown about the intracellular signaling pathways regulating neutrophil activity. The structurally diverse neutrophil-priming agents platelet-activating factor, TNF-alpha, and the substance P analog [D-Arg(6), D-Trp(7,9),N(me)Phe(8)]-substance P(6-11) (SP-G) stimulated a rapid increase in sphingosine kinase activity in freshly isolated human neutrophils. This activity was blocked by preincubation with the sphingosine kinase inhibitor N,N-dimethylsphingosine (DMS). DMS also inhibited the increase in intracellular calcium concentration stimulated by platelet-activating factor, fMLP, and SP-G. This suggests that the increase in intracellular calcium concentration by these agents is dependent on sphingosine kinase activation and the generation of sphingosine-1-phosphate. Changes in cell polarization and the augmentation of the fMLP-induced superoxide anion generation, by all priming agents were also inhibited by DMS, while only the superoxide anion release was blocked by the phosphatidylinositol 3-kinase inhibitor LY294002. Moreover, SP-G and GM-CSF inhibited constitutive neutrophil apoptosis which was completely blocked by DMS. These results suggest a novel role for sphingosine kinase in the regulation of neutrophil priming.     

D-cystathionine ketimine and L-cystathionine ketimine enhance superoxide generation by human neutrophils in a different manner

The effects of d-cystathionine ketimine (D-CK) and l-cystathionine ketimine (L-CK) on the stimulus-induced superoxide generation by human neutrophils were compared. When the cells were preincubated with D-CK, the superoxide generation induced by arachidonic acid (AA), phorbol 12-myristate 13-acetate (PMA), and N-formyl-methionyl-leucyl-phenylalanine (fMLP) were enhanced, showing a dependence on D-CK concentration. The rate of enhancement by D-CK was AA > PMA > fMLP. On the contrary, L-CK largely enhanced the fMLP-induced superoxide generation, whereas it showed no effect on those induced by AA and PMA. The superoxide generations induced by AA and PMA in the D-CK-treated cells were suppressed by staurosporine, while those in the L-CK-treated cells were not affected. Genistein suppressed the fMLP-induced superoxide generation in the L-CK-treated cells more efficiently than that in the D-CK-treated cells. D-CK enhanced seryl phosphorylation of 16. 5-kDa protein in human neutrophils, while L-CK enhanced tyrosyl phosphorylation of 45-kDa protein.     

2-Benzyloxybenzaldehyde inhibits formyl-methionyl-leucyl-phenylalanine stimulation of phospholipase D activation in rat neutrophils

2-Benzyloxybenzaldehyde (CCY1a) inhibited the formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated phospholipase D (PLD)-mediated products, phosphatidic acid (PA) and phosphatidylethanol (PEt) formation in rat neutrophils in a concentration-dependent manner with IC₅₀ values of 15.8±2.5 and 13.9±2.0 μM, respectively. The underlying cellular signaling mechanism of CCY1a inhibition was investigated. CCY1a inhibited the plateau phase but not the initial Ca²⁺ spike of fMLP-stimulated Ca²⁺ signal. CCY1a did not inhibit the [Ca²⁺]_i change in Ca²⁺-free medium in response to fMLP, but inhibited the [Ca²⁺]_i change by the subsequent addition of Ca²⁺. In addition, CCY1a treatment attenuated the fMLP-induced protein tyrosine phosphorylation. The membrane translocation of ADP-ribosylation factor (ARF) and Rho A proteins in neutrophils stimulated with fMLP was attenuated by CCY1a in a concentration-dependent manner. In a cell-free system, neither the membrane association of ARF and Rho A caused by GTPγS nor the phorbol myristate acetate-stimulated membrane translocation of Rho A was suppressed significantly by CCY1a. These results indicate that the attenuation of protein tyrosine phosphorylation, blockade of Ca²⁺ entry, and the suppression of ARF and Rho A membrane translocation are probably obligatory for the CCY1a inhibition of PLD activity in rat neutrophils in response to fMLP.     

Resveratrol inhibits the formation of phosphatidic acid and diglyceride in chemotactic peptide- or phorbol ester-stimulated human neutrophils

Resveratrol (trans-3,5,4'-trihydroxystilbene, Res) is a naturally occurring antioxidant found in grape berry skins and red wine. It has anti-inflammatory effects. In this study, we examined the effect of Res on the formation of phosphatidic acid (PA) and diglyceride (DG), in human neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine (fMLP) or by phorbol 12-myristate 13-acetate (PMA). We measured the masses of PA and DG by using a nonradioactive method. Our results showed that Res inhibited the formation of PA in a concentration dependent manner with an IC(50) value of 42.4 and 60.9 microM in fMLP- and PMA-stimulated cells, respectively. Res also suppressed the formation of phosphatidylethanol (PEt), thereby implying inhibition of phospholipase D (PLD) activity. In addition, Res inhibited the formation of both diacylglycerol (DAG) and ether-linked acylglycerol (EAG) induced by fMLP and by PMA. Our results suggest that Res inhibition of PLD activity may contribute to its anti-inflammatory effects.     

Tyrosine and calcium/calmodulin kinases are common signaling components in the generation of reactive oxygen species in human lymphocytes

This study examined the signaling mechanism involved in the generation of reactive oxygen species (ROS) in human lymphocytes activated by formyl-Met-Leu-Phenylalanine (fMLP; 200 nmol/L) or phorbol-myristate-acetate (PMA; 100 nmol/L). ROS were monitored spectrophotometrically using dichlorofluorescin diacetate. fMLP and PMA significantly increased ROS above the control levels (p<0.05 and 0.001, respectively). These increases were significantly inhibited by catalase, sodium azide, and dimethylsulfoxide but not by superoxide dismutase, suggesting that the ROS apparently included hydrogen peroxide, singlet oxygen and hydroxyl ion but not superoxide anion. PMA-induced responses were reduced by tyrphostin (p<0.01), ST-638 (p<0.05), KN-62 (p<0.001), bisindolylmaleimide (p<0.001), RO-31-8220 (p<0.001), and by LY-83583 (p<0.001), suggesting significant involvement of tyrosine kinase, calcium/calmodulin kinase II, protein kinase C and guanylyl cyclase. fMLP-induced responses were significantly reduced by only tyrphostin (p<0.001), ST-638 (p<0.05), and KN-62 (p<0.01). The results show that tyrosine kinase and calcium/calmodulin kinase II are common signalling components in the production of reactive oxygen species in activated lymphocytes.