Neutrophil migration across monolayers of cytokine-prestimulated endothelial cells: a role for platelet-activating factor and IL-8

In a previous study we observed that neutrophils respond with a rapid rise in [Ca2+]i during adherence to cytokine-activated endothelial cells (EC), caused by EC membrane-associated platelet-activating factor (PAF). In the present study, we investigated whether this form of PAF was important in neutrophil adherence and migration across monolayers of rIL-1 beta- or rTNF alpha-prestimulated EC. PAF receptor antagonists prevented neutrophil migration across cytokine-pretreated EC by approximately 60% (P less than 0.005) without interfering with the process of adherence. The antagonists WEB 2086 and L-652,731 had no effect on neutrophil migration across resting EC induced by formylmethionyl-leucyl-phenylalanine (FMLP). A murine anti-IL-8 antiserum was found to also partially inhibit the neutrophil transmigration across cytokine-activated EC. When the anti-IL-8 antiserum was used in combination with a PAF receptor antagonist, neutrophil migration across cytokine-pretreated monolayers of EC was completely prevented. During transmigration, LAM-1 and CD44 on the neutrophils were down-modulated; both WEB 2086 and anti-IL-8 antiserum partially prevented this down-modulation caused by cytokine-prestimulated EC. Our results indicate that human neutrophils are activated and guided by EC-associated PAF and EC-derived IL-8 during the in vitro diapedesis in between cytokine-stimulated EC.     

FMLP诱导的嗜中性白细胞呼吸爆发与凋亡的关系研究

The relationship between apoptosis of neutrophils and the change of their intracellular free Ca2+ concentration [Ca2+]i was studied. FMLP and A23187 were used to elevate the [Ca2+]i while BAPTA was used to deplete it. Fluorescence microscope, flow cytometry and gel electrophoresis were used to study the percentage of cell apoptosis and the change of f-actin during apoptosis. The results showed that the apoptosis was obviously inhibited by fMLP and A23187, while accelerated by BAPTA. The detection of f-actin showed that the f-actin depolymerized obviously during apoptosis. The elevation of [Ca2+]i inhibit the actin depolymerization while depletion of [Ca2+]i accelerated it. This result indicated that the apoptosis of neutrophil was obviously inhibited by [Ca2+]i elevation but accelerated by [Ca2+]i depletion.     

Modulation of cytosolic-free calcium transients by changes in intracellular calcium-buffering capacity: correlation with exocytosis and O2-production in human neutrophils

The intracellularly trapped fluorescent calcium indicator, quin 2, was used not only to monitor changes in cytosolic-free calcium, [Ca2+]i, but also to assess the role of [Ca2+]i in neutrophil function. To increase cytosolic calcium buffering, human neutrophils were loaded with various quin 2 concentrations, and [Ca2+]i transients, granule content release as well as superoxide [O2-] production were measured in response to the chemotactic peptide formyl-methionyl-leucyl- phenylalanine (fMLP) and the calcium ionophore ionomycin. Receptor- mediated cell activation induced by fMLP caused a rapid rise in [Ca2+]i. The extent of [Ca2+]i rise and granule release were inversely correlated with the intracellular concentration of quin 2, [quin 2]i. These effects of [quin 2]i were more pronounced in the absence of extracellular Ca2+. The initial rate and extent of fMLP-induced O2- production were also inhibited by [quin 2]i. The rates of increase of [Ca2+]i and granule release elicited by ionomycin were also inversely correlated with [quin 2]i in Ca2+-containing medium. As the effects of ionomycin, in contrast to those of fMLP, are sustained, the final increase in [Ca2+]i and granule release were not affected by [quin 2]i. A further reduction of fMLP effects was seen when intracellular calcium stores were depleted by incubating the cells in Ca2+-free medium with ionomycin. The specificity of quin 2 effects on cellular calcium were confirmed by loading the cells with Anis/AM, a structural analog of quin 2 with low affinity for calcium which did not inhibit granule release. In addition, functional responses to phorbol myristate acetate (PMA), which stimulates neutrophils without raising [Ca2+]i, were not affected by [quin 2]i. The findings indicate that rises in [Ca2+]i control the rate and extent of granule exocytosis and O2-generation in human neutrophils exposed to the chemotactic peptide fMLP.     

Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils

Cytosolic free calcium concentration, [Ca2+]i, and exocytosis of azurophil granules (beta-glucuronidase), specific granules (vitamin B12-binding protein), and secretory vesicles (gelatinase) were measured concomitantly in intact human neutrophils under steady state [Ca2+]i. The cells were loaded with the fluorescent calcium indicator quin2 in the presence or absence of extracellular Ca2+, and steady state [Ca2+]i levels ranging from 20 to greater than 2,000 nM were obtained by adding the Ca2+ ionophore ionomycin at various concentrations of extracellular calcium. The extent of exocytosis from the three granule populations was found to be a function of [Ca2+]i. The minimal [Ca2+]i that caused significant release (threshold [Ca2+]i) was approximately 200-300 nM and was similar for all three compartments. Marked differences, however, were found when the [Ca2+]i for half-maximal exocytosis (EC50) was determined. In the absence of cytochalasin B the EC50 was 1,100 +/- 220 nM and 1,600 +/- 510 nM for specific granules and secretory vesicles, respectively, and approximately 6,000 nM for azurophil granules. Cytochalasin B did not affect the threshold [Ca2+]i but decreased the EC50 and enhanced the rate of exocytosis. In the presence of cytochalasin B the EC50 was approximately 600 nM both for secretory vesicles and specific granules, and approximately 2,600 nM for azurophil granules. The addition of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine dramatically changed the [Ca2+]i dependency of granule secretion: It decreased the threshold [Ca2+]i to less than 20 and less than 50 nM, and the EC50 to 50 and 200 nM for specific and azurophil granules, respectively, and it significantly increased the rate of exocytosis. Thus, the additional signal(s) provided by receptor activation markedly lower(s) the Ca2+ requirement of the exocytotic process. Furthermore, these results indicate that the secretion from three different granule populations within the same cell type are differently modulated by [Ca2+]i.     

fMet-Leu-Phe-induced activation of phospholipase D in human neutrophils. Dependence on changes in cytosolic free Ca2+ concentration and relation with respiratory burst activation.

In this study, we have investigated the Ca2+ requirements for the activation of phospholipase D by the tripeptide fMet-Leu-Phe (fMLP) in human neutrophils. EGTA inhibited the activation of phospholipase D (PLD) by 55% (n = 4). When the initial transient rise in [Ca2+]i was prevented by loading the cells with limited amounts of the Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), PLD activation was inhibited by 92% (n = 4). In the presence of both chelators, PLD activation was only 4% of control. In electropermeabilized neutrophils, too, the activation of PLD after the addition of fMLP strongly depends on the Ca2+ concentration, being almost absent with 100 nM free Ca2+ present and reaching maximum activation with a free [Ca2+] of 500 nM. We subsequently investigated the relationship between PLD activation and the activation of the respiratory burst. In neutrophils loaded with BAPTA/AM (10 microM), in which PLD activation was almost absent, a respiratory burst could be induced by fMLP, albeit with a much longer lag time. A respiratory burst could also be elicited by fMLP in electropermeabilized neutrophils incubated with 100 nM free Ca2+. This response, however, was strongly enhanced in the presence of 1 microM Ca2+. Our results indicate that changes in [Ca2+]i are essential for the activation of PLD by fMLP, but probably do not constitute the sole activation signal. In addition, our data provide evidence that PLD activation is important, but not necessary, for activation of the neutrophil respiratory burst.     

Cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils

Cytosolic free calcium ([Ca2+]i) and fusion of secondary granules with the phagosomal membrane (phagosome-lysosome fusion, P-L fusion) were assessed in single adherent human neutrophils during phagocytosis of C3bi-opsonized yeast particles. Neutrophils were loaded with the fluorescent dye fura2/AM and [Ca2+]i was assessed by dual excitation microfluorimetry. Discharge of lactoferrin, a secondary granule marker into the phagosome was verified by immunostaining using standard epifluorescence, confocal laser scanning and electron microscopy. In Ca2(+)-containing medium, upon contact with a yeast particle, a rapid rise in [Ca2+]i was observed, followed by one or more Ca2+ peaks (maximal value 1,586 nM and median duration 145 s): P-L fusion was detected in 80% of the cells after 5-10 min. In Ca2(+)-free medium the amplitude, frequency and duration of the [Ca2+]i transients were decreased (maximal value 368 nM, mostly one single Ca2+ peak and median duration 75 s): P-L fusion was decreased to 52%. Increasing the cytosolic Ca2+ buffering capacity by loading the cells with MAPT/AM led to a dose-dependent inhibition both of [Ca2+]i elevations and P-L fusion. Under conditions where basal [Ca2+]i was reduced to less than 20 nM and intracellular Ca2+ stores were depleted, P-L fusion was drastically inhibited while the cells ingested yeast particles normally. P-L fusion could be restored in Ca2(+)-buffered cells containing ingested particles by elevating [Ca2+]i with the Ca2(+)-ionophore ionomycin. The present findings directly indicate that although the ingestion step of phagocytosis is a Ca2(+)-independent event, [Ca2+]i transients triggered upon contact with opsonized particles are necessary to control the subsequent fusion of secondary granules with the phagosomal membrane.     

Differential up-regulation of specific and azurophilic granule membrane markers in electropermeabilized neutrophils.

We have developed an alternative method to study the degranulation in electropermeabilized human neutrophils by measuring the up-regulation of the specific membrane markers CD63 (residing in the azurophilic granules of resting neutrophils) and CD67 (present in specific granules). The expression of these marker proteins was measured by the binding of specific antibodies to paraformaldehyde-fixed cells and subsequent flow cytometry. We first investigated whether the changes in CD63 and CD67 expression after stimulation of intact cells were comparable with earlier measurements of neutrophil degranulation, in which the release of soluble marker proteins was measured. These experiments indicated that this new method compares favourably with earlier studies, both with respect to kinetics and stimulus dependency. Subsequently, we applied this method (which does not include centrifugation of the cells) to study degranulation in electropermeabilized neutrophils. In permeabilized neutrophils, a clear up-regulation of the specific granule marker CD67 was observed upon incubation with a free Ca2+ concentration of 1 microM, a value of the cytosolic free Ca2+ concentration occurring in formylmethionyl-leucyl-phenylalanine (FMLP)-activated neutrophils. The azurophilic granule marker CD63 required GTP-gamma-S besides 1 microM Ca2+ for a significant up-regulation. Hence, our study indicates a different requirement for intracellular signals of the two main types of granules in human neutrophils.     

Activation of protein kinase C in human neutrophils attenuates agonist-stimulated rises in cytosolic free Ca2+ concentration by inhibiting bivalent-cation influx and intracellular Ca2+ release in addition to stimulating Ca2+ efflux.

Stimulation of fura-2-loaded human neutrophils with formylmethionyl-leucyl-phenylalanine (FMLP) or ionomycin elevated the cytosolic free Ca2+ concentration, [Ca2+], to a maintained elevated level. Activation of protein kinase C (C-kinase) with phorbol 12-myristate 13-acetate, 4 beta-phorbol 12,13-didecanoate or dioctanoylglycerol caused decreases in [Ca2+]i from this level. 4 alpha-Phorbol didecanoate, which does not activate C-kinase, had no effect. These results confirm previous reports that C-kinase activation decreases neutrophil [Ca2+]i by stimulating removal of Ca2+ from the cytosol. Further experiments showed that activation of C-kinase attenuated the component of the FMLP-stimulated [Ca2+]i rise that was dependent on external Ca2+. C-kinase activation also inhibited FMLP-stimulated entry of the quenching cation, Mn2+, used as an indicator of bivalent-cation entry. In contrast, C-kinase activation caused only a partial inhibition of FMLP-stimulated release of Ca2+ from intracellular stores. 4 alpha-Phorbol didecanoate was ineffective in inhibiting Ca2+ entry, Mn2+ entry and intracellular Ca2+ release. Addition of FMLP also stimulated a decrease in the ionomycin-elevated [Ca2+]i, and this effect was blocked by staurosporine, a protein kinase inhibitor. These results show that, in addition to stimulating Ca2+ efflux, C-kinase activation in neutrophils inhibits FMLP-stimulated entry of bivalent cations, and partially inhibits intracellular release of Ca2+. Further, FMLP itself can modulate [Ca2+]i by activation of C-kinase.     

Studies on molecular regulation of phagocytosis and activation of the NADPH oxidase in neutrophils. IgG- and C3b-mediated ingestion and associated respiratory burst independent of phospholipid turnover and Ca2+ transients

The role of messengers derived from hydrolysis of phosphoinositides and other phospholipids, of the basal level of [Ca2+]i and of the increase in [Ca2+]i in phagocytosis and respiratory burst was investigated, using normal neutrophils and neutrophils Ca2(+)-depleted by pretreatment with Quin2/AM and EGTA. 1) Phagocytosis and respiratory burst in control neutrophils challenged with yeast opsonized with IgG or C3b/bi were associated with a stimulation of the production of inositol phosphates, diacylglycerol, phosphatidic acid, arachidonic acid, and rise in [Ca2+]i. 2) In Ca2(+)-depleted neutrophils (basal [Ca2+]i 10 to 20 nM) the phagocytosis of yeast-IgG was similar to that in control neutrophils, the respiratory burst was slightly depressed (-30%), while the increase in [Ca2+]i and production of inositol phosphates, diacylglycerol, and phosphatidic and arachidonic acid did not occur. 3) In Ca2(+)-depleted neutrophils the phagocytosis of yeast-C3b/bi was slightly lower than that in control neutrophils, and the respiratory burst, related to the same number of particles ingested, was depressed by about 60%, whereas the increase in [Ca2+]i and production of inositol phosphates, diacylglycerol, phosphatidic acid, and arachidonic acid release did not occur. These findings demonstrate that transmembrane signaling pathways involving the hydrolysis of phosphoinositides by phospholipase C and D and of other phospholipids by phospholipase C and Az, and the rise in [Ca2+]i are not essential processes for triggering the ingestion of yeast particles opsonized with IgG and C3b/bi and the activation of the NADPH oxidase.     

Superoxide responses to formyl-methionyl-leucyl-phenylalanine in primed neutrophils. Role of intracellular and extracellular calcium.

For superoxide (O2-) responses of human neutrophils stimulated by FMLP, experiments were designed to assess the requirement of extracellular calcium [( Ca2+]o) for priming of O2- responses by platelet-activating factor (PAF), PMA, or ionomycin. Although priming by PMA did not require [Ca2+]o, there was, as expected, a requirement for [Ca2+]o for the optimal priming effects of PAF and ionomycin. The ED50 value for [Ca2+]o in the priming function of PAF was 105 microM. The [Ca2+]o-dependent priming with ionomycin was bimodal with two ED50 values for [Ca2+]o of 90 microM and 3.2 mM. Optimal priming by PAF required at least 4-min exposure of cells to [Ca2+]o. Cells primed by PAF exhibited faster initial rates of O2-production after addition of FMLP, but the duration of O2- production was not prolonged. Whereas PAF-primed responses to FMLP are usually associated with increases in intracellular calcium [( Ca2+]i) after addition of FMLP, two conditions were found in which O2- responses to FMLP in PAF-primed cells occurred in the absence of any detectable increase in [Ca2+]i. When cells were loaded with the calcium chelator, bis-(O-aminophenoxy)-ethane-H,N,N',N'-tetraacetic acid, and then primed with PAF, normal amounts of inositol 1,4,5-trisphosphate were formed, but no increase in [Ca2+]i occurred after addition of FMLP even though the cells exhibited a fully primed O2- response; in Ca2(+)-depleted and ionomycin-permeabilized cells that were primed with PAF and then stimulated with FMLP, O2- was generated in amounts comparable to reference control (primed) cells, but there was suppressed production of inositol 1,4,5-trisphosphate and no increase in [Ca2+]i after addition of FMLP to PAF-primed cells. These data confirm the requirement of [Ca2+]o for optimal priming of neutrophils by PAF and ionomycin (but not cells primed by PMA) and indicate that, under certain conditions, generation of O2- in response to FMLP in PAF-primed neutrophils can occur independent of any increase in [Ca2+]i.     

A role for calcium and protein kinase C in agonist-stimulated adhesion of human neutrophils.

Stimulated adherence of human neutrophils to plastic and changes in cytosolic free Ca2+ concn. [( Ca2+]i) were measured in the same cell preparations. [Ca2+]i-activation curves were constructed to compare the relation between [Ca2+]i and adhesion in response to ionomycin and formylmethionyl-leucyl-phenylalanine (FMLP). This showed that FMLP-stimulated adhesion required less increase in [Ca2+]i than did ionomycin's effect, a result suggesting that an additional stimulatory component might be involved in the response to FMLP. Protein kinase C activation was a possibility, and activation of protein kinase C with a phorbol ester (PMA) was found to stimulate adhesion with no change in [Ca2+]i. A low concentration of PMA was found to synergize with ionomycin to stimulate a greater adhesion response than with each alone, and the [Ca2+]i-activation curve for ionomycin in the presence of PMA was shifted towards that for FMLP. Thus, synergy between [Ca2+]i and protein kinase C (each of which is sufficient alone) probably explains the stimulatory effects of FMLP on adhesion of neutrophils.     

Complete dissociation between the activation of phosphoinositide turnover and of NADPH oxidase by formyl-methionyl-leucyl-phenylalanine in human neutrophils depleted of Ca2+ and primed by subthreshold doses of phorbol 12,myristate 13,acetate

Evidences have been provided by many laboratories that the activation of the NADPH oxidase in neutrophils by formyl-methionyl-leucyl-phenylalanine (FMLP) is strictly linked to a transduction pathway that involves the stimulation, via GTP binding protein, of the phosphoinositide turnover and the increase in [Ca2+]i. The results presented in this paper demonstrate that FMLP can activate the NADPH oxidase by triggering a transduction pathway completely independent of phosphoinositide turnover and Ca2+ changes. In fact: i) Ca2+-depleted neutrophils do not respond to FMLP with the activation of phosphoinositide hydrolysis and NADPH oxidase. Both the responses are restored by the addition of exogenous Ca2+. ii) In Ca2+-depleted neutrophils phorbol-myristate-acetate (PMA) activates the NADPH oxidase. iii) The pretreatment of Ca2+-depleted neutrophils with non stimulatory doses of PMA restores the activation of the NADPH oxidase but not of the turnover of phosphoinositides by FMLP. This priming effect of PMA and the role of this phosphoinositide and Ca2+-independent pathway for the stimulation of the NADPH oxidase by receptors mediated stimuli are discussed.     

Regulation of zymogen granule exocytosis by Ca2+, cAMP, and PKC in pancreatic acinar cells

The effect of cAMP and PKC on zymogen granule exocytosis was investigated by simultaneously measuring cytosolic Ca2+ concentration ([Ca2+]c) and individual zymogen granule exocytosis in isolated mouse pancreatic acini. When acinar cells were stimulated with acetylcholine (ACh, 10 microM), exocytic events were detected through granule-attached apical membranes with [Ca2+]c rise. Application of secretin, forskolin (an adenylate cyclase activator), or PMA (a PKC activator) alone did not elicit any [Ca2+]c rise or zymogen granule exocytosis, but co-stimulation with ACh led to exocytosis in that the total number of secreted granules increased markedly without a significant difference in [Ca2+]c rises. When we evoked exocytosis by [Ca2+]c ramps, pretreatment with forskolin or PMA elicited exocytosis at lower [Ca2+]c levels. These results indicate that PKC or cAMP alone could not directly elicit zymogen granule exocytosis, but that they increase the total releasable pool by rendering zymogen granules more sensitive to Ca2+.     

Effect of olvanil (N-vanillyl-cis-9-octadecenoamide) on cytosolic Ca2+ increase in renal tubular cells

In canine renal tubular cells, effect of olvanil, a presumed cannabinoid and vanilloid receptor modulator, on intracellular Ca2+ concentration ([Ca2+]i) was measured by using fura-2. Olvanil (5-100 microM) caused a rapid and sustained [Ca2+]i rise in a concentration-dependent manner. Olvanil-induced [Ca2+]i rise was prevented by 70 and 90% by removal of extracellular Ca2+ and La3+, respectively, but was not changed by dihydropyridines, verapamil and diltiazem. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of olvanil on [Ca2+]i was abolished; also, pretreatment with olvanil partly reduced thapsigargin-induced [Ca2+]i rise. U73122, an inhibitor of phoispholipase C, abrogated ATP-, but partly inhibited olvanil-, induced [Ca2+]i rise. Two cannabinoid receptor antagonists (AM251 and AM281; 5 microM) and a vanilloid receptor antagonist (capsazepine; 100 microM) did not alter olvanil (50 microM)-induced [Ca2+]i rise. These results suggest that olvanil rapidly increases [Ca2+]i in renal tubular cells, by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release via mechanism(s) independent of stimulation of cannabinoid and vanilloid receptors.     

Mediation by platelet-activating factor of 12-hydroxyeicosatetraenoic acid-induced cytosolic free calcium concentration elevation in neutrophils

12(R)-hydroxyeicosatetraenoic acid (HETE) shows biphasic increase in cytosolic free calcium concentration ([Ca2+]i) in rabbit and human neutrophils; the initial transient phase and the continuous falling phase. 12(S)-HETE was less potent in both species. BN50739, a platelet-activating factor (PAF) receptor antagonist, inhibited both phases of 12(R)-HETE-induced [Ca2+]i rise but did not affect leukotriene B4 (LTB4)-induced [Ca2+]i rise. N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a PAF synthesis inhibitor, and manoalide, a phospholipase A2 inhibitor, reduced 12(R)-HETE-induced [Ca2+]i rise. These blockers inhibited the continuous phase of [Ca2+]i rise induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP) with little effect on the initial phase. It had no significant effect on LTB4-induced [Ca2+]i rise. SC-41930, a LTB4-receptor antagonist, did not block 12-HETE-induced [Ca2+]i rise. In 12(R)-HETE-, FMLP- and LTB4-stimulated cells, accumulations of cell-associated PAF and released PAF were detected but not in unstimulated cells. BN50739 did not affect the accumulation of cell-associated PAF and release of PAF in 12(R)-HETE-stimulated cells. These results suggest that 12(R)-HETE-induced and partially, FMLP-induced, but not LTB4-induced [Ca2+]i rise are mediated by PAF, which is produced and released by stimulation of the cells by 12(R)-HETE and FMLP, respectively.     

Effect of leukotriene B4, prostaglandin E2 and arachidonic acid on cytosolic-free calcium in human neutrophils

Changes in cytosolic free calcium [Ca2+]i and release of beta-glucuronidase in response to leukotriene B4 (LTB4) were measured in intact neutrophils loaded with the fluorescent Ca2+ indicator, quin 2. LTB4 (10(-10) M or higher) caused a rapid rise in [Ca2+]i due to influx from the extracellular medium and release from intracellular pools as well as enzyme release. PGE2 (3 microM) did not alter [Ca2+]i whereas arachidonic acid (10 microM) raised [Ca2+]i. Pretreatment of cells with the chemotactic peptide FMLP inhibited the subsequent rise of [Ca2+]i induced by LTB4. Since chemotactic peptides activate the lipoxygenase pathway of arachidonic acid metabolism, it may be speculated that endogenous LTB4 generation is involved in neutrophil activation.     

Auranofin modulated cytoplasmic free calcium in neutrophils by mobilizing intracellular calcium and inhibiting protein kinase

The effect of the lipophilic gold compound, auranofin (AUR) on the calcium homeostasis of human neutrophils treated with or without n-formyl-methionyl-leucyl-phenylalanine (FMLP) was investigated. In agreement with previous reports, FMLP induced a rapid release of intracellular Ca2+ stores followed by a smaller influx of extracellular Ca2+. AUR and staurosporine enhanced while phorbol 12-myristate 13-acetate suppressed the secondary influx of Ca2+. Mn2(+)-quenching-of-fluorescence studies indicate that phorbol 12-myristate 13-acetate incubation blocked cation entry. AUR or staurosporine potentiation of FMLP effects on cytoplasmic free Ca2+ [( Ca2+]i) was attributed to suppression of negative feedback effects of protein kinase C. AUR (5-45 microM) per se induced a slow release of internal Ca2+ stores followed by a delayed influx of extracellular Ca2+. Control studies showed that AUR did not induce the formation of inositol 1,4,5-trisphosphate, lyse cells, or promote dye leakage. Dithiothreitol suppressed the AUR effect. AUR triggered biphasic but smaller increases in [Ca2+]i of neutrophil cytoplasts. Studies with permeabilized neutrophils showed that AUR directly released Ca2+ from internal stores. By comparison, gold sodium thiomalate, which had no effect on intact cells, also released Ca2+ from permeabilized cells. Present results indicate that AUR modulated [Ca2+]i directly by mobilized Ca2+ from multiple storage sites and indirectly by inhibiting protein kinase C.     

Cilostazol suppresses adhesion of human neutrophils to HUVECs stimulated by FMLP and its mechanisms

The interaction between neutrophils and endothelial cells (ECs) is of great importance in many physiological and pathological progresses. Although cilostazol (CLZ), a novel selective phosphodiesterase (PDE) type 3 inhibitor, has been proved to be useful in vasodilatation and inhibition of platelet aggregation, its effect on adhesion is not clearly known. In this study, we examined the effects and investigated the mechanisms of cilostazol on neutrophil adhesion to human umbilical endothelial cells (HUVECs) triggered by N-formyl-methionyl-leucyl-phenylal-anine (FMLP), a chemotactic peptide. The soluble vascular cell adhesive molecule-1 (sVCAM-1) release from FMLP (10 microM)-stimulated HUVECs was determined by ELISA kits. Fluo-2, a fluorescent indicator, was used to investigate intracellular free calcium concentration ([Ca2+]i) in HUVECs. HL-60 cells were induced to be neutrophilic by DMSO and loaded with Fluo-3, another fluorescent indicator, to detect [Ca2+]i, and CLA was used as a chemiluminescent indicator to determine superoxide production in neutrophilic cells. The result showed that CLZ (1-100 microM) significantly inhibited neutrophil adhesion to FMLP-stimulated HUVECs. In HUVECs, CLZ obviously downregulated sVCAM-1 level, while it had no meaningful influence [Ca2)]i. But in neutrophils, FMLP-activated superoxide generation and [Ca2+]i increase were found being inhibited by exposure to CLZ . Furthermore, we also demonstrated that Ca2+ increase was preceded to the superoxide generation in neutrophils. The results suggest that CLZ involves in adhesion reactions between neutrophil and ECs, partly via VCAM-1 expression in ECs, and decreasing [Ca2+]i induced activation of neutrophils, which means a lot to prevent atherosclerosis and other cardiovascular diseases.     

Activation of the oxygen-radical-generating system in granules of intact human neutrophils by a calcium ionophore (ionomycin).

Subcellular fractionation studies were performed on human neutrophils stimulated with ionomycin (a Ca(2+)-specific ionophore). The results of these studies revealed NADPH-oxidase activity, without any additive, both in the plasma membrane and in the specific granule fractions. After comparing these results with the NADPH oxidase activity induced by the ionophore in intact neutrophils, in differentiated HL-60 cells and in neutrophil cytoplasts, we conclude that ionomycin preferentially activates the NADPH oxidase pool located in the membrane of specific granules. Furthermore, we suggest that incorporation of granule membrane into the plasma membrane makes the associated NADPH oxidase less sensitive to activation induced by a rise in [Ca(2+)]i.     

Enhancement by staurosporine of platelet-activating factor formation in N-formyl peptide-challenged human neutrophils is mediated by intracellular platelet-activating factor binding sites.

Staurosporine potentiates the formation of platelet-activating factor (PAF) and causes a sustained elevation of intracellular Ca2+ ([Ca2+]i). WEB 2086, a specific PAF-receptor antagonist, inhibits both potentiation of PAF formation and elevation of [Ca2+]i by 78% and 65%, respectively. Moreover, the PAF produced by FMLP and/or Staurosporine was completely retained in the cell. This suggests that the effect of staurosporine in FMLP-stimulated neutrophils may be mediated by the action of endogenously produced PAF, which in turn leads to an increase in [Ca2+]i and PAF formation. We conclude that PAF is the major product of human neutrophils which reacts via specific intracellular PAF binding sites to stimulate the phospholipase A2, and its synthesis is under control of a staurosporine-sensitive protein kinase.