Mechanism of arachidonic acid release in human polymorphonuclear leukocytes

Previous studies have demonstrated that [³H]arachidonic acid is released from prelabeled human neutrophil phospholipids when the cells are stimulated by calcium ionophore A23187 or by opsonized zymosan. Neither lysophospholipid generated by phospholipase A₂ activity, diacylglycerol nor monoacylglycerol produced via phospholipase C/diacylglycerol lipase action have been identified following neutrophil challenge. The inability to detect any intermediates during the release of arachidonate is due to either rapid reacylation of lysophospholipid or conversion of diacylglycerol (monoacylglycerol) to cellular acylglycerols. The addition of exogenous [¹⁴C]fatty acid at the time of challenge was employed to determine the involvement of either phospholipase A₂ or phospholipase C activities. Neutrophil stimulation with calcium ionophore A23187 resulted in an incorporation of exogenous [¹⁴C]arachidonate into phosphatidylinositol and phosphatidylcholine, those phospholipids which specifically release arachidonate. When the saturated fatty acid, [¹⁴C]stearate, replaced [¹⁴C]arachidonate, very little [¹⁴C]fatty acid was incorporated into any of the phospholipid species. Lipid phosphorus measurements revealed no significant mass change in any phospholipid class following ionophore challenge. Production of [¹⁴C]phosphatidic acid was not detected, as would be expected if diacylglycerol kinase and de novo phospholipid metabolism were significantly involved.     

Salmonella typhimurium porins stimulate platelet-activating factor synthesis by human polymorphonuclear neutrophils.

Porins, a family of hydrophobic proteins located in the outer membrane of cell-wall of Gram-negative bacteria, were shown to stimulate the synthesis and release of platelet-activating factor (PAF), a 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphorylcholine mediator of inflammation and endotoxic shock produced by polymorphonuclear neutrophils. PAF synthesis was independent either from contamination by LPS or generation of TNF. Experiments with labeled precursors demonstrated that PAF was synthesized via the remodeling pathway that involves acetylation of 1-O-alkyl-sn-glyceryl-3-phosphorylcholine generated from 1-O-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase A2 (PLA2) activity. Porins, indeed, induced a sustained PLA2-dependent mobilization of [14C]arachidonic acid that was inhibited by p-bromodiphenacylbromide. p-Bromodiphenacylbromide, an inhibitor of PLA2, also blocked PAF synthesis by preventing the mobilization of 2-lyso-PAF, the substrate for PAF-specific acetyltransferase. The addition of 2-lyso-PAF restored PAF synthesis. The activity of acetyl CoA:2-lyso-PAF acetyltransferase was transiently increased in porin-stimulated PMN and the [3H]acetyl group was incorporated in the synthetized PAF after cell preincubation with [3H]acetyl CoA. The activation of PAF synthesis by porins as well as its release were dependent on extracellular Ca2+. Porins by forming trans-membrane channels determined a sustained influx of 45Ca2+ into the cytosol. As shown by inhibitors of Ca(2+)-calmodulin complexes, calmodulin mediated the Ca(2+)-dependent activation of enzymes involved in PAF synthesis.     

Phagocytosis-induced release of arachidonic acid from human neutrophils

The phospholipids of human neutrophils were labeled with [³H] arachidonic acid and [¹⁴C] palmitic acid. Phagocytosis of opsonized zymosan resulted in rapid release of free arachidonic acid but not of palmitic acid. Arachidonic acid was not released when the cells were exposed to unopsonized zymosan, zymosan-activated serum, or phorbol myristate acetate. These observations suggest that phagocytosis of opsonized zymosan results in the activation of a phospholipase A₂.     

The omega-hydroxylation of arachidonic acid by human polymorphonuclear leukocytes

Incubations of [1-14C]arachidonic acid with unstimulated human polymorphonuclear leukocytes resulted in the formation of four new metabolites in a previously described reverse-phase HPLC system. Three of these metabolites were largely suppressed in a CO/O2 (80/20, by vol.) atmosphere indicating a cytochrome-P450-dependent monooxygenase reaction. In agreement with this assumption is their NADPH/O2-dependent formation in the microsomal fraction. One metabolite was identified by gas chromatography/mass spectrometry analysis as omega-hydroxy-arachidonic acid and the two others were secondary products identified as omega-carboxy-arachidonic acid and 5,20-dihydroxy-E,Z,Z,Z-6,8,11,14-eicosatetraenoic acid. Since the affinity for arachidonate of the omega-monooxygenase was quite low and the presence of LTB4 suppressed the omega-hydroxylation of arachidonate, we conclude that the known LTB4 omega-monooxygenase is responsible for the formation of omega-hydroxy-arachidonate. It is unlikely, however, that significant concentrations of these metabolites are formed by activated polymorphonuclear leukocytes in vivo. The fourth metabolite remains tightly associated with the leukocytes but has not been further characterized.     

Metabolism of platelet-activating factor by arachidonic acid-depleted rat polymorphonuclear leukocytes

1-O-[3H]Alkyl-2-acetyl-sn-glycero-3-phosphocholine ([3H]PAF) and 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine ([3H]lyso-PAF) when incubated with rat polymorphonuclear leukocytes (PMN) were rapidly metabolized to 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine ([3H]alkyl-acyl-GPC) containing long chain acyl groups in the sn-2 position. The specificity and the absolute requirements of arachidonate (20:4) for acylation into PAF and lyso-PAF were investigated by comparing the rate of [3H]PAF and [3H]lyso-PAF metabolism by control rat PMN with that by rat PMN depleted of 20:4. Comparable rates of metabolism of [3H]PAF and [3H]lyso-PAF by both control and 20:4-depleted PMN were observed at all the concentrations of PAF and lyso-PAF studied. The nature of the fatty acyl group incorporated into the sn-2 position of the [3H]alkyl-acyl-GPC formed was analyzed by argentation chromatography. Dienoic fatty acids were the major fatty acid incorporated into the alkyl-acyl-GPC by both control and 20:4-depleted PMN at all the incubation times studied. At 3 min of incubation with [3H]PAF and [3H]lyso-PAF, control PMN had small but significant amounts of [3H]alkyl-acyl-GPC containing tetraenoic fatty acids, the concentration of which gradually increased as the incubation time progressed. On the other hand, under similar conditions, 20:4-depleted PMN had only trace amounts of the [3H]alkyl-acyl-GPC with tetraenoic fatty acid and the concentration of which remained at the low level throughout the incubation time. At 3 min of incubation, the 20:4-depleted PMN had small but significant amounts of [3H]alkyl-acyl-GPC with saturated fatty acids, the amount of which declined by 10 min and remained at that level as the incubation time progressed. While the concentration of [3H]alkyl-acyl-GPC with dienoic fatty acids in the 20:4-depleted cells gradually increased with the progress of incubation time, these molecular species of GPC in the control PMN remained more or less constant. In spite of a very high concentration (equivalent to that of 20:4 in control PMN) of eicosatrienoic acid (20:3 delta 5,8,11) in the 20:4-depleted PMN, no significant amounts of [3H]alkyl-acyl-GPC with trienoic fatty acid were formed by these cells. The rate of metabolism of [3H]PAF and [3H]lyso-PAF by the resident macrophages isolated from control and 20:4-depleted rats was similar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Measurement of arachidonic acid release from human polymorphonuclear neutrophils and platelets: comparison between gas chromatographic and radiometric assays

a simple gas chromatographic method for the assay of phospholipase A2 (PLA2) has been described in which arachidonic acid released from endogenous phospholipid pools is measured following its extraction and derivatization to pentafluorobenzyl esters. Using this assay, PLA2 activities in control and calcium ionophore-stimulated human neutrophils, as well as in control, thrombin, and calcium ionophore stimulated human platelets, have been measured. These values are compared with those obtained by monitoring the release of radioactivity from [3H]- or [14C]arachidonic acid prelabeled cells. While the radiometric assay measures only the release of exogenously incorporated radioactive arachidonic acid, the gas chromatographic assay measures arachidonic acid released from all the endogenous pools. Thus, the apparent increase in PLA2 activity in stimulated cells measured by the gas chromatographic assay is four- to fivefold higher than that by the radiometric assay. Inclusion of fatty acid free bovine serum albumin in the reaction buffer significantly increases the amount of arachidonic acid that is measured by gas chromatography. The gas chromatographic method has also been successfully utilized for measuring PLA2 activity in cell-free preparations derived from physically disrupted human neutrophils.     

Biosynthesis of platelet-activating factor (PAF) in human polymorphonuclear leucocytes. The role of lyso-PAF disposal and free arachidonic acid.

Tumour necrosis factor (TNF) is a potent mitogen for some fibroblast cell lines. Here we have examined the TNF-mediated changes in protein phosphorylation in Swiss 3T3 and human FS-4 fibroblasts, and compared them with changes observed after the treatment of cells with other mitogens, such as platelet-derived growth factor (PDGF) and bombesin. TNF stimulated the rapid phosphorylation of two 41,000-Mr and two 43,000-Mr cytosol proteins on tyrosine, threonine and/or serine, as did PDGF, epidermal growth factor and fibroblast growth factor; the increased levels of this mitogen-induced protein-tyrosine phosphorylation correlated well with the extent of mitogen-induced DNA synthesis as determined by the percentage of labelled nuclei. In contrast, bombesin, which is an even better mitogen for Swiss 3T3 cells than TNF, stimulated the tyrosine phosphorylation of 41,000-Mr and 43,000-Mr proteins only to a limited extent. On the other hand, bombesin and PDGF stimulated the rapid serine phosphorylation of an 80,000-Mr acidic protein, a major substrate for protein kinase C; increased phosphorylation of the 80,000-Mr protein was not observed at all when cells were stimulated with TNF. These results suggest significant differences among the mitogenic signalling pathways of TNF, PDGF and bombesin as regards the involvement of protein kinases; the mitogenic signalling pathway of TNF involves the activation of tyrosine kinase, but not of protein kinase C, whereas bombesin seems to transduce its mitogenic signal mainly through the activation of protein kinase C, and the activation of both kinases seems to be involved in the mitogenic signalling pathway of PDGF.     

Stimulus-specific induction of phospholipid and arachidonic acid metabolism in human neutrophils

Phospholipid remodeling resulting in arachidonic acid (AA) release and metabolism in human neutrophils stimulated by calcium ionophore A23187 has been extensively studied, while data obtained using physiologically relevant stimuli is limited. Opsonized zymosan and immune complexes induced stimulus-specific alterations in lipid metabolism that were different from those induced by A23187. [3H]AA release correlated with activation of phospholipase A2 (PLA2) but not with cellular activation as indicated by superoxide generation. The latter correlated more with calcium-dependent phospholipase C (PLC) activation and elevation of cellular diacylglycerol (DAG) levels. When cells that had been allowed to incorporate [3H]AA were stimulated with A23187, large amounts of labeled AA was released, most of which was metabolized to 5-HETE and leukotriene B4. Stimulation with immune complexes also resulted in the release of [3H]AA but this released radiolabeled AA was not metabolized. In contrast, stimulation with opsonized zymosan induced no detectable release of [3H]AA. Analysis of [3H]AA-labeled lipids in resting cells indicated that the greatest amount of label was incorporated into the phosphatidylinositol (PI) pool, followed closely by phosphatidylcholine and phosphatidylserine, while little [3H]AA was detected in the phosphatidylethanolamine pool. During stimulation with A23187, a significant decrease in labeled PI occurred and labeled free fatty acid in the pellet increased. With immune complexes, only a small decrease was seen in labeled PI while the free fatty acid in the pellets was unchanged. In contrast, opsonized zymosan decreased labeled PI, and increased labeled DAG. Phospholipase activity in homogenates from human neutrophils was also assayed. A23187 and immune complexes, but not zymosan, significantly enhanced PLA2 activity in the cell homogenates. On the other hand, PLC activity was enhanced by zymosan and immune complexes. Stimulated increases in PLC activity correlated with enhanced superoxide generation induced by the stimulus.     

Arachidonate metabolites, platelet-activating factor, and the mobilization of protein kinase C in human polymorphonuclear neutrophils

In contrast to our previous report (Biochem. Biophys. Res. Comm. 134:587, 1986), we now find that protein kinase C (PKC) is mobilized in human polymorphonuclear neutrophils (PMN) stimulated with platelet-activating factor (PAF) or leukotriene (LT)B4. Thus nanomolar concentrations of each compound caused PMN to lose cytosolic, PKC-specific protein phosphorylating activity, as well as receptors for phorbol myristate acetate (PMA). Smaller gains in membrane-associated PMA receptors accompanied these changes. Diacylglycerol and PMA had very similar effects on PKC. However, unlike these direct PKC activators, PAF and LTB4 induced only moderate decreases in cytosolic PKC; acted only on PMN pretreated with cytochalasin B; did not mobilize PKC in disrupted PMN or activate PKC in a cell-free system; and with respect to PAF, induced responses that partially reversed within 30 min. Furthermore, PAF, LTB4, and several of their structural analogues mobilized PKC at concentrations correlating closely with their respective affinities for cellular LTB4 or PAF receptors. Thus PAF and LTB4 acted by indirect and apparently receptor-mediated mechanisms. Four observations indicated that the cytochalasin B-dependent degranulating actions of PAF and LTB4 involved PKC. First, PKC mobilization and degranulation occurred at the same stimulus concentrations. Second, 5-hydroxyicosatetraenoate dramatically enhanced both PKC mobilization and degranulation when elicited by PAF; it had relatively little influence on LTB4-induced responses. Third, PAF-induced mobilization (t1/2 less than 7 sec) preceded degranulation (t1/2 approximately 20 sec). Finally, a PKC blocker, polymyxin B, was similarly effective in inhibiting degranulation responses to PAF, LTB4, and PMA. Because stimulated PMN may produce and use PAF, LTB4, and 5-hydroxyicosatetraenoate as secondary intracellular mediators, our results implicate PKC as a central and potentially critical regulator of function.     

Stress-induced platelet-activating factor synthesis in human neutrophils

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a potent inflammatory mediator produced by cells in response to physical or chemical stress. The mechanisms linking cell injury to PAF synthesis are unknown. We used liquid chromatography-tandem mass spectrometry to investigate stress-induced PAF synthesis in human neutrophils. PAF synthesis induced by extracellular pH 5.4 correlated with the activation of a stress-activated kinase, p38 mitogen-activated protein kinase (MAPK), and was blocked by the p38 MAPK inhibitor SB 203580. A key enzyme of PAF synthesis, acetyl-CoA:lysoPAF acetyltransferase, which we have previously shown is a target of p38 MAPK, was also activated in an SB 203580-sensitive fashion. Another MAPK pathway, extracellular signal-regulated kinase-1/2 (ERK-1/2), was also activated. Surprisingly, the pharmacological blockade of the ERK-1/2 pathway with PD 98059 did not block, but rather enhanced, PAF accumulation. Two unexpected actions of PD 98059 may underlie this phenomenon: an augmentation of stress-induced p38 MAPK phosphorylation and an inhibition of PAF catabolism. The latter effect did not appear to be due to a direct inhibition of PAF acetylhydrolase. Finally, similar results were obtained using another form of cellular stress, hypertonic sodium chloride. These data are consistent with a model in which stress-induced PAF accumulation is regulated positively by p38 MAPK and negatively by ERK-1/2. Such a model contrasts with the PAF accumulation induced by other forms of stimulation, which we and others have found is up-regulated by both p38 MAPK and ERK-1/2.     

Differential metabolism of exogenous and endogenous arachidonic acid in human neutrophils.

Leukotrienes can be produced by cooperative interactions between cells in which, for example, arachidonate derived from one cell is oxidized to leukotriene A(4) (LTA(4)) by another and this can then be exported for conversion to LTB(4) or cysteinyl leukotrienes (cys-LTs) by yet another. Neutrophils do not contain LTC(4) synthase but are known to cooperate with endothelial cells or platelets (which do have this enzyme) to generate cys-LTs. Stimulation of human neutrophils perfusing isolated rabbit hearts resulted in production of cys-LTs, whereas these were not seen with perfused hearts alone or isolated neutrophils. In addition, the stimulated, neutrophil-perfused hearts generated much greater amounts of total LTA(4) products, suggesting that the hearts were supplying arachidonate to the neutrophils and, in addition, that this externally derived arachidonate was preferentially used for exported LTA(4) that could be metabolized to cys-LTs by the coronary endothelium. Stable isotope-labeled arachidonate and electrospray tandem mass spectrometry were used to differentially follow metabolism of exogenous and endogenous arachidonate. Isolated, adherent neutrophils at low concentrations (to minimize transcellular metabolism between them) were shown to generate higher proportions of nonenzymatic LTA(4) products from exogenous arachidonate (deuterium-labeled) than from endogenous (unlabeled) sources. The endogenous arachidonate, on the other hand, was preferentially used for conversion to LTB(4) by the LTA(4) hydrolase. This result was not because of saturation of the LTA(4) hydrolase, because it occurred at widely differing concentrations of exogenous arachidonate. Finally, in the presence of platelets (which contain LTC(4) synthase), the LTA(4) synthesized from exogenous deuterium-labeled arachidonate was converted to cys-LTs to a greater degree than that from endogenous sources. These experiments suggest that exogenous arachidonate is preferentially converted to LTA(4) for export (not intracellular conversion) and raises the likelihood that there are different intracellular pathways for arachidonate metabolism.     

Synthesis of platelet-activating factor by polymorphonuclear neutrophils stimulated with interleukin-8.

Human interleukin-8 (IL-8) was evaluated for its capability to induce the synthesis and release of platelet-activating factor (PAF) from human polymorphonuclear neutrophils (PMN). IL-8 promotes in a dose-dependent fashion (1-100 ng/ml) a rapid synthesis of PAF, which is only partially released. The synthesis of PAF is preceded by the activation of acetyl-CoA: 1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyl-transferase, suggesting that IL-8 activates the "remodeling pathway" of PAF synthesis. By thin layer chromatography and reverse-phase high pressure liquid chromatography, we demonstrated that PAF synthesized by human PMN stimulated with IL-8 is heterogeneous: the 2-acetylated phospholipids having the biological and physicochemical characteristics of PAF include the 1-O-alkyl form, which is produced in large extent (51%), and the 1-acyl form (20%). The analysis of the individual molecular species of radyl chain indicated nine peaks, 16:0 and 18:0 being the predominant forms. These results identify PAF as a direct product of IL-8 stimulation in PMN.     

Distribution of arachidonic acid in choline- and ethanolamine-containing phosphoglycerides in subfractionated human neutrophils

Human neutrophils were fractionated on Percoll gradients and the various subcellular fractions were analyzed for phospholipid and fatty acid composition. The results showed that plasma membranes and azurophilic granules were enriched with ethanolamine-(PE) relative to choline-(PC) containing phosphoglycerides. A remarkable degree of uniformity existed throughout the gradient with respect to the subclass composition of the subcellular PC and PE components. In each fraction 50-60% of the PC was diacyl, 40-45% was 1-O-alkyl-2-acyl (ether linked), and 2-5% was 1-O-alk-1'-enyl-2-acyl (plasmalogenic). For PE, 20-25% was diacyl, 7-12% ether linked, and 64-76% plasmalogenic. When neutrophils were incubated for 15 min with [1-14C]arachidonic acid and subfractionated most of the PC-associated label was intracellularly localized. A similar result was observed in PE, however, when the cells were allowed to stand for 2 h in fatty acid-free buffer following the 15 min of labeling and then subfractionated there was a sizable migration of [14C]arachidonate into plasma membrane PE. In all cases the diacyl subclass was labeled most heavily after 15 min but after an additional 2 h of incubation in fatty acid-free buffer there was a direct transfer of label to the ether- and plasmalogenic-linked PC and PE subclasses. It was also found that arachidonoyl-coenzyme A 1-acyl-lysophosphatide acyltransferase activity was inherent in all three major membrane types but was enriched in the endoplasmic reticulum/secondary granule fraction. Arachidonate consistently accounted for roughly 5% of the PC and 17% of the PE fatty chain composition in each subcellular fraction. These findings demonstrate that, despite the uniform arachidonate and PC and PE subclass composition within the various neutrophil subcellular fractions, the bulk of the PC- and PE-associated arachidonate is localized in intracellular membranes.     

Role of cytochrome b-559 in arachidonic acid activation of resting human neutrophils

Whether or not cytochrome b-559 is a necessary component of NADPH oxidase activity in neutrophils is still controversial. In highly purified plasma membranes isolated from resting neutrophils and lacking cytochrome b, addition of arachidonic acid induced an NADPH oxidase activity. This activity was similar to that of plasma membranes isolated from phorbol myristate acetate (PMA)-stimulated cells which possessed cytochrome b. Addition of arachidonic acid to the latter plasma membranes did not alter the oxidase activity. It can be concluded that plasma membranes isolated from resting neutrophils have, in the presence of arachidonic acid, an NADPH oxidase activity similar to that of PMA-stimulated cells, except that it is independent of cytochrome b-559.     

Prostaglandin binding sites in human polymorphonuclear neutrophils

Prostaglandin (PG) E2 (greater than or equal to 1.6 nM) and PGD2 (greater than or equal to 16 nM) inhibited polymorphonuclear neutrophil (PMN) degranulation responses to leukotriene (LT) B4 and platelet-activating factor (PAF) whereas PGF2 alpha was bioinactive. [3H]PGE2 and [3H]PGD2 bound to PMN and isolated, plasmalemma-enriched PMN membranes. Binding was time-dependent, specific, saturable, and reversible. Competitive studies indicated that the two PGs bound to distinctly different sites. PMN had high (Kd = 1 nM; Rt = 150/cell) and low (Kd = 100 nM; Rt = 5800/cell) affinity PGE2 binding sites. Only a single type of PGD2 binding site (Kd = 13 nM; Rt = 5100/cell) was detected. We conclude that PGE2 and PGD2 bind to their respective, plasmalemmal receptors to attenuate PMN function. The PGs may act as endogenous stop signals to limit the action of concurrently formed excitatory signals, eg., LTB4 and PAF.     

Effect of fatty acid anilides on the generation of arachidonic acid by human polymorphonuclear leukocytes

The addition of oleoylanilide or linoleylanilide to human polymorphonuclear leukocytes induces a time- and dose-dependent generation of arachidonic acid. Half-maximal effect is caused by a dose of 0.2 mg linoleylanilide/ml. Fatty acid anilides also produce a time- and dose-dependent inhibition of the synthesis of triacylglycerol. Half-maximal effect is caused by 1 microgram linoleylanilide/ml. These results indicate that fatty acid anilides, which have been found in the illegal cooking oil which intoxicated thousands of Spaniards, alter lipid metabolism in human polymorphonuclear leukocytes.     

Lipoxygenbase-derived products of arachidonic acid mediate stimulation of hexose uptake in human polymorphonuclear leukocytes

The titration of metal-freed bovine α-lactalbumin with Mg²⁺ ions causes a two-stepped decrease in the tryptophan fluorescence quantum yield and a pronounced spectral shift towards shorter wavelengths, which seems to be a result of the binding of two magnesium ions to the protein molecule. The magnesium binding constants evaluated from the fluorimetric Mg²⁺-titration are 2·10³ and 2·10² M^?1. Mg²⁺ ions in millimolar concentrations almost do not influence the binding of Ca²⁺ ions to the protein.     

Intracellular localization of platelet-activating factor synthesis in human neutrophils

Human neutrophils were homogenized and fractionated on a continuous sucrose gradient to assess the subcellular location of acetyl-CoA: lyso-PAF acetyltransferase and of newly synthesized PAF (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Acetyltransferase activity showed two subcellular locations in resting neutrophils. One of them cofractionated with plasma membrane and endoplasmic reticulum markers, whereas another major location corresponded to a region of the gradient enriched in tertiary granules. No PAF was detected in resting neutrophils, but PAF synthesis was induced by cell stimulation with ionophore A23187. Most of the newly synthesized PAF was found cell-associated, showing a bimodal subcellular distribution similar to that found for acetyltransferase activity in activated cells. PAF and acetyltransferase were located in a light membrane fraction, enriched in plasma membrane and endoplasmic reticulum, and in an ill-defined region of the gradient between the specific and azurophilic granules in A23187-stimulated cells. These data support the involvement of the acetyltransferase pathway in the synthesis of PAF induced by ionophore A23187, and demonstrate the synthesis and accumulation of newly synthesized PAF in a light membrane fraction as well as in an intracellular dense organelle upon neutrophil activation.