Activation of the neutrophil by calcium-mobilizing ligands. I. A chemotactic peptide and the lectin concanavalin A stimulate superoxide anion generation but elicit different calcium movements and phosphoinositide remodeling

The relevance of phosphoinositide remodeling to calcium movements and to the physiological response of superoxide anion (O2-) generation was probed in neutrophils stimulated by the chemotactic peptide fMet-Leu-Phe and the lectin concanavalin A. fMet-Leu-Phe and concanavalin A triggered O2- generation but elicited different patterns of calcium mobilization and phosphoinositide remodeling. fMet-Leu-Phe (10(-7) M) triggered a rise in cytosolic calcium by mobilization of intracellular calcium (fura-2) and increased calcium permeability (45Ca uptake), while concanavalin A (100 micrograms/ml) elicited a rise in cytosolic calcium, primarily by uptake of extracellular calcium (45Ca uptake). fMet-Leu-Phe triggered rapid breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate and phosphatidylinositol, and generation of inositol 1,4,5-trisphosphate (IP3). In contrast concanavalin A triggered breakdown of phosphatidylinositol, but not PIP2, nor was there a significant increase in IP3. However, both fMet-Leu-Phe and concanavalin A triggered a rapid biphasic increase in levels of labeled diacylglycerol (in [3H]arachidonate or [14C]glycerol prelabeled cells) and a 3-fold increase in [32P] phosphatidic acid. These results are concordant with a role for PIP2 breakdown and generation of IP3 specifically in intracellular calcium mobilization but not for other aspects of the signaling pathway for O2- generation. Calcium permeability changes were associated with elevated diacylglycerol and [32P]phosphatidic acid, although a cause and effect relationship is not apparent. Ligands such as concanavalin A enhance cytosolic calcium and trigger generation of O2- without significant PIP2 remodeling; elevated diacylglycerol and cytosolic calcium are the common events associated with ligand-induced O2- generation.     

Changes in phosphatidylinositol and phosphatidic acid in stimulated human neutrophils. Relationship to calcium mobilization, aggregation and superoxide radical generation

Human neutrophils aggregate and release mediators of inflammation, such as active oxygen species and lysosomal enzymes, when exposed to the chemoattractant, fMet-Leu-Phe, or the tumor promotor, phorbol myristate acetate. In order to 'stage' events which may lead to such neutrophil responses, we determined the temporal relationship between stimulus-induced changes in the endogenous phospholipids phosphatidylinositol (PI) and phosphatidic acid, the mobilization of calcium, and the onset of aggregation and generation of superoxide anion during the initial 2 min of cell activation. Within 5 s after addition of fMet-Leu-Phe (10(-7) M) neutrophils accumulated phosphatidic acid and the levels of PI decreased, as determined by two-dimensional thin-layer chromatography and phosphorus determinations. By 5 s, phosphatidic acid levels rose approximately 3.5-fold and at 15 s the loss of PI exceeded the quantity of phosphatidic acid generated. In response to phorbol myristate acetate (1 microgram/ml), however, changes in PI or phosphatidic acid were not observed until after 60 s. Accumulation of phosphatidic acid in fMet-Leu-Phe-stimulated cells was not inhibited by chelation of extracellular calcium. Neutrophils exposed to either fMet-Leu-Phe or phorbol myristate acetate also showed rapid decrements in fluorescence of cell-associated chlorotetracycline (used as an indirect probe of mobilization of intracellular membrane-associated calcium) and took up 45Ca2+ from the extracellular medium (under 60 s). The results indicate that changes in calcium mobilization, together with the alterations in phospholipid metabolism (under 5 s) anteceded aggregation and the generation of O2-. (10-15 s) induced by fMet-Leu-Phe. In contrast, when neutrophils were exposed to phorbol myristate acetate, changes in PI and phosphatidic acid (over 60 s) were observed after the mobilization of calcium (under 5 s) and the onset of O2-. generation and aggregation (30-35 s).     

Effect of staurosporine on fMet-Leu-Phe-stimulated human neutrophils: dissociated release of inositol 1,4,5-trisphosphate, diacylglycerol and intracellular calcium.

Staurosporine, a microbial alkaloid, enhances inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DG) production rapidly and dose-dependently in fMet-Leu-Phe (FMLP)-stimulated human neutrophils showing maximal effects at 1 microM concentration. The IP3 increase was specific for staurosporine as three other putative protein kinase C (PKC) inhibitors, H7, sphingosine and palmitoylcarnitine were unable to enhance the IP3 generation in FMLP-stimulated human neutrophils. Staurosporine, at concentrations 0.3-1.0 microM, did not affect the initial mobilization of FMLP-induced intracellular Ca2+ (Ca2+i), although a sustained elevation of cytosolic Ca2+ level was observed within 5 min. This effect could not be suppressed, even by 1 microM phorbol-myristate 12,13-acetate (PMA). Whereas lower concentrations of staurosporine (less than or equal to 100 nM) were unable to affect FMLP-induced IP3 production, DG accumulation and Ca2+i, the PMA-inhibited initial Ca2+i signal and IP3 formation triggered by FMLP were almost completely restored. At higher concentrations (greater than or equal to 300 nM) staurosporine reversed the inhibitory effect of other protein kinases, distinct from the PMA-inducible one, which may be responsible for the phosphatidyl inositol 4,5-bisphosphate (PIP2) breakdown, thus causing accumulation of IP3 and DG and an elevation of C2+i level. Whereas IP3 declined to basal level within 5 min, the DG level remained elevated during the same period. This phenomenon is attributed to phospholipase D (PLD) stimulation by staurosporine, which augments the DG synthesis, in part through PA degradation via phosphatidic acid (PA) phosphohydrolase.     

Activation of NADPH oxidase and phospholipase D in permeabilized human neutrophils. Correlation between oxidase activation and phosphatidic acid production.

A major function of human neutrophils (PMN) during inflammation is formation of oxygen radicals through activation of the respiratory burst enzyme, NADPH oxidase. Stimulus-induced production of both phosphatidic acid (PA) and diglyceride (DG) has been suggested to mediate oxidase activity; however, transductional mechanisms and cofactor requirements necessary for activation are poorly defined. We have utilized PMN permeabilized with Staphylococcus aureus alpha-toxin to elucidate the signal pathway involved in eliciting oxidase activity and to investigate whether PA or DG act as second messengers. PMN were permeabilized in cytoplasmic buffer supplemented with ATP and EGTA for 15 min before addition of NADPH and various cofactors. Oxidase activation was assessed by superoxide dismutase inhibitable reduction of ferricytochrome C; PA and DG levels were measured by radiolabeled product formation or by metabolite mass formation. Both superoxide (O2-) and PA formation were initiated by 10 microM GTP gamma S; addition of cytosolic levels of calcium ions (Ca2+, 120 nM) enhanced O2- and PA formation 1.5-2 fold. DG levels showed little change during these treatments. PA formation preceded O2- production and varying GTP gamma S levels had parallel effects on O2- and PA formation. However, while PA formation and oxidase activation occurred in tandem at Ca2+ levels of < 1 microM, higher calcium enhanced PA formation but inhibited O2- production. Removal of ATP completely blocked O2- production but had little effect on PA formation; in contrast, if ATP was replaced with ATP gamma S, parallel production of PA and O2- occurred in the absence of other cofactors. Finally, while inhibition of PA production by ethanol pretreatment led to inhibition of O2- formation in PMN treated with GTP gamma S alone, in cells stimulated with a combination of GTP gamma S and Ca2+, ethanol continued to inhibit PA formation but had no effect on O2- production. Our results do not support a role for DG in the signal transduction path leading to oxidase activation and, while we show a close correlation between oxidase activation and PA production under many physiologic conditions, we also demonstrate that PA is not sufficient to induce oxidase activation and O2- formation can occur when PA production is inhibited.     

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.     

Changes in phosphatidylinositol and phosphatidic acid in stimulated human neutrophils: Relationship to calcium mobilization,aggregation and superoxide radical generation

Human neutrophils aggregate and release mediators of inflammation, such as active oxygen species and lysosomal enzymes, when exposed to the chemoattractant, fMet-Leu-Phe, or the tumor promotor, phorbol myristate acetate. In order to ‘stage’ events which may lead to such neutrophil responses, we determined the temporal relationship between stimulus-induced changes in the endogenous phospholipids phosphatidylinositol (PI) and phosphatidic acid, the mobilization of calcium, and the onset of aggregation and generation of superoxide anion during the initial 2 min of cell activation. Within 5 s after addition of fMet-Leu-Phe (10^?7 M) neutrophils accumulated phosphatidic acid and the levels of PI decreased, as determined by two-dimensional thin-layer chromatography and phosphorus determinations. By 5 s, phosphatidic acid levels rose approximately 3.5-fold and at 15 s the loss of PI exceeded the quantity of phosphatidic acid generated. In response to phorbol myristate acetate (1 μg/ml), however, changes in PI or phosphatidic acid were not observed until after 60 s. Accumulation of phosphatidic acid in fMet-Leu-Phe-stimulated cells was not inhibited by chelation of extracellular calcium. Neutrophils exposed to either fMet-Leu-Phe or phorbol myristate acetate also showed rapid decrements in fluorescence of cell-associated chlorotetracycline (used as an indirect probe of mobilization of intracellular membrane-associated calcium) and took up ⁴⁵Ca²⁺ from the extracellular medium (under 60 s). The results indicate that changes in calcium mobilization, together with the alterations in phospholipid metabolism (under 5 s) anteceded aggregation and the generation of O^?₂ (10–15 s) induced by fMet-Leu-Phe. In contrast, when neutrophils were exposed to phorbol myristate acetate, changes in PI and phosphatidic acid (over 60 s) were observed after the mobilization of calcium (under 5 s) and the onset of O^?₂ generation and aggregation (30–35 s).     

Role of phospholipase D-derived diradylglycerol in the activation of the human neutrophil respiratory burst oxidase. Inhibition by phosphatidic acid phosphohydrolase inhibitors.

An agonist-activated phospholipase D/phosphatidic acid phosphohydrolase (PAH) pathway was recently demonstrated in human neutrophils, and evidence suggests that phosphatidic acid (PA) and/or diradylglycerol (DG) generated from this pathway participates in activation of the O2(-)-generating respiratory burst. We have used a series of cationic amphiphilic compounds (sphingosine, propranolol, chlorpromazine, and desipramine) and antibiotics (clindamycin, trimethoprim, and roxithromycin) all of which inhibit the respiratory burst, to investigate the role of the phospholipase D/PAH pathway in neutrophil activation. The phosphatidylcholine (PC) pool in intact cells was first labeled using [3H]-1-O-alkyl-lysoPC; released [3H]-PA and [3H]-DG were then quantified after the addition of either chemo-attractant or PMA. Using either agonist, all compounds showed a dose-dependent inhibition of [3H]-DG generation which correlated with inhibition of O2- generation, but compounds failed to inhibit directly the NADPH oxidase in a cell-free system. For either activator, a plot of the ID50 values for O2- generation vs those for DG generation was linear over four orders of magnitude. In many cases, inhibition of [3H]-DG generation corresponded to an increase in [3H]-PA, implicating PAH as the locus of inhibition. Superoxide generation was inhibited under conditions where PA was either elevated or minimally affected. Neither O2- release nor DG generation showed any selectivity for stereoisomers of propranolol, suggesting that this inhibition does not act via a specific binding site on PAH. No evidence was obtained for an effect of the inhibitors on PA mobility as monitored by electron spin resonance studies of spin-labeled PA in a model membrane system. Data are consistent with an effect of the inhibitors at the level of the interaction of PAH with the membrane and/or its substrate. These data imply that DG produced via the phospholipase D/PAH pathway functions in the activation or maintenance of the respiratory burst.     

Phospholipid metabolism in human neutrophils activated by N-formyl-methionyl-leucyl-phenylalanine. Degranulation is not required for release of arachidonic acid: studies with neutrophils and neutrophil-derived cytoplasts.

Neutrophils respond to chemoattractants by aggregating, degranulating, remodelling of phospholipids and releasing arachidonic acid. To determine whether ligand-induced remodelling of phospholipids depends on redistribution of intracellular organelles (degranulation), we compared phospholipid remodelling of human neutrophils with that of neutrophil-derived cytoplasts. Cytoplasts, organelle-depleted vesicles of cytosol surrounded by plasmalemma, cannot degranulate. Without a stimulus, [3H]arachidonate was incorporated preferentially into phosphatidylinositol (PI) and phosphatidylcholine (PC). Exposure of cytoplasts and neutrophils prelabelled with [3H]arachidonate or [14C]glycerol to fMet-Leu-Phe (10(-7) M) induced rapid changes in distribution of label and mass of individual phospholipids: [3H]arachidonate in phosphatidic acid (PA) increased 500% (120 s), [14C]glycerol incorporation and mass of PA approached 200% of unstimulated values, and [3H]arachidonate in PI decreased continuously; these data are compatible with activity of a PI/PA cycle. However, the mass of PI in both preparations and [14C]glycerol label in intact neutrophils increased initially (5 s), suggesting net synthesis and mobilization of more than one pool of PI. Heterogeneity of PC pools was also observed: [3H]arachidonate was lost from PC immediately upon addition of stimulus, whereas mass and [14C]glycerol values increased. Thus, net phospholipid synthesis, redistribution of arachidonate and activation of the PI/PA cycle are immediate responses of the neutrophil to receptor occupancy by chemoattractants. Furthermore, the similarity in response to fMet-Leu-Phe of neutrophils and granule-free cytoplasts indicates that these processes are independent of degranulation.     

Superoxide generation by either 1-oleoyl-2-acetylglycerol or A23187 in human neutrophils is enhanced by indomethacin

Indomethacin at a concentration (10(-4) M) which depressed the effect on O2- generation by fMet-Leu-Phe, markedly enhanced O-2 generation by both 1-oleoyl-2-acetylglycerol and the calcium ionophore, A23187. These results are explicable in terms of the hypothesis that synergism between cytosolic calcium and protein kinase C is involved in signal transduction for the respiratory burst in the human neutrophil.     

Divergent effects of propranolol on neutrophil superoxide release: involvement of phosphatidic acid and diacylglycerol as second messengers.

Relatively high levels of propranolol (170 microM) markedly attenuated the generation of 1,2 diacylglycerol in neutrophils stimulated with either FMLP plus cytochalasin B or with 20.0 mM NaF. This effect resulted from inhibition of phosphatidic acid phosphohydrolase as it was accompanied by a corresponding increase in the recovery of phosphatidic acid in organic extracts of stimulated cells. Although propranolol enhanced phosphatidic acid levels in neutrophils treated with FMLP alone, the drug had only a slight inhibitory influence on diglyceride generation in these cells. The effect of propranolol on enhancement of PA levels in neutrophils treated with FMLP alone strongly correlated with enhancement of FMLP-induced O2- generation. However, propranolol induced a similar dose-dependent inhibition of O2- generation in neutrophils stimulated with either FMLP + cytochalasin B or with 20.0 mM NaF. These results are consistent with the hypothesis that both phosphatidic acid and diacylglycerol are required for optimal initiation of neutrophil O2- release.     

Tumor necrosis factor alpha priming of phospholipase D in human neutrophils. Correlation between phosphatidic acid production and superoxide generation.

Tumor necrosis factor alpha (TNF) primes human neutrophils (PMN) for enhanced superoxide (O2-) production if cells are subsequently stimulated with the chemotactic peptide, n-formyl-Met-Leu-Phe (fMLP). fMLP activates phospholipase D to form phosphatidic acid (PA), and a correlation may exist between PA production and O2- generation in PMN. Therefore, we assessed the ability of TNF to prime phospholipase D activation in PMN stimulated with fMLP. TNF (100 units/ml) pretreatment primed enhanced PA production in PMN challenged with 1 microM fMLP, in the absence of cytochalasin B, as demonstrated by increased production of tritiated PA from PMN label with 1-O-[9',10'-3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine ([3H]LPAF) and by increased PA mass. PA was formed via activation of phospholipase D and occurred with minimal production of diglycerides. Production of O2- was also enhanced in identically treated cells, and we demonstrated a direct correlation between enhanced PA formation and O2- production. Conversely, ethanol inhibition of PA formation led to a comparable reduction in O2- generation. This report of priming of phospholipase D by physiological agonists is the only natural system where enhanced PA formation has been dissociated from diglyceride formation. Our results suggest a link between PA production and NADPH oxidase activation in human PMN.     

The role of cytosolic free calcium in the generation of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate in HL-60 cells. Differential effects of chemotactic peptide receptor stimulation at distinct Ca2+ levels

The generation of the two inositol trisphosphate (IP3) isomers, 1,4,5-IP3 and 1,3,4-IP3, and its relation to changes in the cytosolic free calcium concentration, [Ca2+]i, in response to the chemotactic peptide fMet-Leu-Phe was studied in the human promyelocytic cell line HL-60, induced to differentiate with dimethyl sulfoxide. Stimulation by fMet-Leu-Phe within seconds transiently elevates 1,4,5-IP3 to peak values averaging 8-fold basal levels, and leads to a concomitant rise in [Ca2+]i and to degranulation. These responses are followed by a slower and more sustained rise in 1,3,4-IP3. Alterations in [Ca2+]i modulate differentially the generation of the two IP3 isomers. At [Ca2+]i lower than 30 nM, no IP3 is generated upon fMet-Leu-Phe stimulation. Working at normal resting [Ca2+]i, but preventing the fMet-Leu-Phe induced transient rise in [Ca2+]i (by prior depletion of intracellular Ca2+ stores and working in calcium-free medium) the fMet-Leu-Phe stimulation of 1,3,4-IP3 levels is attenuated, whereas the response of 1,4,5-IP3 is not significantly altered. Maintained elevation of [Ca2+]i to micromolar levels with the Ca2+ ionophore ionomycin generates enhanced 1,3,4-IP3 levels in the absence of fMet-Leu-Phe, whereas the fMet-Leu-Phe stimulation of 1,4,5-IP3 generation is markedly inhibited. Pertussis toxin selectively abolishes the fMet-Leu-Phe-induced IP3 production, whereas ionomycin stimulation of 1,3,4-IP3 generation is unaffected. These findings indicate that in intact cells: receptor-triggered phosphatidylinositol bisphosphate phosphodiesterase activation has a minimal Ca2+ requirement, but does not depend on a previous or concomitant rise in [Ca2+]i; Ca2+ elevations above micromolar levels decrease the fMet-Leu-Phe-induced generation of 1,4,5-IP3; and 1,3,4-IP3 generation is not directly linked to receptor activation and appears to result both from increased [Ca2+]i and 1,4,5-IP3 levels.     

Bombesin stimulation of inositol 1,4,5-trisphosphate generation and intracellular calcium release is amplified in a cell line overexpressing the N-ras proto-oncogene.

Human neutrophils and HL-60 leukaemic cells possess an NADPH oxidase which catalyses superoxide (O2-) formation and is activated by the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe). In dibutyryl cyclic AMP-differentiated HL-60 cells, ATP and UTP in the presence of cytochalasin B activated O2- formation with EC50 values of 5 microM and efficacies amounting to 30% of that of fMet-Leu-Phe. The potency order of purine nucleotides in activating O2- generation was ATP = adenosine 5'-O-(3-thiotriphosphate) greater than ITP greater than dATP = ADP. Pyrimidine nucleotides activated NADPH oxidase in the potency order UTP greater than dUTP greater than CTP = TTP = UDP. Pertussis toxin completely prevented activation of NADPH oxidase by fMet-Leu-Phe and UTP, whereas the effect of ATP was only partially inhibited. ATP and UTP enhanced O2- generation induced by fMet-Leu-Phe by up to 8-fold, and primed the cells to respond to non-stimulatory concentrations of fMet-Leu-Phe. Activation of NADPH oxidase by UTP but not by ATP was inhibited by various activators of adenylate cyclase. In dimethyl sulphoxide-differentiated HL-60 cells and in human neutrophils, ATP and UTP per se did not activate NADPH oxidase, but they potentiated the effect of fMet-Leu-Phe. Our results suggest that purine and pyrimidine nucleotides act via purino- and novel pyrimidinoceptors respectively, which are coupled to guanine nucleotide-binding proteins leading to the activation of NADPH oxidase. As ATP and UTP are released from cells under physiological and pathological conditions, these nucleotides may play roles as intercellular signal molecules in the activation of O2- formation.     

The roles of degranulation and superoxide anion generation in neutrophil aggregation

Human neutrophils when exposed to appropriate stimuli aggregate, generate O(2) and secrete lysosomal constituents. To determine whether a causal relationship may exist between these responses neutrophils were exposed to either N-formyl-methionyl-leucyl-phenylalanine, phorbol myristate acetate, or the two calcium ionophores, A23187 and prostaglandin Bx. Each agent elicited all of the above responses. The concentrations required to elicit the aggregation of 30 . 10(6) neutrophils/ml were comparable to that required for O(2) generation or lysozyme release. In a series of experiments designed to dissociate these responses, cells were suspended in a concentration too dilute (3 . 10(6) neutrophils/ml) to permit aggregation to occur. O(2) generation and lysozyme release was measurable and varied in a dose-dependent fashion to the concentration of stimulus. In a second series of experiments, neutrophils were treated with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid to inhibit degranulation without affecting O(2) generation. Aggregation was inhibited in a parallel fashion with lysozyme release. When detectable O(2) was removed from the medium by superoxide dismutase and catalase, aggregation and lysozyme release unaffected showing that aggregation can not be due to the presence of O(2) or its products in the extracellular medium. Neither aggregation of resting cells nor augmentation of fMet-Leu-Phe-induced aggregation was observed when cells were exposed to either supernatants of degranulated neutrophils or constituents of specific granules (lysozyme, lactoferrin). Kinetic analysis showed that in the absence of cytochalasin B degranulation preceded aggregation, while in its presence aggregation preceded degranulation.     

Leishmania donovani: surface membrane acid phosphatase blocks neutrophil oxidative metabolite production

We show that a purified preparation of the prominent tartrate-resistant acid phosphatase (E.C.3.1.3.2), isolated from the external surface of the intracellular parasite Leishmania donovani (promastigote form), inhibits toxic oxidative metabolite production of neutrophils. Preincubation of a neutrophil suspension (2.5 X 10(6) cells/ml) for 15 min at 37 C with 250 units (1 unit equals 1 nmole of 4-methylumbelliferyl phosphate cleaved per hr at pH 5.5) of the acid phosphatase in Krebs-Ringer phosphate buffer (pH 7.4) decreased O2 consumption, O2- production, and H2O2 production of N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated neutrophils to 15-25% of control values. The acid phosphatase also affected concanavalin A-stimulated O2-production by neutrophils, but had no effect on the rate of phorbol myristic acetate-stimulated O2- production, chemotactic peptide binding, degranulation, or membrane depolarization. Addition of an acid phosphatase inhibitor (Complex E; (NH4)6[P2Mo18O62] X 9H2O) to suspensions of opsonized promastigotes and neutrophils resulted in a threefold or greater enhancement of O2- production. These results suggest a possible pathophysiologic role for the acid phosphatase of L. donovani promastigotes.     

Calcium signals in neutrophils can be divided into three distinct phases

Rabbit neutrophils were loaded with the fluorescence probe indo-1 and cytosolic free calcium levels were monitored during chemotactic peptide (fMet-Leu-Phe) stimulation. The fMet-Leu-Phe-induced calcium signal consisted of three consecutive phases: (1) an initial peak that was independent of extracellular calcium, (2) a secondary shoulder that required extracellular calcium but was totally blocked by hyperosmolality and (3) a final plateau of elevated calcium that was dependent on extracellular calcium but insensitive to hyperosmolality.     

Stimulation of respiratory burst by cyclocommunin in rat neutrophils is associated with the increase in cellular Ca2+ and protein kinase C activity

In this study, the underlying mechanisms of stimulation by cyclocommunin, a natural pyranoflavonoid, of respiratory burst in rat neutrophils was investigated. Cyclocommunin evoked a concentration-dependent stimulation of superoxide anion (O2*-) generation with a slow onset and long lasting profile. The maximum response (16.4+/-2.3 nmol O2*-/10 min per 10(6) cells) was observed at 3-10 microM cyclocommunin. Cyclocommunin did not activate NADPH oxidase in a cell-free system. Cells pretreated with pertussis toxin or n-butanol did not affect the cyclocommunin-induced O2*- generation. However, a protein kinase inhibitor staurosporine and EGTA greatly reduced the O2*-generation caused by cyclocommunin. Treatment of neutrophils with phorbol 12-myristate 13-acetate (PMA), but not with formylmethionyl-leucyl-phenylalanine (fMLP), for 20 min significantly reduced the O2*- generation following the subsequent stimulation of cells with cyclocommunin. Cyclocommunin did not affect the cellular mass of phosphatidic acid (PA). Neither the tyrosine kinase inhibitor, genistein, nor the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, affected cyclocommunin-induced O2*- generation. The enzyme activities of neutrophil cytosolic and membrane-associated protein kinase C (PKC) were both increased significantly with 100 microM cyclocommunin. The membrane-associated PKC-theta and PKC-beta were increased following the stimulation of neutrophils with 30 and 100 microM cyclocommunin, respectively. Cyclocommunin reduced the [3H]phorbol 12,13-dibutyrate ([3H]PDB) binding to cytosolic PKC in a concentration-dependent manner. Cyclocommunin (> or =3 microM) significantly evoked a slow and long lasting [Ca2+]i elevation in neutrophils, and a phospholipase C (PLC) inhibitor U73122 greatly inhibited these Ca2+ responses. Moreover, the increase in cellular inositol bis- and trisphosphate (IP2 and IP3) levels were observed in neutrophils stimulated with 30 microM cyclocommunin for 3 min. Collectively, these results indicate that the stimulation of respiratory burst by cyclocommunin is probably mediated by the synergism of PKC activation and [Ca2+]i elevation in rat neutrophils.     

Roles for beta II-protein kinase C and RACK1 in positive and negative signaling for superoxide anion generation in differentiated HL60 cells

beta-Protein kinase (PKC) is essential for ligand-initiated assembly of the NADPH oxidase for generation of superoxide anion (O(2)). Neutrophils and neutrophilic HL60 cells contain both betaI and betaII-PKC, isotypes that are derived by alternate splicing. betaI-PKC-positive and betaI-PKC null HL60 cells generated equivalent amounts of O(2) in response to fMet-Leu-Phe and phorbol myristate acetate. However, antisense depletion of betaII-PKC from betaI-PKC null cells inhibited ligand-initiated O(2) generation. fMet-Leu-Phe triggered association of a cytosolic NADPH oxidase component, p47(phox), with betaII-PKC but not with RACK1, a binding protein for betaII-PKC. Thus, RACK1 was not a component of the signaling complex for NADPH oxidase assembly. Inhibition of beta-PKC/RACK1 association by an inhibitory peptide or by antisense depletion of RACK1 enhanced O(2) generation. Therefore, betaII-PKC but not betaI-PKC is essential for activation of O(2) generation and plays a positive role in signaling for NADPH oxidase activation in association with p47(phox). In contrast, RACK1 is involved in negative signaling for O(2) generation. RACK1 binds to betaII-PKC but not with the p47(phox).betaII-PKC complex. RACK1 may divert betaII-PKC to other signaling pathways requiring beta-PKC for signal transduction. Alternatively, RACK1 may sequester betaII-PKC to down-regulate O(2) generation.     

Reversible activation of the neutrophil superoxide generating system by hexachlorocyclohexane: correlation with effects on a subcellular superoxide-generating fraction

gamma-Hexachlorocyclohexane was found to exert profound effects on the phosphatidylinositol cycle, cytosolic calcium level, and the respiratory burst of human neutrophils. Exposure of neutrophils prelabelled with 32P to 4 X 10(-4) M gamma-hexachlorocyclohexane almost tripled radioactivity in phosphatidic acid and correspondingly decreased radioactivity in phosphatidylinositol 4,5 bisphosphate. Under similar conditions, gamma-hexachlorocyclohexane evoked the generation of superoxide at a rate of over 11 nmol/min/10(6) cells and more than doubled cytosolic-free calcium concentration as monitored by Quin-2 fluorescence. Because intermediates of the phosphatidylinositol cycle, via increases in available calcium levels or activated protein kinase C, are considered potential second messengers for activation of the NADPH-dependent O-2-generating system, we compared neutrophil responses to gamma-hexachlorocyclohexane with responses to phorbol myristate acetate, an activator of protein kinase C with well known effects on neutrophils. Like phorbol myristate acetate, gamma-hexachlorocyclohexane induced neutrophil degranulation but was not an effective chemotactic stimulus. The ability of gamma-hexachlorocyclohexane to induce a pattern of oxidative activation in neutrophil cytoplasts similar to that in intact cells indicated that concurrent degranulation was not required for sustained O-2 generation in response to this agent. When neutrophils or neutrophil cytoplasts exposed to gamma-hexachlorocyclohexane were centrifuged and resuspended in stimulus-free medium, O-2 generation ceased entirely but could be reinitiated by addition of the same stimulus. This finding was in contrast to the continued O-2 production by phorbol myristate acetate-stimulated neutrophils similarly washed and resuspended in stimulus-free medium. Unlike subcellular fractions of phorbol myristate acetate-stimulated neutrophils, corresponding fractions prepared from gamma-hexachlorocyclohexane-stimulated neutrophils contained almost no detectable NADPH-dependent O-2-generating activity. Subcellular oxidase activity was not recovered when cells and membrane fractions were continuously exposed to gamma-hexachlorocyclohexane during disruption and fractionation after cell stimulation, nor could it be induced by the addition of the stimulus to the subcellular fractions. Thus, the stimulus dependence of continuous neutrophil superoxide release evoked by gamma-hexachlorocyclohexane does not merely reflect a physical interaction of the agonist with the enzyme system involved.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phospholipase D activation by platelet-activating factor, leukotriene B4, and formyl-methionyl-leucyl-phenylalanine in rabbit neutrophils. Phospholipase D activation is involved in enzyme release.

Lipid chemoattractants, such as platelet-activating factor and leukotriene B4, as well as the peptide chemoattractant FMLP, were found to stimulate [3H]phosphatidic acid ([3H]PA) formation in 1-O-[3H]octadecyl-lyso platelet-activating factor-labeled rabbit neutrophils. The stimulation of [3H]PA formation appears to result from the activation of phospholipase D (PLD), because in the presence of ethanol, chemoattractant stimulation produced [3H]phosphatidylethanol, the characteristic compound produced by PLD at the expense of [3H]PA formation. The PLD activation by all chemoattractants tested was primed by cytochalasin B and revealed a similar time dependence. However, lipid chemoattractants were less potent as compared with FMLP, and the maximal stimulation by the former was lower than that by the latter. From these results, it is concluded that the mechanism of PLD activation by lipid chemoattractants is similar to, but different from, that by FMLP. Cytochalasin B stimulated degranulation and [3H]PA formation in agonist-stimulated neutrophils, and their stimulations were well correlated. Ethanol inhibited both agonist-stimulated [3H]PA formation and degranulation in a concentration-dependent manner, but the inhibition in degranulation was much less than that in [3H]PA formation. These results suggest that PLD activation is involved in degranulation, but another signaling pathway may also be required for full stimulation of degranulation. When the radiolabeled neutrophils were stimulated by chemoattractants for 5 min, 1,2-[3H]diglyceride was found to accumulate. The accumulation was inhibited by either ethanol or the phosphatidate phosphohydrolase inhibitor propranolol, which indicates that PA produced by PLD can be converted to 1,2-diglyceride by phosphatidate phosphohydrolase. Under these conditions, propranolol did not inhibit degranulation stimulated by chemoattractants. These results indicate that PA produced by PLD is more important than its metabolite diglyceride for the degranulation of rabbit neutrophils.