Human granulocyte-macrophage colony-stimulating factor and other cytokines prime human neutrophils for enhanced arachidonic acid release and leukotriene B4 synthesis

Human recombinant granulocyte-macrophage CSF (GM-CSF) "primes" neutrophils for enhanced biologic responses to a number of secondary stimuli. Here, we examined the properties of neutrophil priming by GM-CSF and other growth factors such as human rTNF and granulocyte CSF. Although GM-CSF has a negligible direct effect on [3H]arachidonic acid release, it enhances or "primes" neutrophils for three- to fivefold increased release of [3H]arachidonic acid, induced by 1.0 microM A23187 and the chemotactants FMLP, platelet-activating factor, and leukotriene B4 (LTB4) (all 0.1 microM). The priming effects of GM-CSF were concentration- and time-dependent (maximum 100 pM, 1 h at 23 degrees C), and consistent with the determined dissociation constant of the human GM-CSF receptor. Indomethacin (10(-8) M), cycloheximide (100 micrograms/ml), and pertussis toxin (200 ng/ml, 2 h at 37 degrees C) had no effect on GM-CSF-, A23187, or platelet-activating factor-induced [3H]arachidonic acid release. The lipoxygenase inhibitor, nordihydroguaiaretic acid, however, totally abolished A23187-induced [3H]arachidonic acid release from both diluent- and GM-CSF-treated neutrophils. Consistent with this observation, we found that GM-CSF-pretreated neutrophils synthesize increased levels of LTB4 after stimulation with A23187 and chemotactic factors. GM-CSF enhances neutrophil arachidonic acid release and LTB4 synthesis, and thereby may amplify the inflammatory response to chemotactic factors and other physiologically relevant stimuli.     

Phosphatidylinositol 3'-kinase-mediated calcium mobilization regulates chemotaxis in phosphatidic acid-stimulated human neutrophils

Phosphatidylinositol 3'-kinase (PI 3'-kinase) plays an important role in the migration of hepatocytes, endothelial cells and neoplastic cells to agonists which activate cellular tyrosine kinases. We examined the PI 3'-kinase-dependent chemotactic responses of neutrophilic leukocytes induced by phosphatidic acid (PA) in order to clarify mechanisms by which the enzyme potentially influences cellular migration. Western analysis of immunoprecipitates indicated that PA induced the tyrosine phosphorylation of three distinct proteins involved in functional activation which co-immunoprecipitated in PA-stimulated cells. These proteins were identified as lyn, syk and the 85 kDa regulatory subunit of PI 3'-kinase. Chemotactic responses to PA but not to several other neutrophil agonists were inhibited by the PI 3'-kinase inhibitors wortmannin and LY294002. Chemotactic inhibition resulted from upstream inhibition of calcium mobilization. Chelation of extracellular calcium by ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) did not affect the PA-induced chemotaxis, whereas chelation of intracellular calcium by 1, 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) attenuated this response. Thus, changes in intracellular Ca(2+) levels that can be effected by Ca(2+) mobilized from intracellular stores in the absence of Ca(2+) influx regulate PA-induced chemotaxis. Furthermore, PI 3'-kinase inhibition blunted the agonist-dependent generation of inositol 1,4,5-trisphosphate (IP(3)), suggesting that PI 3'-kinase exerted its effects on calcium mobilization from intracellular sources by mediating activation of phospholipase C (PLC) in PA-stimulated cells. Moreover, the PI 3'-kinase inhibitor LY294002 also inhibited phosphorylation of syk in PA-stimulated cells. We, therefore, propose that products of PI 3'-kinase confined to the inner leaflet of the plasma membrane play a role in activation of syk, calcium mobilization and induction of chemotactic migration.     

Diethylstilbestrol inhibits phospholipase D activity and degranulation by stimulated human neutrophils

In the present study the effects of diethylstilbestrol on phospholipase D activity and degranulation by human neutrophils were examined. Diethylstilbestrol is a synthetic estrogen and has structural similarity to resveratrol. Resveratrol is a natural polyphenolic antioxidant and has been shown to inhibit the activity of phospholipase D in stimulated neutrophils. Phospholipase D catalyzes the hydrolysis of phosphatidylcholine to yield phosphatidic acid and choline. It also catalyzes the transfer of the phosphatidyl group to ethanol forming phosphatidylethanol at the expense of phosphatidic acid. Phospholipase D activation is associated with degranulation by neutrophils stimulated with chemotactic peptide, formyl-methionyl-leucyl-phenylalanine. The results show that diethylstilbestrol at 100 microM induced a complete inhibition of phosphatidic acid formation in neutrophils, the latter activated by chemotactic peptide. In the presence of ethanol, diethylstilbestrol dose dependently reduced phosphatidylethanol formation induced by chemotactic peptide or by phorbol 12-myristate 13-acetate, indicative of diethylstilbestyrol inhibition of phospholipase D activity. The results also demonstrate that diethylstilbestrol inhibited degranulation by chemotactic peptide-stimulated neutrophils. In comparison to resveratrol, diethylstilbestrol exhibits a stronger inhibition on PA formation, phospholipase D activity and degranulation. These findings suggest that diethylstilbestrol-like resveratrol, may have anti-inflammatory effect in vitro.     

Granulocyte-macrophage colony-stimulating factor increases the synthesis of leukotriene B4 by human neutrophils in response to platelet-activating factor. Enhancement of both arachidonic acid availability and 5-lipoxygenase activation.

Granulocyte-macrophage CSF (GM-CSF) primes human neutrophils for increased functional responsiveness to a variety of inflammatory agonists. In the present report, we have investigated the effect of human GM-CSF on the ability of platelet-activating factor (PAF) to induce the synthesis of 5-lipoxygenase products in human neutrophils. Human neutrophils stimulated with PAF in the range of 10(-5) to 10(-7) M for 15 min released small quantities of leukotriene B4 and its omega-oxidation products, 20-OH- and 20-COOH-leukotriene B4 in amounts that were detectable by enzyme immunoassay. Preincubation of normal peripheral blood neutrophils with human rGM-CSF enhanced the synthesis of the 5-lipoxygenase products in a time- and dose-dependent manner. Treatment with GM-CSF enabled their detection in response to lower concentrations of PAF (greater than or equal to 10(-9) M). The PAF receptor antagonist BN52021 inhibited the synthesis of 5-lipoxygenase products by GM-CSF-treated neutrophils in response to PAF. In addition to its effect on PAF-induced leukotriene synthesis, GM-CSF also augmented intracellular calcium mobilization by PAF. This observation prompted us to examine the effect of GM-CSF on two calcium-dependent events that are essential for leukotriene synthesis, arachidonic acid liberation, and 5-lipoxygenase activation. GM-CSF by itself, did not directly activate either of these two processes, however, it consistently and markedly enhanced the ability of PAF to do so. These results indicate that preincubation of peripheral blood neutrophils with GM-CSF enhances the ability of PAF to stimulate leukotriene synthesis by increasing both arachidonic acid availability and 5-lipoxygenase activation in response to PAF. These observations provide additional evidence of an important role for GM-CSF in the modulation of inflammatory responses to endogenous agonists through enhancement of the production of potent cellular inflammatory mediators such as leukotrienes.     

Granulocyte-macrophage colony-stimulating factor primes phospholipase D activity in human neutrophils in vitro: role of calcium, G-proteins and tyrosine kinases.

The addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) to human peripheral blood neutrophils primes phospholipase D (PLD) to subsequent stimulation by N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA). The present investigation was directed at the elucidation of the pathway(s) involved in the regulation of the activity of PLD in untreated as well as in GM-CSF-primed neutrophils. Pretreatment with pertussis toxin (PT) totally inhibited fMLP-induced activation of PLD in control or GM-CSF-treated cells. PT did not affect the activation of PLD by PMA but inhibited the priming effect of GM-CSF. Activation of PLD by fMLP was dose-dependently inhibited by erbstatin, an inhibitor of tyrosine kinases. Furthermore, pre-incubation with GM-CSF accelerated the tyrosine phosphorylation response to fMLP (as analysed by protein immunoblot with antiphosphotyrosine antibodies). In PMA-stimulated neutrophils, erbstatin antagonized the priming effect of GM-CSF on PLD without affecting the direct effects of the phorbol ester. Buffering cytoplasmic calcium with the chelator BAPTA inhibited fMLP-induced activation of PLD as monitored by the formation of phosphatidylethanol. The stimulation of PLD by PMA was partially attenuated in BAPTA-loaded cells while the priming effect of GM-CSF was abolished. Thus, priming of human neutrophil PLD by GM-CSF may be mediated by G-proteins, by increases in the levels of cytosolic free calcium, and by stimulation of protein kinase C and/or tyrosine kinase(s).     

Cholesterol-modulating agents selectively inhibit calcium influx induced by chemoattractants in human neutrophils

The effects of cholesterol-perturbing agents on the mobilization of calcium induced upon the stimulation of human neutrophils by chemotactic factors were tested. Methyl-beta-cyclodextrin and filipin did not alter the initial peak of calcium mobilization but shortened the duration of the calcium spike that followed the addition of fMet-Leu-Phe. These agents also inhibited the influx of Mn(2+) induced by fMet-Leu-Phe or thapsigargin. Methyl-beta-cyclodextrin and filipin completely abrogated the mobilization of calcium induced by 10(-10) m platelet-activating factor, which at this concentration depends to a major extent on an influx of calcium as well as the influx of calcium induced by 10(-7) m platelet-activating factor. On the other hand, methyl-beta-cyclodextrin and filipin enhanced the mobilization of calcium induced by ligation of FcgammaRIIA, an agonist that did not induce a detectable influx of calcium. Finally, methyl-beta-cyclodextrin and filipin enhanced the stimulation of the profile of tyrosine phosphorylation, the activity of phospholipase D (PLD), and the production of superoxide anions induced by fMet-Leu-Phe. These results suggest that the calcium channels utilized by chemotactic factors in human neutrophils are either located in cholesterol-rich regions of the plasma membrane, or that the mechanisms that lead to their opening depend on the integrity of these microdomains.     

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.     

Granulocyte-macrophage colony-stimulating factor primes neutrophils by activating a pertussis toxin-sensitive G protein not associated with phosphatidylinositol turnover

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine which produces diverse biological effects in target cells of myeloid origin. GM-CSF enhances the production of superoxide anion (O2-) by mature neutrophils in response to chemotactic peptides such as formyl-methionyl-leucyl-phenylalanine (fMLP), but alone it has no effect on this system. This process has been termed "priming." fMLP activates neutrophils via a pertussis toxin-sensitive GTP-binding protein, leading to the rapid production of the second messengers diacylglycerol (DAG) and inositol trisphosphate, via phosphatidylinositol turnover, and arachidonic acid (AA) by a presumptive phospholipase A2-mediated mechanism. All three second messengers may lead to the generation of O2-. We investigated the effect of priming of GM-CSF on these systems. GM-CSF had no effect on fMLP-stimulated DAG and inositol trisphosphate levels, nor did it amplify the response to exogenously added phorbol ester (to mimic the action of DAG) or calcium ionophore. Neutrophils primed with the cytokine showed a small, but significant, enhancement of fMLP-stimulated AA release. Compared with unprimed controls, primed neutrophils also showed a significant increase in O2- production when stimulated with either AA or the nonhydrolyzable GTP analogue, GTP-gamma-S. The magnitude of enhanced O2- production was similar to that observed after fMLP treatment of primed cells. All of these effects, including the increased sensitivity to AA treatment, were inhibited by pertussis toxin. These data show that GM-CSF primes neutrophils by modulating the activity of at least one pertussis toxin-sensitive G protein coupled to a metabolic pathway that mobilizes and utilizes arachidonic acid.     

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

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

Phosphatidylinositol 3′-kinase-mediated calcium mobilization regulates chemotaxis in phosphatidic acid-stimulated human neutrophils

Phosphatidylinositol 3′-kinase (PI 3′-kinase) plays an important role in the migration of hepatocytes, endothelial cells and neoplastic cells to agonists which activate cellular tyrosine kinases. We examined the PI 3′-kinase-dependent chemotactic responses of neutrophilic leukocytes induced by phosphatidic acid (PA) in order to clarify mechanisms by which the enzyme potentially influences cellular migration. Western analysis of immunoprecipitates indicated that PA induced the tyrosine phosphorylation of three distinct proteins involved in functional activation which co-immunoprecipitated in PA-stimulated cells. These proteins were identified as lyn, syk and the 85 kDa regulatory subunit of PI 3′-kinase. Chemotactic responses to PA but not to several other neutrophil agonists were inhibited by the PI 3′-kinase inhibitors wortmannin and LY294002. Chemotactic inhibition resulted from upstream inhibition of calcium mobilization. Chelation of extracellular calcium by ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA) did not affect the PA-induced chemotaxis, whereas chelation of intracellular calcium by 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA) attenuated this response. Thus, changes in intracellular Ca²⁺ levels that can be effected by Ca²⁺ mobilized from intracellular stores in the absence of Ca²⁺ influx regulate PA-induced chemotaxis. Furthermore, PI 3′-kinase inhibition blunted the agonist-dependent generation of inositol 1,4,5-trisphosphate (IP₃), suggesting that PI 3′-kinase exerted its effects on calcium mobilization from intracellular sources by mediating activation of phospholipase C (PLC) in PA-stimulated cells. Moreover, the PI 3′-kinase inhibitor LY294002 also inhibited phosphorylation of syk in PA-stimulated cells. We, therefore, propose that products of PI 3′-kinase confined to the inner leaflet of the plasma membrane play a role in activation of syk, calcium mobilization and induction of chemotactic migration.     

Characterization of the priming effects of human granulocyte-macrophage colony-stimulating factor on human neutrophil leukotriene synthesis

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is an in vitro and in vivo stimulator of human bone marrow myelomonocytic precursor cells and mature granulocyte and macrophage effector cells. We have compared the effect of GM-CSF on the synthesis of 5-lipoxygenase products induced by the chemotactic peptide fMet-Leu-Phe and the calcium ionophore A23187 in human neutrophils. Although GM-CSF alone did not stimulate detectable synthesis of products of the 5-lipoxygenase pathway, pre-incubation of neutrophils with 200 pM GM-CSF for 1 hour at 23 degrees C enhanced synthesis of leukotriene B4, its all-trans isomers and omega-oxidation products, and 5-hydroxyeicosatetraenoic acid in response to both the calcium ionophore A23187 (1.5 microM), and the chemotactic peptide fMet-Leu-Phe (0.1 microM). This priming effect of GM-CSF was maximal after a 60 min incubation at 23 degrees C, or after a 30 min preincubation at 37 degrees C. The effect of GM-CSF was maximal using a concentration of 1 nM. Enhancement of the leukotriene synthesis stimulated by A23187 was only observed when the cells were stimulated by the ionophore for periods of 3 minutes or less. In contrast, the enhancing effect of GM-CSF was still apparent when cells were exposed to fMet-Leu-Phe for as long as 15 minutes. Furthermore, the enhancing effect of GM-CSF was ablated when neutrophils were stimulated with A23187 and exogenous arachidonic acid. However, co-addition of exogenous arachidonic acid with fMet-Leu-Phe did not entirely mask the effect of GM-CSF. Possible mechanisms of action of GM-CSF are discussed.     

Regulation of the respiratory burst by cyclic 3',5'-AMP, an association with inhibition of arachidonic acid release.

The mechanism of cAMP regulation of the respiratory burst was studied with HL-60 cells that had been DMSO-differentiated to a neutrophil-like cell. To evaluate the effects of known cAMP concentrations, cells were permeabilized with streptolysin-O. Chemotactic peptide (FMLP)-stimulated NADPH oxidase activity was inhibited by cAMP at concentrations higher than 3 microM. Because intracellular calcium was buffered, inhibitory actions of cAMP were not mediated by modulation of calcium concentration. Effects of cAMP on chemotactic peptide signal transduction mediated by phospholipase C, phospholipase D, and phospholipase A2 were then determined. Neither inositol phosphate generation (phospholipase C) nor phosphatidylethanol generation (phospholipase D activity in presence of 1.6% ethanol) induced by FMLP were significantly affected by cAMP. In contrast, cAMP potently inhibited FMLP-induced arachidonic acid mobilization (phospholipase A2). NADPH oxidase activity induced by exogenous arachidonic acid was not inhibited by cAMP. These results indicate that cAMP-mediated inhibition of arachidonic acid mobilization may be important in regulation of the respiratory burst.     

Activation of phospholipase D: a signaling system set in motion by perturbation of the T lymphocyte antigen receptor/CD3 complex.

A number of cellular signaling systems are called into play by interaction of the T lymphocyte antigen receptor/CD3 complex with its cognate antigen. Well-described signaling systems include phosphoinositide turnover, tyrosine phosphorylation, protein kinase C activation, and increased cytosolic calcium. We have explored the possibility that another recently described signaling system, activation of phospholipase D, may be operative. Data presented here demonstrate that stimulation of Jurkat T cells with anti-CD3 antibodies or phorbol esters resulted in activation of phospholipase D, as measured by production of phosphatidylethanol and phosphatidic acid. The combination of anti-CD3 antibody plus phorbol ester led to a greater than additive production of phosphatidylethanol and to the additive production of phosphatidic acid (in the absence of ethanol). Phorbol esters as a second stimulus with anti-CD3 antibody led to a additive increase in cellular diacylglycerol content but provided no increased production of inositol phosphates, suggesting that diacylglycerol production in these cells results from hydrolysis of noninositol containing lipids as well as from phosphinositides. Exogenous addition of phosphatidic acid led to increases in cytosolic calcium that, depending on the concentration used, resulted from release of an intracellular store of calcium and influx of extracellular calcium. Changes in cytosolic calcium occurred in the absence of inositol phosphates production. These studies establish a role for increased phospholipase D activity in T lymphocyte activation.     

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).     

Tumor necrosis factor-alpha priming of phospholipase A2 activation in human neutrophils. An alternative mechanism of priming.

The cytokine, TNF-alpha, interacts with human neutrophils (PMN) via specific membrane receptors and primes leukotriene B4 (LTB4) production in PMN for subsequent stimulation by calcium ionophores. We have further examined the effects of TNF-alpha on arachidonic acid (AA) release, LTB4 production, and platelet-activating factor (PAF) formation in PMN by prelabeling cells with either [3H]AA or [3H]lyso-PAF, priming with human rTNF-alpha, and then stimulating with the chemotactic peptide, FMLP. TNF-alpha, alone, had little effect; minimal AA release, LTB4 or PAF production occurred after PMN were incubated with 0 to 1000 U/ml TNF-alpha. However, when PMN were first preincubated with 100 U/ml TNF-alpha for 30 min and subsequently challenged with 1 microM FMLP, both [3H] AA release and LTB4 production were elevated two- to threefold over control values. Measurement of AA mass by gas chromatography and LTB4 production by RIA confirmed the radiolabeled results. TNF-alpha priming also increased PAF formation after FMLP stimulation. These results demonstrate that TNF-alpha priming before stimulation with a physiologic agonist can enhance activation of phospholipase A2 (PLA2) resulting in increased AA release and can facilitate the activities of 5-lipoxygenase (LTB4 production) and acetyltransferase (PAF formation). Reports in the literature have hypothesized that the priming mechanism involves either production of PLA2 metabolites, increased diglyceride (DG) levels, or enhanced cytosolic calcium levels induced by the priming agent. We investigated these possibilities in TNF-alpha priming of PMN and report that TNF-alpha had no direct effect on PLA2 activation or metabolite formation. Treatment of PMN with TNF-alpha did not induce DG formation and, in the absence of cytochalasin B, no increased DG production (measured by both radiolabel techniques and mass determinations) occurred after TNF-alpha priming followed by FMLP stimulation. TNF-alpha also had no effect on basal cytosolic calcium and did not enhance intracellular calcium levels after FMLP stimulation. These results suggest that an alternative, as yet undefined, mechanism is active in TNF-alpha priming of human PMN.     

Deactivation of the effects of F-Met-Leu-Phe and leukotriene B4 on calcium mobilization in rabbit neutrophils

Preincubation of rabbit neutrophils with the synthetic chemotactic factor f-Met-Leu-Phe has been found to diminish the ability of these cells to mobilize calcium upon subsequent stimulation by f-Met-Leu-Phe or by leukotriene B₄. The preexposure of the neutrophils to leukotriene B₄ on the other hand results in a diminished subsequent response to itself but an unaltered response to f-Met-Leu-Phe. These results demonstrate that deactivation can be observed at the level of calcium mobilization, strengthen the postulated second messenger role of calcium in neutrophils and imply that neutrophil activation by chemotactic factors can bypass the arachidonic acid metabolic pathway.     

Granulocyte-macrophage colony stimulating factor up-regulates CCR1 in human neutrophils

Neutrophils (polymorphonuclear leukocytes; PMN) are phagocytic cells instrumental in the clearance of infectious pathogens. Human PMN are commonly thought to respond primarily to chemokines from the CXC family. However, recent findings suggest that under specific cytokine activation conditions, PMN can also respond to some CC chemokines. In this study, the effect of GM-CSF, a well-characterized PMN priming and maturation factor, on CC-chemokine receptor (CCR) expression in PMN was investigated. Constitutive expression of CCR1 and CCR3 mRNA in PMN was detected by ribonuclease protection assay. Following incubation of PMN with GM-CSF (0.01-10 ng/ml; 6 h) CCR1 mRNA expression was rapidly (approximately 1 h) up-regulated. In contrast, no significant induction of CCR2, CCR3, CCR4, or CCR5 mRNA was observed. CCR1 protein was also up-regulated by GM-CSF stimulation. GM-CSF-induced up-regulation of CCR1 showed functional consequences because GM-CSF-treated PMN, but not control cells, responded to the CC chemokines macrophage inflammatory protein-1alpha, monocyte chemoattractant protein-3, and RANTES in assays of chemotactic migration and intracellular calcium mobilization. These results suggest that PMN activated by the proinflammatory cytokine GM-CSF can change their receptor expression pattern and become responsive to CC chemokines.     

Unique inhibitory profile of platelet activating factor induced calcium mobilization, polyphosphoinositide turnover and granule enzyme secretion in rabbit neutrophils towards pertussis toxin and phorbol ester

Platelet activating factor has been found to increase the intracellular level of free calcium (as monitored by the fluorescent calcium indicator quin-2) and to stimulate the turnover of the polyphosphoinositides in rabbit neutrophils. Calcium mobilization induced by platelet activating factor, in contrast to previous reports with chemotactic factors, is unaffected by pertussis toxin; on the other hand, stimulated polyphosphoinositol hydrolysis and granule enzyme secretion are potently antagonized under the same conditions. The calcium, as well as the secretory responses to the lipid mediator are largely dependent on the presence of extracellular calcium. Internal contributions to the quin-2 signal are only detectable at relatively high concentrations of platelet activating factor. Calcium mobilization and secretion stimulated by platelet activating factor are inhibited following a short incubation with phorbol 12-myristate 13-acetate. These results are discussed in terms of the possibility that platelet activating factor activates neutrophils via dual pathways, the first involving direct interaction with phorbol ester inhibitable calcium channels and the other the stimulation in a manner dependent on a guanine nucleotide binding protein of the phospholipase C specific for polyphosphoinositides.     

Effect of ethanol on ATP-induced phospholipases C and D and serine base exchange in glioma C6 cells

The effect of extracellular ATP, a nucleotide receptor agonist in the central nervous system, was investigated in glioma C6 cells on the intracellular Ca2+ level and the formation of phosphatidylethanol and phosphatidic acid in the presence and absence of ethanol (150 mM). In the cells prelabeled with [14C]palmitic acid, 100 microM ATP induced both the hydrolysis and the transphosphatidylation reactions leading to the formation of [14C]phosphatidic acid; addition of ethanol generated [14C]phosphatidylethanol. However, ATP-mediated increase in the level of [14C]phosphatidic acid was not inhibited by ethanol. Furthermore, ethanol augmented ATP-induced transient and sustained increase in the intracellular Ca2+ concentration, whereas ethanol alone did not produce any change in the intracellular Ca2+ level. These results indicate that in glioma C6 cells, ATP induces activation of polyphosphoinositide-specific phospholipase C and phospholipase D and that ethanol enhances this effect. In the present investigation we have also shown that long-term (2 days) ethanol treatment, at concentration relevant to chronic alcoholism (100 mM), decreased the incorporation of [14C]serine into phosphatidylserine. Since the effect of ethanol on ATP-induced activities of phospholipase C and phospholipase D and on serine base-exchange in glioma C6 cells differs significantly from that in cultured neuronal cells, these results may contribute to a better understanding of the mechanisms of ethanol action in cells of glial origin.