Prostaglandin E1 inhibits N-formyl-methionyl-leucyl-phenylalanine-mediated depolarization responses by decreasing the proportion of responsive cells without affecting chemotaxin-induced forward light scatter changes

Hypaque-Ficoll-purified human polymorphonuclear neutrophils (PMN) equilibrated with the membrane potential-sensitive probe 3,3'dipentyloxacarbocyanine [di-O-C(5)(3)] were incubated with buffer or cytochalasin B (cyto B) followed by incubation with prostaglandin E1 (PGE1) (0 to 10(-5) M) for 5 min at 37 degrees C. The cells were then stimulated with N-formyl-methionyl-leucyl-phenylalanine (FMLP) (0 to 10(-5) M). Changes in forward light scatter (FWD-SC), 90 degrees scatter (90 degrees -SC), and fluorescence intensity were measured by flow cytometry to determine the effects of PGE1 on FMLP-induced shape change, secretion, and membrane potential responses, respectively. In other experiments, the effects of PGE1 preincubation on FMLP +/- cyto B and phorbol myristate acetate-induced (O2) production were measured by superoxide dismutase-inhibitable cyto c reduction. PGE1 had no direct effects on the FWD-SC, 90 degrees-SC, or resting potential fluorescence of unstimulated or cyto B-pretreated PMN. PGE1 produced a dose-dependent inhibition of the proportion of depolarizing PMN in response to FMLP, which was maximal at 10(-6) M (42.1 +/- 6.9% inhibition, p less than 0.005), but was apparent at 10(-8) M. The PGE1-induced inhibition was maximal after 30 sec of incubation at 37 degrees C and was caused by a decrease in the maximal percentage of depolarizing PMN without a significant change in the FMLP dose-response curve (Km = 2.43 vs 3.62 X 10(-8) M, control vs PGE1-treated) or an inhibition in the degree of depolarization by the responding subpopulation. PGE1 also inhibited the loss of 90 degrees-SC induced by FMLP in cyto B-pretreated cells (secretion response) (46.2 +/- 16.5% inhibition of the maximal 90 degrees-SC loss, n = 5, p less than 0.005), but did not affect the increase in FWD-SC seen with FMLP-induced PMN activation or the ability of cyto B to recruit more PMN to depolarize. PGE1 also inhibited FMLP +/- cyto B-induced O2 production in a dose-dependent fashion; phorbol myristate acetate-induced O2 production was also slightly inhibited, but only at high PGE1 concentrations. The data indicate that PGE1 inhibits FMLP-induced cell activation by a mechanism that involves a step distal to the recruitment of unresponsive PMN by cyto B, and that PGE1 is capable of inhibiting depolarization responses without affecting FMLP-induced shape change, providing more support for a dissociation between the two activation pathways.     

Stimulation of human polymorphonuclear leukocyte phosphatidylinositol-4-phosphate kinase by concanavalin A and formyl-methionyl-leucyl-phenylalanine is calcium-independent. Correlation with maintenance of actin assembly.

Chemoattractants directly stimulate the enzyme activity that synthesizes phosphatidylinositol-4,5-bisphosphate (PIP2), phosphoinositol-4-monophosphate (PIP) kinase. The present study determined whether stimulation of this enzyme correlates with actin assembly by assessing the calcium dependence of this reaction. Incubation of neutrophils with 5 to 100 micrograms/ml Con A caused a concentration-dependent increase in PIP kinase activity ranging from 1.38- to 3.4-fold. The effective concentration which stimulated PIP kinase by 50% (17 micrograms/ml, EC50) corresponded with the EC50 for Con A-induced superoxide production (32 micrograms/ml). Like chemoattractants, the increase in PIP kinase by Con A was characterized by a 2.6-fold increase in the maximum velocity (Vmax) of the enzyme, and no change in the Km for ATP. The kinetics of FMLP- and Con A-induced filamentous actin formation preceded stimulation of PIP kinase and was sustained over the same time period that this increased enzyme activity was noted. Although transmembrane signaling by FMLP and Con A requires an increase in intracellular calcium for some polymorphonuclear leukocyte (PMN) functional responses, calcium depletion of PMN by incubation with 100 microM Quin 2 A/M and 5 mM EGTA did not prevent the stimulation of PIP kinase by FMLP or Con A. In addition, calcium depletion did not prevent the increase in filamentous actin formation by FMLP and Con A in PMN. These findings demonstrate that Con A increases PIP kinase activity in human PMN and that PIP kinase stimulation and maintenance of actin assembly are independent of calcium fluxes in these cells. Because PIP2 controls the function of the actin-regulatory proteins, profilin and gelsolin, changes in the synthetic rate of PIP2 through regulation of PIP kinase may provide a molecular basis for the prolonged stimulation of actin assembly in human PMN by agonists such as Con A and FMLP.     

Deactivation of guinea pig pulmonary alveolar macrophage responses to N-formyl-methionyl-leucyl-phenylalanine: chemotaxis, superoxide generation, and binding

Incubation of pulmonary alveolar macrophages (PAM) with the synthetic chemotactic tripeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP) results in deactivation of PAM chemotaxis. The chemotactic response to 10(-8) M FMLP was inhibited 85% after 30 min of preincubation with 10(-6) M FMLP and 48% by 10(-8) M FMLP. Only the higher dose of FMLP (10(-6) M) caused deactivation of the chemotactic response to C5a (20%). Preincubation with partially purified C5a at a concentration of 100 microliter/ml produced a 32% inhibition of the PAM response to 10(-8) M FMLP. In contrast, preincubation with FMLP had no significant effect on superoxide generation, either at baseline or after stimulation. Levels of intracellular cyclic adenosine-3',5'-monophosphate (cAMP) increased in response to PGE1 in the presence of 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, but FMLP failed to induce a change in cAMP levels. Studies of 3H-FMLP binding were consistent with two populations of membrane receptors with different affinities. Preincubation of PAM with FMLP did not result in a reduction of maximal binding. We conclude that FMLP induces deactivation of PAM chemotaxis, but cross-deactivation occurs only after high dose treatment. Unlike the PMN, macrophage chemotactic activation is not accompanied by an elevation in cAMP levels. These observations suggest that PAM chemotaxis is influenced by prior exposure to chemotactic stimuli, but other aspects of the PAM response diverge from that of PMN. The mechanism of deactivation of PAM does not appear to result from a shift in the dose-response curve or decreased availability of membrane receptors, but may involve uncoupling of post-receptor cellular responses.     

Human neutrophil laminin receptors: activation-dependent receptor expression

Activated human polymorphonuclear leukocytes (PMN) isolated from peripheral blood specifically bind 125I-laminin after stimulation with phorbol 12-myristate 13-acetate (PMA) or f-Met-Leu-Phe (FMLP) at 37 degrees C. Changes in laminin receptor expression are stimulus dose dependent at both chemotactic (10(-10) M to 10(-6) M) concentrations of FMLP, and secretory (greater than 5 ng/ml) levels of PMA. In the presence of cytochalasin B (5 micrograms/ml), 10(-7) M FMLP activation stimulates specific laminin binding, with an apparent Kd = 3.9 X 10(-9) M and 6.47 X 10(5) binding sites/cell, reaching equilibrium within 10 min at 4 degrees C. This observed activation-dependent change in laminin receptor expression is not due to interference by endogenous laminin, because no fluorescein-visualized anti-laminin antibody bound to cells without added glycoprotein, regardless of the level of activation. Levels of neutrophil lysozyme release, which show a PMA dose dependence similar to that of receptor binding activity, suggest that granule-plasma membrane fusion may be significant during increases in receptor expression. A lack of receptor stimulation by PMA from a granule-deficient patient or in granule-depleted cytoplasts from normal donors additionally supports this hypothesis. Electroblot transfer and autoradiography of subcellular fractions from unstimulated PMN reveals the presence of a 68,000 dalton laminin-binding component in the secondary/tertiary granule (beta) fraction, which may represent an intracellular laminin receptor pool.     

Modulation of the heterogeneous membrane potential response of neutrophils to N-formyl-methionyl-leucyl-phenylalanine (FMLP) by leukotriene B4: evidence for cell recruitment

Individual human neutrophils (PMN) isolated by Hypaque-Ficoll gradient sedimentation, dextran sedimentation, or buffy coat preparation were assessed for the effects of leukotriene B4 (5S,12R dihydroxy 6,14-cis-8, 10 trans eicosatetraenoic acid (LTB4)-pretreatment on N-formylmethionyl-leucyl-phenylalanine (FMLP)-mediated membrane potential or oxidative responses by using flow cytometry and a lipophilic probe of membrane potential (di-pentyl-oxacarbocyanine, di-O-C(5)3), or the nitroblue tetrazolium dye (NBT) reduction test, respectively. Although exposure to LTB4 (10(-7) M) had no effect on the membrane potential of resting PMN and little effect on oxidant production, pretreating PMN with LTB4 followed by FMLP (10(-6) M) demonstrated a significant enhancement in the proportion of depolarizing PMN over that seen with FMLP alone (p = 0.0014, N = 9). This recruitment of previously unresponsive cells by LTB4 was dose and time dependent, with the maximal relative increase in the proportion of depolarizing cells occurring at LTB4 concentrations of 10(-8) to 10(-7) M and within 1 min of LTB4 addition. The recruitment effect persisted despite vigorous washing of the cells. LTB4 also increased the proportion of NBT-positive PMN in response to FMLP. Although LTB4 alone did not depolarize PMN it did induce a light scatter shift indicative of cell activation. 3H-FMLP binding studied at 0 degree C comparing buffer and LTB4-treated PMN indicated no significant change in the number or affinity of FMLP binding. The data provide evidence for the recruitment of a greater proportion of cells into a FMLP-responsive state as a mechanism for the enhanced functional response of PMN pretreated with LTB4, as well as for a dissociation of the membrane potential and light scattering responses of cells to this pro-inflammatory LT. The mechanism of recruitment remains unclear, but it most likely involves the modulation of a post-FMLP binding step.     

Stimulus-specific effects of endotoxin on superoxide production by rabbit polymorphonuclear leukocytes

The release of superoxide (O2-) by polymorphonuclear leukocytes (PMN) is an important function that contributes to microbial death. Controversy exists as to the effect of bacterial endotoxin (lipopolysaccharide, or LPS) on the production of O2-. We have injected rabbits with 25 micrograms Escherichia coli LPS intravenously and studied PMN function 18 to 24 hours later. Relative to PMN from saline-injected controls, PMN from LPS-treated rabbits released markedly greater amounts of O2- in response to 10 ng/ml phorbol myristate acetate (PMA) as measured by nmol cytochrome C reduced in 20 minutes (40.8 +/- 7.8 for LPS-treated PMN versus 10.1 +/- 1.6 for control, p less than 0.01). LPS injection, however, significantly reduced O2- release in response to C (complement) 5a (1.4 +/- 0.6 nmole/20 minutes for LPS-treated PMN versus 5.6 +/- 1.3 nmole/20 minutes for control, p less than 0.01). O2- release in response to a third stimulus, n-formyl-methionyl-leucyl-phenylalanine (10(-7) to 10(-9) M), was not affected by LPS. O2- release in response to PMA was enhanced over a wide range of PMA concentrations (10 to 300 ng/ml). Kinetic studies over 30 minutes indicated that, after a brief initial latency in measurable response, LPS enhanced responsiveness to PMA at all time points observed. The reduced responsiveness to C5a corresponds to a previously reported down regulation of receptors for this ligand after intravenous LPS. The observations indicate that intravenous LPS can alter a critical function of PMN for at least 24 hours in a stimulus-specific manner.     

Characterization of N-formyl-methionyl-leucyl-phenylalanine receptors on human neutrophils. Effects of isolation and temperature on receptor expression and functional activity

The study of polymorphonuclear neutrophil (PMN) surface receptor expression provides a means for the assessment of PMN function and state of cellular activation. In this study, we characterized binding of the chemotactic peptide FMLP to whole PMN, with particular attention to those variables that may account for the wide variation reported in the literature. These included avoidance of oxidized FMLP as a radioligand contaminant, determination of the optimal cold ligand concentration necessary for achieving minimal nonspecific binding throughout the range of radioligand concentrations used in saturation experiments (greater than or equal to 5 x 10(-5) M), avoidance of radioligand concentrations that equal or exceed receptor saturation and are not suitable for Scatchard analysis (greater than or equal to 60 to 80 nM), and avoidance of inadvertent receptor mobilization due to room temperature PMN isolation techniques and cell warming. PMN isolated and maintained at 4 degrees C expressed a single, high affinity population of FMLP receptors (approximately 6000 receptors per cell) with a KD of 15.5 nM. These characteristics, and in particular the single-affinity nature of the expressed FMLP receptor site, were derived from saturation experiments and confirmed with agonist competition studies. PMN subjected to room temperature isolation or 37 degrees C warming exhibited a 2.5-fold increase in FMLP receptor expression (approximately 15,000 receptors per cell) without changes in receptor affinity. These latter PMN, in correlation with increased receptor expression, had increased initial, maximal rates of FMLP-induced superoxide generation (10.2 vs 6.3 nmol/min/10(6) PMN for cells isolated and maintained at 4 degrees C) as a manifestation of their functional activation. The avoidance of inadvertent cellular activation during PMN isolation is essential to studies of PMN function, activation and the role of FMLP receptor expression/mobilization in these processes.     

Novel synthesis of S-nitrosoglutathione and degradation by human neutrophils.

S-nitrosoglutathione (SNO-GSH), a stable derivative of nitric oxide, is an endothelium-derived relaxation factor, which provokes vasodilation, inhibits platelet aggregation, and inhibits neutrophil (PMN) superoxide anion (O2+) generation. We have established a novel method for synthesis of S-nitrosoglutathione using a column containing S-nitrosothiol covalently attached to agarose. S-nitrosoglutathione was a product as assessed after separation using C-18 reverse-phase HPLC and absorption spectroscopy. We examined the stability of SNO-GSH in the presence or absence of PMN. The half-life (mercuric acid diazotization) of SNO-GSH in Hepes was greater than 60 min. The addition of resting PMN did not affect the T1/2 of SNO-GSH. PMN exposed to N-fMet-Leu-Phe (FMLP, 10(-7) M) reduced measurable SNO-GSH (15 microM) at 5 min (48 +/- 5.0% control, P less than 0.05). Incubation (5 min, 37 degrees C) of PMN with 10 microM tenidap (an anti-inflammatory drug which inhibits PMN activation) before addition of FMLP blocked the PMN-dependent degradation of SNO-GSH (42 +/- 3 vs 78 +/- 1.3% control, P = 0.01). We confirmed the recovery of SNO-GSH through measurements by bioassay (platelet aggregation) and HPLC analysis. The degradation of S-nitrosothiols by activated neutrophils may reverse the inhibitory effect of S-nitrosothiols on PMN functions and contribute to tissue injury at sites of inflammation.     

A comparison of the effects of intact and deacylated lipopolysaccharide on human polymorphonuclear leukocytes

Enzymatic deacylation of LPS markedly reduces its activity in the dermal Shwartzman reaction. Inasmuch as polymorphonuclear leukocytes (PMN) are involved in the genesis of tissue injury in Shwartzman reactions, we have investigated the effects of deacylated LPS (dLPS) on PMN. Compared to LPS, dLPS was ineffectual as a stimulus of both PMN adherence and release of secondary granule enzymes, and dLPS inhibited specific LPS-induced adherence. Neither LPS nor dLPS caused release of the primary granule enzymes, myeloperoxidase, and elastase. Unlike LPS, dLPS failed to prime PMN for superoxide release when a second stimulus (FMLP, 10(-6) M was given. The mechanism of the LPS induced increase in PMN adherence was investigated, and we found that LPS significantly increased the amount of the adhesive glycoprotein CD11b on the surface of the PMN. dLPS had no effect on CD11b expression. Our results suggest that enzymatic deacylation of LPS profoundly alters its ability to stimulate PMN and deacylation of LPS by inflammatory cells in vivo might be an important mechanism limiting the toxic effects of LPS.     

IL-8 production by human polymorphonuclear leukocytes. The chemoattractant formyl-methionyl-leucyl-phenylalanine induces the gene expression and release of IL-8 through a pertussis toxin-sensitive pathway.

IL-8 is a novel chemotactic cytokine, produced by a variety of blood and tissue cells, that has marked activating effects on polymorphonuclear leukocytes (PMN). We report that IL-8 is produced and released by human PMN after stimulation with the chemotactic agonist FMLP. Release of IL-8 in response to FMLP was transient and not influenced by PMN adherence or by the absence of serum in the medium. Maximum yields were usually obtained with 10 nM FMLP within 2 h of stimulation (0.5-3.5 ng/ml/7 x 10(6) cells, range of 17 different donors). IL-8 release was dependent on FMLP-induced de novo protein synthesis because it was inhibited by cycloheximide, was paralleled by enhanced expression of IL-8 mRNA and was potentiated from two- to sixfold after preincubation of PMN with cytochalasin B. The FMLP effect was direct and not dependent on LPS or on contaminating monocytes, which showed only low responsiveness to FMLP. Pretreatment of PMN with pertussis toxin prevented FMLP-dependent IL-8 production, the effect being evident both at the level of mRNA expression and protein secretion. In addition, two other chemoattractans, platelet-activating factor and C5a, were found capable to induce release of IL-8 by PMN. The results of this study suggest that chemotactically stimulated PMN may be able to amplify the recruitment process of PMN to the inflammatory site by releasing IL-8. As a long-lived cytokine, IL-8 could markedly prolong the attractant effect.     

Protease-modulation of neutrophil superoxide response

Although prior studies with mAb have defined an endogenous chymotrypsin-like protease in the neutrophil (polymorphonuclear leukocyte (PMN)) membrane that is associated with initiation of superoxide response to inflammatory stimuli, it is not known whether extracellular proteases (in the inflammatory milieu) can also influence PMN activation. This study examined the ability of four neutral proteases: cathepsin G, elastase, chymotrypsin, and trypsin, to modify PMN superoxide response to FMLP, PMA, and arachidonate. In response to 1 microM FMLP, PMN treated with cathepsin G, chymotrypsin, or elastase showed 64%, 60%, and 32% increases, respectively, in superoxide generation when compared with control, untreated cells (p less than 0.05 for each). These increments were dependent on intact enzymatic function of the proteases, were greatest when enzyme and stimulus were added concurrently, and persisted after PMN were washed free of enzyme. Enhancement of superoxide response was not stimulus specific; in response to 10 ng/ml PMA, cells treated with cathepsin G showed a 84%, and elastase a 57%, increase in superoxide generation (p less than 0.05 for both) with a marked reduction in the time required for onset of this response. For cell activation with 80 microM arachidonate, treatment with elastase produced a 180% increase in superoxide production (p less than 0.025). Neutrophils incubated with trypsin demonstrated significant decreases in superoxide response to PMA (-34%, p less than 0.05) and arachidonate (-39%, p less than 0.01). The enzymes themselves were not stimuli for superoxide production nor were they scavengers for superoxide in cellfree system. We conclude that local release of the PMN primary-granule neutral proteases, cathepsin G, and elastase within inflammatory sites can augment neutrophil effector function by up-regulating oxidative response to defined inflammatory stimuli. This autocrine/paracrine function may provide a significant increase in antimicrobial activity, but may also enhance the potential for host tissue injury.     

A derivative of wheat germ agglutinin specifically inhibits formyl-peptide-induced polymorphonuclear leukocyte chemotaxis by blocking re-expression (or recycling) of receptors

We examined the mechanism of action of a derivative of wheat germ agglutinin (WGA-D) which specifically and irreversibly inhibits N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced polymorphonuclear leukocyte (PMN) chemotaxis. At a concentration that completely inhibited PMN chemotaxis, WGA-D had no effect on either the uptake or release of [3H]-FMLP by PMN. Similarly, WGA-D did not affect either the short-term binding to, or internalization by, PMN of a fluoresceinated FMLP analog. WGA-D did interfere, however, with the re-expression (or recycling) of FMLP receptors by PMN that had been preincubated with 1 microM FMLP for 10 min at 4 degrees C. This effect was specific for WGA-D, because it was not observed when concanavalin A was used. Scatchard plot analysis of FMLP binding to PMN after receptor re-expression demonstrated that WGA-D-treated PMN had a significant diminution in the number of high affinity receptors. WGA-D-mediated inhibition of FMLP receptor re-expression was associated with inhibition of FMLP-induced PMN chemotaxis, but had no effect on either FMLP-induced PMN superoxide anion generation or degranulation. Studies using [125I]-WGA-D demonstrated that PMN did not internalize WGA-D spontaneously. PMN did internalize [125I]-WGA-D, however, when stimulated with FMLP. Internalization of WGA-D by FMLP-stimulated PMN was rapid, dependent on the concentration of FMLP, and specific. Internalization of [125I]-WGA-D by PMN did not occur when highly purified human C5a, instead of FMLP, was used as a stimulus. Subcellular fractionation studies demonstrated that [125I]-WGA-D and [3H]-FMLP were co-internalized by PMN, and segregated to a compartment co-migrating with Golgi markers. Western blot analysis, using PMN plasma membranes, demonstrated that WGA-D bound to a single membrane glycoprotein that migrated with an apparent m.w. of 62,000. The data indicate that WGA-D, perhaps by binding to the FMLP receptor, inhibits FMLP-induced PMN chemotaxis by blocking the re-expression (or recycling) of a population of receptors required for continuous migration.     

Modulation of platelet-activating factor (PAF) synthesis and release from human polymorphonuclear leukocytes (PMN): role of extracellular albumin

Human neutrophilic polymorphonuclear leukocytes (PMN) stimulated with N'-formyl-methionyl-leucyl-phenylalanine (FMLP) in the presence of cytochalasin B but in the absence of human serum albumin (HSA) synthesized only small amounts of platelet-activating factor (PAF) that attained maximum levels within 60-120 s after stimulation; in addition, no release of PAF occurred. However, in the presence of 2.5 mg HSA/ml, there was a threefold increase in PAF synthesis, 30-40% of which was released within 5 min after FMLP stimulation. In the presence of 50 mg HSA/ml there was at least a fourfold increase in PAF synthesis and release, with maximal synthesis occurring 10-20 min after stimulation. Thus, the presence of HSA during PMN stimulation not only induced an albumin dose-dependent increase in PAF release but significantly augmented the synthesis of PAF. In contrast to PAF synthesis and release, the presence or absence of HSA had no effect upon lysosomal enzyme secretion from FMLP-stimulated PMN, which was maximal within 30-60s after stimulation. These results demonstrate that HSA plays an essential role in vitro in the synthesis and release of PAF from human PMN, and support the hypothesis that there is a cyclic PAF synthesis-release coupling mechanism in the stimulated human PMN.     

Mechanism of polymorphonuclear leukocyte activation by myristate. Involvement of calcium ion and protein kinase C

The stimulative effects of myristate on the superoxide generation and depolarization of membrane potential of polymorphonuclear leukocytes (PMN) are particularly strong, yet myristate does not affect the intracellular free Ca2+ level ([Ca2+]i) in the presence of 1 microM free calcium in calcium-EGTA buffer. The half maximum concentration of myristate was 10 microM. Myristate inhibited the transitory changes in [Ca2+]i induced by formylmethionyl-leucyl-phenylalanine (FMLP), but stimulated further the FMLP-induced superoxide generation; these effects are similar to those of phorbol myristate acetate (PMA). The myristate-induced superoxide generation was partially inhibited by H-7, a specific inhibitor of protein kinase C. Myristate stimulated the activity of Ca2+- and phospholipid-dependent protein kinase (protein kinase C) in a concentration-dependent manner in the presence of 10(-6) M Ca2+. The Ka was 100 microM. These results suggested that there is no relation between the superoxide generation and the [Ca2+]i change in PMNs and that the effects of myristate are similar to those of PMA against PMN.     

Tumor necrosis factor-alpha enhances platelet activation via cathepsin G released from neutrophils

In this paper we show that TNF-alpha enhances platelet activation. Experiments were performed on a human polymorphonuclear neutrophil (PMN)-platelet cooperation system in which PMN, stimulated by FMLP, release cathepsin G (Cat.G), a serine proteinase responsible for the activation of nearby platelets. Pretreatment of the mixed cell suspension with 5 ng/ml TNF-alpha resulted in a strong platelet activation (37.7 +/- 3.2% aggregation; 46.0 +/- 14.4% serotonin release) in response to a weak concentration of FMLP (1.25 x 10(-8) M) inducing by itself only 7.7 +/- 4.0% of aggregation and 3.8 +/- 4.1% of serotonin release (mean +/- SD; n = 10). This effect was concentration dependent (maximum between 5 and 10 ng/ml) and was optimal for a brief preincubation time (5 min). Under these experimental conditions the target of TNF-alpha was PMN, as shown by beta-glucuronidase release. The observed potentiation was modified neither by 0.1 mM acetyl salicylic acid (a cyclo-oxygenase inhibitor) nor by 0.1 mM BN 52021 (a platelet-activating factor antagonist), while such a phenomenon was fully inhibited by 20 micrograms/ml eglin C, a strong and specific inhibitor of the human granulocytic proteinases, elastase and Cat.G. In fact, full inhibition was also observed with 300 nM alpha-1-antichymotrypsin, a specific inhibitor of Cat.G. This clear-cut evidence of Cat.G involvement was substantiated by the enhancement of Cat.G release from FMLP-activated PMN primed with TNF-alpha. These results demonstrate that the priming of PMN by TNF-alpha may modulate the activation of other inflammatory cells, particularly of platelets. It is hypothesized that this phenomenon could contribute to pulmonary pathologies, and more specifically to the adult respiratory distress syndrome, a disease for which PMN, platelet and TNF-alpha involvement has been proposed.     

Methylxanthine bronchodilators potentiate multiple human neutrophil functions

Methylxanthines, including the bronchodilators theophylline and aminophylline, in high concentrations (greater than 10(-4) M) inhibit cyclic nucleotide phosphodiesterase activity and in low, clinically relevant concentrations (10(-5) to 10(-4) M) are antagonists of extracellular adenosine receptors. The effect of therapeutic concentrations of methylxanthines on human neutrophil functions stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP) was examined. Preincubation of cytochalasin B-treated neutrophils with 10(-5) M to 3 X 10(-3) M methylxanthine resulted in a biphasic, concentration-dependent effect on neutrophil aggregation, lysosomal enzyme release, and superoxide anion formation. At 10(-5) to 10(-4) M, theophylline and aminophylline potentiated neutrophil aggregation, lysosomal enzyme release (30 to 50%, p less than 0.005), and superoxide anion formation (30 to 60%, p less than 0.005). 1-Methyl-3-isobutylxanthine at these same concentrations potentiated only neutrophil aggregation and lysosomal enzyme release (30 to 40%, p less than 0.005). The three methylxanthines inhibited each response up to 90% at concentrations greater than 10(-4) M. 8-Phenyltheophylline, which does not inhibit phosphodiesterase activity, produced only potentiation. Preincubation of neutrophils with adenosine deaminase mimicked the methylxanthine potentiation, whereas addition of adenosine (3 X 10(-8) to 3 X 10(-7) M) reversed the methylxanthine-induced potentiation in a concentration-dependent manner. These results indicate that therapeutic concentrations of methylxanthines may potentiate neutrophil activation in vivo by competing with circulating adenosine for neutrophil adenosine receptors.     

Polymorphonuclear leukocytes cap a derivative of wheat germ agglutinin upon stimulation with formyl peptide and C5a but not leukotriene B4

Previously, we reported that a derivative of wheat germ agglutinin (termed WGA-D) specifically inhibits human polymorphonuclear leukocyte (PMN) chemotaxis to FMLP by blocking reexpression (or recycling) of formyl peptide receptors. WGA-D (? formyl peptide receptor probe) binds to a protein on the PMN membrane that exhibits the same m.w. as the formyl peptide receptor. Since clustering (i.e., capping) of ligand-receptor complexes most likely precedes their internalization, we examined the ability of normal and stimulated PMN to cap fluoresceinated WGA-D. We found that, in contrast to capping of fluoresceinated Con A, PMN cap WGA-D in a chemotactic factor-specific fashion. Fluoresceinated WGA-D (5.0 to 20 micrograms/ml) alone did not induce either PMN shape changes (i.e., activation) or capping. Both FMLP (1 to 1000 nM) and human C5a (0.1 to 1.0 nM) induced PMN to polarize and to cap bound WGA-D, in a concentration-dependent fashion. Interestingly, leukotriene B4 (LTB4) (5.0 nM), while inducing the same degree of PMN polarization as FMLP (100 nM) and C5a (0.5 nM), failed to induce PMN to cap bound WGA-D. In contrast, FMLP (100 nM), C5a (0.5 nM), and LTB4 (5.0 nM) induced PMN to cap bound fluoresceinated Con A (10 micrograms/ml) to the same extent. The effect of suboptimal concentrations of FMLP and C5a on capping of WGA-D by PMN was additive. LTB4 did not enhance either FMLP or C5a-induced capping of WGA-D by PMN. Also, FMLP and C5a (but not LTB4) were capable of inducing both desensitization and cross-desensitization of WGA-D capping by PMN. Studies using rhodamine-labeled WGA-D and a fluoresceinated analog of FMLP revealed that both capped to the same place on the PMN membrane. Thus, the data suggest that WGA-D binds to a site on the PMN membrane that is either the FMLP receptor or very closely associated with it.     

cAMP and human neutrophil chemotaxis. Elevation of cAMP differentially affects chemotactic responsiveness.

Neutrophils (PMN) treated with cAMP elevating agents were evaluated for their chemotactic responsiveness to FMLP and leukotriene B4 (LTB4). PGE1 and isoproterenol, increased PMN cyclic AMP production and inhibited chemotaxis to both FMLP and LTB4. In contrast, forskolin, which activates adenylate cyclase directly, inhibited chemotaxis to FMLP but not to LTB4. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), was required for inhibition of PMN chemotaxis to FMLP by forskolin, PGE1, and isoproterenol. Isoproterenol and PGE1 inhibited PMN chemotaxis to LTB4 in the absence of IBMX and chemotaxis was further inhibited in the presence of IBMX. PMN cAMP levels were stimulated 2- to 3-fold with isoproterenol, 6- to 10-fold with PGE1, and 5- to 7-fold with forskolin over basal levels in the presence of IBMX. These observations demonstrate that total cellular cAMP concentration is not correlated with inhibition of PMN chemotaxis to all stimuli; forskolin, which increased cyclic AMP 5- to 7-fold over basal levels, did not inhibit chemotaxis to LTB4, whereas isoproterenol, which increased cyclic AMP only 2- to 3-fold over basal levels, inhibited chemotaxis to LTB4. PMN cAMP extrusion was determined under basal conditions and in the presence of PGE1, isoproterenol, or forskolin. PMN extruded cAMP under all conditions examined.     

Modulation of platelet-activating factor (PAF) synthesis and release from human polymorphonuclear leukocytes (PMN): role of extracellular Ca2+

Extracellular Ca2+ regulated the synthesis and release of platelet-activating factor (PAF) from human polymorphonuclear leukocytes (PMN) stimulated with N'-formyl-methionyl-leucyl-phenylalanine (FMLP) in the presence of cytochalasin B. Maximum PAF synthesis and release required the presence of 0.14 mM Ca2+ whereas 1.4 mM Ca2+ was necessary for maximum lysosomal enzyme secretion. The synthesis of PAF occurred within 2.5 min after PMN stimulation in the presence of 1.4 mM Ca2+; however, PAF release did not occur until 5 min after stimulation. Peak PAF release occurred by 7.5 min but accounted for only 30-40% of the total amount of PAF synthesized, the remainder being retained on or within the PMN. Stimulation of PMN in the presence of 0.01 M EDTA or EGTA decreased PAF synthesis and release by greater than 95%. In the absence of extracellular Ca2+, stimulated PMN synthesized PAF in amounts that were 10-30% of maximum, but there was no release of the newly synthesized PAF. At Ca2+ concentrations greater than 0.01 mM, there was a dose-dependent (up to 0.14 mM) increase in PAF synthesis that was associated with the initiation and concomitant increase in the amount of PAF released. These data suggest the presence of a PAF synthesis-release coupling mechanism in which the extracellular Ca2+-dependent release of PAF stimulates additional PAF synthesis.     

Recombinant human granulocyte colony-stimulating factor enhances superoxide release in human granulocytes stimulated by the chemotactic peptide

Recombinant human granulocyte colony-stimulating factor (G-CSF) by itself was not an effective stimulus for inducing the release of superoxide (O-2) in human granulocytes. However, G-CSF was able to prime human granulocytes, and enhanced O-2 release stimulated by the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). The preincubation with G-CSF for 5-10 min at 37 degrees C was sufficient for priming the cells. The optimal enhancing effect was obtained at 25 ng/ml of G-CSF. The enhancement of O-2 release by G-CSF was observed over the complete range of effective concentrations of FMLP (10(-8)-10(-6) M). These findings indicate that G-CSF is a potent activator of mature granulocyte functions.