Stimulus-specificity of the chemotactic deactivation of human neutrophilis by lipoxygenase products of arachidonic acid

Preincubation of human neutrophils with chemotactic concentrations of 5(S)-hydroxy-eicosatetraenoic acid (5-HETE) or 5(S), 12(R)-dihydroxy-6, 14 cis-8, 10 trans-eicosatetraenoic acid (leukotriene B₄) induces a state of preferential chemotactic unresponsiveness to the homologous factor, termed deactivation, and less suppression of the responses to other chemotactic stimuli. The ratio of the concentration required for maximal chemotactic deactivation of neutrophils to that which stimulates chemotaxis optimally is greater for 5-HETE and leukotriene B₄ than for peptide and protein factors. In contrast to other chemotactic factors, 5-hydroperoxy-eicosatetraenoic acid (5-OOHETE) induces neutrophil chemotactic deactivation that is independent of the nature of the subsequent stimulus and is more slowly reversible after elimination of the fluid-phase deactivating factor. The unique characteristics of the chemotactic deactivation of human neutrophils by 5-OOHETE may be attributable in part to its endogenous metabolism to potent deactivating factors or to covalent derivatization of subcellular structures of the neutrophils by the highly reactive 5-OOHETE.     

Mechanism involved in the mobilization of neutrophil calcium by 5-hydroxyeicosatetraenoate

5-Hydroxyeicosatetraenoate (5-HETE), like leukotriene B4 and platelet-activating factor, stimulated human polymorphonuclear neutrophils to mobilize intracellular calcium. The three compounds acted through mechanisms that were inhibited by pertussis toxin, cholera toxin, and PMA. Each agonist, furthermore, desensitized (or down-regulated) the neutrophil's calcium mobilization response to a second challenge with the same agonist. However, 5-HETE and leukotriene B4 had little or no activity in cross-desensitizing neutrophil responses to each other or to platelet-activating factor. Furthermore, 5-HETE interfered minimally or not at all with the binding of radiolabeled leukotriene B4 and platelet-activating factor to their respective receptors on neutrophils. Thus, 5-HETE mobilizes neutrophil calcium by a mechanism different from those used by leukotriene B4 and platelet-activating factor. This mechanism appears to involve specific 5-HETE receptors that couple to pertussis toxin-inhibitable, GTP-binding proteins.     

Lipoxygenase products of arachidonic acid modulate biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human neutrophils via phospholipase A2

When human neutrophils, previously labeled in their phospholipids with [14C]arachidonate, were stimulated with the Ca2+-ionophore, A23187, plus Ca2+ in the presence of [3H]acetate, these cells released [14C]arachidonate from membrane phospholipids, produced 5-hydroxy-6,8,11,14-[14C]eicosatetraenoic acid (5-HETE) and 14C-labeled 5S,12R-dihydroxy-6-cis,8,10-trans, 14-cis-eicosatetraenoic acid ([14C]leukotriene B4), and incorporated [3H]acetate into platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Ionophore A23187-induced formation of these radiolabeled products was greatly augmented by submicromolar concentrations of exogenous 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), 5-HETE, and leukotriene B4. In the absence of ionophore A23187, these arachidonic acid metabolites were virtually ineffective. Nordihydroguaiaretic acid (NDGA) and several other lipoxygenase/cyclooxygenase inhibitors (butylated hydroxyanisole, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 1-phenyl-2-pyrazolidinone) caused parallel inhibition of [14C]arachidonate release and [3H]PAF formation in a dose-dependent manner. Specific cyclooxygenase inhibitors, such as indomethacin and naproxen, did not inhibit but rather slightly augmented the formation of these products. Furthermore, addition of 5-HPETE, 5-HETE, or leukotriene B4 (but not 8-HETE or 15-HETE) to neutrophils caused substantial relief of NDGA inhibition of [3H]PAF formation and [14C]arachidonate release. As opposed to [3H]acetate incorporation into PAF, [3H]lyso-PAF incorporation into PAF by activated neutrophils was little affected by NDGA. In addition, NDGA had no effect on lyso-PAF:acetyl-CoA acetyltransferase as measured in neutrophil homogenate preparations. It is concluded that in activated human neutrophils 5-lipoxygenase products can modulate PAF formation by enhancing the expression of phospholipase A2.     

Requirement of free arachidonic acid for leukotriene B4 biosynthesis by 12-hydroperoxyeicosatetraenoic acid-stimulated neutrophils

Stimulation of human neutrophils with 12-hydroperoxyeicosatetraenoic acid (12-HPETE) led to formation of 5S, 12S-dihydroxyeicosatetraenoic acid (DiHETE), but leukotriene B4 (LTB4) or 5-hydroxyeicosatetraenoic acid (5-HETE) was not detectable by reversed-phase high-performance liquid chromatography analysis. N-formylmethionylleucylphenylalanine (FMLP) induced the additional synthesis of small amounts of LTB4 in 12-HPETE-stimulated neutrophils. The addition of arachidonic acid greatly increased the synthesis of LTB4 and 5-HETE by neutrophils incubated with 12-HPETE. In experiments using [1-14C]arachidonate-labeled neutrophils, little radioactivity was released by 12-HPETE alone or by 12-HPETE plus FMLP, while several radiolabeled compounds, including LTB4 and 5-HETE, were released by A23187. These findings demonstrate that LTB4 biosynthesis by 12-HPETE-stimulated neutrophils requires free arachidonic acid which may be endogenous or exogenous.     

Unique aspects of the modulation of human neutrophil function by 12-L-hydroperoxy-5,8,10,14-eicosatetraenoic acid

12-L-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-OOHETE), a labile intermediate generated by the lipoxygenation of arachidonic acid in platelets, and 12-L-hydroxy-5,8,10.14-eicosatetraenoic acid (12-OHETE), the reduction product of 12-OOHETE, were examined for their effects on human neutrophil function in vitro. 12-OOHETE elicited a maximal neutrophil chemotactic response at 4 microgram/ml, that exceeded by over 50% the maximal chemotactic response to 10-20 microgram/ml of 12-OHETE. Similarly 12-OOHETE was more potent than 12-OHETE in evoking neutrophil chemokinetic responses and in enhancing the expression of C3b receptors on neutrophils. The concentration of guanosine 3':5' cyclic monophosphate (cGMP) in neutrophils was increased to the same plateau level by 5 ng/ml of 12-OOHETE and by 50 ng/ml of 12-OHETE. Elevations in the concentration of cGMP were maintained for 30 min or longer by a single dose of 12-OOHETE, but fell between 10 and 20 min after the introduction of 12-OHETE. The release of neutrophil lysosomal enzymes by the chemotactic fragments of C5 was augmented substantially by 12-OOHETE, while 12-OHETE had only a marginal effect. The non-chemotactic methyl ester of 12-OHETE failed to inhibit the chemotactic responses to 12-OOHETE at molar ratios that suppressed comparable response to 12-OHETE by 42-86%. Thus 12-OOHETE is more potent than 12-OHETE in the stimulation of some human neutrophil functions and in the elevation of the cellular concentration of cGMP. Furthermore, 12-OOHETE may activate neutrophils by pathways not available to 12-OHETE.     

Unique aspects of the modulation of human neutrophil function by 12-L-hydroperoxy-5,8,10,14-eicosatetraenoic acid

12-L-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-OOHETE), a labile intermediate generated by the lipoxygenation of arachidonic acid in platelets, and 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-OHETE), the reduction product of 12-OOHETE, were examined for their effects on human neutrophil function . 12-OOHETE elicited a maximal neutrophil chemotactic response at 4 μg/ml, that exceeded by over 50% the maximal chemotactic response to 10–20 μg/ml of 12-OHETE. Similarly 12-OOHETE was more potent than 12-OHETE in evoking neutrophil chemokinetic responses and in enhancing the expression of C3b receptors on neutrophils. The concentration of guanosine 3′:5′ cyclic monophosphate (cGMP) in neutrophils was increased to the same plateau level by 5 ng/ml of 12-OOHETE and by 50 ng/ml of 12-OHETE. Elevations in the concentration of cGMP were maintained for 30 min or longer by a single dose of 12-OOHETE, but fell between 10 and 20 min after the introduction of 12-OHETE. The release of neutrophil lysosomal enzymes by the chemotactic fragments of C5 was augmented substantially by 12-OOHETE, while 12-OHETE had only a marginal effect. The non-chemotactic methyl ester of 12-OHETE failed to inhibit the chemotactic responses to 12-OOHETE at molar ratios that suppressed comparable responses to 12-OHETE by 42–86%. Thus 12-OOHETE is more potent than 12-OHETE in the stimulation of some human neutrophil functions and in the elevation of the cellular concentration of cGMP. Furthermore, 12-OOHETE may activate neutrophils by pathways not available to 12-OHETE.     

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.     

Stereospecificity of leukotriene B4 and structure-function relationships for chemotaxis of human neutrophils

The chemotactic activity of leukotriene B4 (5S, 12R Dihydroxy 6, 14 cis, 8, 10 trans eicosatetraenoic acid) (LTB4) was examined by using a sensitive Boyden-chamber assay. The activity of LTB4 was compared to other biosynthetic stereoisomers: 5S, 12R Dihydroxy 6, 8, 10 trans 14 cis eicosatetraenoic acid (6-trans LTB4); 5S, 12S Dihydroxy 6, 8, 10 trans 14 cis eicosatetraenoic acid (12-epi-6-trans LTB4), 5S, 12S DiHETE; the metabolic product 20-Hydroxy LTB4 (20-OH LTB4); methylated LTB4 (Methyl-LTB4), and the related monoHETE 5-HETE and 12-HETE. The compounds were purified by several steps of reverse phase and straight phase HPLC. The LTB4 exhibits measurable chemotactic activity at 10(-9) M with maximal activity at 10(-7) M and an ED50 of 10(-8) M. The LTB4 isomers and monoHETE were less chemotactic than previously reported. The monoHETE (5-HETE and 12-HETE), the isomer 12-epi-6-trans LTB4, and 5S, 12S DiHETE fail to attract neutrophils at levels between 10(-6) and 10(-5) M. If these compounds are chemotactic, then activity is at least four orders of magnitude less than that of LTB4. The isomer 6-trans LTB4 at 10(-6) to 10(-5) M induced chemotaxis with an extrapolated ED50 value of 10(-5) M, indicating that a trans for cis change in configuration at position 6 reduces the chemotactic activity of LTB4 by 1000-fold. Conversely, the metabolic product 20-OH LTB4 is at least as active as the native compound LTB4. Methylation of the carboxyl group of LTB4 reduces its chemotactic activity by two orders of magnitude. These results indicate a high degree of stereospecificity for the LTB4 receptor with strict dependence on hydroxyl group, and triene configuration and considerable dependence on the carboxyl group. Modification at the aliphatic omega end of the LTB4 molecule has a minimal effect on function, suggesting that the hydrophobicity of this portion of the molecule is not important for optimal activity. Furthermore, we propose that metabolic products of LTB4 may be of greater importance than LTB4 as physiologic inflammatory mediators in vivo.     

Selective feed-back inhibition of the 5-lipoxygenation of arachidonic acid in human T-lymphocytes

Purified human T-lymphocytes exhibit 5-lipoxygenase activity as demonstrated by the conversion of arachidonic acid to 5-hydroxy-eicosatetraenoic acid (5-HETE), 5(S),12(R)-di-hydroxy-eicosa-6,14 cis-8,10 trans-tetraenoic acid (leukotriene B₄), and 5,12-di-HETE isomers of leukotriene B₄ that lack a 6-cis double bond. The concentrations of leukotriene B₄, 5-HETE, 11-HETE and 15-HETE in suspensions of T-lymphocytes were increased significantly by concanavalin A and by the calcium ionophore A23187. Preincubation of T-lymphocytes with 15-HETE at μM concentrations, characteristic of suspensions of stimulated lymphocytes, inhibited selectively the increases in the levels of 5-HETE and leukotriene B₄, but not of 11-HETE and prostaglandin E₂.     

Formation of leukotrienes and hydroxy acids by human neutrophils and platelets exposed to monosodium urate

Monosodium urate (MSU) crystals stimulate the production of arachidonic acid metabolites by human neutrophils and platelets. Neutrophils exposed to MSU generated leukotriene B (LTB), 6-trans-LTB4, 12-epi-6-trans-LTB4, and 5S, 12S DHETE from endogenous sources of arachidonate. In addition to these metabolites both monohydroxyeicosatetraenoic acids (i.e., 5-HETE) and omega-oxidation products (i.e., 2O -COOH LTB4) were formed by neutrophils exposed to MSU. Addition of exogenous arachidonic acid led to increased formation of each of these metabolites. When neutrophils were treated with colchicine (10 microM), LTB4 but not 5-HETE formation was impaired. (1-14C)Arachidonate-labeled platelets exposed to MSU released (1-14C)-arachidonate, (14C)-12 HETE, (14C)-HHT and (14C)-thromboxane B2. Results indicate that MSU stimulates arachidonic acid metabolism in both human neutrophils and platelets. Moreover, they suggest not only that metabolites of arachidonate may be considered as possible candidates for mediators of inflammation in crystal-associated diseases, but that colchicine blocks the formation of LTB4.     

Inhibition of leukotriene formation in human leukocytes by halothane

The effects of an inhalation anesthetic, halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) on the formation of 5-lipoxygenase metabolites such as leukotriene B4, 5(S)-hydroxyeicosatetraenoic acid (5-HETE), 6-trans-isomers of leukotriene B4 and leukotriene C4 were studied in human leukocytes stimulated with calcium ionophore A23187. Halothane inhibited the formation of all these metabolites dose dependently and the formation was restored by removal of the drug. The anesthetic also reversibly inhibited the release of [3H]arachidonic acid from neutrophils with a half-inhibition concentration of less than 0.19 mM. The formation of 5-lipoxygenase metabolites was not inhibited by the anesthetic when leukocytes were stimulated with the ionophore in the presence of exogenous arachidonic acid. These observations indicate that the inhibitory effect of halothane on the formation of 5-lipoxygenase metabolites in leukocytes is mainly due to the inhibition of arachidonic acid release.     

12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid is a more potent neutrophil chemoattractant than the 12(R) epimer in the rat cornea

12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid [12(R)-HETE] is reported to be more potent than its epimer 12(S)-HETE as a chemoattractant for human neutrophils in vitro and following topical application to the skin. To assess the in vivo neutrophil chemoattractant potencies of 12(S)-HETE and 12(R)-HETE in the rat, we injected 1 microgram, 5 micrograms, or 10 micrograms of these eicosanoids into the corneal stroma. Rats were killed 12-15 hours after injection, and the number of neutrophils in the stroma was counted in a histological section of the cornea including the injection site. The number of neutrophils was significantly increased in corneas injected with 5 micrograms (+103% of control) or 10 micrograms (+456% of control) of 12(S)-HETE and in those injected with 10 micrograms of 12(R)-HETE (+111% of control). The neutrophilic infiltrate in corneas injected with 1 microgram or 5 micrograms of 12(S)-HETE was not significantly different from that in corneas injected with 1 microgram of leukotriene B4. The data for the 10 micrograms injections indicate that 12(S)-HETE is a more potent neutrophil chemoattractant than 12(R)-HETE in the rat cornea. Our results suggest that species or tissue specificity may determine the relative potencies of 12-HETE epimers as chemoattractants for neutrophils, and that 12(S)-HETE may be an important inflammatory mediator in the rat cornea.     

Effect of various lipoxygenase metabolites of arachidonic acid on degranulation of polymorphonuclear leukocytes

Lipoxygenase metabolites of guinea pig peritoneal polymorphonuclear leukocytes stimulated with 10 microM A23187 plus arachidonic acid were isolated and identified. These metabolites were compared with each other and to chemically synthesized arachidonate metabolites for their ability to stimulate leukocyte degranulation. 5(S),12(R)-Dihydroxy-6,8,10-(cis/trans/trans)14-cis-eicosatetraenoic acid (leukotriene B4) produced a significant release of lysozyme, but not beta-glucuronidase or beta-N-acetylglucosaminidase at low concentrations (EC50 = 6.5 x 10(-9) M), while the leukocyte nonenzymatically generated 5,12-or 5,6-dihydroxyeicosatetraenoic acids had no effect at these concentrations. Higher concentrations (1--10 microM) of all the dihydroxyeicosatetraenoic acids, 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and its hydroperoxy precursor stimulated significant lysozyme release which was greater than that produced by 15-hydroxy-5,8,11-13-eicosatetraenoic acid, arachidonic acid, or its acetylene analogue, 5,8,11,14-eicosatetraynoic acid. Micromolar concentrations of leukotriene B4 and 5-HETE also stimulated significant release of beta-N-acetylglucosaminidase above controls, but not beta-glucuronidase. These results suggest that leukotriene B4 may play a role in regulating the release of certain granule-bound enzymes from polymorphonuclear leukocytes.     

Cellular regulatory role of leukotriene B4: its effects on cation homeostasis in rabbit neutrophils

We have found that exogenous leukotriene B4 modifies calcium homeostasis in rabbit neutrophils in a manner essentially analogous to that of the chemotactic peptide f-Met-Leu-Phe. Leukotriene B4 causes a rapid and dose-dependent increase in membrane permeability to calcium and a release of calcium from previously unexchangeable intracellular pool(s). The net result of these changes is to transiently elevate the intracellular level of exchangeable calcium. A stereoisomer of leukotriene B4 with greatly reduced secretory activity toward neutrophils (5S, 12S-di HETE) is essentially without effect on the rate of 45Ca uptake at concentrations equal to those that produce near maximal enhancement by leukotriene B4. Leukotriene B4, in addition to its effects on calcium metabolism, also increases the rate of 22Na influx into rabbit neutrophils. The relationships between the action of leukotriene B4 on calcium homeostasis and the neutrophil-directed activities of arachidonic acid and its lipoxygenase metabolites are discussed     

A novel leukotriene produced by stimulation of leukocytes with formylmethionylleucylphenylalanine

Stimulation of human polymorphonuclear leukocytes with the chemotactic peptide formylmethionylleucylphenylalanine led to the formation of a novel leukotriene: 5(S),12(R)-dihydroxy-6,8,10,14-eicosatetraen-1,20-dioic acid. This dihydroxydicarboxylic acid is derived from omega-oxidation of 5(S),12(R),dihydroxy-6,8,10,14-eicosatetradienoic acid (leukotriene B4). The intermediate 5(S),12(R),20-trihydroxy-6,8,10,14-eicosatetraenoic acid was also isolated from these incubations. The two metabolites of leukotriene B4 exhibit chemotactic properties for human polymorphonuclear leukocytes but are less active in this respect than the parent compound.     

12-Lipoxygenase from bovine polymorphonuclear leukocytes, an enzyme with leukotriene A4-synthase activity

Bovine polymorphonuclear leukocytes exhibit a 12-lipoxygenase activity upon sonication. In contrast to bovine platelet 12-lipoxygenase and other 12-lipoxygenases, this enzyme is unable to convert 5(S)-HETE (5(S)-hydroxy,6-trans-8,11,14-cis-eicosatetraenoic acid) or 5(S)-HPETE (5(S)-hydroperoxy,6-trans-8,11,14-cis-eicosatetraenoic acid) into 5(S),12(S)-dihydroxy-6,10-trans,8,14-cis-eicosatetraenoic acid. Surprisingly, the formation of leukotriene A4-derived products namely leukotriene B4 and the leukotriene B4-isomers 12-epi,6-trans- leukotriene B4 and 6-trans-leukotriene B4, was observed upon incubation of this enzyme with 5(S)-HPETE. Hence, the 12-lipoxygenase from bovine polymorphonuclear leukocytes possesses leukotriene A4-synthase activity.     

Characterization of the secretory activity of leukotriene B4 toward rabbit neutrophils

We have described in det ail the secretory activity of leukotriene B4 toward rabbit neutrophils. Leukotriene B4 rapidly and vigorously degranulates rabbit neutrophils. This activity is stereospecific, cytochalasin B-dependent, and is enhanced by extracellular calcium. Pretreatment with leukotriene B4 deactivates rabbit neutrophils, i.e., cells so treated do not respond to stimulation by an additional bolus of leukotriene B4. In addition, the secretory activity of leukotriene B4 is sharply dependent on the simultaneous presence of cytochalasin B. Rabbit neutrophils therefore exhibit the previously described desensitization to the effect of cytochalasin B. In these and other discussed respects the characteristics of the leukotriene B4-induced degranulation of rabbit neutrophils are strikingly similar to those of the chemotactic factors. These results support the hypothesis that leukotriene B4 mediates, at least in part, the secretory, and possibly other, activities of chemotactic factors.     

Human neutrophil chemotactic and degranulating activities of leukotriene B5 (LTB5) derived from eicosapentaenoic acid

Leukotriene B4 exhibited 10- to 30-fold greater chemotactic potency for human neutrophils than eicosapentaenoic acid-derived leukotriene B5, as assessed in modified Boyden micropore filter chambers. In contrast, leukotrienes B4 and B5 were equipotent stimuli of human neutrophil lysosomal degranulation in vitro, as quantified by the release of beta-glucosaminidase. Analyses of competitive inhibition of the binding of [3H] leukotriene B4 to neutrophils indicated that leukotriene B4 binds with a 500-fold greater association constant than leukotriene B5 to a subclass of high-affinity receptors, which appears to transduce chemotactic responses efficiently, while leukotrienes B4 and B5 bind equally well to low-affinity receptors.     

Metabolism of arachidonic acid in neutrophils from alloxan-diabetic rabbits

The production of 5-lipoxygenase products from arachidonic acid was investigated in polymorphonuclear leukocytes (PMNL) isolated from non-diabetic and alloxan-induced diabetic rabbits: (i) production of 5-hydroxyeicosatetraenoic acid, leukotriene B4, and the two 6-trans-leukotriene B4 isomers were significantly decreased in the PMNL of diabetic rabbits when compared to non-diabetic rabbits; (ii) production of LTB4 and 5-HETE from diabetic PMNL required the addition of Ca2+ and A23187 to a greater degree than control incubations; and (iii) the availability of substrate in the PMNL of diabetics was not a limiting factor for 5-lipoxygenase product formation. Alternative pathways of arachidonic acid metabolism were also evaluated: the recovery of exogenous leukotriene B4 and 5-hydroxyeicosatetraenoic acid were identical using PMNL from control and diabetic rabbits and peptido-leukotrienes were not detected by radioimmunoassay. The data suggest that the activity of 5-lipoxygenase and the production of 5-hydroperoxyeicosatetraenoic acid in the diabetic PMNL may be limiting factors since the formation of leukotriene B4, leukotriene B4 isomers, and 5-hydroxyeicosatetraenoic acid are depressed in PMNL of diabetic rabbits. Alternative pathways do not account for the conversion of arachidonic acid to other products nor are the elimination pathways for LTB4 and 5-HETE different. Decreased formation of 5-hydroxyeicosatetraenoic acid and leukotriene B4 could predispose diabetic subjects to infection due to a decrease in mediators leading to the local accumulation of PMNL in the inflammatory response.     

Direct demonstration of increased intracellular concentration of free calcium in rabbit and human neutrophils following stimulation by chemotactic factor

An increase in the level of intracellular free calcium concentration in rabbit and human neutrophils stimulated by chemotactic factors has been demonstrated directly using the calcium-sensitive fluorescent probe quin-2. Addition of f-Met-Leu-Phe (10(-9) M), C5a (3 x 10(-9) M) or leukotriene B4 (6 x 10(-8) M) to the neutrophils induces a rapid increase in the intracellular concentration of free calcium that reaches a maximum value 15 seconds following stimulation. At concentrations of f-Met-Leu-Phe less than 10(-8) M the enhancement is dose dependent with an ED50 of 8 x 10(-11) M and is significantly reduced in the presence of EGTA in the suspending medium.