Inhibition of leukotriene B4-induced neutrophil migration by lipoxin A4: structure-function relationships.

Lipoxins are trihydroxytetraene metabolites which are derived from arachidonic acid through an interaction between different lipoxygenase pathways. Previous work has shown that lipoxin A4 (LXA4) inhibits the chemotactic responsiveness of neutrophils (PMN) to leukotriene B4. We have now assessed the structural determinants of the lipoxin A4 molecule which are necessary for its inhibitory activity, using structural analogs of LXA4 prepared by chemical synthesis. Our results indicate the importance of two adjacent free hydroxyl groups in either the R or the S configuration; one hydroxyl group has to be in the C-6 position, but the other hydroxyl group can be in either the C-5 or the C-7 position for the conferment of inhibitory activity.     

Comparative effect of leukotriene B4 and leukotriene B5 on calcium mobilization in human neutrophils

Leukotriene B4 (LTB4) is reported to exert its biological activity in neutrophils through the increase in cytosolic free calcium that follows binding to its specific receptor. Leukotriene B5 has been shown to be far less active than LTB4. Therefore we compared the capacity of LTB4 and LTB5 to stimulate the rise in cytosolic free calcium using fura-2-loaded human neutrophils, to assess the relationship between the calcium mobilizing activity and biological potency of LTB4 and LTB5. At any concentration tested, LTB5 was less active than LTB4 in increasing cytosolic free calcium. ED50 for LTB4 and LTB5 were 5 X 10(-10) M and 5 X 10(-9) M, respectively. The difference in the binding affinities of LTB4 and LTB5 to the LTB4 receptor has been reported to explain the difference in their biological activities. In the present study we further demonstrated that the calcium mobilizing activity of LTB4 and LTB5 also correlates the different biological activity of the two compounds.     

Cytoplasmic pH change induced by leukotriene B4 in human neutrophils

Leukotriene B4 induced a biphasic change in the cytoplasmic pH of human neutrophils: an initial rapid acidification followed by an alkalinization. The acidification was slightly reduced by the removal of extracellular Ca2+, but the subsequent alkalinization was not. The leukotriene B4-induced alkalinization was dependent on extracellular Na+ and pH, and was inhibited by amiloride and its more potent analogue, 5-(N,N-hexamethylene)amiloride. These characteristics indicate that the cytoplasmic alkalinization is mediated by the Na+-H+ exchange. Oxidation products of leukotriene B4, 20-hydroxyleukotriene B4, 20-carboxyleukotriene B4, and (5S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) also stimulated the Na+-H+ exchange, but higher concentrations were required. Treatment of the cells with pertussis toxin inhibited both phases of the leukotriene B4-induced pHi change, while cholera toxin did not affect the pHi change. The alkalinization induced by leukotriene B4 was inhibited by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, but was not inhibited by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide which has a less inhibitory effect on protein kinase C. Acidification was not affected by the drugs. These findings suggest that a GTP-binding protein sensitive to pertussis toxin and protein kinase C are involved in the activation of the Na+-H+ exchange stimulated by leukotriene B4.     

A cell-free preparation of human neutrophils catalyzing NADPH-dependent conversion of leukotriene B4

The sonicate of human neutrophils converted leukotriene B4 to a polar product in aerobic condition in the presence of NADPH at a rate comparable to that of the intact cells. NADH could scarcely replace NADPH. The conversion was not observed in anaerobic conditions and was inhibited by carbon monoxide (CO/O2 = 4/1) or by 1 mM p-chlormercuribenzoate, while it was not affected by 1 mM KCN, 5 mM NaN3, 200 micrograms/ml catalase, 100 mM mannitol, and 10 micrograms/ml superoxide dismutase. These observations suggest that the myeloperoxidase-H2O2-halide system and active oxygen species are not involved in the reaction. The activity was observed in the 100,000xg supernatant from the homogenate, in which cytochrome P-450 was not detected.     

Perturbation of beta-glucan receptors on human neutrophils initiates phagocytosis and leukotriene B4 production

Normal human neutrophils were stimulated with the yeast cell wall product, zymosan, and examined for two biologic responses, ingestion of particles and production of leukotriene B4 (LTB4), under conditions that were comparable and optimal for the quantitation of each response. Monolayers of adherent neutrophils ingested unopsonized zymosan particles, at particle-to-cell ratios of 12.5:1 to 125:1, in a dose- and time-related manner. At a ratio of 125:1, the percentages of neutrophils ingesting greater than or equal to 1 and greater than or equal to 3 zymosan particles reached plateau levels of 55 +/- 6 and 32 +/- 9% (mean +/- SD, n = 8), respectively, within 30 min. At this same ratio, neutrophils during gravity sedimentation with zymosan particles synthesized LTB4 in a time-dependent manner for at least 45 min. The maximum amount of immunoreactive LTB4 released into supernatants was 3.8 +/- 1.2 ng per 10(6) neutrophils (mean +/- SD, n = 5) and the corresponding total immunoreactive LTB4 was 6.2 +/- 1.9 ng per 10(6) neutrophils. Treatment of 2 x 10(7) suspended neutrophils with 250 micrograms of trypsin for 20 min before concurrent assessment of neutrophil phagocytosis and LTB4 production reduced both of these responses by about 50%. Pretreatment of neutrophils with 800 micrograms/ml of soluble yeast beta-glucan inhibited their ingestion of zymosan by 84% (mean +/- SD, n = 3), with 50% inhibition occurring with 100 micrograms/ml of soluble beta-glucan; 800 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. Pretreatment of neutrophils with 400 micrograms/ml of soluble yeast beta-glucan inhibited neutrophil synthesis of LTB4 by 90%, with 50% occurring with 200 micrograms/ml; 400 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. The presence of 1.25 micrograms/ml of cytochalasin B during incubation with zymosan particles reduced neutrophil phagocytosis from 65 to 6%, and neutrophil synthesis of LTB4 from total levels of 6.0 +/- 0.3 ng/10(6) cells to zero (mean +/- SD, n = 3). Pretreatment with either cytochalasin B or vinblastine did not alter neutrophil generation of LTB4 induced by calcium ionophore. Neutrophils pretreated with vinblastine, at 4 x 10(-6) to 4 x 10(-4) M, and then maintained at one-half these concentrations during incubation with unopsonized zymosan particles exhibited no diminution in particle ingestion, but were markedly reduced in zymosan-induced synthesis of LTB4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Leukocyte inhibitory factor activates human neutrophils and macrophages to release leukotriene B4 and thromboxanes

Recent evidence has proved that cytokines can stimulate the production of 5-lipoxygenase products. Leukotriene B4 (LTB4) is a major mediator of leukocyte activation in acute inflammatory reactions, which produce chemotaxis, lysosomal enzyme release, and cell aggregation. Leukocyte inhibitory factor (LIF) also causes biological responses related to inflammation, i.e., LIF directly induces specific granule secretion by polymorphonuclears (PMNs) and potentiates many formyl-methionyl-leucyl-phenylalanine (FMLPs) mediated responses. Since arachidonic acid products are important mediators of inflammation, we have studied the effects of LIF on the arachidonic acid cascade products LTB4 and thromboxane A2 (TxA2). Resuspended at a final concentration of greater than 95% polymorphonuclear PMNs were isolated and tested with some cytokines on the release of LTB4 and TxA2. Peripheral blood mononuclear cells were isolated and seeded in Petri dishes and incubated for 60 min. Adherent macrophages were used for the cytokine stimulation study. Both types of leukocytes were treated with LIF, interleukin 6 (IL 6), and granulocyte-monocyte colony stimulating factor (GM-CSF) at different concentrations, and test agents A23187 and FMLP. Radioimmunoassay for LTB4 and TxB2 was determined by the resulting supernatants. Treatment of PMNs and macrophages with LIF at different concentrations proved to generate significant increases in LTB4 and TxA2 production. This was compared with IL 6 and GM-CSF, which had no effects. In these experiments, TxA2 generations could not be attributed to platelet contamination of PMN suspensions. The quantity of platelet contamination was not sufficient to influence how much TxB2 was produced. The similarities of LIF to other arachidonate stimulating cytokines suggest a similar mode of action in producing hematologic changes typical of tissue injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential inside-out activation of beta2-integrins by leukotriene B4 and fMLP in human neutrophils

We have investigated how LTB4, an endogenous chemoattractant encountered early in the inflammatory process, and fMLP, a bacteria-derived chemotactic peptide emanating from the site of infection, mediate inside-out regulation of the beta2-integrin. The role of the two chemoattractants on beta2-integrin avidity was investigated by measuring their effect on beta2-integrin clustering and surface mobility, whereas their effect on beta2-integrin affinity was measured by the expression of a high affinity epitope, a ligand-binding domain on beta2-integrins, and by integrin binding to s-ICAM. We find that the two chemoattractants modulate the beta2-integrin differently. LTB4 induces an increase in integrin clustering and surface mobility, but only a modest increase in integrin affinity. fMLP evokes a large increase in beta2-integrin affinity as well as in clustering and mobility. Lipoxin, which acts as a stop signal for the functions mediated by pro-inflammatory agents, was used as a tool for further examining the inside-out mechanisms. While LTB4-induced integrin clustering and mobility were inhibited by lipoxin, only a minor inhibition of fMLP-induced beta2-integrin avidity and no inhibition of integrin affinity were detected. The different modes of the inside-out regulation of beta2-integrins suggest that distinct mechanisms are involved in the beta2-integrin modulation induced by various chemoattractants.     

Chemotaxis of human neutrophils and eosinophils towards leukotriene B4 and its 20-w-oxidation products in vitro

Peripheral blood neutrophils and eosinophils from 70 patients and controls were studied for their in vitro chemotactic and chemokinetic responses towards synthetic leukotriene B4 (LTB4), 20-OH-LTB4 and 20-COOH-LTB4. All three factors induced chemotaxis and chemokinesis of cells. 20-OH-LTB4 was always less and 20-COOH-LTB4 even less active than the parent compound. Cells from patients with atopic eczema and T cell lymphoma moved less than cells from normal controls or from patients with psoriasis. In the presence of LTB4, 20-OH-LTB4 and buffer alone, more eosinophils than neutrophils moved to the lower side of the filter, while this did not occur with platelet activating factor as chemoattractant. Studies of neutrophil and eosinophil chemotaxis in the presence of LTB4 should therefore always take into account a high variability of the quantitative response which is donor and disease dependent.     

Selective localization of recognition complexes for leukotriene B4 and formyl-Met-Leu-Phe within lipid raft microdomains of human polymorphonuclear neutrophils

Neutrophilic polymorphonuclear leukocytes contain glycosphingolipid- and cholesterol-enriched lipid raft microdomains within the plasma membrane. Although there is evidence that lipid rafts function as signaling platforms for CXCR chemokine receptors, their role in recognition systems for other chemotaxins such as leukotriene B4 (LTB4) and fMLP is unknown. To address this question, human neutrophils were extracted with 1% Brij-58 and fractionated on sucrose gradients. B leukotriene receptor-1 (BLT-1), the primary LTB4 receptor, partitioned to low density fractions, co-isolating with the lipid raft marker, flotillin-1. By contrast, formyl peptide receptor (FPR), the primary fMLP receptor, partitioned to high density fractions, co-isolating with a non-raft marker, Cdc42. This pattern was preserved after the cells were stimulated with LTB4 or fMLP. Fluorescence resonance energy transfer (FRET) was performed to confirm the proximity of BLT-1 and FPR with these markers. FRET was detected between BLT1 and flotillin-1 but not Cdc42, whereas FRET was detected between FPR and Cdc42, but not flotillin-1. Pretreating neutrophils with methyl-beta-cyclodextrin, a lipid raft-disrupting agent, suppressed intracellular Ca(2+) mobilization and ERK1/2 phosphorylation in response to LTB4 but had no effect on either of these responses to fMLP. We conclude that BLT-1 is physically located within lipid raft microdomains of human neutrophils and that disrupting lipid raft integrity suppresses LTB4-induced activation. By contrast, FPR is not associated with lipid rafts, and fMLP-induced signaling does not require lipid raft integrity. These findings highlight the complexity of chemotaxin signaling pathways and offer one mechanism by which neutrophils may spatially organize chemotaxin signaling within the plasma membrane.     

Platelet activating factor and leukotriene B4 induce hyperpolarization of human endothelial cells but depolarization of neutrophils

We studied one expression of cell activation in neutrophils (PMN) and endothelial cells (EC), membrane potential changes [assessed by the fluorescent dye, di-C-O5(3)]. Human neutrophils responded with depolarization after exposure to fMLP, LTB4, A23187, PAF and PMA. In contrast, only PAF and LTB4 induced membrane potential changes in human umbilical vein EC, which responded with increased fluorescence, possibly indicating membrane hyperpolarization. These discordant responses may reflect processes of significance for interactions between EC and PMN.     

Colchicine inhibits ionophore-induced formation of leukotriene B4 by human neutrophils: the role of microtubules

Neutrophils which ingest particles (serum-treated zymosan, monosodium urate crystals) or are exposed to calcium ionophore A23187 generate leukotriene B4 (LTB4). Earlier work has shown that cells exposed to colchicine before exposure to monosodium urate crystals produce less LTB4; the formation of 5-HETE is unaffected. To determine whether inhibition by colchicine of LTB4 generation was stimulus-specific and was mediated by microtubule integrity, the effects of colchicine (10 microM, 60 min) on the release of lipoxygenase products from neutrophils exposed to ionophore A23187 (10 microM, 5 min) were examined. In the presence of exogenous arachidonic acid (100 microM, 15 min), colchicine decreased LTB4 to 48% +/- 11.7 of control and 5-HETE to 60.5% +/- 5.7 of control (mean +/- SEM); 15-HETE was also decreased to 61% +/- 10.3 of control. In the absence of exogenous arachidonate, LTB4 was decreased to 22.2% +/- 11.7 of control and 5-HETE to 13% +/- 4.8 of control. Lumicolchicine did not significantly affect formation of 5-HETE or LTB4. However, vinblastine sulfate (20 microM, 60 min), another microtubule-disruptive agent, decreased the formation of both 5-lipoxygenase products. The effects of colchicine and vinblastine were not due to impairment of cell viability because the release of cytoplasmic lactic dehydrogenase was unaffected. Ultrastructural analysis of centriolar microtubules showed that decrements in microtubule numbers of colchicine- and vinblastine-treated cells paralleled decrements in 5-lipoxygenase products. These pharmacologic manipulations suggested that functional microtubules might be required for optimal lipoxygenase activity. Consequently, we prepared neutrophil-derived cytoplasts, devoid of an intact microtubule system. No significant decreases in the 5- or 15-lipoxygenase products were found when cytoplasts were exposed to colchicine in the presence of exogenous arachidonate and A23187. The data show that colchicine inhibits the formation of lipoxygenase products from neutrophils stimulated with A23187, most likely via its effect on microtubules, the integrity of which appears necessary for full expression of 5- and 15-lipoxygenases.     

Leukotriene B3, leukotriene B4 and leukotriene B5; binding to leukotriene B4 receptors on rat and human leukocyte membranes

Specific high-affinity binding sites for [3H]-leukotriene B4 have been identified on membrane preparations from rat and human leukocytes. The rat and human leukocyte membrane preparations show linearity of binding with increasing protein concentration, saturable binding and rapid dissociation of binding by excess unlabelled leukotriene B4. Dissociation constants of 0.5 to 2.5 nM and maximum binding of 5000 fmoles/mg protein were obtained for [3H] leukotriene B4 binding to these preparations. Displacement of [3H]-leukotriene B4 by leukotriene B4 was compared with displacement by leukotriene B3 and leukotriene B5 which differ from leukotriene B4 only by the absence of a double bond at carbon 14 or the presence of an additional double bond at carbon 17, respectively. Leukotriene B3 was shown to be equipotent to leukotriene B4 in ability to displace [3H]-leukotriene B4 from both rat and human leukocyte membranes while leukotriene B5 was 20-50 fold less potent. The relative potencies for the displacement of [3H]-leukotriene B4 by leukotrienes B3, B4 and B5 on rat and human leukocyte membranes were shown to correlate well with their potencies for the induction of the aggregation of rat leukocytes and the chemokinesis of human leukocytes.     

Binding and metabolism of leukotriene B4 by neutrophils and their subcellular organelles

The subcellular distribution of leukotriene (LT)B4 binding and metabolizing sites was investigated in human neutrophils. Cells were disrupted by nitrogen cavitation and fractionated by Percoll density gradient centrifugation to yield cytoplasm, membranes, azurophilic granules, and specific granules. Only membrane fractions contained high affinity [3H]LTB4 binding sites. Binding of radiolabeled ligand to membranes was rapid, reversible, and saturable; it was blocked by a series of LTB4 analogues at concentrations corresponding to their respective potencies in 1) blocking [3H]LTB4 binding to whole cells and 2) stimulating neutrophil degranulation responses. In contrast, [3H]LTB4 was metabolized by fractions enriched with markers for cytoplasm plus endoplasmic reticulum. The metabolic activity was sedimented by ultracentrifugation, enhanced by NADPH, and inhibited at 4 degrees C. The cell-free system, like intact cells, metabolized [3H]LTB4 to omega-oxidized product rapidly and quantitatively at 37 degrees C but was inactive at 4 degrees C. Whole cells converted radiolabel to 20-hydroxy (approximately 30% of product) and 20-carboxy (approximately 70% of product) derivatives; the cell-free system formed principally 20-hydroxy-[3H]LTB4. These products were less bioactive than LTB4. Nevertheless, metabolism of LTB4 played little role in limiting the cells' response to the ligand: neutrophils completed degranulation and became desensitized to LTB4 within 3-5 min of exposure. Within this time frame, they oxidized less than 30% of the stimulus, and the extracellular fluid of these neutrophil suspensions was fully capable of activating fresh cells. We conclude that neutrophils transmit bioactions of LTB4 via a specific receptor integrally associated with their plasmalemma and/or endoplasmic reticulum. They inactivate the stimulus via a particulate omega-oxidase. At the level of the individual cell, receptor down-regulation, rather than ligand metabolism, appears to limit functional responses such as degranulation.     

Na+/H+ exchange modulates the production of leukotriene B4 by human neutrophils.

Human neutrophils produce various compounds of the 5-lipoxygenase pathway, including (5S)-hydroxyeicosatetraenoic acid, leukotriene B4, its 6-trans isomers and omega-oxidation metabolites of LTB4, when the cells are stimulated with the Ca2+ ionophore A23187. The elevation in the extracellular pH (pHo) facilitated the cytoplasmic alkalinization induced by the ionophore as determined fluorometrically using 2',7'-bis(carboxyethyl)carboxyfluorescein and enhanced the production of all the 5-lipoxygenase metabolites. The production decreased when the alkalinization was blocked by the decrease in the pHo, the removal of the extracellular Na+ or the addition of specific inhibitors of the Na+/H+ exchange, such as 5-(NN-hexamethylene)amiloride, 5-(N-methyl-N-isobutyl)amiloride and 5-(N-ethyl-N-isopropyl)amiloride. The alkalinization of the cytoplasm with methylamine completely restored the production suppressed by the removal of Na+ from the medium. These findings suggest that the change in the cytoplasmic pH (pHi) mediated by the Na+/H+ exchange regulates the production of the lipoxygenase metabolites. The site of the metabolism controlled by the pHi change seemed to be the 5-lipoxygenase, because the production of all the metabolites decreased in parallel and the release of [3H]arachidonic acid from the neutrophils in response to the ionophore was not affected by the pHi change. Furthermore, the production of the 5-lipoxygenase metabolites stimulated by A23187 with or without exogenous arachidonic acid showed a similar pHo-dependence and the production induced by N-formylmethionyl-leucylphenylalanine (chemotactic peptide) with exogenous arachidonic acid also decreased when the cytoplasmic alkalinization was inhibited.     

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 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.     

The effect of eicosapentaenoic acid on leukotriene B production by human neutrophils

Eicosapentaenoic acid, which is a major fatty acid in fish oil, previously has been shown to competitively inhibit the cyclooxygenase-catalyzed metabolism of arachidonic acid in platelets. In the present study the effect of eicosapentaenoic acid on the production of leukotriene B via the lipoxygenase pathway in human neutrophils was examined. Eicosapentaenoate was incorporated into complex lipids of neutrophils at the same rate as arachidonate; release of the two homologous fatty acids in response to calcium ionophore A23187 was equivalent and both fatty acids were metabolized to a leukotriene B. The products derived from eicosapentaenoic acid were identified as leukotriene B5 and its stereoisomers. Eicosapentaenoate was a less favorable substrate for leukotriene B5 synthesis (94 ng/10(7) cells/5 min at 20 microM exogenous fatty acid) than arachidonate was for leukotriene B4 (401 ng under the same conditions). However, eicosapentaenoate or an oxygenated product inhibited arachidonate metabolism since at equimolar concentrations of eicosapentaenoate and arachidonate leukotriene B4 production was decreased by 68%. The inhibitory effect occurred at the level of leukotriene A hydrolase. The biological activity of eicosapentaenoate -derived products was tested; leukotriene B5 was found to have only approximately 10% of the potency of leukotriene B4 in inducing the aggregation of neutrophils, and the stereoisomers of leukotriene B5 were inactive. These data suggest that diets enriched in eicosapentaenoic acid affect neutrophils by decreasing the quantity of leukotriene B and by the production of a less potent leukotriene.     

Effect of leukotriene B4, prostaglandin E2 and arachidonic acid on cytosolic-free calcium in human neutrophils

Changes in cytosolic free calcium [Ca2+]i and release of beta-glucuronidase in response to leukotriene B4 (LTB4) were measured in intact neutrophils loaded with the fluorescent Ca2+ indicator, quin 2. LTB4 (10(-10) M or higher) caused a rapid rise in [Ca2+]i due to influx from the extracellular medium and release from intracellular pools as well as enzyme release. PGE2 (3 microM) did not alter [Ca2+]i whereas arachidonic acid (10 microM) raised [Ca2+]i. Pretreatment of cells with the chemotactic peptide FMLP inhibited the subsequent rise of [Ca2+]i induced by LTB4. Since chemotactic peptides activate the lipoxygenase pathway of arachidonic acid metabolism, it may be speculated that endogenous LTB4 generation is involved in neutrophil activation.