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.     

Specific leukotriene formation by purified human eosinophils and neutrophils

Human granulocytes isolated from peripheral blood have been described to synthesize both LTB4 and LTC4 from arachidonic acid. We have observed that the amount of LTC4 produced by human granulocyte preparations is strongly dependent on the relative amount of eosinophils. To investigate a possibly significant difference in leukotriene synthesis of the eosinophilic and neutrophilic granulocytes, we developed a purification method to isolate both cell types from granulocytes obtained from the blood of healthy donors. Leukotrienes were generated by incubation of the purified cells with arachidonic acid, calcium ionophore A23187, calcium-chloride and reduced glutathione. Surprisingly, eosinophils were found to produce almost exclusively the spasmogenic LTC4. In contrast, neutrophils produce almost exclusively the chemotactic LTB4, its omega-hydroxylated metabolite 20-hydroxy-LTB4 and two non-enzymically formed LTB4 isomers.     

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.     

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.     

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.     

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.     

The endocannabinoid 2-arachidonoyl-glycerol activates human neutrophils: critical role of its hydrolysis and de novo leukotriene B4 biosynthesis

Although endocannabinoids are important players in nociception and obesity, their roles as immunomodulators remain elusive. The main endocannabinoids described to date, namely 2-arachidonoyl-glycerol (2-AG) and arachidonyl-ethanolamide (AEA), induce an intriguing profile of pro- and anti-inflammatory effects. This could relate to cell-specific cannabinoid receptor expression and/or the action of endocannabinoid-derived metabolites. Importantly, 2-AG and AEA comprise a molecule of arachidonic acid (AA) in their structure and are hydrolyzed rapidly. We postulated the following: 1) the released AA from endocannabinoid hydrolysis would be metabolized into eicosanoids; and 2) these eicosanoids would mediate some of the effects of endocannabinoids. To confirm these hypotheses, experiments were performed in which freshly isolated human neutrophils were treated with endocannabinoids. Unlike AEA, 2-AG stimulated myeloperoxidase release, kinase activation, and calcium mobilization by neutrophils. Although 2-AG did not induce the migration of neutrophils, it induced the release of a migrating activity for neutrophils. 2-AG also rapidly (1 min) induced a robust biosynthesis of leukotrienes, similar to that observed with AA. The effects of 2-AG were not mimicked nor prevented by cannabinoid receptor agonists or antagonists, respectively. Finally, the blockade of either 2-AG hydrolysis, leukotriene (LT) B(4) biosynthesis, or LTB(4) receptor 1 activation prevented all the effects of 2-AG on neutrophil functions. In conclusion, we demonstrated that 2-AG potently activates human neutrophils. This is the consequence of 2-AG hydrolysis, de novo LTB(4) biosynthesis, and an autocrine activation loop involving LTB(4) receptor 1.     

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     

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.     

Stimulation of human myelopoiesis by leukotriene B4

Cultivation of human mononuclear bone marrow cells for 10 days in the presence of leukotriene B4 (8 X 10(-8) - 3 X 10(-6)M) led to an increase in the formation of granulocyte-macrophage colonies. The increase varied between 19 and 122% when compared to control cells. 5S, 12S-Dihydroxy-6, 8, 10, 14-eicosatetraenoic acid (5S, 12S-DHETE), an isomer of leukotriene B4, did not stimulate colony formation. Preincubation of the cells with 5S, 12S-DHETE inhibited the stimulatory action of leukotriene B4 on the proliferation of bone marrow cells. The present study indicates that leukotriene B4 amplifies the stimulation caused by the colony stimulating factor(s) and may play a role in modulating granulocyte and macrophage poiesis by a positive feedback mechanism.     

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.     

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)     

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.     

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.     

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)     

The leukotriene B4 paradox: neutrophils can, but will not, respond to ligand-receptor interactions by forming leukotriene B4 or its omega-metabolites.

Leukotriene B4 (5S,12R-dihydroxy-6,14-cis,8,10-trans-eicosatetraenoic acid, LTB4) is released from neutrophils exposed to calcium ionophores. To determine whether LTB4 might be produced by ligand-receptor interactions at the plasmalemma, we treated human neutrophils with serum-treated zymosan (STZ), heat-aggregated IgG and fMet-Leu-Phe (fMLP), agonists at the C3b, Fc and fMLP receptors respectively. STZ (10 mg/ml) provoked the formation of barely detectable amounts of LTB4 (0.74 ng/10(7) cells); no omega-oxidized metabolites of LTB4 were found. Adding 10 microM-arachidonate did not significantly increase production of LTB4 or its metabolites. Addition of 50 microM-arachidonate (an amount which activates protein kinase C) before STZ caused a 40-fold increase in the quantity of LTB4 and its omega-oxidation products. Neither phorbol myristate acetate (PMA, 200 ng/ml) nor linoleic acid (50 microM), also activators of protein kinase C, augmented generation of LTB4 by cells stimulated with STZ. Neither fMLP (10(-6) M) nor aggregated IgG (0.3 mg/ml) induced LTB4 formation (less than 0.01 ng/10(7) cells). Moreover, cells exposed to STZ, fMLP, or IgG did not form all-trans-LTB4 or 5-hydroxyeicosatetraenoic acid; their failure to make LTB4 was therefore due to inactivity of neutrophil 5-lipoxygenase. However, adding 50 microM-arachidonate to neutrophil suspensions before fMLP or IgG triggered LTB4 production, the majority of which was metabolized to its omega-oxidized products (fMLP, 20.2 ng/10(7) cells; IgG, 17.1 ng/10(7) cells). The data show that neutrophils exposed to agonists at defined cell-surface receptors produce significant quantities of LTB4 only when treated with non-physiological concentrations of arachidonate.     

The conversion of leukotriene C4 to isomers of leukotriene B4 by human eosinophil peroxidase

The smooth muscle contractile and vasoactive mediator leukotriene C₄ (5(S)-hydroxy-6(R)-sulfido-glutathionyl-eicosatetraenoic acid; LTC₄) is converted by phorbol ester-stimulated human eosinophils to two isomers of leukotriene B₄, 5(S),12(R)-6,8,10 trans-14 cis-eicosatetraenoic acid (5(S),12(R)-“all-trans”-LTB₄) and 5(S),12(S)-“all-trans”-LTB₄, which are leukocyte chemotactic factors lacking the humoral functions of LTC₄. Optimal conversion of LTC₄ to the “all-trans” isomers of LTB₄ by intact eosinophils and soluble eosinophil peroxidase requires both H₂O₂ and halide ions. Oxidative metabolism of leukotrienes may represent an important regulatory function of eosinophils in hypersensitivity reactions.     

Intracellular retention of the 5-lipoxygenase pathway product, leukotriene B4, by human neutrophils activated with unopsonized zymosan

The cellular and extracellular distribution of leukotriene B4 (LTB4) generated in human neutrophilic polymorphonuclear leukocytes (PMN) stimulated with unopsonized zymosan has been compared with that generated in PMN activated by the calcium ionophore. The amounts of extracellular and intracellular LTB4 were quantitated by radioimmunoassay. The authenticity of the immunoreactive LTB4 was confirmed by the elution of a single immunoreactive peak after reverse phase-high performance liquid chromatography (RP-HPLC) at the retention time of synthetic LTB4, by the identical elution time of a peak of radiolabeled product derived from [3H]arachidonic acid-labeled PMN with the immunoreactive product, and by the comparable chemotactic activity on a weight basis of immunoreactive LTB4 and synthetic LTB4 standard. Under optimal conditions of stimulation by unopsonized zymosan, more than 78% of the generated immunoreactive LTB4 remained intracellular, whereas with optimal activation by the ionophore, less than 8.6% of immunoreactive LTB4 was retained. Resolution by RP-HPLC of the products from the supernatants and cell extracts of [3H]arachidonic acid-labeled PMN stimulated with unopsonized zymosan and those stimulated with calcium ionophore allowed identification and measurement of 5-hydroxyeicosatetraenoic acid (5-HETE), 6-trans-LTB4, LTB4, and omega oxidation products of LTB4 by radioactivity. With zymosan stimulation of PMN, 5-HETE and the 6-trans-LTB4 diastereoisomers were not released, LTB4 was partially released, and the omega oxidation products of LTB4 were preferentially extracellular in distribution. In contrast, with ionophore stimulation, only 5-HETE had any duration of intracellular residence being equally distributed intra- and extracellularly throughout the 30-min period of observation; 6-trans-LTB4, LTB4, and the omega oxidation products of LTB4 were retained at less than 19%. The respective distributions of 5-HETE after zymosan and ionophore stimulation were not altered by the introduction of albumin to the reaction mixtures to prevent reacylation, or by hydrolysis of the cell extract to uncover any product that had been reacylated. The finding that stimulation of PMN with unopsonized zymosan results in the cellular retention of 5-lipoxygenase products suggests that release of these metabolites may be an event that is regulated separately from their generation.