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.     

Subhemolytic doses of Escherichia coli hemolysin evoke large quantities of lipoxygenase products in human neutrophils

Polymorphonuclear leukocytes (PMN) have been identified as preferred target cells for Escherichia coli hemolysin in human blood (Bhakdi, S., Greulich, S., Muhly, M., Ebersp?cher, B., Becker, H., Thiele, A., and Hugo, F. (1989) J. Exp. Med. 169, 737-754). Leukotriene and 5-hydroxyeicosatetraenoic acid generation was investigated in human PMN challenged with E. coli hemolysin in the absence or presence of free arachidonic acid or eicosapentaenoic acid (EPA). In the absence of exogenous free fatty acids, E. coli hemolysin (0.01-10 hemolytic units/ml) induced moderate generation of leukotriene B4 (LTB4) and its omega-oxidation products. The presence of free arachidonic acid (10 microM) during E. coli hemolysin (0.1 hemolytic unit/ml) challenge evoked the generation of large quantities of these products (greater than 100 pmol/1.5 x 10(7) PMN). In parallel, large amounts of 5-hydroxyeicosatetraenoic acid and nonenzymatic LTA4 hydrolysis products appeared. Product release peaked or plateaued 5-10 min after E. coli hemolysin challenge. The presence of exogenous EPA upon E. coli hemolysin challenge resulted in the exclusive generation of LTB5 and metabolites, LTA5 decay products and 5-hydroxyeicosapentaenoic acid. Dose and time dependences corresponded to those with arachidonic acid provision, and the total of EPA-derived products surpassed that of arachidonic acid metabolites in corresponding experiments approximately 2-fold. Increasing the time between free fatty acid provision and E. coli hemolysin challenge resulted in a rapid decline in the generation of arachidonic acid or EPA metabolites. Thus, subhemolytic doses of E. coli hemolysin evoke marked PMN eicosanoid generation that is dependent on exogenous free fatty acid supply, with total amounts approximating those found in calcium ionophore-stimulated neutrophils.     

Release of leukotrienes by human monocytes on stimulation of their phagocytic receptor for particulate activators

The respective capacities of adherent human monocytes to metabolize endogenous arachidonic acid into leukotrienes C4 (LTC4) and B4 (LTB4) in response to activation with an ionophore, A23187, or to phagocytosis of unopsonized zymosan particles and IgG-sensitized sheep erythrocytes ( EsIgG ) were compared under optimal conditions for each stimulus. Resolution of the cellfree supernatant, after ionophore activation, by reverse-phase high performance liquid chromatography (RP-HPLC) identified only two products which eluted at the retention times of LTC4 and LTB4. There was correspondence between their quantitation by integrated optical density and radioimmunoassay, and the recoveries from the initial supernatant were 80% by radioimmunoassay. Activation of adherent monocytes from 12 donors by ionophore and by zymosan particles released 68.1 ng and 10.0 ng LTB4 and 29.5 ng and 2.1 ng LTC4, respectively. With trypsin pretreatment, the monocytes responded fully to ionophore activation but were inhibited in their response to zymosan particles as assessed by phagocytosis and leukotriene release, indicating that the zymosan stimulus acted through a trypsin-sensitive membrane receptor. When the response of adherent monocytes from nine donors to zymosan particles and to EsIgG were compared at identical particle concentrations and with similar numbers of ingesting monocytes, zymosan elicited LTB4 release (mean 6.7 ng) from all and LTC4 (mean 1.5 ng) from eight donors, while EsIgG caused low level release of LTB4 (mean 0.7 ng) from six and LTC4 from only one of the donors. Neither zymosan nor ionophore stimulation led to the metabolism of exogenously added [3H]LTB4 or [3H]LTC4 as assessed by RP-HPLC of the cellfree supernatants and by quantitation of the eluted labeled products. Thus, transmembrane activation of adherent monocytes by their receptor for particulate activators, in contrast to stimulation of their IgG-Fc receptor, reproducibly releases substantial quantities of LTB4 and LTC4, and may represent an important mechanism for regulating the microenvironment in the nonimmune host.     

Calcium-dependent eicosanoid metabolism by concanavalin A-stimulated human monocytes in vitro. Synergism with phorbol ester indicates separate regulation of leukotriene B4 synthesis and release.

Human monocytes obtained by counter-current centrifugal elutriation released arachidonic acid when challenged in vitro with Con A, as well as with other soluble (PMA or ionomycin) or particulate stimuli (serum-treated zymosan). Cyclo-oxygenase metabolites were the principal eicosanoids detected in the supernatants of Con A-stimulated, [3H]arachidonate-labeled monocytes, 5-Lipoxygenase (5-LO) products, such as leukotriene B4 (LTB4), were conspicuously absent. Release of arachidonate and its metabolites in response to Con A was dependent on the presence of extracellular Ca2+, but not Mg2+. In contrast to serum-treated zymosan challenge, which resulted in increased inositol trisphosphate and LTB4 release, Con A-induced inositol phospholipid hydrolysis in monocytes was limited to phosphatidylinositol or phosphatidylinositol monophosphate. Despite an inability to augment LTB4 release, Con A or PMA induced a loss of 5-lipoxygenase from a cytosolic compartment that was similar to that achieved with a calcium ionophore (ionomycin), a potent stimulus for LTB4 generation. When cell-associated LTB4 was evaluated, evidence for increased LTB4 production was obtained in response to either stimulus (PMA greater than Con A). In combination, however, PMA and Con A treatment resulted in monocyte LTB4 release comparable with that observed with the calcium ionophore or STZ. LTB4 release in response to all stimuli tested was inhibited by MK-886, a drug that binds to 5-lipoxygenase-activating protein. These results indicate the following: 1) Phospholipase A2 activation and attendant arachidonic acid release induced by agents that increase intracellular Ca2+ and/or generate diacylglycerol results in increased synthesis and release of PG and increased synthesis of leukotrienes, but not necessarily leukotriene release. 2) 5-LO translocation, which may occur independently of increased intracellular Ca2+, may be necessary for LTB4 generation but is insufficient for its release. 3) 5-Lipoxygenase-activating protein activity is necessary for 5-LO activation and LTB4 release in response to all stimuli investigated here. 4) Phorbol ester, an activator of protein kinase C, may synergize with agents such as Con A (which by themselves induce a minimal intracellular Ca2+ rise), so as to result in the release of LTB4. Thus, Con A may represent a class of surface receptor-aggregating agents that initiates inflammatory changes or immunomodulation associated with liberation of PG and might predispose to release of other inflammatory mediators, such as leukotrienes, in the presence of additional signals including protein kinase activation.     

Leukotriene B4 triggers the in vitro and in vivo release of potent antimicrobial agents

Leukotriene B(4) (LTB(4)) is a bioactive lipid derived from the metabolism of arachidonic acid. Mainly produced by polymorphonuclear leukocytes (PMN) and macrophages, LTB(4) triggers several functional responses important in host defense, including the secretion of lysosomal enzymes, the activation of NADPH oxidase activity, NO formation, and phagocytosis. We report that LTB(4), but not structural analogs thereof, stimulates primed human PMN to release molecules having potent antimicrobial activities. Exposure of bacteria (Escherichia coli and Staphylococcus aureus) or viruses (herpes simplex virus type 1 and HIV type 1) to supernatants of LTB(4)-activated PMN led to > or =90% reduction in infectivity. ELISA and mass spectroscopy analysis of proteins released from LTB(4)-activated PMN have identified several antimicrobial proteins, including alpha-defensins, cathepsin G, elastase, lysozyme C, and LL-37, that are likely to participate in the killing of microorganisms. In addition to these in vitro observations, i.v. injections of LTB(4) (50 microg/kg) to monkeys led to an increase in alpha-defensin plasmatic levels and enhanced ex vivo antimicrobial activities of plasma. These results demonstrate the ability of LTB(4) to cause the release of potent antimicrobial agents from PMN in vitro as well as in vivo and add further support to the important role of LTB(4) in host defense.     

Enhanced 5-lipoxygenase activity in lung macrophages compared to monocytes from normal subjects

We compared lipoxygenase activities of lung macrophages obtained from bronchoalveolar lavage to activities of blood monocytes purified by using discontinuous plasma/Percoll density gradients and adherence to tissue culture plastic in five normal subjects. Cells were incubated with ionophore A23187 (10(-9) to 10(-5) M) or arachidonic acid (0.12 to 80 microM) for 1 to 60 min at 37 degrees C to construct dose-response and time-dependence curves of lipoxygenase product generation. Products were identified and were quantified by using high-pressure liquid chromatography and ultraviolet spectroscopy. Under all conditions of product generation, both macrophages and monocytes generated predominantly (5S,12R)-dihydroxy-(6Z, 8E, 10E, 14Z)-eicosatetraenoic acid (leukotriene B4 (LTB4] and (5S)-hydroxy-(6E, 8Z, 11Z, 14Z) - eicosatetraenoic acid (5 - HETE), but, in each subject, macrophages invariably released greater amounts of LTB4 and 5-HETE than monocytes. In response to A23187, macrophages released a maximum of 183 +/- 96 pmol of LTB4 and 168 +/- 108 pmol of 5-HETE per 10(6) cells (mean +/- SEM), whereas monocytes released only 16 +/- 1 and 18 +/- 8 pmol per 10(6) cells of LTB4 and 5-HETE, respectively. After adding arachidonic acid, macrophages released a maximum of 52 +/- 21 pmol of LTB4 and 223 +/- 66 pmol of 5-HETE, whereas monocytes released no detectable products. The results suggest that mononuclear phagocyte maturation in the lung may be accompanied by an enhanced ability to generate 5-lipoxygenase products.     

Release of leukotrienes C4 and B4 and prostaglandin E2 from human monocytes stimulated with aggregated IgG, IgA, and IgE

Purified human peripheral blood monocytes were stimulated with aggregated human myeloma proteins of different classes or the calcium ionophore A23187 and the release of leukotrienes C4 and B4 (LTC4, LTB4), and prostaglandin E2 (PGE2) into the supernatant was determined. The ionophore induced release of 10 +/- 5 ng LTC4/10(6) cells and 25 +/- 8 ng LTB4/10(6) cells. Aggregated IgG, IgA, and IgE, but not IgM or monomeric immunoglobulins (Ig), induced release of LTC4 and LTB4 that was approximately 10 to 20% of that induced by ionophore. In addition, IgG, IgA, and IgE, but not IgM, induced release of PGE2 (range 0.015 to 0.22 ng/10(6) cells). Aggregated Ig induced LTC4, LTB4, and PGE2 release in a dose-dependent manner; maximal leukotriene (LT) release was observed by 30 min, in contrast to PG release, which continued to increase up to 2.5 hr. Both ionophore- and Ig-induced LTC4 and LTB4 release were completely inhibited by removal of calcium from the media and by preincubation of cells with nordihydroguaiaretic acid. Indomethacin inhibited Ig-induced PGE2 release by 80%. Phagocytosis of the Ig aggregates was not required for LT or PGE2 release, since release was not inhibited by cytochalasin B. Release of LTC4, LTB4, and PGE2 induced by IgG, IgA, and IgE, but not IgM, correlated with the presence or absence of monocyte Fc receptors (FcR) as determined by rosette assays. The data suggest that IgG, IgA, and IgE immune complexes mostly likely induce monocyte arachidonic acid metabolism via cross-linking of FcR. The ability of monocytes to release eicosanoids in the absence of phagocytosis suggests that interaction of monocytes with immobilized immune complexes, such as those deposited in blood vessel walls or glomerular basement membranes, could initiate metabolism of arachidonic acid by monocytes. Such a mechanism could contribute to inflammatory reactions characterized by mononuclear cell infiltrates.     

Arachidonic acid and 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid modulate human polymorphonuclear neutrophil activation by monocyte derived neutrophil activating factor

Exposure of human polymorphonuclear neutrophils (PMN) to human monocyte derived neutrophil activating factor(s) (NAF) resulted in a concentration-dependent extracellular release of granule constituents. NAF also induced the generation of 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid [Leukotriene B4 (LTB4)] by PMNs which was enhanced in the presence of exogenous arachidonic acid (AA). In contrast to its enhancing effect on LTB4 production, AA inhibited NAF-stimulated PMN degranulation. 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE), a product of the 15-lipoxy-genation of AA in PMNS, caused a concentration-dependent suppression of degranulation and LTB4 generation by PMNs in contact with NAF. 15-HETE also inhibited the rise in cytosolic-free calcium [( Ca2+]i) observed in NAF activated PMNs. These data suggest that AA and a 15-lipoxygenase product modulate the NAF-associated activation pathway in human PMNs.     

Phospholipid and arachidonic acid metabolism in zymosan-stimulated human monocytes: modulation by cAMP

Receptor-ligand interaction in mononuclear phagocytes is intimately linked to alterations in membrane phospholipids and release of arachidonic acid (AA). In addition, synthesis of bioactive lipids from released AA can result in further modification of cell responses. Upon challenge with opsonized zymosan, [3H]-arachidonic acid ([3H]-AA)-labeled human monocytes released 25 +/- 2% of their incorporated radiolabel within 30 min. Pretreatment of the monocytes with 5 X 10(-4) M isobutylmethylxanthine (IBMX) or 1 X 10(-3) M dibutyryl cyclic AMP (d-cAMP) inhibited total [3H]-AA release in the presence of zymosan by 47% and 42%, respectively. Analysis of incorporated [3H]-AA in cellular phospholipid pools indicated that significant amounts of label were lost from both phosphatidylcholine (PC) and phosphatidylinositol (PI) during zymosan stimulation. Treatment with d-cAMP substantially inhibited the loss of label from PC, but had no affect on PI. HPLC analysis of cell supernatants from zymosan-treated cells indicated that 5-HETE was the predominant metabolite generated from [3H]-AA, and its production was depressed during treatment with d-cAMP. Phospholipase activity in human monocyte homogenates was not effected by d-cAMP or IBMX at the highest concentrations used, whether these were added directly to the homogenate or by pretreatment of whole cells, demonstrating that inhibition required an intact cell. These results suggest that human monocytes exposed to opsonized zymosan release AA via two mechanisms and that modulation by cAMP is indirectly effecting a phospholipase directed towards PC.     

Enhancement by monokines of leukotriene generation by human eosinophils and neutrophils stimulated with calcium ionophore A23187

Human blood eosinophils and neutrophils that had been incubated with the supernatants of cultures of lipopolysaccharide (LPS)-stimulated blood mononuclear cells demonstrated respective enhanced abilities to produce immunoreactive leukotriene C4 (LTC4) and immunoreactive leukotriene B4 (LTB4) after activation by the calcium ionophore A23187. Under optimal conditions, the enhancing effect was observed with the eosinophils (n = 21) and the neutrophils (n = 14) from all but one donor of each type of granulocyte. Enhancement was maximum when granulocytes were preincubated with a 1/3 dilution of LPS-stimulated mononuclear cell culture supernatants for 1 to 2.5 min and were then stimulated with 2.5 microM ionophore for 1 to 2 min (neutrophils) or 15 min (eosinophils). Maximal enhancement ranged from 20 to 4500% for LTC4 generation by eosinophils (geometric mean, 87%) and from 30 to 1600% for LTB4 generation by neutrophils (geometric mean, 105%). There was no enhancement of leukotriene biosynthesis when the LPS-stimulated mononuclear cell culture supernatants and ionophore were added simultaneously to the granulocytes. The enhancing activity for LTC4 generation by eosinophils was removed by washing the cells after the addition of the LPS-stimulated mononuclear cell culture supernatants and before the introduction of ionophore. This enhancing activity was produced by Ig-, Leu-1- adherent blood mononuclear cells, which are presumed to be monocytes; supernatants of adherent cells augmented A23187-induced LTC4 generation by eosinophils from 21 to 2300% (geometric mean, 402%) in 11 experiments and LTB4 generation by neutrophils from 7 to 200% (geometric mean, 60%) in 10 experiments. There was an inverse correlation between the percent enhancement and the LTC4 levels produced by stimulated eosinophils in the absence of the monokine(s) (r = -0.79, p less than 0.01), but not between percent enhancement and the LTB4 levels generated by ionophore-activated neutrophils in the control buffer. The activity of the monocyte-derived enhancing material on each type of granulocyte was relatively heat stable. Enhancement of eosinophil production of LTC4 was associated with an acidic group of monocyte-derived molecules having isoelectric points of 4.2 to 4.3, 4.5 to 4.6, and 4.9, and exhibiting marked heterogeneity in size.(ABSTRACT TRUNCATED AT 400 WORDS)     

Assessment of leukotriene B4 synthesis in human lymphocytes by using high performance liquid chromatography and radioimmunoassay methods

Leukotrienes (LT), mainly LTB4, have been shown recently to affect several functions of human lymphocytes in vitro, and they are regarded as putative modulators of the immune response. Although it is recognized that human neutrophils, eosinophils, monocyte-macrophages, and mast cells can generate LTs, the synthesis of 5-lipoxygenase products by lymphocytes is still the subject of a controversy. Human peripheral blood mononuclear leukocytes, nylon wool-purified lymphocytes, CD4+, CD4- T cells, large granular lymphocytes, and various fractions of pure lymphocyte preparations obtained by counter flow centrifugal elutriation were stimulated for 10 min to 24 hr with ionophore A23187, phytohemagglutinin, concanavalin A, or lipopolysaccharide with or without exogenous arachidonic acid (AA); supernatants were analyzed by reverse-phase high performance liquid chromatography (HPLC) coupled with radioimmunoassay (RIA) methods for the presence of LTB4. Pure human lymphocyte preparations, which were shown to be free of monocytes, did not release any detectable amount of LTB4. Increasing percentage of contaminating monocytes was clearly paralleled by increasing amounts of LTB4. Murine thymocytes, interleukin 2-dependent CTLL2 cytotoxic lymphocytes, EL4 thymoma cells, and human Jurkatt cells were also found to be unable to generate detectable amounts of LTB4 after stimulation with ionophore A23187, phytohemagglutinin, phorbol myristate acetate, recombinant interleukin 1, or interleukin 2 with or without exogenous AA. The addition of increasing numbers of adherence-purified monocytes to Jurkatt cells was followed by increased synthesis of LTB4. In conclusion, the present study indicates that the synthesis of LTB4 by pure human lymphocyte preparations or some human and animal lymphoid cell lines is not detectable by combined HPLC-RIA methods in any of the conditions used.     

Selective inhibition of leukotriene B4 biosynthesis in rat pulmonary alveolar macrophages by dietary selenium deficiency

Weanling male Fisher 344 rats were maintained on low selenium basal and Se-supplemented diets for 38 weeks. A several fold reduction in the glutathione peroxidase activity of the lung and liver tissues in rats maintained on low Se basal diet established their Se-deficient status. Analysis of the supernatants from resting pulmonary alveolar macrophage suspensions showed negligible extracellular release of PGE2, TXB2 and LTB4 in both diet groups. A challenge with opsonized zymosan particles increased the release of the same three arachidonic acid metabolites by several fold in both diet groups. The differences between the two diet groups with respect to the secretion of the products of the cyclooxygenase pathway, PGE2 and TXB2 were negligible. By contrast, a significant reduction in the extracellular release of LTB4 was observed in cells from animals on low selenium basal diet. These results suggest a selective inhibition of LTB4 biosynthesis in pulmonary alveolar macrophages by dietary deficiency of selenium.     

Differential augmentation by recombinant human tumor necrosis factor-alpha of neutrophil responses to particulate zymosan and glucan

Zymosan (Z) and its major insoluble carbohydrate component beta-linked glucan activate human neutrophils (PMN) through a trypsin-sensitive recognition mechanism. This mechanism is believed to involve the PMN CR3R. Both Z and glucan generated dose and time-dependent release of the secondary lysosomal granule marker vitamin B12 binding protein, leukotriene B4 (LTB4) and superoxide from PMN and were phagocytosed with similar dose-dependent kinetics. The PMN superoxide and LTB4 responses to glucan; however, were consistently greater than those to the same doses of Z. The phagocytosis of both particles was significantly reduced after partial digestion with beta-laminarinase but not beta-glucosidase or alpha-mannosidase suggesting a recognition mechanism dependent on intact beta-1,3-glucosidic bonds in both particles. TNF-alpha (rhTNF-alpha) promoted a time- and dose-dependent increase in the expression of PMN CR3 up to 60 min. The increased expression of CR3 was paralleled by the release of the secondary lysosomal granule marker vitamin B12-binding protein. This granule contains a population of CR3R in its boundary membrane and it is the fusion of this membrane with the plasma membrane that may represent the mechanism by which CR3 expression is increased. Preincubation of PMN with 10(-9)M rhTNF-alpha augmented phagocytosis, LTB4, and superoxide generation by PMN in response to activation by Z. In contrast, none of the responses to glucan was significantly increased after incubation with rhTNF-alpha. These differences suggest a lack of absolute homology between the recognition mechanisms for zymosan and glucan and that there is a component of the recognition mechanism for zymosan that is independent of that for glucan and is up-regulated after rhTNF-alpha pretreatment.     

Essential fatty acid deficiency inhibits the in vivo generation of leukotriene B4 and suppresses levels of resident and elicited leukocytes in acute inflammation

Essential fatty acid (EFA) deficiency exerts an anti-inflammatory effect in several models of inflammation. In an effort to understand underlying mechanisms, the effect of EFA deficiency on the generation of eicosanoids and the elicitation of leukocytes in a model of acute inflammation was examined. Acute inflammation was induced by the i.p. injection of zymosan in mice. The injection of zymosan in normal mice was followed by a short burst of eicosanoid synthesis lasting 2 hr. Leukotriene (LT)B4, LTC4, LTD4, and LTE4, thromboxane B2, and 6-keto-prostaglandin F1 alpha were detected using high pressure liquid chromatography and specific radioimmunoassays. This initial phase of eicosanoid production was followed by a more prolonged infiltration of leukocytes (predominantly polymorphonuclear neutrophils (PMN)) lasting 48 hr with little eicosanoid synthesis. When challenged with zymosan, EFA-deficient mice exhibited a marked decrease in the production of eicosanoids during the early phase. No LTB could be detected at all. The number of resident peritoneal macrophages in EFA-deficient mice was also substantially decreased, and the influx of PMN during the inflammatory response was markedly diminished. In order to establish that the generation of eicosanoids during the early phase of this model of acute inflammation played a causal role in the later infiltration of PMN, the effect of the mixed lipoxygenase/cyclooxygenase inhibitor, BW755C, on LTB formation and PMN influx in this model of inflammation was assessed in control animals. BW755C completely blocked LTB synthesis and inhibited the subsequent influx of PMN. In conclusion, EFA deficiency inhibits eicosanoid generation, depresses levels of resident macrophages, and markedly diminishes the influx of PMN in the acute inflammatory response. The decrease in PMN influx appears to result from the inhibition of the antecedent generation of LTB.     

Arachidonic acid metabolism among human mononuclear leukocytes. Lipoxygenase-related pathways

Human peripheral blood mononuclear cells were isolated and assessed for the presence of contaminating polymorphonuclear leukocytes and platelets. Incubations of these cell isolates were performed in the presence or absence of the calcium ionophore A23187 and/or 1-14C-labeled or unlabeled arachidonic acid. Using reverse phase high pressure liquid chromatography with simultaneous monitoring of ultraviolet light absorption at 229 and 280 nm and, where appropriate, of radioactivity, our studies reveal that human peripheral blood mononuclear cells generate leukotrienes C4 and B4 (LTC4 and LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) following stimulation with A23187. The ratio of LTC4 to LTB4 was approximately 10-fold greater among the mononuclear cells than among similar incubations of polymorphonuclear leukocytes. Furthermore, the mononuclear cells failed to metabolize LTB4 into the omega-hydroxy or omega-carboxy derivatives that were always present in, and very characteristic of incubations of polymorphonuclear leukocytes. Depletion of monocytes from the mononuclear cells by double adherence resulted in virtual loss of the generation of 5-lipoxygenase-derived products by the remaining nonadherent cells, supporting the conclusion that the monocytes and not the lymphocytes were the source of LTC4, LTB4, and 5-HETE. The presence of both 12-HETE and the cyclooxygenase-derived 12-hydroxyheptadecatrienoic acid correlated with the degree of platelet contamination, suggesting that the platelets account for the presence of these compounds.     

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)     

Omega-oxidation is the major pathway for the catabolism of leukotriene B4 in human polymorphonuclear leukocytes

Leukotriene B4 (LTB4), formed by the 5-lipoxygenase pathway in human polymorphonuclear leukocytes (PMN), may be an important mediator of inflammation. Recent studies suggest that human leukocytes can convert LTB4 to products that are less biologically active. To examine the catabolism of LTB4, we developed (using high performance liquid chromatography) a sensitive, reproducible assay for this mediator and its omega-oxidation products (20-OH- and 20-COOH-LTB4). With this assay, we have found that human PMN (but not human monocytes, lymphocytes, or platelets) convert exogenous LTB4 almost exclusively to 20-OH- and 20-COOH-LTB4 (identified by gas chromatography-mass spectrometry). Catabolism of exogenous LTB4 by omega-oxidation is rapid (t1/2 approximately 4 min at 37 degrees C in reaction mixtures containing 1.0 microM LTB4 and 20 X 10(6) PMN/ml), temperature-dependent (negligible at 0 degrees C), and varies with cell number as well as with initial substrate concentration. The pathway for omega-oxidation in PMN is specific for LTB4 and 5(S),12(S)-dihydroxy-6,8,10,14-eicosatetraenoic acid (only small amounts of other dihydroxylated-derivatives of arachidonic acid are converted to omega-oxidation products). Even PMN that are stimulated by phorbol myristate acetate to produce large amounts of superoxide anion radicals catabolize exogenous leukotriene B4 primarily by omega-oxidation. Finally, LTB4 that is generated when PMN are stimulated with the calcium ionophore, A23187, is rapidly catabolized by omega-oxidation. Thus, human PMN not only generate and respond to LTB4, but also rapidly and specifically catabolize this mediator by omega-oxidation.     

Neutrophils and mast cells. I. Human neutrophil-derived histamine-releasing activity

Histamine release occurs during the late phase allergic reaction concomitantly with neutrophil (PMN) infiltration. To determine whether PMN might release a factor capable of causing histamine release, supernatants generated by incubating human PMN in the presence or absence of specific activators were added to rat basophilic leukemia cells (RBL) and histamine release was measured. PMN supernatants from 17 of 21 donors induced noncytotoxic histamine release. Neutrophil-derived histamine-releasing activity, termed HRA-N, was dose-dependent and supernatants from greater than or equal to 10(7) PMN/ml caused 6 to 27% net histamine release from RBL. PMN supernatants induced histamine release as effectively as did intact PMN cocultured with RBL. The capacity of various donors to generate HRA-N was not related to atopic status or gender but was inversely related to the proportion of eosinophils (EOS) contaminating the PMN isolate (the larger the proportion of EOS, the lower the histamine release). Addition of EOS to PMN during the generation of HRA-N completely inhibited histamine-releasing activity. HRA-N was not released from mononuclear cells or platelets contaminating the PMN preparation. HRA-N release was not increased by the presence of either serum-treated zymosan or phorbol myristate acetate, agents that caused dose-related release of PMN granule enzymes. Indeed, HRA-N was released from unstimulated PMN in the complete absence of granule enzyme release. HRA-N release was detectable by 15 min and the majority of release occurred between 45 and 60 min of incubation. Thus, the data indicate that HRA-N is released spontaneously from human PMN and that HRA-N release is independent of primary or secondary PMN granule release. It is attractive to suggest that release of HRA-N by PMN might act to recruit mast cells or basophils into participating in acute inflammatory reactions.     

Noncytolytic terminal complement complexes may serve as calcium gates to elicit leukotriene B4 generation in human polymorphonuclear leukocytes

Complement effects on human polymorphonuclear leukocytes (PMN) have generally been ascribed to the anaphylatoxin C5a, which induces degranulation, superoxide anion generation, migration, and cell aggregation via interaction with membrane receptors. We here report that complement activation on the surface of antibody-sensitized human PMN provokes generation of the potent lipid mediator leukotriene B4 (LTB4) in strict dependence on complement component C8, but in the absence of detectable C9. The kinetics of LT generation are rapid, comparable with those observed after challenge with the calcium-ionophore A23187. LTB4 release is a distinct event that is dissociable from cytotoxicity as assessed by lactate dehydrogenase (LDH) release (dependent on C9) and from superoxide generation (independent of C8 and C9). It is dose dependent on extracellular calcium and is not observed in the absence of calcium. It is inhibited by substances interfering with calcium-calmodulin function (trifluoperazine and W7), but not by blockers of physiologic calcium channels (nimodipine, verapamil, and D 888). Addition of purified C8 to cells bearing C5b-7 induces a severalfold increase in their passive permeability to 45calcium. Sieving experiments with the use of marker molecules of different sizes collectively indicate the existence of small hydrophilic channels consisting exclusively or predominantly of C5b-8 complexes, which allow passive transmembrane flux of small molecules with Mr less than 200. Thus, noncytolytic terminal complement complexes may serve as a biological bypass gate for calcium in PMN membranes, triggering the arachidonic acid cascade with generation of LTB4 at doses well below the threshold required to invoke overt cell damage.     

Metabolism of exogenous arachidonic acid by mouse peritoneal macrophages

On incubation of resident mouse peritoneal macrophages with arachidonic acid several hydroxyacyl derivatives detectable in cellular supernatants are formed. As main products monohydroxyarachidonic acids (monoHETE's) were identified. In addition, smaller amounts of dihydroxyarachidonic acids (diHETE's) were formed. A detailed analysis of cell culture supernatants by reversed phase HPLC, normal phase HPLC in combination with UV-spectroscopy and combined gas-chromatography/masspectrometry revealed the presence of 5-, 8-, 12- and 15- monoHETE's, two distinct 5,12-diHETE's, several 8,15-diHETE's and 14,15-diHETE. Among the 5,12-diHETE's, only small amounts of a compound with the characteristics of LTB4 were detected. Under the conditions employed, the cyclooxygenase products PGE2 and PGI2 (as 6-keto-PGF1 alpha) were only minor metabolites. In contrast, when macrophage cultures were stimulated with the phagocytic stimulus zymosan, PGI2, PGE2 and LTC4 were found as the major conversion products of arachidonic acid, whereas mono- and diHETE's were not formed in detectable amounts.