Platelet-activating factor induces leukotriene C4 synthesis by purified human eosinophils

Platelet-activating factor, at a concentration of 10 microM, was capable of inducing leukotriene C4 synthesis by eosinophils of healthy donors, i.e. (3.1 +/- 0.3) x 10(6) molecules leukotriene C4/cell (n = 31, mean +/- SEM, cell purity 87 +/- 2%). Reversed-phase high performance liquid chromatography analysis demonstrated the exclusive synthesis of leukotriene C4. At a concentration of 1 microM, platelet-activating factor was capable of significantly enhancing the calcium ionophore A23187, the opsonized zymosan or the arachidonic acid induced leukotriene C4 synthesis by eosinophils. These results show that PAF is capable of inducing and enhancing the leukotriene C4 formation by human eosinophils.     

Effects of immune complexes, zymosan preparations and ionophore A 23187 on leukotriene formation in human leukocytes

Incubation of human leukocytes with opsonized zymosan or IgG immune complexes led to a time dependent release of leukotrienes (LT) B4 and C4. After 3-4 min, the levels of LTB4 were 93 and 35 pmol/3*10(7) cells, respectively [corrected]. These amounts were 2-4 times lower than those released by leukocytes stimulated with the calcium ionophore A 23187. The levels of LTC4 were 8 and 20 times lower than those of LTB4 after incubation with opsonized zymosan or immune complexes, respectively. Heat-inactivation of the serum prior to zymosan coating decreased the effect of opsonized zymosan. Uncoated zymosan was an even weaker stimulus of leukotriene formation. These results suggest that both complement factors and immunoglobulins play a pivotal role in activating leukotriene synthesis in a mixed suspension of human leukocytes.     

Leukotriene C4 formation by enriched human basophil preparations from normal and asthmatic subjects.

Numbers of circulating basophils are increased in asthmatic subjects, compared to normal subjects. Basophil enriched cell preparations from normal and asthmatic subjects were challenged in vitro with the calcium ionophore A23187, anti-IgE, or opsonized zymosan to study leukotriene C4 formation, histamine release, and prostaglandin D2 formation. No prostaglandin D2 formation by basophils was observed. Furthermore, opsonized zymosan was not capable of inducing any mediator formation or release from basophils. At optimal stimulation conditions no differences were found between basophils from normal and asthmatic subjects concerning A23187 or anti-IgE induced leukotriene C4 formation or histamine release. A23187 and anti-IgE induced leukotriene C4 formation were in the range of 1-20 and 0.6-4.8 pmol/10(6) basophils respectively.     

Synthesis and release of leukotriene C4 by human eosinophils

When human peripheral blood eosinophils isolated to 92.5% +/- 6.9 purity were stimulated with either the calcium ionophore A23187 or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), immunoreactive leukotriene C4 (LTC4) was initially localized intracellularly and was subsequently released to the external medium in kinetically distinguishable steps. Eosinophils were stimulated with 2.5 microM A23187 in the presence of 20 mM L-serine, a hypochlorous acid scavenger that prevents the oxidative metabolism of sulfidopeptide leukotrienes. Total production of immunoreactive LTC4, the sum of intra- and extracellular LTC4, was complete within 5 to 10 min. At 5, 10, and 30 min, 65.9% +/- 15.2, 42.3% +/- 24.3, and 5.5% +/- 3.9, respectively, of the total amount of LTC4 measured remained intracellular as detected after the media and cells were separated and the latter was extracted with methanol. The time course for the intracellular synthesis and extracellular release of immunoreactive LTC4 from eosinophils pretreated with 5 micrograms/ml cytochalasin B and stimulated with 0.5 microM FMLP was like that obtained with ionophore, although the total LTC4 production was only approximately 10%. The identity of the intracellular LTC4 was confirmed by elution with reverse-phase high pressure liquid chromatography followed by scanning UV spectroscopy, radioimmunoassay, and bioassay. Eosinophils that were stimulated with A23187 in the absence of L-serine metabolized newly synthesized LTC4 to 6-trans-LTB4 diastereoisomers and subclass-specific diastereoisomeric sulfoxides that were identified only in the extracellular medium. Thus the response of purified eosinophils to two different stimuli demonstrates a transient intracellular accumulation of biologically active LTC4, the distinct extracellular release, and the apparent limitation of oxidative metabolism to the extracellular location.     

Leukotriene C4 production from human eosinophils in vitro. Role of eosinophil chemotactic factors on eosinophil activation

We studied the role of naturally occurring eosinophil chemotactic factors on leukotriene (LT)C4 production from highly purified (87.1 +/- 2.4%) normodense eosinophils. Platelet activating factor (PAF) directly induced LTC4 production from eosinophils in a dose (10(-9) to 10(-5) M) and a time-dependent manner. PAF (10(-5) M) induced 0.74 +/- 0.08 ng of LTC4 production/10(6) eosinophils. However, lyso-PAF, eosinophil chemotactic factor of anaphylaxis, and LTB4 failed to induce LTC4 production within the tested range. Furthermore, the pre-incubation of eosinophils with 5 micrograms/ml of cytochalasin B did not alter the chemotactic factor-induced LTC4 production. When eosinophils were stimulated by the submaximal concentration (1 microgram/ml) of calcium ionophore A23187, the pre-incubation of eosinophils with 10(-6) M or 10(-5) M of PAF, or 10(-5) M of eosinophil chemotactic factor of anaphylaxis significantly enhanced LTC4 production up to 163.9 +/- 17.5% (p less than 0.05), 279.2 +/- 32.9% (p less than 0.01) and 165.2 +/- 21.2% (p less than 0.05) of the control, respectively. However, the pre-incubation with lyso-PAF or LTB4 failed to enhance A23187-induced LTC4 production. The pre-incubation of eosinophils with phosphatidyl serine also failed to enhance A23187-induced LTC4 production. However, the direct stimulation of protein kinase C by PMA enhanced the submaximal concentration of A23187-induced LTC4 production from eosinophils up to 179.5 +/- 20.9% (p less than 0.05) of the control. Our findings indicate that PAF and ECF-A work not only as chemotactic factors but also induce a functionally active state of eosinophils probably through their post-receptor mechanisms, and contribute to the inflammatory processes.     

Evaluation of inhibitors of eicosanoid synthesis in leukocytes: possible pitfall of using the calcium ionophore A23187 to stimulate 5' lipoxygenase

The effect on arachidonate metabolism of two compounds (BW755C and benoxaprofen) which have been reported to inhibit 5' lipoxygenase in leukocytes has been evaluated in human polymorphonuclear leukocytes (PMN) stimulated with the calcium ionophore A23187 and serum-treated zymosan (STZ). The syntheses of leukotriene B4 (LTB4) and thromboxane B2 (TXB2) from endogenous substrate were determined by specific radioimmunoassays as indicators of 5' lipoxygenase and cyclo-oxygenase activity in the PMN respectively. Benoxaprofen inhibited the synthesis of leukotriene B4 by human PMN stimulated with the calcium ionophore A23187, but it was approximately 5 times less potent than BW755C. However, benoxaprofen (IC50 1.6 X 10(-4)M) was approximately 100 times less potent than BW755C (IC50 1.7 X 10(-6)M) at inhibiting leukotriene B4 synthesis induced by serum-treated zymosan. Both drugs inhibited thromboxane synthesis by leukocytes stimulated with A23187 or serum-treated zymosan at similar concentrations (approximately 5 X 10(-6)M). The data obtained using STZ as stimulus are consistent with previous in vivo studies and indicate that benoxaprofen is a relatively selective inhibitor of cyclo-oxygenase. However, this selectivity was far less apparent when A23187 was used as a stimulus to release the eicosanoids which suggests that this inhibition could be via an indirect mechanism and therefore A23187 should be used with caution as a stimulus of 5' lipoxygenase for evaluating inhibitors of eicosanoid synthesis.     

Interleukin-5 potentiates sulfidopeptide leukotriene production by human eosinophils

Interleukin-5 (IL-5) has been shown to be a selective eosinophil growth and differentiation factor. In the present study, the effect of recombinant human IL-5 on human eosinophil sulfidopeptide leukotriene production was investigated. IL-5 did not affect leukotriene synthesis in unstimulated eosinophils. However, IL-5 potentiated leukotriene synthesis by eosinophils stimulated with serum treated zymosan (STZ) or the calcium ionophore A23187 by 69% and 135%, respectively. The priming effect of IL-5 was dose dependent, with significant stimulation occurring at 1 000 U/ml for STZ and 100-1 000 U/ml for A23187. Pre-incubation with IL-5 did not increase leukotriene synthesis further.     

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)     

Human peritoneal macrophages synthesize leukotrienes B4 and C4

Macrophages were isolated from the dialysis fluid of patients undergoing continuous ambulatory peritoneal dialysis and separated by gradient centrifugation and purification on 50% Percoll. The cells were prelabeled with [14C]arachidonic acid for 1.5 h. The labeled cells were then incubated with calcium ionophore A23187 (1 microM), serum-treated zymosan (200 micrograms/ml), and a lipoxygenase inhibitor, nordihydroguairetic acid (1 X 10(-5) M). The arachidonate metabolites in the medium were separated on Sep-Pak columns, and finally purified by reverse-phase high-pressure liquid chromatography (HPLC). The labeled products co-chromatographed with authentic leukotriene B4 and leukotriene C4 standards. Serum-treated zymosan and A23187 significantly stimulated and nordihydroguairetic acid significantly inhibited leukotriene synthesis. Leukotriene D4 was not detected, which suggests that these cells contain low gamma-glutamyltranspeptidase or high dipeptidase activity. These results establish, for the first time, that human peritoneal macrophages synthesize the lipoxygenase products, leukotriene B4 and leukotriene C4.     

Generation of leukotrienes by human monocytes pretreated with cytochalasin B and stimulated with formyl-methionyl-leucyl-phenylalanine

The N-formylated tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) initiated the generation of immunoreactive C-6 sulfidopeptide leukotrienes and of leukotriene B4 (LTB4) in a dose-dependent manner from monolayers of human monocytes pretreated for 10 min with 5 micrograms/ml of cytochalasin B. The EC50 for the immunoreactive C-6 sulfidopeptide leukotrienes was 10(-8) M FMLP and for immunoreactive LTB4 was 5 X 10(-8) M FMLP. The maximal response to FMLP occurred within 10 min, and the sum of the two classes of leukotrienes generated was about 1/6 that obtained from monocytes stimulated with calcium ionophore A23187. The requirement for cytochalasin B in order for FMLP, but not the calcium ionophore, to stimulate leukotriene generation is compatible with the ability of cytochalasin B to augment in other cells certain stimulus-specific transmembrane responses that are not dependent on the integrity of the cytoskeleton. Resolution by reverse phase high performance liquid chromatography of the products released from monocytes pretreated with cytochalasin B and stimulated with FMLP or calcium ionophore yielded a single peak of immunoreactive LTB4 eluting at the same retention time as the synthetic standard; immunoreactive C-6 sulfidopeptide leukotrienes eluted at the retention times of leukotriene C4 (LTC4) and leukotriene D4 (LTD4). [3H]LTB4 was not metabolically altered by monocytes pretreated with cytochalasin B and activated with FMLP in comparison with cells treated with buffer alone, whereas [3H]LTC4 was partially converted to [3H]LTD4. The leukotriene-generating response of monolayers of human monocytes pretreated with cytochalasin B to FMLP is receptor-mediated, as indicated by the inactivity of the structural analog N-acetyl-methionyl-leucyl-phenylalanine and by the capacity of the FMLP receptor antagonist carbobenzoxyphenylalanyl-methionine to inhibit the agonist action of FMLP in a dose-response fashion.     

Enhancement of human eosinophil cytotoxicity and leukotriene synthesis by biosynthetic (recombinant) granulocyte-macrophage colony-stimulating factor

Culture medium conditioned by activated human T lymphocytes enhances the in vitro cytotoxicity of purified human eosinophils toward Schistosoma mansoni larvae, suggesting the existence of a mechanism for T lymphocyte regulation of eosinophil function. Here we show that purified biosynthetic (recombinant) human T lymphocyte granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced markedly two eosinophil functions: cytotoxicity toward schistosomula by a mean of 676%, and calcium ionophore A23187-induced generation of leukotriene C4 (LTC4) by a mean of 135%. Augmentation of each eosinophil function by GM-CSF was time- and dose-dependent, with a dose-response relationship at concentrations between 1 and 20 pM. Tumor necrosis factor (TNF) enhanced eosinophil cytotoxicity with slower kinetics, a different dose-dependence relationship, and to a lower maximum, as compared with GM-CSF. There was no detectable effect of TNF on calcium ionophore A23187-induced generation of LTC4. The effect of GM-CSF on arachidonic acid metabolism to LTC4 reached a plateau with 60 min of incubation before stimulation with ionophore, and was characterized by an initial augmentation of the intracellular level of LTC4 and a subsequent increment in extracellular LTC4. Thus, GM-CSF can serve as a mediator for T lymphocyte regulation of functions of mature eosinophils. It is also the first defined macromolecule known to enhance metabolism of membrane-derived arachidonic acid via the 5-lipoxygenase pathway.     

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.     

Selective inhibition of leukotriene C4 synthesis in human neutrophils by ethacrynic acid

Addition of glutathione S-transferase inhibitors, ethyacrynic acid (ET), caffeic acid (CA), and ferulic acid (FA) to human neutrophils led to inhibition of leukotriene C4 (LTC4) synthesis induced by calcium ionophore A23187. ET is the most specific of these inhibitors for it had little effect on LTB4, PGE2 and 5-HETE synthesis. The inhibition of LTC4 was irreversible and time dependent. ET also had little effect on 3H-AA release from A23187-stimulated neutrophils.     

Modulation of alveolar macrophage-derived 5-lipoxygenase products by the sulfhydryl reactant, N-ethylmaleimide

The sulfhydryl reactant N-ethylmaleimide (NEM) stimulates the release and cyclooxygenase metabolism of arachidonic acid in rat alveolar macrophages. Because both 5-lipoxygenation and leukotriene (LT) C4 synthesis represent sulfhydryl-dependent steps in the 5-lipoxygenase pathway, we examined the effect of NEM on 5-lipoxygenase, as well as cyclooxygenase, metabolism in resting and agonist-stimulated cells by reverse-phase high performance liquid chromatography and radioimmunoassay. NEM at 5-10 microM stimulated the synthesis of thromboxane, but not prostaglandin E2 or the 5-lipoxygenase products LTC4, LTB4, or 5-hydroxyeicosatetraenoic acid from endogenously released arachidonate. In the presence of exogenous fatty acid, however, NEM stimulated the synthesis of large quantities of LTB4. The effect of NEM on arachidonate metabolism stimulated by the calcium ionophore A23187 and the particulate zymosan was also investigated. NEM augmented arachidonate release and thromboxane synthesis stimulated by A23187 but inhibited A23187-induced LTC4 synthesis with an IC50 of approximately 4.3 microM. This inhibitory effect closely paralleled the ability of NEM to deplete intracellular glutathione (IC50 approximately 4.3 microM). Preincubation with the intracellular cysteine delivery agent L-2-oxothiazolidine-4-carboxylate augmented intracellular glutathione concentration and A23187-stimulated LTC4 synthesis and attenuated the capacity of NEM to deplete glutathione and inhibit LTC4 synthesis. While LTB4 and 5-hydroxyeicosatetraenoic synthesis were unaffected at these low NEM concentrations, LTB4 synthesis was inhibited at high concentrations (IC50 approximately 210 microM). Zymosan-induced eicosanoid synthesis was modulated by NEM in a similar fashion. Thus, NEM is an agonist of arachidonate metabolism with the capacity to modulate the spectrum of macrophage-derived eicosanoids by virtue of specific biochemical interactions with substrates and enzymes of the 5-lipoxygenase pathway.     

Effects of immune complexes, zymosan preparations and ionophore A 23187 on leukotriene formation in human leukocytes

Incubation of human leukocytes with opzonized zymosan or IgG immune complexes led to a time dependent release of leukotrienes (LT) B₄ and C₄. After 3–4 min, the levels of LTB₄ and LTC₄ were 93 and 35 pmol/310⁷ cells, respectively. These amounts were 2–4 times lower than those released by leukocytes stimulated with the calcium ionophore A 23187. The levels of LTC₄ were 8 and 20 times lower than those of LTB₄ after incubation with opsonized zymosan or immune complexes, respectively. Heat-inactivation of the serum prior to zymosan coating decreased the effect of opsonized zymosan. Uncoated zymosan was an even weaker stimulus of leukotriene formation. These results suggest that both complement factors and immunoglobulins play a pivotal role in activating leukotriene synthesis in a mixed suspension of human leukocytes.     

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.     

Calcium ionophore enables soluble agonists to stimulate macrophage 5-lipoxygenase

The regulation of arachidonic acid conversion by the 5-lipoxygenase and the cyclooxygenase pathways in mouse peritoneal macrophages has been studied using particulate and soluble agonists. Particulate agonists, zymosan and latex, stimulated the production of cyclooxygenase metabolites as well as the 5-lipoxygenase product, leukotriene C4. In contrast, incubation with the soluble agonist phorbol myristate acetate or exogenous arachidonic acid led to the production of cyclooxygenase metabolites but not leukotriene C4. We tested the hypothesis that the 5-lipoxygenase, unlike the cyclooxygenase, requires activation by calcium before arachidonic acid can be utilized as a substrate. Addition of phorbol myristate acetate to macrophages in the presence of calcium ionophore (A23187) at a concentration which alone did not stimulate arachidonate metabolism resulted in a synergistic increase (50-fold) in leukotriene C4 synthesis compared to phorbol ester or A23187 alone. No such effect on the cyclooxygenase pathway metabolism was observed. Exogenous arachidonic acid in the presence of A23187 produced similar results yielding a 10-fold greater synthesis of leukotriene C4 over either substance alone without any effects on the cyclooxygenase metabolites. Presumably, calcium ionophore unmasked the synthesis of leukotriene C4 from phorbol myristate acetate-released and exogenous arachidonate by elevating intracellular calcium levels enough for 5-lipoxygenase activation. These data indicate that once arachidonic acid is released from phospholipid by an agonist, it is available for conversion by both enzymatic pathways. However, leukotriene synthesis may not occur unless intracellular calcium levels are elevated either by phagocytosis of particulate agonists or with calcium ionophore.     

Effect of leukotrienes, bradykinin and calcium ionophore (A 23187) on bovine endothelial cells: release of prostacyclin

Incubation of the bovine endothelial cell line, CPAE, with the calcium ionophore (A23187), bradykinin (BK), leukotriene D4 (LTD4) or leukotriene C4 (LTC4) resulted in concentration dependent increases in prostacyclin release measured as 6-keto-prostaglandin F1 alpha. The kinetics of induction of prostacyclin synthesis differed among the agents studied. Statistically significant increases in prostacyclin were observed one minute after treatment, with A23187, at slightly longer times with bradykinin and after approximately three minutes with the leukotrienes. Two other leukotrienes were tested. Both leukotriene B4 and leukotriene E4 (LTE4) were inactive at concentrations up to 10 microM. The induction of prostacyclin synthesis by LTC4 and LTD4 was inhibited by cycloheximide and actinomycin-D. The effect of BK was inhibited by cycloheximide but not by actinomycin-D. Induction by A23187 was not inhibited by either actinomycin-D or cycloheximide. The results suggest that these agents induced the increases in prostacyclin synthesis by different mechanisms.     

In vivo formation of leukotriene E5 by murine peritoneal cells

Resident mouse peritoneal cells, stimulated in vivo with opsonized zymosan, produced leukotriene C4 and E4, with LTE4 being the major (80-90%) product. When mice were placed on diets containing increasing amounts of fish oil, four additional sulfidopeptide leukotrienes (SP-LT), LTC5, LTE5, 11-trans LTC5 and 11-trans LTE5, were identified. The identity of LTE5 was confirmed by spectrophotometric, chromatographic and enzymatic methods. When equivalent amounts of n-6 and n-3 polyunsaturated fatty acids (PUFA) were included in the diet, the stimulated peritoneal cells (in vivo) produced higher quantities of LTE5 (30.2 +/- 5.4 ng/10(6) cells) than LTE4 (22.8 +/- 7.3 ng/10(6) cells). In addition, in vitro studies demonstrated a 60% reduction in LTC4 (42.0 +/- 10.8 ng/10(6) cells to 16.7 +/- 6.2 ng/10(6) cells) and the appearance of LTC5 (2.1 +/- 0.9 ng/10(6) cells) in resident macrophages (stimulated with A23187) from mice maintained on a fish oil diet compared to mice fed the control diet. This study demonstrated that formation of the pentaenyl SP-LT in vivo, in particular LTE5, by peritoneal cells can significantly contribute to the endogenous SP-LT pool in response to an inflammatory stimulus following a dietary regimen containing fish oil.     

Histaminase release from human eosinophils.

Phagocytosis of opsonized zymosan by human eosinophils results in a dose-dependent noncytotoxic release of histaminase as well as arylsulfatase and beta-glucuronidase. The calcium ionophore A23187 also stimulates release of eosinophil histaminase at concentrations of ionophore which barely release arylsulfatase and beta-glucuronidase. Zymosan-induced histaminase release from eosinophils but not from neutrophils was abolished or markedly reduced in the presence of cytochalasin B, suggesting a difference in the mechanisms of histaminase release from the two granulocyte cell types.