Endothelial cell leukotriene C4 synthesis results from intercellular transfer of leukotriene A4 synthesized by polymorphonuclear leukocytes

Leukotriene (LT) synthesis and metabolism were studied in porcine aortic endothelial cells. Leukotrienes were identified by combinations of guinea pig lung parenchymal strip bioassay, radioimmunoassay, and UV spectrophotometry with high performance liquid chromatography. Endothelial cells stimulated with the calcium ionophore, A23187, were unable to convert arachidonic acid to detectable levels of LTA4-derived products including the biologically active metabolites, LTB4 or LTC4. However, these cells readily converted exogenous LTA4 to the potent slow-reacting substance, LTC4. Smaller quantities of 11-trans-LTC4 and LTD4 were also observed. LTB4 was not detectable in these incubations nor was LTB4 metabolism observed. The possible intercellular transfer of LTA4 between polymorphonuclear leukocytes (PMNL) and endothelial cells was tested since PMNL release LTA4 when stimulated and have significant contact with endothelium. When A23187-stimulated neutrophils were coincubated with endothelial cells, a significant increase in LTC4 levels was detected over PMNL alone. LTC4 is formed by the enzymatic conjugation of glutathione (GSH) with LTA4. Therefore in some experiments, endothelial cells were prelabeled with [35S]cysteine to allow intracellular synthesis of [35S]GSH. When unlabeled PMNL were added, as a source of LTA4 to the prelabeled endothelial cells, substantial levels of [35S] LTC4 were recovered. The data indicate that endothelial cells synthesize LTC4 from LTA4. They also demonstrate a specific PMNL-endothelial cell interaction in which endothelial cell LTC4 synthesis results from the intercellular transfer of LTA4 produced by PMNL.     

Eosinophil peroxidase-mediated inactivation of leukotrienes B4, C4, and D4

The slow-reacting substance (SRS) bioactivity of leukotrienes C4 (LTC4) and D4 (LTD4) was rapidly decreased by incubation with eosinophil peroxidase (EPO), H2O2, and iodide, bromide, or to a lesser degree, chloride, LTB4 chemotactic activity was also decreased by the EPO-H2-H2-halide system, although at a slower rate. Myeloperoxidase could substitute for EPO in these reactions. Leukotriene inactivation was greatly decreased or abolished by deletion of any of the components of the system or by the addition of the hemeprotein inhibitors, azide, cyanide, or aminotriazole, indicating a requirement for peroxidase. The H2O2 concentration employed in the above studies was 10(-4) M. H2O2 at higher concentrations (5 x 10(-4) to 10(-2) M) inactivated LTC4 and LTD4 in the absence of EPO and a halide but had no effect on the chemotactic activity of LTB4. We have previously shown that horse eosinophils stimulated with the calcium ionophore A23187 generate SRS. In the present study, eosinophils stimulated in this way were found to release extracellularly both H2O2 and EPO. Incubation of eosinophils with azide that inhibits EPO, and catalase that degrades H2O2, significantly increased the amount of SRS activity detected in the extracellular medium after A23187 stimulation. These findings suggests eosinophils may play an important modulating role in hypersensitivity reactions both by the production of leukotrienes and by their inactivation through the release of H2O2 and EPO.     

Metabolism of arachidonic acid through the 5-lipoxygenase pathway in normal human peritoneal macrophages

Peritoneal macrophages (PM), obtained from 39 healthy women with normal laparoscopy findings, were stimulated with the ionophore A23187 or/and arachidonic acid (AA) both in adherence and in suspension. AA lipoxygenase metabolites were determined by reversed-phase HPLC. The major metabolites identified were 5-hydroxyeicosatetraenoic acid (5-HETE), leukotriene (LT)B4 and LTC4. The 20-hydroxy-LTB4, 20-carboxy-LTB4, and 15-HETE were not detected. Incubations of adherent PM with 2 microM A23187 induced the formation of LTB4, 110 +/- 19 pmol/10(6) cells, 5-HETE, 264 +/- 53 pmol/10(6) cells and LTC4, 192 +/- 37 pmol/10(6) cells. When incubated with 30 microM exogenous AA, adherent PM released similar amounts of 5-HETE (217 +/- 67 pmol/10(6) cells), but sevenfold less LTC4 (27 +/- 12 pmol/10(6) cells) (p less than 0.01). In these conditions LTB4 was not detectable. These results indicate that efficient LT synthesis in PM requires activation of the 5-lipoxygenase/LTA4 synthase, as demonstrated previously for blood phagocytes. When stimulated with ionophore, suspensions of Ficoll-Paque-purified PM produced the same lipoxygenase metabolites. The kinetics of accumulation of the 5-lipoxygenase/LTA4 synthase products in A23187-stimulated adherent cells varied for the various metabolites. LTB4 reached a plateau by 5 min, whereas LTC4 levels increased up to 60 min, the longest incubation time studied. Levels of 5-HETE were maximal at 5 min, and then slowly decreased with time. Thus, normal PM, in suspension or adherence, have the capacity to produce significant amounts of 5-HETE, LTB4, and LTC4. The profile of lipoxygenase products formed by the PM and the reactivity of this cell to AA and ionophore A23187 are similar to those of the human blood monocyte, but different from those of the human alveolar macrophage.     

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.     

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)     

Effects of leukotrienes and f-Met-Leu-Phe on oxidative metabolism of neutrophils and eosinophils

We assessed the effects of several leukotrienes and of f-Met-Leu-Phe on oxygen consumption in neutrophils and on the initial burst of chemiluminescence (CL) in both neutrophils and eosinophils. It was found that f-Met-Leu-Phe initiated 2.6 times higher oxygen consumption in neutrophils than did leukotriene B4 (LTB4). f-Met-Leu-Phe also stimulated five to 10 times more CL from both types of granulocytes than LTB4, which was at least five times more potent than its omega-hydroxylated metabolite, 20-OH-LTB4, whereas the corresponding 20-COOH derivative was effective only in eosinophils. The double dioxygenation product 5(S), 12(S)- DHETE caused no CL. Neutrophils from patients with chronic granulomatous disease did not respond with CL to any of the agents. The peak of CL occurred 50 to 60 sec after the addition of fMLP, whereas the LTB4-associated peak occurred after 5 to 6 sec and then rapidly subsided. The treatment of cells with sodium azide to inhibit the myeloperoxidase system did not change the kinetics or the rapid decline of the LTB4-induced CL. The CL response to LTB4 could be inhibited to 85% by 0.5 microgram/ml of superoxide dismutase, to 72% by 200 mg/ml of catalase, and to 50% by 80 microM of mannitol. The corresponding figures for f-Met-Leu-Phe-induced CL were 80, 58, and 16%, suggesting that, although a substantial part of the CL appears to be due to superoxide ion production, other oxygen radicals are involved in luminol-enhanced CL production. Thus, in contrast to some previous reports that leukotrienes do not stimulate an oxidative metabolic response in granulocytes despite their potent activity as chemotactic factors, our studies show that leukotrienes are definite inducers of granulocyte oxidative metabolism.     

Arachidonic acid metabolism by canine tracheal epithelial cells. Product formation and relationship to chloride secretion

Canine tracheal epithelial cells freshly isolated from mongrel dog trachea were used to study relationships between arachidonic acid metabolism and chloride ion movement. High performance liquid chromatography (HPLC) analysis of the cell incubation media after the addition of A23187 showed the presence of prostaglandin H synthase and lipoxygenase-derived metabolites. The major prostaglandin H synthase metabolite identified by HPLC, gas chromatography, and mass spectrometry was prostaglandin (PG) D2. The major lipoxygenase metabolites were leukotriene (LT) C4 and LTB4. LTB4 was identified by HPLC, UV spectroscopy, and gas chromatography. Straight phase HPLC of the methyl esters indicated only a minor formation of LTB4 isomers. LTC4 was identified by HPLC, UV spectroscopy, and conversion to LTD4 by gamma-glutamyl transpeptidase. Analysis by radioimmunoassays indicated approximately 1-2 ng of LTB4 and peptide LT formed by 10(6) cells after A23187 stimulation. The addition of ionophore A23187 caused a rapid release of arachidonic acid metabolites which was completed within 5 min of stimulation. Cl- secretion was measured in parallel studies of excised tracheas in Ussing chambers. Cl- secretion occurred at 2-3 min after the addition of ionophore, and the most rapid change occurred with the highest PGD2 concentrations. Indomethacin produced a concentration-dependent inhibition of PGD2 formation and Cl- movement. The addition of PGE2, PGD2, and PGH2 effectively stimulated Cl- secretion. LTC4 also stimulated Cl- secretion, but the stimulation was inhibited by indomethacin. These results indicate that canine tracheal epithelial cells metabolize arachidonic acid via both prostaglandin H synthase and lipoxygenase enzymes. It appears that endogenous PGD2 formation is the important variable controlling the Cl- ion movement in canine trachea.     

Synthesis and metabolism of leukotrienes by human endothelial cells: influence on prostacyclin release

The synthesis and metabolism of leukotrienes (LTs) by endothelial cells was investigated using reverse-phase high-performance liquid chromatography. Cells were incubated with [14C]arachidonic acid. LTA4 or [3H]LTA4 and stimulated with ionophore A23187. The cells did not synthesize leukotrienes from [14C]arachidonic acid. LTA4 and [3H]LTA4 were converted to LTC4, LTD4, LTE4 and 5,12-diHETE. Endothelial cells metabolized [3H]LTC4 to [3H]LTD4 and [3H]LTE4. The metabolism of [3H]LTC4 was inhibited by L-serine-borate complex, phenobarbital and acivicin in a concentration-related manner, with maximal inhibition occurring at a concentration of 0.1 M, 0.01 M and 0.01 M, respectively. LTC4, LTB4 and LTD4 stimulated the synthesis of prostacyclin, measured by radioimmunoassays as 6-keto-PGF1 alpha. The stimulation by LTC4 was greater than that by LTD4 or LTB4. LTE4, 14,15-LTC4 and 14,15-LTD4 failed to stimulate the synthesis of prostacyclin. LTD4 and LTB4 also stimulated the release of PGE2, whereas LTC4 did not. Serine-borate and phenobarbital inhibited LTC4-stimulated synthesis of prostacyclin in a concentration-related manner. They also inhibited the release of prostacyclin by histamine, A23187 and arachidonic acid. Acivicin had no effect on the release of prostacyclin by LTC4, histamine or A23187. Furthermore, FPL-55712, an LT receptor antagonist, inhibited LTC4-stimulated prostacyclin synthesis but had no effect on histamine-stimulated release of prostacyclin or PGE2. Indomethacin inhibited both LTC4- and histamine-stimulated release. The results show that (a) endothelial cells metabolize LTA4, LTC4 and LTD4 but do not synthesize LTs from arachidonic acid; (b) LTC4 act directly at the leukotriene receptor to stimulation prostacyclin synthesis; (c) the presence of the glutathione moiety at the C-6 position of the eicosatetraenoic acid skeleton is necessary for leukotriene stimulation of prostacyclin release; and (d) the metabolism of LTC4 to LTD4 and LTE4 does not appear to alter the ability of LTC4 to stimulate the synthesis of PGI2.     

Platelet-activating factor and leukotriene biosynthesis in whole blood. A model for the study of transcellular arachidonate metabolism

As a model to perhaps better indicate potential in vivo tissue inflammatory events, the generation of leukotriene (LT)B4, 20-OH-LTB4, sulfidopeptide LT, and platelet-activating factor (PAF) from human whole blood stimulated with zymosan was compared with that produced by isolated human neutrophils suspended either in buffer or plasma. Several reports have shown that substantial LTB4 biosynthesis could be induced after addition of zymosan to whole blood, but little was known concerning the generation of other important lipid mediators, or the cellular source of these. We have shown that, in spite of some subject variation, the zymosan-induced production of 20-OH-LTB4, LTB4, and LTE4 reached maxima within 30 to 60 min with 1.1, 2.8, and 0.60 ng/10(6) neutrophils, respectively. These concentrations would be sufficient to induce significant biologic effects. Studies with isolated cell mixtures suggested that the neutrophil was the primary source of the lipid mediators or their precursors in this system, although a number of other cell types contributed as accessory cells to the final amounts and mix of mediators produced. The ratio of neutrophils to accessory cells in mixed cell experiments dramatically modified the metabolic pattern of leukotriene generation. The concentration of LTB4 was increased in the presence of RBC and that of LTE4 when platelets were present. These results suggested that cellular cooperation and transcellular biosynthesis played a key role in the overall production of eicosanoids such as LTB4 and LTC4. The concomitant synthesis of PAF in isolated cells and in whole blood was also determined as another member of the complex lipid mediator network. Maximal production of cell-associated PAF was observed within 30 min after the initiation of phagocytosis and reached levels of 3 to 5 ng PAF/10(6) neutrophils. When other cells were present in a coincubation system, the time course for production of PAF was not altered, but maximal concentration of PAF was lower, perhaps as a result of enhanced PAF metabolism. Study of eicosanoids and other lipid mediator production in mixed cell populations provides insight into those events occurring within tissues, where cross-cell signaling and transcellular biosynthesis may occur.     

Leukotriene generation and small airway smooth muscle responsiveness in human lung

The hypothesis was tested that endogenous leukotriene (LT) production in the lung causes desensitisation of airway smooth muscle to LT. The synthesis of LTB4, C4, D4 and E4 by human lung tissue, obtained at thoracotomies, after stimulation with Ca-ionophore was assessed by HPLC. Functional studies of small airway smooth muscle from the same tissue specimens were carried out using LTC4 and methacholine as the contracting agents. Generation of LTB4, C4, D4 and E4 was 453 +/- 82, 84 +/- 15, 71 +/- 27 and 40 +/- 16 pmol/g fresh tissue respectively (mean +/- S.E.M., n = 10). All airway smooth muscle preparations responded to LTC4 in a concentration dependent way with a -log EC20 of 8.56 +/- 0.13, a -log EC50 of 7.95 +/- 0.08 and a Tmax of 82 +/- 11 mg force/mg tissue weight, corresponding to 79 +/- 4% of the maximal response to methacholine (mean +/- S.E.M.; 27 preparations from 10 patients). No correlations were found between any of the functional parameters (-logEC20, -logEC50, Tmax to LTC4 and methacholine) and the amounts of LT's generated by the lung tissue. Furthermore airway smooth muscle contractility was not significantly reduced after repeated exposure of bronchiolar strips to LTC4 in vitro. These findings suggest that the responsiveness of human peripheral airway smooth muscle to LT is not related to the capacity of the lung tissue to synthetize LT.     

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.     

Activation of NADPH oxidase in human neutrophils. Synergism between fMLP and the neutrophil products PAF and LTB4

The induction of the respiratory burst in human neutrophils by combinations of fMLP and either PAF or LTB4 was studied. Pretreatment with PAF (0.0001 to 10 uM), which by itself did not elicit the burst, greatly enhanced the rate and extent of fMLP-induced superoxide production. A synergism of a different kind was observed with the reversed stimulus sequence: Pretreatment with fMLP made the neutrophils capable to respond to PAF with superoxide production. A moderate enhancement of the fMLP response was also obtained following pretreatment with LTB4. The response of the cells to LTB4, however, was not influenced by fMLP, and no synergism was observed between the two neutrophil products PAF and LTB4. The results of this study demonstrate a marked synergism between fMLP and PAF and suggest that PAF may function as an amplifier of the respiratory burst response of stimulated neutrophils.     

Attenuated zymosan-induced peritoneal vascular permeability and IgE-dependent passive cutaneous anaphylaxis in mice lacking leukotriene C4 synthase

Leukotriene C(4) synthase (LTC(4)S), the terminal 5-lipoxygenase pathway enzyme that is responsible for the biosynthesis of cysteinyl leukotrienes, has been deleted by targeted gene disruption to define its tissue distribution and integrated pathway function in vitro and in vivo. The LTC(4)S (-/-) mice developed normally and were fertile. LTC(4)S activity, assessed by conjugation of leukotriene (LT) A(4) methyl ester with glutathione, was absent from tongue, spleen, and brain and > or = 90% reduced in lung, stomach, and colon of the LTC(4)S (-/-) mice. Bone marrow-derived mast cells (BMMC) from the LTC(4)S (-/-) mice provided no LTC(4) in response to IgE-dependent activation. Exocytosis and the generation of prostaglandin D(2), LTB(4), and 5-hydroxyeicosatetraenoic acid by BMMC from LTC(4)S (-/-) mice and LTC(4)S (+/+) mice were similar, whereas the degraded product of LTA(4), 6-trans-LTB(4), was doubled in BMMC from LTC(4)S (-/-) mice because of lack of utilization. The zymosan-elicited intraperitoneal extravasation of plasma protein and the IgE-mediated passive cutaneous anaphylaxis in the ear were significantly diminished in the LTC(4)S (-/-) mice. These observations indicate that LTC(4)S, but not microsomal or cytosolic glutathione S-transferases, is the major LTC(4)-producing enzyme in tissues and that its integrated function includes mediation of increased vascular permeability in either innate or adaptive immune host inflammatory responses.     

Biosynthesis of eicosanoids and transcellular metabolism of leukotrienes in murine bone marrow cells

Leukotriene B(4) (LTB(4)) biosynthesis by polymorphonuclear leukocytes (PMNs) is an important factor of inflammatory responses. PMNs also release LTA(4), an unstable intermediate that can be taken up by neighboring cells and metabolized into LTC(4). Most studies of LT synthesis have been carried out using human PMNs, but very little information is available about mouse PMNs. Mouse bone marrow PMNs were found to synthesize eicosanoids upon stimulation with A23187, fMLP, or zymosan. The major eicosanoids produced are LTB(4) and 5-hydroxyeicosatetraenoic acid, with some nonenzymatic products of LTA(4) hydrolysis. No cysteinyl leukotrienes were produced, in contrast to what was observed with human blood neutrophil preparations. Human megakaryoblast-like MEG-01 cells synthesized thromboxane B(2) and prostaglandin E(2) in response to A23187 but produced no 5-lipoxygenase (5-LO)-derived eicosanoids. When mouse bone marrow cells (mBMCs) and MEG-01 cells were stimulated during coincubation, LTC(4) and LTD(4) were produced. Mouse peritoneal macrophages from 5-LO-deficient mice were able to synthesize LTC(4) when incubated with mBMCs from wild-type mice, demonstrating transcellular exchange of LTA(4) from mBMCs into murine peritoneal macrophages. These data demonstrate that murine bone marrow PMNs are a valid model for the study of LT biosynthesis, which now offers the possibility to investigate specific biochemical pathways through the use of transgenic mice.     

Cyclooxygenase and lipoxygenase metabolite synthesis by polymorphonuclear neutrophils: in vitro effect of dipyrone

Functional activity of polymorphonuclear neutrophils (PMN) is associated with the metabolism of Arachidonic Acid (AA) released from membrane phospholipids. In this study the in vitro effect of dipyrone, a non steroidal anti-inflammatory drug, on the production of AA metabolites through cyclooxygenase (CO) and lipoxygenase (LO) pathways by stimulated PMN has been investigated. PMN isolated by counterflow centrifuge elutriator were greater than 98% pure and viable. Metabolite production was evaluated by RIA of Thromboxane A2 (TxA2), Prostaglandin E2 (PGE2), Leukotriene B2 (LTB4) and Leukotriene C4 (LTC4) after PMN stimulation with calcium ionophore A 23187 (20 microM). The levels of beta-thromboglobulin (RIA) lower than 5 ng/ml allowed us to rule out activation of residual contaminant platelets. In these experimental conditions, in the absence of dipyrone the products (ng/10(6) cells) of AA metabolism were LTB4 (3.51 +/- 0.22), LTC4 (0.81 +/- 0.08), TxB2 (0.144 +/- 0.025) and PGE2 (0.150 +/- 0.017). Incubation with dipyrone induced changes of PGE2 and TXB2 production in a dose dependent fashion (r = 0.83 and r = 0.87, p less than 0.001), obtaining already at the lowest drug concentration (5 micrograms/ml) a significant inhibition (33 and 40% for TxB2 and PGE2 p less than 0.005). No significant changes of LTB4 and LTC4 production have been observed. The results of this study indicate that dipyrone relevantly affects CO metabolite synthesis by stimulated PMN at concentrations comparable to those reached in therapeutic use. The inhibition of PGE2 synthesis which is present in inflamed tissues and actively participates in inflammatory reactions, could contribute to the therapeutic anti-inflammatory action of dipyrone.     

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.     

Synthesis and metabolism of leukotrienes in gamma-glutamyl transpeptidase deficiency

Leukotrienes (LTs) are active lipid mediators derived in the 5-lipoxygenase pathway. LTC(4), the primary cysteinyl LT, is cleaved by gamma-glutamyl transpeptidase (GGT), resulting in LTD(4). We studied the synthesis and metabolism of LTs in three patients with GGT deficiency. LTs were analyzed in urine, plasma, and monocytes after HPLC separation by enzyme immunoassays, radioactivity detection, and electrospray tandem mass spectrometry. Analysis of LTs in urine revealed increased concentrations of LTC(4) (12.8-17.9 nmol/mol creatinine; controls, <0.005 nmol/mol creatinine), whereas LTE(4) was below the detection limit (<0.005 nmol/mol creatinine; controls, 32.2 +/- 8.6 nmol/mol creatinine). In plasma of one patient, LTC(4) was found to be increased (17.3 ng/ml; controls, 9.6 +/- 0.4 ng/ml), whereas LTD(4) and LTE(4) were below the detection limit (<0.005 ng/ml). LTB(4) was found within normal ranges. In contrast to controls, the synthesis of LTD(4) and LTE(4) in stimulated monocytes was below the detection limit (<0.1 ng/10(6) cells; controls, 37.1 +/- 4.8 cells and 39.4 +/- 5.6 ng/10(6) cells, respectively). The formation of [(3)H]LTD(4) from [(3)H]LTC(4) in monocytes was completely deficient (<0.1%; controls, 85 +/- 7%). Our data demonstrate a complete deficiency of LTD(4) biosynthesis in patients with a genetic deficiency of GGT. GGT deficiency represents a new inborn error of cysteinyl LT synthesis and provides a unique model in which to study the pathobiological coherence of LT and glutathione metabolism.     

Generation of leukotriene C4, leukotriene B4, and prostaglandin D2 by immunologically activated rat intestinal mucosa mast cells

Mucosal mast cells (MMC) were isolated from the intestine of Nippostrongylus brasiliensis-infected rats and then activated with Ag or with anti-IgE in order to assess their metabolism of arachidonic acid to leukotriene (LT) C4, LTB4, and prostaglandin D2 (PGD2). After challenge of MMC preparations of 19 +/- 1% purity with five worm equivalents of N. brasiliensis Ag, the net formation of immunoreactive equivalents of LTC4, LTB4, and PGD2 was 58 +/- 8.3, 22 +/- 4.5, and 22 +/- 3.4 ng/10(6) mast cells, respectively (mean +/- SE, n = 7). When MMC preparations of 56 +/- 9% purity were activated by Ag, the net generation of immunoreactive equivalents of LTC4, LTB4, and PGD2/10(6) MMC was 107 +/- 15, 17 +/- 5.4, and 35 +/- 18 ng, respectively. These data indicate that the three eicosanoids originated from the MMC rather than from a contaminating cell. Analysis by reverse phase HPLC of the C-6 sulfidopeptide leukotrienes present in the supernatants of the activated MMC preparations of lower purity revealed LTC4, LTD4, and LTE4. In a higher purity MMC preparation only LTC4 was present, suggesting that other cell types in the mucosa are able to metabolize LTC4 to LTD4 and LTE4. The release of histamine and the generation of eicosanoids from intestinal MMC and from peritoneal cavity-derived connective tissue-type mast cells (CTMC) isolated from the same N. brasiliensis-infected rats were compared. When challenged with anti-IgE, these MMC released 165 +/- 41 ng of histamine/10(6) mast cells, and generated 29 +/- 3.6, 12 +/- 4.2, and 4.7 +/- 1.0 ng (mean +/- SE, n = 3) of immunoreactive equivalents of LTC4, LTB4, and PGD2/10(6) mast cells, respectively. In contrast, CTMC isolated from the same animals and activated with the same dose of anti-IgE released approximately 35 times more histamine (5700 +/- 650 ng/10(6) CTMC), generated 7.5 +/- 2.3 ng of PGD2/10(6) mast cells, and failed to release LTC4 or LTB4. These studies establish, that upon immunologic activation, rat MMC and CTMC differ in their quantitative release of histamine and in their metabolism of arachidonic acid to LTC4 and LTB4.     

Leukotriene B4 level in neutrophils from allergic and healthy subjects stimulated by low concentration of calcium ionophore A23187. Effect of exogenous arachidonic acid and possible endogenous source.

Peripheral blood neutrophils from patients with allergic rhinitis and from normal subjects were incubated for 5 min at 37 degrees C with 0.15 microM calcium ionophore A23187 in the absence or presence of exogenous arachidonic acid (2.5 to 10 microM). In neutrophils from allergic patients, the leukotriene B4 (LTB4) level was significantly increased by exogenous arachidonic acid in a concentration-dependent manner (16.2 +/- 4.2 and 38.1 +/- 6.8 pmol/5 min per 2 X 10(6) cells in the absence and presence of 10 microM arachidonic acid, respectively; P less than 0.005; n = 8). The LTB4 level in neutrophils from healthy subjects was only 0.97 +/- 0.17 pmol/5 min per 2 x 10(6) cells (n = 5) and was not enhanced by exogenous arachidonate. When cells from allergic patients were challenged in the presence of exogenous [1-14C]arachidonic acid, released LTB4 was radiolabeled and the incorporated radioactivity increased with the labeled arachidonate concentration. Labeled LTB4 was never detectable after incubating neutrophils from normal donors with exogenous labeled arachidonate. When neutrophils were incubated with [1-14C]arachidonate for 1 h, the different lipid pools of the two cell populations were labeled but both types of neutrophils produced unlabeled LTB4 in response to ionophore stimulation. The hydrolysis of choline and ethanolamine phospholipids into diacyl-, alkenylacyl- and alkylacyl-species revealed that solely the alkylacyl-subclass of phosphatidylcholine was unlabeled. We conclude (i) that neutrophils from allergic patients stimulated by low ionophore concentration produce more LTB4 than neutrophils from healthy subjects and incorporate exogenous arachidonate, (ii) that endogenous arachidonate converted to LTB4 by the 5-lipoxygenase pathway may provide only from 1-O-alkyl-2-arachidonoyl-glycero-3-phosphocholine.