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.     

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.     

Neutrophil degranulation: evidence pertaining to its mediation by the combined effects of leukotriene B4, platelet-activating factor, and 5-HETE

Stimulus-activated polymorphonuclear neutrophils (PMN) produce leukotriene B4 (LTB4), 5-hydroxyeicosatetraenoate (5-HETE), and platelet-activating factor (PAF). Each of these lipids promotes PMN degranulation; in combination they have additive and potentiating effects that result in prominent degranulation responses at relatively low concentrations. Thus, the combined interactions of LTB4, 5-HETE, and PAF may mediate responses in PMN activated by other stimuli. This possibility was examined by measuring the responses of PMN made insensitive to one or more of these lipids. Cells were pretreated with LTB4, 5-HETE, and/or PAF for 8 min; exposed for 2 min to cytochalasin B (which is required for lipid-induced degranulation); and then challenged. PMN challenged with only buffer released minimal amounts of granule-bound enzymes. Furthermore, the lipid-pretreated cells were hyporesponsive to challenge with 1) various combinations of these same lipids or 2) ionophore A23187. The relative potencies of the lipids in producing hyporesponsiveness to themselves or A23187 were: 5-HETE less than PAF less than or equal to LTB4 less than PAF + LTB4 less than PAF + LTB4 + 5-HETE. For both types of challenge, reduced responsiveness occurred in cells pretreated with greater than 0.1 nM LTB4 and/or greater than 0.2 nM PAF, persisted in cells washed after lipid pretreatment, and did not develop in cells pretreated with various combinations of bioinactive structural analogues of the lipids. Thus, PAF, LTB4, and 5-HETE interacted to desensitize PMN, and the degranulating actions of A23187 required cells that were fully responsive to each of the three lipids. This supports the concept that the lipids act together in mediating certain of the ionophore's effects. However, lipid-desensitized PMN degranulated fully when challenged with C5a, a formylated oligopeptide, or phorbol myristate acetate. Degranulation responses, therefore, may proceed through various pathways, only some of which involve the lipid products studied here.     

Development of a radioimmunoassay for 15-HETE and its application to 15-HETE production by reticulocytes

Mono-hydroxy-eicosatetraenoic acids (HETE's) are frequently the principal lipoxygenase-derived products in a number of cell types. This paper describes the development of a selective and sensitive radioimmunoassay procedure for 15-HETE, a metabolite which has previously been shown to be both an activator and inhibitor of leukotriene formation in various cells. Initially, rabbits were immunized with 15-HETE conjugated to bovine serum albumin. After seven months, the anti-plasma showed significant binding of tritiated 15-HETE (40-45% binding with a 1:600 dilution of the anti-plasma) which was displaceable by cold 15-HETE. The sensitivity of the assay was approximately 20 pg. of 15-HETE. The anti-plasma exhibited very little (less than 1%) cross-reactivity with arachidonic acid, 5-, 8-, 9-, 11- and 12-HETE's, HHT, TXB2, PGE2 and 6-Keto-PGF1 alpha. Significant cross-reactivity was observed with 5,15-diHETE (53%), 8, 15-diHETE (6.6%), and several other 15-hydroxy-eicosanoids. Rabbit reticulocytes have a very active 15S-lipoxygenase and converted arachidonic acid (final concentration 7 microM) principally to 15-HETE. Unstimulated reticulocytes were found to release negligible amounts of 15-HETE as determined by radioimmunoassay. Treatment of these cells with the calcium ionophore A23187 (0.16 to 4.0 micrograms/ml) elicited a level of 15-HETE release (8 - 14 ng/ml) that was twenty to forty times less than that obtained with exogenous arachidonic acid (2.5 micrograms/ml). The radioimmunoassay reported here may be useful for identifying factors which stimulate cellular release of 15-HETE and other 15-hydroxy-eicosanoids from endogenous arachidonic acid.     

Formation of leukotrienes and hydroxy acids by human neutrophils and platelets exposed to monosodium urate

Monosodium urate (MSU) crystals stimulate the production of arachidonic acid metabolites by human neutrophils and platelets. Neutrophils exposed to MSU generated leukotriene B (LTB), 6-trans-LTB4, 12-epi-6-trans-LTB4, and 5S, 12S DHETE from endogenous sources of arachidonate. In addition to these metabolites both monohydroxyeicosatetraenoic acids (i.e., 5-HETE) and omega-oxidation products (i.e., 2O -COOH LTB4) were formed by neutrophils exposed to MSU. Addition of exogenous arachidonic acid led to increased formation of each of these metabolites. When neutrophils were treated with colchicine (10 microM), LTB4 but not 5-HETE formation was impaired. (1-14C)Arachidonate-labeled platelets exposed to MSU released (1-14C)-arachidonate, (14C)-12 HETE, (14C)-HHT and (14C)-thromboxane B2. Results indicate that MSU stimulates arachidonic acid metabolism in both human neutrophils and platelets. Moreover, they suggest not only that metabolites of arachidonate may be considered as possible candidates for mediators of inflammation in crystal-associated diseases, but that colchicine blocks the formation of LTB4.     

The production of 5-HETE and leukotriene B in rat neutrophils from carrageenan pleural exudates

Rat neutrophils isolated from three-hour carrageenan pleural exudates actively metabolize arachidonic acid into three major metabolites, HHT, 11-HETE and 15-HETE. However, in the presence of the calcium ionophore, A23187, or the non-ionic detergent, BRIJ 56, these cells also produce 5-HETE and LTB. The production of these lipoxygenase products is calcium dependent. While non-steroidal anti-inflammatory drugs do not affect 5-HETE or LTB production, BW 755C and ETYA inhibit formation of these metabolites from exogenously added arachidonic acid.     

The production of 5-HETE and leukotriene B in rat neutrophils from carrageenan pleural exudates

Rat neutrophils isolated from three-hour carrageenan pleural exudates actively metabolize arachidonic acid into three major metabolites, HHT, 11-HETE and 15-HETE. However, in the presence of the calcium ionophore, A₂₃₁₈₇, or the non-ionic detergent, BRIJ 56, these cells also produce 5-HETE and LTB. The production of these lipoxygenase products is calcium dependent. While non-steroidal anti-inflammatory drugs do not affect 5-HETE or LTB production, BW 755C and ETYA inhibit formation of these metabolites from exogenously added arachidonic acid.     

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

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

Lipoxin generation by human megakaryocyte-induced 12-lipoxygenase.

Eicosanoid biosynthesis was examined with a human megakaryocytic cell line (Dami). Megakaryocytes incubated with [1-14C]arachidonic acid and either ionophore A23187 or thrombin generated both thromboxane and 12-hydroxyheptadecatrienoic acid (HHTrE). Exposure to phorbol myristate acetate (PMA) for 1 through 9 days induced differentiation and revealed an increase in the conversion of [1-14C]arachidonate to cyclooxygenase- and lipoxygenase (LO)-derived products. The LO-derived product was identified as 12S-HETE by its physical characteristics including GC/MS and chiral column SP-HPLC. PMA-treated Dami cells did not generate 5-HETE, leukotrienes or lipoxins from exogenous arachidonic acid while they did convert leukotriene A4 (LTA4) to lipoxin A4, lipoxin B4 and their respective all-trans isomers. In addition, COS-M6 cells transfected with a human 12-lipoxygenase cDNA and incubated with either arachidonic acid or LTA4 generated 12-HETE and lipoxins, respectively. The lipoxin profile generated by transfected COS-M6 cells incubated with LTA4 was similar to that generated by the PMA-treated Dami cells. Results indicate that human megakaryocytes can transform arachidonate and LTA4 to bioactive eicosanoids and that the 12-lipoxygenase appears upon further differentiation of these cells. In addition, they indicate that the 12-LO of human megakaryocytes and the 12-LO expressed by transfected COS cells can generate both lipoxins A4 and B4. Together they suggest that the human 12-LO can serve as a model of LX-synthetase activity with LTA4.     

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.     

15-Hydroxyeicosatetraenoic acid (15-HETE) receptors. Involvement in the 15-HETE-induced stimulation of the cryptic 5-lipoxygenase in PT-18 mast/basophil cells.

The mechanisms of stimulation of the inactive 5-lipoxygenase in mast/basophil PT-18 cells by microM 15-hydroxyeicosatetraenoic acid (15-HETE) was investigated. Treatment of PT-18 cells with pM 15-[3H]HETE at 4 degrees for 3 h resulted in the cell association of 10% of the ligand: two-thirds was incorporated into cellular lipids and a third was bound to specific 15-HETE cellular binding sites. Binding data analysis indicated a single class of 15-HETE binding sites with a Kd of 162 nM and a Bmax of 7.1 x 10(5) sites/cell. Unlabeled 15-HETE, 12-HETE, and 5,15-diHETE inhibited the binding of 15-[3H]HETE to cells, whereas LTB4 and PGF2 alpha were relatively ineffective. 2.4 microM 15-HETE (unlabeled) prevented 50% 15-[3H]HETE incorporation. Examination of the effects of 15-HETE methyl ester, 12-HETE, 5,15-diHETE, and pertussis toxin on both the 15-HETE-induced 5-lipoxygenase activation and 15-HETE cell association processes indicated a preponderant correlation of this activation process with specific 15-HETE binding rather than 15-HETE incorporation into phospholipids. In addition, 5,15-diHETE itself stimulated the inactive 5-lipoxygenase and eight times more [3H]diHETE was bound to cells than became incorporated into cellular lipids. The results support the involvement of low affinity 15-HETE receptors, rather than 15-HETE incorporation into cellular lipids, in the 15-HETE-induced stimulation of the 5-lipoxygenase in PT-18 cells.     

15-Hydroxyeicosatetraenoic Acid Inhibits Superoxide Anion Generation by Human Neutrophils: Relationship to Lipoxin Production

Human neutrophils can aggregate, degranulate, and release mediators of inflammation including oxygen radicals and lipoxygenase (LO)-derived products of arachidonic acid. The regulation of 5– and 15-lipoxy-genases appears to be important since their products (e.g. leukotrienes and lipoxins) display unique spectra of bioactions. Addition of 15-HETE. a product of the 15-LO, to neutrophils in suspension dramatically shifted the LO products generated and led to a dose-dependent increase in lipoxins, while the production of leukotriene B₄ and its μ-oxidation products (i.e. 20-COOH-LTB₄ and 20-OH-LTB₄) was inhibited. Exogenous 15-HETE also dose-dependently inhibited the generation of superoxide anions induced by either the chemotactic peptide f-met-leu-phe or the divalent cation ionophore A23187. Neither lipoxin A, nor lipoxin B₄ (10^?8?10^?6M) inhibited O₂^?_? generation induced by either f-met-leu-phe or A23187. These results indicate that in addition to serving as a substrate for lipoxin generation, 15-HETE also inhibits superoxide anion generation by human neutrophils. Together they provide further evidence to suggest that products of the 15-lipoxygenase may serve a regulatory role at inflammatory loci.     

Human B lymphocytes possess 5-lipoxygenase activity and convert arachidonic acid to leukotriene B4.

Incubation of cell sonicates from monoclonal B cells with arachidonic acid led to the formation of leukotriene (LT) B4 and 5-hydroxy-eicosatetraenoic acid (5-HETE). In contrast, stimulation of intact B cells with the calcium ionophore A23187 +/- arachidonic acid did not, under similar conditions, lead to formation of LTB4. The identification of these products was based on reverse phase- and straight phase-HPLC analysis, UV-spectroscopy and gas chromatography-mass spectrometry. Cell sonicates of highly enriched human tonsillar B lymphocytes also converted arachidonic acid to LTB4 and 5-HETE. Activation of these cells with B cell mitogen and cytokines for three days led to an upregulation of 5-lipoxygenase activity. This study provides evidence for the biosynthesis of LTB4 from arachidonic acid in B cell lines and in normal human tonsillar B lymphocytes.     

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.     

Stimulation of lipoxin synthesis from leukotriene A4 by endogenously formed 12-hydroperoxyeicosatetraenoic acid in activated human platelets

Human platelets are devoid of 5-lipoxygenase activity but convert exogenous leukotriene A₄ (LTA₄) either by a specific LTC₄ synthase to leukotriene C₄ or via a 12-lipoxygenase mediated reaction to lipoxins. Unstimulated platelets mainly produced LTC₄, whereas only minor amounts of lipoxins were formed. Platelet activation with thrombin, collagen or ionophore A23187 increased the conversion of LTA₄ to lipoxins and decreased the leukotriene production. Maximal effects were observed after incubation with ionophore A23187, which induced synthesis of comparable amounts of lipoxins and cysteinyl leukotrienes (LTC₄, LTD₄ and LTE₄). Chelation of intra- and extracellular calcium with quin-2 and EDTA reversed the ionophore A23187-induced stimulation of lipoxin synthesis from LTA₄ and inhibited the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) from endogenous substrate. However, calcium did not affect the 12-lipoxygenase activity in the 100 000 × g supernatant of sonicated platelet suspensions. Furthermore, the stimulatory effect on lipoxin formation induced by platelet agonists could be mimicked in intact platelets by the addition of low concentrations of arachidonic acid, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) or 13-hydroperoxyoctadecadienoic acid (13-HPODE). The results indicate that the elevated lipoxin synthesis during platelet activation is due to stimulated 12-lipoxygenase activity induced by endogenously formed 12-HPETE.     

Evidence for LTB4/12-HETE binding sites in a human epidermal cell line

We identified leukotriene B4 (LTB4)/12-hydroxyeicosatetraenoic acid (12-HETE) binding sites in a squamous cell cancer-derived human epidermal cell line. Analysis of the binding data revealed a single class of binding sites with a dissociation constant of 0.16 microM and a Bmax of 3.8 x 10(6) sites per cell. Competitive binding assays with various eicosanoids at 37 degrees C showed nearly equal binding of 12(S)-HETE, 12(R)-HETE and LTB4. 5(S)-HETE and LTB4-analogs bound with lesser affinity. Specific LTB4 binding at 37 degrees C could also be demonstrated in freshly isolated normal human keratinocytes. Since lipoxygenase-derived eicosanoids are thought to play an important role in hyperproliferative and inflammatory skin diseases, the identification of LTB4/12-HETE binding sites in keratinocytes could have implications for the development of new drugs controlling these disease processes.     

Enhanced superoxide anion generation but reduced leukotriene B4 productivity in thioglycollate-elicited peritoneal macrophages

Lipoxygenase metabolism of arachidonic acid was compared between peritoneal macrophages from untreated rats and those from rats on day 7 after intraperitoneal injection of thioglycollate broth (TG). Resident macrophages (M phi) from untreated rats produced mainly LTB4 (303 +/- 25 pmol/5 x 10(6) cells) and 5-HETE (431 +/- 56 pmol/5 x 10(6) cells) when stimulated with 5 micrograms/ml calcium ionophore A23187 for 20 min at 37 degrees C. On the other hand, TG-elicited M phi generated less amounts of lipoxygenase metabolites (157 +/- 10 pmol LTB4 and 319 +/- 19 pmol 5-HETE/5 x 10(6) cells) with the same stimulus. Then, leukotriene productivity was examined by using subcellular fractions of each M phi lysate and an unstable epoxide intermediate, leukotriene A4. LTA4 hydrolase activity was mainly contained in soluble fractions from the both groups of M phi. The cytosol fraction from the resident M phi exhibited the following specific and total activity; 2.2 +/- 0.1 nmol LTB4/mg protein/5 min and 12.2 +/- 0.5 nmol LTB4/5 min per 10(8) cells. On the contrary, the cytosol fraction from the TG-elicited M phi showed 1.9 +/- 0.1 nmol LTB4/mg protein/5 min and 9.6 +/- 0.3 nmol LTB4/5 min per 10(8) cells. The resident M phi, however, generated 0.14 +/- 0.04 nmol O2-/min/4 x 10(5) cells whereas the TG-elicited M phi did 0.49 +/- 0.13 nmol O2-/min/4 x 10(5) cells when stimulated with wheat germ lectin. These results suggest that the TG-elicited macrophages show enhanced superoxide production but generate less lipoxygenase metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of lipoxygenase products from arachidonic acid metabolism in stimulated murine eosinophils

The presence of arachidonic acid lipoxygenase pathways in murine eosinophils was demonstrated by the isolation and identification of several lipoxygenase products from incubations of these cells. The most abundant arachidonate metabolite from murine eosinophils stimulated with ionophore A23187 and exogenous arachidonic acid was 12-S-hydroxyeicosatetraenoic acid (12-S-HETE), and the next most abundant was 15-HETE. Two families of leukotrienes were also recovered from these incubations. One family comprised the hydrolysis products of leukotriene A4, and the other included products derived from the 14,15-oxido analog of leukotriene A4 (14,15-leukotriene A4). Two double oxygenation products of arachidonate were also identified. These compounds were a 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) and a 5,12-dihydroxyeicosatetraenoic acid (5,12-diHETE). Eosinophil stimulation promoter is a murine lymphokine which enhances the migration of eosinophils. When murine eosinophils were incubated with eosinophil stimulation promoter in concentrations sufficient to produce a migration response, a 2-3-fold increase in the production of 12-HETE was observed compared to unstimulated cells. Coupled with the recent demonstration that arachidonic acid lipoxygenase inhibitors suppress the migration response to eosinophil stimulation promoter and that 12-HETE induces a migration response, this observation provides further evidence in support of the hypothesis that eosinophil stimulation promoter stimulation of eosinophils results in the generation of lipoxygenase products which modulate the migratory activity of the cells.     

Phospholipase A2 activation in human neutrophils. Differential actions of diacylglycerols and alkylacylglycerols in priming cells for stimulation by N-formyl-Met-Leu-Phe

Both 1,2-diacyl- and 1-O-alkyl-2-acylglycerols are formed during stimulation of human neutrophils (PMN), and both can prime respiratory burst responses for stimulation by the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP); however, mechanisms of priming are unknown. Arachidonic acid (AA) release through phospholipase A2 activation and metabolism by 5-lipoxygenase are important activities of PMN during inflammation and could be involved in the process of primed stimulation. Therefore, we have examined the ability of diacyl- and alkylacylglycerols to act as priming agents for AA release and metabolism in human neutrophils. After prelabeling PMN phospholipids with [3H]AA, priming was tested by incubating human PMN with the diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), or its alkylacyl analog, 1-O-delta 9-octadecenyl-2-acetylglycerol (EAG) before stimulating with fMLP. fMLP (1 microM), OAG (20 microM), or EAG (20 microM) individually caused little or no release of labeled AA. However, after priming PMN with the same concentrations of either OAG or EAG, stimulation with 1 microM fMLP caused rapid (peak after 1 min) release of 6-8% of [3H]AA from cellular phospholipids; total release was similar with either diglyceride. Priming cells with OAG also enhanced conversion of released AA to leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) upon subsequent fMLP stimulation, but AA metabolites were not increased in EAG-primed PMN. If fMLP was replaced with the calcium ionophore A23187 (which directly causes release of AA and production of LTB4 and 5-HETE), priming by both diglycerides again enhanced release of [3H]AA, but only OAG priming increased lipoxygenase activity. Indeed, EAG pretreatment markedly reduced LTB4 and 5-HETE production. Thus, both diglycerides prime release of AA from membrane phospholipids but have opposite actions on the subsequent metabolism of AA.     

Stereochemical requirements for substrate specificity of LTB4 20-hydroxylase

LTB4 20-hydroxylase (P-450LTB) is the cytochrome P-450 in the microsomes of human polymorphonuclear leukocytes that catalyzes the omega-oxidation of leukotriene B4 (LTB4) to 20-OH LTB4. The activity of P-450LTB for LTB4 compared to isomers and analogs of LTB4 at a concentration of 0.3 microM revealed a preference of P-450LTB for both the triene bond configuration of LTB4 and for the chirality of the 5S and 12R hydroxyl groups. 15S-Hydroxyeicosatetraenoic acid, 8(R/S), 15S-dihydroxy-5-cis-9,11,13-trans-eicosatetraenoic acid, 8R,15S-dihydroxy-5,13-cis-9,11-trans-eicosatetraenoic acid, and 5S,15S-dihydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid were each not subject to omega-oxidation, indicating a negative effect of the presence of a 15-hydroxyl group on substrate recognition. At a concentration of 1.5 microM, 12R- and 12S-hydroxyeicosatetraenoic acid were converted to their respective 20-OH derivatives at rates that were 34.2 +/- 11.6% (mean +/- S.D., n = 3) and 3.5 +/- 4.3% (mean +/- S.D., n = 4), respectively, of that of LTB4 to 20-OH LTB4, further indicating that P-450LTB can distinguish the chirality of the 12-hydroxyl group. The lower Km of LTB4 (2.0 microM), as compared to those of its 6-trans-12-epi isomer (3.8 microM) and 5-epi-LTB4 (6.6 microM) confirmed the preference of P-450LTB for the specific triene bond structure of LTB4 and its preference for the chirality of the hydroxyl groups of LTB4 within this structurally related class of molecules. At equal 1.5-microM concentrations, LTB4 completely inhibited the omega-oxidation of all other substrates and partially suppressed that of leukotriene B5, consistent with the lower Km of LTB4 and indicating that P-450LTB catalyzed the omega-oxidation of all substrates. Thus, P-450LTB is a novel cytochrome P-450 of human polymorphonuclear leukocytes with substrate recognition determined by the triene bond configuration and the chirality of the hydroxyl groups.