Arachidonic acid metabolism of the murine eosinophil. II. Involvement of the lipoxygenase pathway in the response to the lymphokine eosinophil stimulation promoter

Eosinophil stimulation promoter (ESP) is a murine lymphokine that enhances the migration of eosinophils. Exogenous arachidonic acid between 0.5 and 2 micrograms/ml potentiated the activity of ESP on murine eosinophil migration, whereas such concentrations did not affect migration in the absence of ESP. Among the lipoxygenase products identified from an enriched population of murine eosinophils, leukotriene B4 (optimal activity at 100 ng/ml) and 12-HETE (optimal activity at 2 micrograms/ml) stimulated migration of these cells. Another lipoxygenase product from these cells 15-HETE inhibited ESP-induced migration; between 5 and 10 micrograms/ml 15-HETE decreased by one-half both stimulated migration and 12-HETE biosynthesis. Structurally diverse drugs at concentrations that inhibited HETE biosynthesis inhibited ESP-induced migration. The concentrations that decreased migration activity by one-half were 5 microM NDGA, 10 microM ETYA, and 150 microM BW755C. Aspirin and indomethacin at concentrations reported to inhibit prostaglandin biosynthesis did not substantially inhibit ESP activity, but concentrations of indomethacin above 20 microM caused concentration-dependent inhibition of migration. The selective lipoxygenases inhibitor 134,7,10,13-eicosatetraynoic acid was more potent than ETYA in inhibition of ESP-induced migration, and the selective cyclooxygenase inhibitor 6,9,12-octadecatriynoic acid did not effect inhibition. These results are consistent with the hypothesis that stimulation of eosinophils by the lymphokine ESP involves the generation of lipoxygenase products from arachidonic acid, which positively and negatively regulate the migratory activities of these cells.     

Identification of 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid as a novel and potent human eosinophil chemotactic eicosanoid.

Incubation of human eosinophils with arachidonic acid led to the formation of a novel and potent eosinophil chemotactic lipid (ECL) (Morita, E., Schr?der, J.-M., and Christophers, E. (1990) J. Immunol. 144, 1893-1900). To test the working hypothesis of whether ECL could have been formed via eosinophil-arachidonic acid 15-lipoxygenase we investigated whether other arachidonic acid 15-lipoxygenases such as soybean lipoxygenase I catalyze formation of a similar ECL. In the presence of hemoproteins and soybean lipoxygenase I arachidonic acid is converted to an ECL, which has physicochemical properties similar to those found for the eosinophil-derived ECL. Purification of this ECL by high performance liquid chromatography revealed that ECL is structurally different from well known eosinophil chemotactic eicosanoids such as leukotriene B4, 5,15-(6E,8Z,11Z,13E)-dihydroxyeicosatetraenoic acid (5,15-diHETE), and (8S,15S)-(5Z,9E,11Z,13E)-dihydroxyeicosatetra eno ic acid ((8S,15S)-diHETE). UV spectra of this ECL with absorbance maxima at 230 and 278 nm revealed the presence of two independent chromophores such as a conjugated oxodiene and a conjugated diene. Catalytic hydrogenation of ECL methyl ester led to the formation of 5,15-dihydroxyarachidic acid methyl ester. Reduction of ECL with sodium borohydride produced a product which is identical with authentic (5S,15S)-(6E,8Z,11Z,13E)-diHETE. Formation of an ECL monomethoxime derivative supports the conclusion that this highly potent eosinophil chemotactic eicosanoid is structurally identical with 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid.     

Evidence for a novel pathway of leukotriene formation in human platelets

The metabolism of arachidonic acid via lipoxygenase-catalyzed reactions in washed human platelets was investigated. In addition to the previously discovered lipoxygenase metabolites, 12-hydroxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid, 8,15-dihydroxyeicosatetraenoic acid and 14,15-dihydroxyeicosatetraenoic acid, several other products were formed. The compounds were all dihydroxylated metabolites of arachidonic acid, containing a conjugated triene structure, and identified as 11,12-dihydroxyeicosatetraenoic acid (two isomers) and 5,12-dihydroxyeicosatetraenoic acid (four isomers). The identification was based on ultraviolet spectroscopy and gas chromatography-mass spectrometry of native and hydrogenated compounds. Stereochemical analysis of the hydroxyl groups of the 5,12-dihydroxyeicosatetraenoic acids and experiments with 18O2 indicated that the compounds were formed by the 12-lipoxygenase pathway, probably via an unstable epoxide.     

Chemokinetic activity of arachidonic and lipoxygenase products on leuocyctes of different species

A number of hydroperoxy (HPETE) and hydroxy (HETE) products of the lipoxygenase pathway of arachidonic acid metabolism are chemotactic and chemokinetic for human neutrophils. We have investigated the relative chemokinetic potency of some of these products on human, rat and rabbit neutrophils. The most potent lipoxygenase product studied was 5,12-dihydroxy-6,8,10-14-eicosatetraenoic acid (5,12-diHETE), which was maximally chemokinetic and chemotactic between 0.1 and 1.0ng/ml for the three species. The 5, 11 and 12-HPETEs and HETEs were chemokinetic, but less active by at least two orders of magnitude, for human and rabbit neutrophils at concentrations between 0.1 and 10micrograms/ml. 15-HPETE and 15-HETE were inactive on human leucoctes, and none of the monosubstituted products studied were chemokinetic for rat neutrophils. These results indicate that 5,12-diHETE may be an important mediator in the local accumulation of leucocytes in the inflammatory response.     

Chemokinetic activity of arachidonic acid lipoxygenase products on leuocyctes of different species

A number of hydroperoxy (HPETE) and hydroxy (HETE) products of the lipoxygenase pathway of arachidonic acid metabolism are chemotactic and chemokinetic for human neutrophils. We have investigated the relative chemokinetic potency of some of these products on human, rat and rabbit neutrophils. The most potent lipoxygenase product studied was 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid (5,12-diHETE), which was maximally chemokinetic and chemotactic between 0.1 and 1.0ng/ml for the three species. The 5, 11 and 12-HPETEs and HETEs were chemokinetic, but less active by at least two orders of magnitude, for human and rabbit neutrophils at concentrations between 0.1 and 10μg/ml. 15-HPETE and 15-HETE were inactive on human leucocytes, and none of the monosubstituted products studied were chemokinetic for rat neutrophils. These results indicate that 5,12-diHETE may be an important mediator in the local accumulation of leucocytes in the inflammatory response.     

COX-2-dependent and -independent biosynthesis of dihydroxy-arachidonic acids in activated human leukocytes

Biosynthesis of 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) in leukocytes involves consecutive oxygenation of arachidonic acid by 5-lipoxygenase (LOX) and 15-LOX in either order. Here, we analyzed the contribution of cyclooxygenase (COX)-2 to the biosynthesis of 5,15-diHETE and 5,11-diHETE in isolated human leukocytes activated with lipopolysaccharide and calcium ionophore A23187. Transformation of arachidonic acid was initiated by 5-LOX providing 5S-HETE as a substrate for COX-2 forming 5S,15S-diHETE, 5S,15R-diHETE, and 5S,11R-diHETE as shown by LC/MS and chiral phase HPLC analyses. The levels of 5,15-diHETE were 0.45 ± 0.2 ng/10⁶ cells (mean ± SEM, n = 6), reaching about half the level of LTB₄ (1.3 ± 0.5 ng/10⁶ cells, n = 6). The COX-2 specific inhibitor NS-398 reduced the levels of 5,15-diHETE to below 0.02 ng/10⁶ cells in four of six samples. Similar reduction was achieved by MK-886, an inhibitor of 5-LOX activating protein but the above differences were not statistically significant. Aspirin treatment of the activated cells allowed formation of 5,15-diHETE (0.1 ± 0.05 ng/10⁶ cells, n = 6) but, as expected, abolished formation of 5,11-diHETE. The mixture of activated cells also produced 5S,12S-diHETE with the unusual 6E,8Z,10E double bond configuration, implicating biosynthesis by 5-LOX and 12-LOX activity rather than by hydrolysis of the leukotriene A₄-epoxide. Exogenous octadeuterated 5S-HETE and 15S-HETE were converted to 5,15-diHETE, implicating that multiple oxygenation pathways of arachidonic acid occur in activated leukocytes. The contribution of COX-2 to the biosynthesis of dihydroxylated derivatives of arachidonic acid provides evidence for functional coupling with 5-LOX in activated human leukocytes.     

Effect of various lipoxygenase metabolites of arachidonic acid on degranulation of polymorphonuclear leukocytes

Lipoxygenase metabolites of guinea pig peritoneal polymorphonuclear leukocytes stimulated with 10 microM A23187 plus arachidonic acid were isolated and identified. These metabolites were compared with each other and to chemically synthesized arachidonate metabolites for their ability to stimulate leukocyte degranulation. 5(S),12(R)-Dihydroxy-6,8,10-(cis/trans/trans)14-cis-eicosatetraenoic acid (leukotriene B4) produced a significant release of lysozyme, but not beta-glucuronidase or beta-N-acetylglucosaminidase at low concentrations (EC50 = 6.5 x 10(-9) M), while the leukocyte nonenzymatically generated 5,12-or 5,6-dihydroxyeicosatetraenoic acids had no effect at these concentrations. Higher concentrations (1--10 microM) of all the dihydroxyeicosatetraenoic acids, 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and its hydroperoxy precursor stimulated significant lysozyme release which was greater than that produced by 15-hydroxy-5,8,11-13-eicosatetraenoic acid, arachidonic acid, or its acetylene analogue, 5,8,11,14-eicosatetraynoic acid. Micromolar concentrations of leukotriene B4 and 5-HETE also stimulated significant release of beta-N-acetylglucosaminidase above controls, but not beta-glucuronidase. These results suggest that leukotriene B4 may play a role in regulating the release of certain granule-bound enzymes from polymorphonuclear leukocytes.     

Increased activity of 5-lipoxygenase in polymorphonuclear leukocytes from asthmatic patients

The formation of 5-lipoxygenase products of arachidonic acid, 5-HETE and 5,12-diHETE, was determined in 100,000 X g supernatant of polymorphonuclear leukocytes from 17 healthy subjects, 17 patients with extrinsic asthma and 15 patients with intrinsic asthma. After the supernatant was incubated with 14C-arachidonic acid in the presence of calcium and indomethacin, the lipoxygenase products of arachidonic acid were separated by thin layer chromatography. The results were expressed as the percentage conversion of 14C-arachidonic acid into the product per 10(7) cells. The formation of 5,12-diHETE, but not of 5-HETE, was significantly increased in the cells from the group of patients with extrinsic asthma (4.38 +/- 0.78%, mean +/- S.E.; p less than 0.01) and intrinsic asthma (6.09 +/- 1.11%; p less than 0.01), when compared to normal subjects (1.74 +/- 0.30%). Both extrinsic and intrinsic asthmatics had significantly enhanced 5-lipoxygenase activity, which was expressed as the sum of percentage conversion of 14C-arachidonic acid into 5-HETE and 5,12-diHETE. The percentage conversion in normal subjects was 4.19 +/- 0.39%, 6.24 +/- 0.84% for 17 patients with extrinsic asthma (p less than 0.05), and 8.59 +/- 1.29% for 15 patients with intrinsic asthma (p less than 0.01). There was no significant difference between these asthmatic groups. These results indicate that 5-lipoxygenase activity is increased in patients with bronchial asthma.     

Biosynthesis and metabolism of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid by human umbilical vein endothelial cells

Incubation of cultured human umbilical vein endothelial cells with [1-14C]arachidonic acid, followed by reverse-phase high-pressure liquid chromatography analysis, results in the appearance of two principal radioactive products besides 6-keto-prostaglandin F1 alpha. The first peak is 12-L-hydroxy-5,8,10-heptadecatrienoic acid, a hydrolysis product of the prostaglandin endoperoxide. The second peak was esterified, converted to the trimethylsilyl ether derivative, and analyzed by gas chromatography-mass spectrometry and shown to be the lipoxygenase product 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). Incubation of the 15-HETE precursor 15(S)-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) with endothelial cells results in the formation of four distinct UV absorbing peaks. UV and gas chromatography-mass spectrometry analysis showed these peaks to be 8,15(S)-dihydroxy-5,8,11,13-eicosatetraenoic acids (8,15-diHETE) differing only in their hydroxyl configuration and cis trans double-bond geometry. Formation of 8,15-diHETE molecules suggests the prior formation of the unstable epoxide molecule 14(S),15(S)-trans-oxido-5,8-Z-14,15-leukotriene A4 or an attack at C-10 of 15-HPETE by an enzyme with mechanistic features in common with a 12-lipoxygenase. The observation that endothelial cells can synthesize both 15-HETE and 8,15-diHETE molecules suggests that this cell type contains both a 15-lipoxygenase and a system that can synthesize 14,15-leukotriene A4.     

Leukotriene formation by a purified reticulocyte lipoxygenase enzyme. Conversion of arachidonic acid and 15-hydroperoxyeicosatetraenoic acid to 14, 15-leukotriene A4

The purified lipoxygenase of rabbit reticulocytes converts arachidonic acid at 0 degrees C to 15-hydroperoxyeicosatetraenoic acid (15-HPETE) and to 12-hydroperoxyeicosatetraenoic acid (12-HPETE) via reactions which involve hydrogen abstraction at C-13 and C-10, respectively. At 37 degrees C the enzyme converts arachidonic acid to additional products which were identified as 13-hydroxy-14,15-epoxy-5,8,11-eicosatrienoic acid, 8,15-dihydroperoxy-5,9,11,13- and 5,15-dihydroperoxy-6, 6,8,11,13-eicosatetraenoic acids (8,15-diHPETE and 5,15-HPETE, respectively) and diastereoisomers of 8,15-dihydroxy-5,9,11,13-eicosatetraenoic acid (8,15-diHPETEs). The 8,15- and 5,15-diHPETEs were formed by double lipoxygenation since each incorporated 2 molecules of 18O2 and since their synthesis from 15-HPETE was blocked under anaerobic conditions. The 8,15-diHETEs each incorporated 18O from 18O2 at C-15 and were found to arise from nonenzymatic hydrolysis of an epoxytriene which was identified as 14,15-leukotriene A4 by trapping in acidic methanol. This compound was a major product of 15-HPETE in anaerobic incubations. The conversion of 15-HPETE to 14,15-leukotriene A4 was inhibited by the lipoxygenase inhibitors nordihydroguairetic acid and 5,8,11,14-eicosatetraynoic acid. The 14,15-leukotriene A4 synthase and 15-lipoxygenase activities were inhibited by 5,8,11,14-eicosatetraynoic acid in a similar time-dependent manner. The results support a mechanism whereby 14,15-leukotriene A4 is synthesized from 15-HPETE by a further enzymatic step carried out by the reticulocyte 15-lipoxygenase via hydrogen abstraction at C-10 and a redox cycle of the non-heme iron atom of the enzyme.     

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.     

Albumin modifies the metabolism of hydroxyeicosatetraenoic acids via 12-lipoxygenase in human platelets

12-Lipoxygenase and cyclooxygenase 1 are the dominating enzymes that metabolize arachidonic acid in human platelets. In addition to the conversion of arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid, 12-lipoxygenase can also utilize 5(S)-hydroxyeicosatetraenoic acid and 15(S)-hydroxyeicosatetraenoic acid to form 5(S), 12(S)-dihydroxyeicosatetraenoic acid and 14(R), 15(S)-dihydroxyeicosatetraenoic acid, respectively. Furthermore, 15(S)-hydroxyeicosatetraenoic acid works as an inhibitor for 12-lipoxygenase. In the present paper we have studied the influence of albumin on the in vitro metabolism of 5 - and 15 -hydroxyeicosatetraenoic acids, and 5,15 -dihydroxyeicosatetraenoic acid by the platelet 12-lipoxygenase. The presence of albumin reduced the formation of 5(S),12(S)- dihydroxyeicosatetraenoic acid from 5(S)-hydroxyeicosatetraenoic acid, however, it had no effect on the 12(S)-hydroxyeicosatetraenoic acid production from endogenous arachidonic acid. In contrast, when 15(S)-hydroxyeicosatetraenoic acid was incubated with activated platelets, the formation of 14(R), 15(S)- dihydroxyeicosatetraenoic acid was stimulated by the presence of albumin. Furthermore, albumin reduced the inhibitory action 15(S)-hydroxyeicosatetraenoic acid had on 12(S)-hydroxyeicosatetraenoic acid formation from endogenous arachidonic acid. However, addition of exogenous arachidonic acid (20 microm) to the incubations inverted the effects of albumin on the conversion of 15(S)-hydroxyeicosatetraenoic acid to 14(R),15(S)- dihydroxyeicosatetraenoic acid and the production of 12(S)-hydroxyeicosatetraenoic acid in these incubations. Based on the Scatchard equation, the estimates of the binding constants to albumin were 1.8 x 10(5) for 15 -HETE, 1.4 x 10(5) for 12-HETE, and 0.9 x 10(5) for 5 -HETE respectively. These results suggest an important role of albumin for the regulation of the availability of substrates for platelet 12-lipoxygenase.     

Increase in the formation of leukotriene B4 and other lipoxygenase products in peritoneal macrophages of adrenalectomized rats

The effect of adrenalectomy on the formation of cyclooxygenase and lipoxygenase products by activated peritoneal rat macrophages was determined. After isolation, the cells were incubated with [1-14C]arachidonic acid and the calcium ionophore A23187 and the metabolites isolated by HPLC chromatography. The main components formed in the controls are 6-keto-prostaglandin F1 alpha, thromboxane B2 and 12-HETE. One peak represents 5,12-di-HETE. Smaller amounts of prostaglandin F2 alpha, prostaglandin E2, prostaglandin D2, leukotriene B4 and 15-HETE are also present. After adrenalectomy, a considerable increase occurs in the amounts of leukotriene B4, 15-HETE and 12-HETE. The increase in the prostaglandins is smaller. The compounds formed from endogenous arachidonic acid are also determined. In the cells of the controls, 6-keto-prostaglandin F1 alpha and thromboxane B2 are produced in higher amounts than leukotriene B4. After adrenalectomy, the formation of leukotriene B4 is much more increased than that of 6-keto-prostaglandin F1 alpha. These effects are most probably related to a diminished amount or inactivation of lipocortin, a glucocorticosteroid-induced peptide with phospholipase A2 inhibitory activity in adrenalectomized animals.     

Inhibition of leukotriene biosynthesis by acetylenic analogs

The monoacetylenic acid, 5,6-dehydroarachidonic acid (5,6-DHA), inhibits the 5-lipoxygenase in RBL-1 extracts in a time-dependent irreversible manner. In intact cell systems, 5,6-DHA is not as effective as ETYA or 15(S)-HEYA in inhibiting the 5-lipoxygenase activities, because 5,6-DHA is metabolized into triglycerides, phospholipids and hydroxylated products. While lipoxygenation of arachidonic acid at C-5 and C-12 is inhibited by 15-HETE, the transformation of arachidonic acid into 5,15-diHETE via 15-HPETE in human leukocytes is relatively insensitive to 15-HETE.     

Arachidonic acid metabolism in the murine eosinophil. III. Effect of nonsteroid anti-inflammatory drugs on lymphokine-directed eosinophil migration in vivo

In vitro studies of murine eosinophils indicated that lymphokine-stimulated metabolism of arachidonic acid by a lipoxygenase pathway was required for a migration response. In this study we tested the effects of drugs that affect arachidonic acid metabolism on lymphokine-dependent eosinophil accumulation in vivo by the use of 111In-labeled eosinophils. Indomethacin at different dosages either stimulated (1 mg/kg) or partially inhibited (5 to 25 mg/kg) eosinophil accumulation. Aspirin had no significant effects in doses up to 50 mg/kg. BW755C, a drug that inhibits both cyclooxygenase and lipoxygenase, dramatically inhibited eosinophil accumulation at 25 to 125 mg/kg. Pretreatment in vitro of 111In-labeled eosinophils with some drugs known to inhibit lipoxygenase reduced their subsequent accumulation at a lymphokine-injected site in vivo, but the high concentrations required for inhibition suggested that the effects of the drugs were at least partially reversible. Pretreatment with indomethacin did not inhibit the ability of 111In-labeled eosinophils to accumulate at the site of lymphokine injection in vivo. It may be anticipated from these results that drugs inhibiting lipoxygenase will have critical effects on manifestations of immunologic reactions that recruit eosinophils.     

Lipoxygenase products of arachidonic acid modulate biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human neutrophils via phospholipase A2

When human neutrophils, previously labeled in their phospholipids with [14C]arachidonate, were stimulated with the Ca2+-ionophore, A23187, plus Ca2+ in the presence of [3H]acetate, these cells released [14C]arachidonate from membrane phospholipids, produced 5-hydroxy-6,8,11,14-[14C]eicosatetraenoic acid (5-HETE) and 14C-labeled 5S,12R-dihydroxy-6-cis,8,10-trans, 14-cis-eicosatetraenoic acid ([14C]leukotriene B4), and incorporated [3H]acetate into platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Ionophore A23187-induced formation of these radiolabeled products was greatly augmented by submicromolar concentrations of exogenous 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), 5-HETE, and leukotriene B4. In the absence of ionophore A23187, these arachidonic acid metabolites were virtually ineffective. Nordihydroguaiaretic acid (NDGA) and several other lipoxygenase/cyclooxygenase inhibitors (butylated hydroxyanisole, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 1-phenyl-2-pyrazolidinone) caused parallel inhibition of [14C]arachidonate release and [3H]PAF formation in a dose-dependent manner. Specific cyclooxygenase inhibitors, such as indomethacin and naproxen, did not inhibit but rather slightly augmented the formation of these products. Furthermore, addition of 5-HPETE, 5-HETE, or leukotriene B4 (but not 8-HETE or 15-HETE) to neutrophils caused substantial relief of NDGA inhibition of [3H]PAF formation and [14C]arachidonate release. As opposed to [3H]acetate incorporation into PAF, [3H]lyso-PAF incorporation into PAF by activated neutrophils was little affected by NDGA. In addition, NDGA had no effect on lyso-PAF:acetyl-CoA acetyltransferase as measured in neutrophil homogenate preparations. It is concluded that in activated human neutrophils 5-lipoxygenase products can modulate PAF formation by enhancing the expression of phospholipase A2.     

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.     

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.     

Metabolism of exogenous arachidonic acid by mouse peritoneal macrophages

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

Hydroperoxide lyase in rabbit leukocytes: conversion of 15-hydroperoxyeicosatetraenoic acid to 15-keto-pentadeca-5,8,11,13-tetraenoic acid

Incubation of 15-HPETE with rabbit peripheral blood leukocytes resulted in the generation of 8,15-diHETE, 14,15-diHETE, 5,15-diHETE, 15-HETE and a polar metabolite with a retention time on RP-HPLC of 9.5 min, U.V. max at 280 nm. Reduction of this polar metabolite with NaBH4 shifted the U.V. max to 233 nm, suggesting the presence of a conjugated dienone system. Electron impact GC/MS analysis on the polar metabolite revealed a structure of a C-15 short chain aldehyde: 15-keto-pentadeca 5,8,11,13-tetraenoic acid. The formation of this new metabolite is proposed to be catalyzed by the enzyme hydroperoxide lyase. Thus, it is possible that the presence of hydroperoxide lyase activity in leukocytes not only provide a new mechanism for the transformation of hydroperoxides it also may provide a de novo protective effect by controlling the level of intracellular arachidonic acid derived hydroperoxides as well as further prevented their clastogenic action and cellular damage.