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.     

Selective feed-back inhibition of the 5-lipoxygenation of arachidonic acid in human T-lymphocytes

Purified human T-lymphocytes exhibit 5-lipoxygenase activity as demonstrated by the conversion of arachidonic acid to 5-hydroxy-eicosatetraenoic acid (5-HETE), 5(S),12(R)-di-hydroxy-eicosa-6,14 cis-8,10 trans-tetraenoic acid (leukotriene B₄), and 5,12-di-HETE isomers of leukotriene B₄ that lack a 6-cis double bond. The concentrations of leukotriene B₄, 5-HETE, 11-HETE and 15-HETE in suspensions of T-lymphocytes were increased significantly by concanavalin A and by the calcium ionophore A23187. Preincubation of T-lymphocytes with 15-HETE at μM concentrations, characteristic of suspensions of stimulated lymphocytes, inhibited selectively the increases in the levels of 5-HETE and leukotriene B₄, but not of 11-HETE and prostaglandin E₂.     

Characterization of leukotriene A4 and B4 bioysnthesis

We have studied LTA₄ and LTB₄ synthesis in a cell-free system from RBL-1 cells. All the enzymes leading to the formation of LTB₄ from arachidonic acid are localized in the soluble fraction (100, 000 x g supernatant) of these cells. The formation of LTA₄ and LTB₄ is complete by 10 min. When we varied the arachidonic acid concentration from 1 to 300 μM, the synthesis of LTB₄ leveled off at 30 μM and of LTA₄ at 100 μM while 5-HETE had not reached a plateau at 300 μM. This enzyme system has the capacity to generate relatively large amounts of 5-HETE and LTA₄ and only a relatively small amount of LTB₄. Therefore, the rate limiting step is not the 5-lipoxygenase, the first step in the pathway, but the conversion of LTA₄ to LTB₄. This is in contrast to cyclooxygenase pathway where the first step is rate limiting. A second addition of arachidonic acid at submaximal concentration for LTA₄ synthesis did not produce any additional LTA₄ or LTB₄. Further study of this phenomenon showed that the 5-lipoxygenase and LTA-synthase were inactivated with time by preincubation with arachidonic acid and that peroxy fatty acids seem to be the inactivating species.     

5-Lipoxygenase from rat PMN lysate

The products of arachidonic acid metabolism in the 15,000xg supernatant of sonicated rat PMN are described. Only products derived from 5-lipoxygenase are observed. These products are 5-HETE and products derived from hydrolysis of LTA₄, particularly LTB₄. Some minor products derived from decomposition of 5-HPETE are also observed. The dependence of the activity of 5-lipoxygenase on enzyme and on substrate concentrations is presented and discussed in terms of a kinetic model that includes enzyme inactivation during turnover and substrate inhibition. The 5-lipoxygenase activity is stimulated by Ca⁺⁺ and ATP.     

Fatty acids induce leukotriene C4 synthesis in macrophages in a fatty acid binding protein-dependent manner

Obesity results in increased macrophage recruitment to adipose tissue that promotes a chronic low-grade inflammatory state linked to increased fatty acid efflux from adipocytes. Activated macrophages produce a variety of pro-inflammatory lipids such as leukotriene C₄ (LTC₄) and 5-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE) suggesting the hypothesis that fatty acids may stimulate eicosanoid synthesis. To assess if eicosanoid production increases with obesity, adipose tissue of leptin deficient ob/ob mice was analyzed. In ob/ob mice, LTC₄ and 12-HETE levels increased in the visceral (but not subcutaneous) adipose depot while the 5-HETE levels decreased and 15-HETE abundance was unchanged. Since macrophages produce the majority of inflammatory molecules in adipose tissue, treatment of RAW264.7 or primary peritoneal macrophages with free fatty acids led to increased secretion of LTC₄ and 5-HETE, but not 12- or 15-HETE. Fatty acid binding proteins (FABPs) facilitate the intracellular trafficking of fatty acids and other hydrophobic ligands and in vitro stabilize the LTC₄ precursor leukotriene A₄ (LTA₄) from non-enzymatic hydrolysis. Consistent with a role for FABPs in LTC₄ synthesis, treatment of macrophages with HTS01037, a specific FABP inhibitor, resulted in a marked decrease in both basal and fatty acid-stimulated LTC₄ secretion but no change in 5-HETE production or 5-lipoxygenase expression. These results indicate that the products of adipocyte lipolysis may stimulate the 5-lipoxygenase pathway leading to FABP-dependent production of LTC₄ and contribute to the insulin resistant state.     

Arachidonate 15-lipoxygenase in human corneal epithelium and 12- and 15-lipoxygenases in bovine corneal epithelium: Comparison with other bovine 12-lipoxygenase

Lipoxygenases of bovine and human corneal epithelia were investigated. The bovine epithelium contained an arachidonate 12-lipoxygenase and a 15-lipoxygenase. The 12-lipoxygenase was found in the microsomal fraction, while the 15-lipoxygenase was mainly present in the cytosol (100 000 × g supernatant). 12S-Hydroxyeicosatetraenoic acid (12S-HETE) and 15S-hydroxyeicosa-tetraenoic acid (15S-HETE) were identified by GC-MS and chiral HPLC. BW A4C, an acetohydroxamic acid lipoxygenase inhibitor, reduced the biosynthesis of 12S-HETE and 15S-HETE by over 90% at 10 μ M. IC₅₀ for the 12-lipoxygenase was 0.3 μM. The bovine corneal 12-lipoxygenase was compared with the 12-lipoxygenases of bovine platelets and leukocytes. All three enzymes metabolized ¹⁴C-labelled linoleic acid and α-linolenic acid poorly (5–16%) in comparison with [^l4C]arachidonic acid. [¹⁴C]Docosahexaenoic acid and [¹⁴C]4,7,10,13,16-docosapentaenoic acid appeared to be less efficiently converted by the corneal enzyme than by the platelet and leukocyte enzymes. Immunohistochemical analysis of the bovine corneal epithelium using a polyconal antibody against porcine leukocyte 12-lipoxygenase gave positive staining. The cytosol of human corneal epithelium converted [¹⁴C]arachidonic acid to one prominent metabolite. The product co-chromatographed with 15S-HETE on reverse phase HPLC, straight phase HPLC and chiral HPLC. Our results suggest that human corneal epithelium contains a 15-lipoxygenase and that bovine corneal epithelium contains both a 15-lipoxygenase and a 12-lipoxygenase. The corneal 12-lipoxygenase appears to differ catalytically from earlier described bovine 12-lipoxygenases.     

A human epithelial cell line, intestine 407, can produce 5-hydroxyeicosatetraenoic acid and leukotriene B4

The present study was carried out to further characterize the role of non-inflammatory cells in the inflammatory process. More specifically, we have investigated whether human epithelial cells can generate inflammatory lipid mediators via activation of the 5-lipoxygenase pathway. The cells were stimulated with the calcium ionophore A23187 (5 μM) for different periods of time, after which the production of eicosanoids was determined by gradient reverse-phase high performance liquid chromatography (RP-HPLC) and rapid spectral detection, permitting continuous ultraviolet spectroscopy. In both non-prelabeled cells and cells prelabeled with [1-^14Carachidonic acid, cell stimulation for 30 min or more resulted in the production of two important 5-lipoxygenase products: 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B₄ (LTB₄). Stimulation for 15 min or less, however, led solely to the formation of 5-HETE. The identities of 5-HETE and LTB₄ were confirmed by HPLC retention times and UV spectra, as well as by gas chromatography-mass spectrometry for 5-HETE and radioimmunoassay for LTB₄. It can therefore be concluded that human epithelial cells in general can produce important inflammatory mediators, which suggests that epithelial cells may play a more active role in the inflammatory process than is normally assumed.     

A calcium-independent 5-lipoxygenase system in mast/basophil PT-18 cells

Mammalian 5-lipoxygenase systems exist in inactive or cryptic states and have to be stimulated in order to metabolize exogenous [14C]arachidonic acid to 5-HETE and leukotrienes. In most cells, both the activation process and the 5-lipoxygenase activity are calcium-dependent. However, the cryptic 5-lipoxygenase system in the murine PT-18 mast/basophil cell line, which can be stimulated by 15-hydroxyeicosatetraenoic acid (15-HETE), is unusual. Studies with fura-2 loaded PT-18 cells indicate that increases in cytosolic calcium do not appear to correlate with enhanced 5-lipoxygenase product formation. Thus, both the calcium ionophore ionomycin and arachidonic acid increase cytosolic calcium levels but have very little effect on [14C]5-HETE formation, whereas 15-HETE induces large increases in [14C]5-HETE production but no concomitant enhancement in cytosolic calcium is observed. Chelation of extracellular calcium by 3 mM EGTA resulted in a 30-40% inhibition of [14C]5-HETE formation induced by 15 HETE, whereas 3 mM EGTA has no appreciable effect on a crude PT-18 5-lipoxygenase homogenate. These results indicate that in PT-18 cells, calcium does not appear to play an important role in either the 15-HETE-induced activation process, or the enzymatic activity of the cryptic 5-lipoxygenase system.     

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.     

Ionophore-stimulated lipoxygenase activity and histamine release in a cloned murine mast cell, MC9

A cloned murine mast cell line designated MC9 expresses a 5-lipoxygenase activity when stimulated with the ionophore A23187. Upon addition of 0.5 uM ionophore, MC9 cells produce 270 ± 43 pmoles 5-HETE, 74 ± 40 pmoles 5,12 di HETEs and 65 ± 31 pmoles LTC₄/10⁶ cells from 37 uM exogenously added [1-¹⁴C]arachidonic acid in two minutes. 5-HETE and 5,12-di HETES, including LTB₄ were identified by GC/MS whereas LTC₄ was confirmed by HPLC mobility, bio-assay, RIA and enzymatic transformation. The principal cyclooxygenase products were PGD₂ and TxB₂ (8.5 ± 2.4 and 5.4 ± 1.2 pmoles/10⁶ cells respectively). Prostanoids were identified by comigration with authentic standards on two-dimensional thin layer chromatograms. Production of arachidonic acid lipoxygenase metabolites stimulated with ionophore proved relatively insensitive to removal of extracellular Ca⁺² and chelation by EGTA. In addition, MC9 5-lipoxygenase required only low micromolar amounts of exogenous arachidonic acid for maximal activity. Whereas production of arachidonic acid metabolites lasted only two to five minutes, histamine release stimulated with ionophore was not initiated until 5 minutes (12 ± 3% cellular histamine) and continued for 30 minutes (37 ± 7% cellular histamine). Although these cells metabolize arachidonic acid differently from the classic peritoneal-derived mast cell, they resemble subpopulations found in certain tissues (such as mucosa) and should be useful in understanding the biochemistry of mast cell mediator release.     

Tumor necrosis factor alpha stimulates arachidonic acid metabolism in human osteoblastic osteosarcomal cells

The effects of tumor necrosis factor alpha (TNF-α) on arachidonic acid (AA) metabolism were investigated by prelabeling the human osteoblastic osteosarcoma cell line, G292, with [³H]AA. TNF-α differentially stimulates cyclooxygenase and lipoxygenase pathways of AA metabolism in a dose response manner in the cells. The highest concentration of TNF-α (10⁻⁸ M) significantly increased the cyclooxygenase pathway, with prostaglandin E₂ (PGE₂) being a major product. However, at the lowest concentration (10⁻¹⁰ M) of TNF-α, 15-hydroxyeicosatetraenoic acid (HETE) production was significantly increased, with no significant effects on the other identifiable products. When the concentration of TNF-α was increased to 10⁻⁹ M leukotriene B₄ (LTB₄), 15-, 12-, and 5-HETE were significantly increased. The calcium ionophore A23187 (10⁻⁶ M) significantly increased 15-HETE production, without significantly affecting cyclooxygenase metabolites. However, a combination of TNF-α (10⁻⁸ M) and A23187 (10⁻⁶ M) caused an inhibitory effect on each agent-induced PGE₂ or 15-HETE production.     

Endogenous hydroxyeicosatetraenoic acids stimulate the human polymorphonuclear leukocyte 15-lipoxygenase pathway

Arachidonic acid metabolism in ionophore A23187-activated human polymorphonuclear leukocytes (PMNs) proceeds predominantly via the 5-lipoxygenase pathway in comparison to metabolism by the 15-lipoxygenase route. Products of both lipoxygenase pathways appear to be involved in the mediation of inflammatory reactions. Pretreatment of polymorphonuclear leukocytes with micromolar amounts of the platelet-derived 12-lipoxygenase product 12-hydroxy-5,8,10,14- eicosatetraenoic acid (12-HETE) prior to the addition of A23187 and [14C]arachidonic acid resulted in the unexpected dose-dependent stimulation of the 15-lipoxygenase pathway, as evidenced by the formation of [14C]15-HETE. A concomitant inhibition of the 5-lipoxygenase pathway was also observed. The structural identity of 15-HETE was confirmed by retention times on straight-phase and reverse-phase high pressure liquid chromatography in comparison with an authentic standard, radioimmunoassay, and chemical derivatization. When other isomeric HETEs were tested, the order of stimulatory potencies was 15-HETE greater than 12-HETE greater than 5-HETE. When arachidonic acid metabolism via the 5-lipoxygenase route was inhibited by nordihydroguaiaretic acid, previously ineffective concentrations of exogenous 12-HETE were now able to stimulate the polymorphonuclear leukocyte 15-lipoxygenase. Thus, blockade of the 5-lipoxygenase pathway appeared to be a prerequisite for the activation of the 15-lipoxygenase. The HETE-induced activation of the 15-lipoxygenase occurred within 1-2 min, was a reversible process, and was enhanced in the presence of A23187. In nine donors tested, up to 14-fold stimulation of [14C]15-HETE production was observed. Our results indicate that endogenous HETEs can have a dual role in the post-phospholipase regulation of arachidonic acid metabolism since they can act as physiological stimulators of the 15-lipoxygenase as well as inhibitors of the 5-lipoxygenase.     

A kinetic scheme of human neutrophil 5-lipoxygenase activity

In animal cells arachidonic acid is metabolized via the 5-, 12- and 15-lipoxygenase pathways. The kinetic mechanism of action of plant (soya) and animal (reticulocyte) 15-lipoxygenases is now well established. 5-Lipoxygenase possesses, in all probability, the most complex mechanism of activity regulation. At present several effectors of neutrophil 5-lipoxygenase, both cytosolic and membrane-bound ones, have been identified. The molecular and kinetic mechanisms of action of the enzyme are still open to question. A kinetic scheme of regulation of synthesis of arachidonic acid 5-lipoxygenase metabolites which does not exclude the presence of two binding sites on the enzyme molecule, is proposed. Within the framework of this kinetic scheme the enzyme activator complex may be the active form of the enzyme. There is evidence that the curve for the time dependence of 5-HETE accumulation in neutrophils stimulated by the Ca2+ ionophore A23187 has a maximum, while the corresponding curve for the LTB4 accumulation is a curve with saturation. It was shown that an increase in the concentration of exogenous arachidonate induces the synthesis of 5-HETE, whereas the concentration of LTB4 remains practically unchanged. The results of mathematical analysis of the above kinetic scheme and a comparison of experimental and calculated values suggest that the reaction effector, Ca2+, plays a crucial regulatory role in the observed kinetic dependencies reflecting the formation of two sequential products of 5-lipoxygenase oxidation of arachidonate. In this way Ca2+ strongly influences the first step of the reaction, i.e., 5-HETE formation; its effect on the second reaction step (5-HETE conversion into LTA4) is far less apparent.     

Modulation of the 5-lipoxygenase activity of MC-9 mast cells: Activation by hydroperoxides

In order to identify regulatory steps in leukotriene synthesis, the biochemical characteristics of a 5-lipoxygenase activity in the 100,000 xg supernatant from sonicates of cells of an IL-3 dependent murine mast cell clone, MC-9 were determined. Principal products from exogenous ¹⁴C-arachidonic acid were identified as leukotriene B₄, diastereomeric 5,12-dihydroxy-eicossatetraenoic acids (5.12 diHETEs) 5-hydroperoxy and hydroxyeicosatetraenoic acids (5-HPETE and 5-HEYE) as well as a novel metabolite 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). The lipoxygenase activity had a pH optimum of 6.9 and was highly dependent upon added Ca⁺⁺. The effective Ca⁺⁺ concentration for 50 per cent activation (EC₅₀) was 3 uM. Activity was also stimulated by ATP (EC₅₀ = 160 uM). The cytosolic 5-lipoxygenase activity exhibited a biphasic concentration dependence for arachidonic acid with maximum product formation occurring at 35 uM (ca. 20 nmole/mg/4 min). The lipoxygenase activity exhibited apparent lag phase kinetics which were more pronounced at low protein concentrations (0.3 mg/ml). In addition, the lag phase was greatly accentuated by the addition of a hydroperoxide scavenging system consisting of glutathione (1 mM) plus glutathione peroxidase (0.4 unit/ml). In contrast, addition of any several hydroperoxides, i.e. 5-,8-,9- or 15-HPETE (EC₅₀ ca. 1 uM), but not the corresponding alcohols (5-HETE and 15-HETE), shortened the lag phase. These results show that the 5-lipoxygenase requires hydroperoxide for activation and that cellular level of hydroperoxides may be an important factor regulating leukotriene synthesis.     

12-Hydroxyeicosatetraenoic acid reduces prostacyclin production by endothelial cells

12-Hydroxyeicosatetraenoic acid (12-HETE), a lipoxygenase product released by activated platelets and macrophages, reduced prostacyclin (PGI₂) formation in bovine aortic endothelial cultures by as much as 70%. Maximal inhibition required 1 to 2 h to occur and after 2 hr, a concentration of 1 μM 12-HETE produced 80% of the maximum inhibitory effect. 5-HETE and 15-HETE also inhibited PGI₂ formation. The inhibition was not specific for PGI₂; 12-HETE reduced the formation of all of the radioactive eicosanoids synthesized from [1-¹⁴C]arachidonic acid by human umbilical vein endothelial cultures. Inhibition occurred in the human cultures when PGI₂ formation was elicited with arachidonic acid, ionophore A23187 or thrombin. These findings suggest that prolonged exposure to HETEs may compromise the antithrombotic and vasodilator properties of the endothelium by reducing its capacity to produce eicosanoids, including PGI₂.     

Ability of 15-hydroxyeicosatrienoic acid (15-OH-20:3) to modulate macrophage arachidonic acid metabolism

Mouse peritoneal macrophages metabolize dihomogammalinolenic acid (20:3n-6) primarily to 15-hydroxy-8,11,13-eicosatrienoic acid (15-OH-20:3). Since the biological properties of this novel trienoic eicosanoid remain poorly defined, the effects of increasing concentrations of 15-OH-20:3 and its arachidonic acid (20:4n-6) derived analogue. 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), on mouse macrophage 20:4n-6 metabolism were investigated. Resident peritoneal macrophages were prelabeled with [3H]-20:4n-6 and subsequently stimulated with zymosan in the presence of either 15-OH-20:3 or 15-HETE (1-30 microM). After 1 hr, the radiolabeled soluble metabolites were analyzed by reverse phase high performance liquid chromatography. 15-OH-20:3 inhibited zymosan-induced leukotriene C4 (IC50 = 2.4 microM) and 5-HETE (IC50 = 3.1 microM) synthesis. In contrast to the inhibition of macrophage 5-lipoxygenase, 15-OH-20:3 enhanced 12-HETE synthesis (5-30 microM) and had no measurable effect on cyclooxygenase metabolism (1-10 microM) i.e., 6-keto-prostaglandin F1 alpha and prostaglandin E2 synthesis. Addition of exogenous 15-HETE produced similar effects. These results suggest that the manipulation of macrophage 15-OH-20:3n-6 levels may provide a measure of cellular control over 20:4n-6 metabolism, specifically, leukotriene production.     

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.     

The identification of hydroxy fatty acids in psoriatic skin

Psoriasis is a common chronic inflammatory and proliferative skin disease characterised by epidermal neutrophil infiltration which may be induced by chemotactic substances in the involved epidermis. Superficial psoriatic scale was shown to contain biologically active amounts of leukotriene B₄ and monohydroxy-eicosatetraenoic acid (HETE)- like material as determined by assay for chemokinetic activity in high performance liquid chromatography (HPLC) fractions of scale extracts. Extracts of scale and chamber fluid from abraded lesional and uninvolved psoriatic skin were purified by HPLC and appropriate fractions were analysed by gas chromatography - mass spectrometry (GC-MS). The following monohydroxy metabolites of arachidonic, linoleic and 11,14-eicosadienoic acids were identified : 15-HETE, 12-HETE, 11-HETE, 9-HETE, 8-HETE, 5-HETE, 13-hydroxy-octadecadienoic acid (13-HODD), 9-HODD and 15-hydroxy-eicosadienoic acid (15-HEDE). The results suggested that 12-HETE, 13-HODD and 9-HODD are the most abundant monohydroxy fatty acids in the psoriatic skin extracts described above. Assays of 13-HODD, 9-HODD and 15-HEDE for chemokinetic activity were negative with concentrations up to 10^?4M. The biological significance of these three compounds in not known, but some of the hydroxylated metabolites of arachidonic acid may, by virtue of their chemotactic properties, be relevant to the pathogenesis of the psoriatic neutrophil infiltrate.     

Structural requirements in hydroxyeicosanoids for the activation of the 5-lipoxygenase in PT-18 mast/basophil cells

We have previously reported that 15-hydroxyeicosatetraenoic acid (15-HETE) stimulated the 5-lipoxygenase in the murine PT-18 mast/basophil cell line to produce leukotriene B4 and 5-HETE from exogenously added arachidonic acid. In order to determine the structural requirements in the HETE molecule that are necessary for the activation of this 5-lipoxygenase, various isomeric HETEs, derivatives and analogs were prepared, purified and tested. The order of stimulatory potencies was: 15-HETE acetate greater than 15-HETE = 15-hydroperoxyeicosatetraenoic acid (15-HPETE) greater than 5-HPETE = 12-HPETE greater than 5-HETE. 15-HETE methyl ester, 12-HETE and prostaglandin E2 were ineffective over the concentration range tested. Several diHETEs were also tested. 5S,15S-DiHETE was somewhat less potent than 15-HETE, whereas both 8S,15S-diHETE and leukotriene B4 were inactive. The calcium ionophore A23187 was much less effective than 15-HETE. These structure-activity studies indicate the importance of the nature, position and location of the various functional groups in the HETE molecule and suggest that a specific recognition site is involved in the activation of the 5-lipoxygenase in PT-18 cells.     

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.