Neutrophil aggregation and degranulation. Effect of arachidonic acid.

In response to aggregating and degranulating stimuli, platelets metabolize endogenous arachidonic acid to bioactive derivatives. These derivatives can stimulate platelets to degranulate and aggregate and, therefore, may be mediators of the platelet response. Because exogenous arachidonic acid also stimulates platelets to degranulate, aggregate, and form these mediators, we examined the effect of adding arachidonic acid to purified human neutrophil suspensions. Micromolar concentrations of arachidonic acid stimulated neutrophils to aggregate but not to degranulate. Cytochalasin B, a potentiator of neutrophil responses to chemotactic factors, also potentiated the arachidonic-acid-induced aggregation response; 5,8,11,14-eicosatetraynoic acid, an inhibitor of arachidonic acid metabolism, blocked this response. Aggregation of neutrophils, was not stimulated by several fatty acids with structural similarity to arachidonic acid. These results suggest that metabolic derivatives of arachidonic acid may be active in stimulating certain neutrophil responses. The role of these derivatives in mediating neutrophil responses to various stimuli needs to be examined.     

Arachidonic acid aggregates neutrophils

Arachidonic acid, but not several structurally similar fatty acids, stimulated neutrophils in suspension to aggregate; this effect was blocked by 5,8,11,14-eicosatetraynoic acid, an inhibitor of arachidonic acid metabolism. Analagous to platelets, arachidonate may be a precursor of active metabolites which mediate neutrophil responses.This work was supported in part by NIH grants AI-09651, HL-05474, HL-22437 and HL-07202.     

Involvement of bivalent cations and arachidonic acid in neutrophil aggregation

Chemotactic factors and arachidonic acid aggregate neutrophils; indomethacin and 5,8,11,14-eicosatetraynoic acid, two blockers of cellular arachidonate metabolism, inhibit these responses. Additionally, A23187, an ionophore which specifically transports bivalent cations into cells, also aggregates the neutrophils, and this response, as well as the response to chemotactic factors, requires the presence of extracellular calcium and magnesium. In this report these relationships were further studied. It was found that human neutrophil aggregation stimulated by arachidonic acid also required calcium and magnesium. Furthermore, cells preincubated with arachidonate for 4 min before exposure to the bivalent cations did not aggregate before or after this exposure and, following this exposure, were refractory to subsequent stimulation by more arachidonate, a chemotactic tripeptide (formylmethionylleucylphenylalanine), or A23187, i.e., these cells had become nonselectively desensitized to the aggregating agents. Finally, indomethacin and 5,8,11,14-eicoasatetraynoic acid inhibited the aggregation response stimulated by A23187 and their potency in doing so paralleled their potency in inhibiting the responses induced by arachidonate or the chemotactic tripeptide. Thus, the neutrophil aggregation responses induced by arachidonate, chemotactic factor, and A23187 were similarly influenced by preincubating the cells with arachidonate, had similar requirements for calcium and magnesium, and were similarly inhibited by blockers of arachidonate metabolism. It appears that bivalent cations and arachidonic acid play essential and, perhaps, interacting roles in the aggregation response to diverse stimuli.Publication 1877 of the Research Institute of Scripps Clinic, La Jolla, California 92037.Supported by research grants: A1 07007 (NIH); Office of Naval Research; Council for Tobacco Research.     

Release of arachidonic acid metabolites by human monocytes or lymphocytes: effect of treatment with interferon on stimulation by phorbol ester or calcium ionophore

The simultaneous production of prostaglandins, leukotrienes, and hydroxyeicosatetraenoic acids was studied in human peripheral blood monocytes obtained by counter-current centrifugal elutriation. Monocytes prelabeled for 4 hr with [3H]arachidonic acid (AA) released label into the surrounding medium in response to treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or calcium ionophore (A23187). High-performance liquid chromatography of monocyte supernatants demonstrated that labeled compounds included those which eluted with authentic standards for thromboxane B2, 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT), 6-keto-prostaglandin F alpha, prostaglandin E2, and 15-hydroxyeicosatetraenoic acid (HETE). 5-HETE and leukotriene B4 (LtB4) were detected only in response to ionophore treatment. Highly purified lymphocytes did not convert AA to autocoids, despite the release of free arachidonate in response to either stimulus. Pretreatment of monocytes with recombinant human interferon (IFN)-gamma or IFN-alpha for 18 hr resulted in enhanced release of labeled arachidonic acid and increased conversion to autocoids after TPA or ionophore stimulation. Absolute amounts of prostaglandin E2 produced in response to TPA or ionophore treatment were increased as well. These results demonstrate the autocoid profile released by stimulated human monocytes and illustrate the effects of IFN treatment on the production of lipoxygenase metabolites of arachidonic acid as well as cyclooxygenase products.     

Uptake and esterification of arachidonic acid by trophozoites of Giardia lamblia

Although trophozoites of Giardia lamblia have not been demonstrated to possess the capacity for synthesis of phospholipids, these protozoan parasites would be exposed to fatty acids within the human small intestine. We have evaluated the metabolic incorporation of arachidonic and palmitic acids by Giardia trophozoites. Trophozoites (2.25 X 10(6)) were incubated with 12 nM [3H]fatty acid for up to 60 min. Uptake of [3H]arachidonate by trophozoites was rapid, increasing from 37% at 1 min to 65% at 10 min. Uptake of palmitate was rapid but less extensive. In contrast to palmitate, almost all of the trophozoite-associated [3H]arachidonate was esterified into phospholipids and neutral lipids. By 1 and 60 min 37% and 82% of [3H]arachidonate, respectively, were incorporated into phospholipids, including phosphatidylinositol and phosphatidylcholine. Peak incorporation of [3H]arachidonate into phosphatidylcholine (30 mmol [3H]fatty acid (mol phospholipid)-1) occurred at 60 min; whereas incorporation into the pool of phosphatidylinositol, which accounted for only 4% of trophozoite phospholipid, was maximal at 10 min (190 mmol [3H]fatty acid (mol phospholipid)-1) and declined significantly thereafter as arachidonic acid was released from phosphatidylinositol. Therefore, Giardia trophozoites not only utilize exogenous fatty acids in the formation of glycerolipids but also preferentially incorporate arachidonic acid into a metabolically active pool of phosphatidylinositol.     

花生四烯酸及其衍生物对肿瘤坏死因子诱导的中性粒细胞超氧基释放的影响

目的:通过两种炎症介质、肿瘤坏死因子(TNFα)和花生四烯酸(arachidonic acid,AA)及其衍生物在人体外周血中性粒细胞产生超氧基的作用,研究其对控制炎症性疾病的发展的作用。方法:采用光泽精依赖性光释放法测定TNFα对高级脂肪酸及其衍生物激发中性粒细胞产生超氧基的能力。结果:TNFα预温细胞极大增强AA激发粒细胞产生超氧基,高级脂肪酸中多聚不饱和脂肪酸,如二十五碳五烯酸(eicosapentaenoic acid)、二十二碳六烯酸(docosahexaenoic acid)、亚麻酸(linoleic acid)、亚油酸(linolenic acid),以及单不饱和脂肪酸单不饱和脂肪酸油酸(oleic acid)也象AA一样受TNFα的影响。AA的羟化物(C_20:4,n-6 hydroxy)羟过氧化物(C_20:4,n-6 hydroperoxy)则不受TNFα的影响。结果还表明:分别是AA的脂氧化物和环氧合酶代谢产物的白三烯(LTB₄)和前列腺素E₂(PGE₂)是两种主要的炎症介质,前者激发粒细胞产生超氧基,后者则抑制。结论:各种免疫反应中起作用的介质联网的复杂性,不饱和脂质和细胞因子的相互影响,可能在疾病过程和免疫应答调节中起着重要作用。     

Arachidonic and eicosapentaenoic acid metabolism in bovine neutrophils and platelets: effect of calcium ionophore

Substitution of dietary fatty acids has potential for altering the inflammatory response. The purpose of the present study was to define the metabolites of arachidonic acid (AA) and eicosapentaenoic acid (EPA) secreted by bovine peripheral blood neutrophils and platelets. High performance liquid chromatography was used to characterize cyclooxygenase and lipoxygenase metabolites secreted in response to the calcium ionophore A23187. Cells were prelabelled with 3H-AA or 3H-EPA prior to challenge with the calcium ionophore. Bovine neutrophils secreted leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) as the major metabolites of AA, as well as the corresponding leukotriene B5 (LTB5) and 5-hydroxyeicosapentaenoic acid (5-HEPE) metabolites of EPA. Peptidoleukotrienes derived from 3H-AA or 3H-EPA were not detected under these conditions. The major tritiated metabolites secreted from bovine platelets were: thromboxane A2, measured as the stable metabolite thromboxane B2 (TXB2); hydroxyheptadecatrienoic acid (HHT) and 12-HETE derived from 3H-AA; and the omega-3 analogs TXB3 and 12-HEPE, derived from 3H-EPA. Preferred substrate specificities existed amongst the AA- and EPA-derived metabolites for the intermediary enzymes involved in the arachidonic acid cascade. These findings support the hypothesis that substitution of membrane-bound AA by EPA has potential for modulation of the host inflammatory response following cellular phospholipid mobilization.     

Rapid uptake and esterification of arachidonic acid and other fatty acids by microfilariae of Brugia malayi

We have examined the differential incorporation and esterification of exogenous fatty acids by microfilariae of the human filarial parasite Brugia malayi. Microfilariae incubated with 2 nM [3H]arachidonic acid over 1 h rapidly took up this fatty acid. Palmitic, oleic and linoleic acids were also incorporated by parasites. In contrast to these other fatty acids, little incorporated arachidonic acid remained as free fatty acid within microfilariae. Arachidonate was rapidly esterified into phospholipids, with 66% of incorporated arachidonate esterified into phospholipids at 1 min. Esterification of other fatty acids into phospholipids was quantitatively lesser and occurred into phosphatidylcholine and phosphatidylethanolamine. Arachidonate was preferentially esterified into phosphatidylinositol, which constituted only 10% of the total parasite phospholipid pool, and into phosphatidylcholine. By 1 min these two phospholipid classes, respectively, comprised 53% and 43% of [3H]arachidonyl-phospholipids. Neither the microfilarial incorporation of arachidonate nor its esterification into parasite phospholipids could be saturated by noncytotoxic concentrations of up to 600 microM. Microfilariae, which in vivo are exposed to arachidonate in blood, can rapidly, avidly and with high capacity incorporate exogenous arachidonate and esterify it preferentially into specific classes of phospholipids, including phosphatidylinositol. Like many mammalian cells, these phylogenetically distinct metazoan parasites possess efficient means for utilizing host-derived arachidonic acid.     

Activation of neutral sphingomyelinase in human neutrophils by polyunsaturated fatty acids.

Although unesterified polyunsaturated fatty acids (PUFA) have been shown to elicit marked changes in neutrophil function, the associated signal transduction processes require clarification. In this study we examined the effect of PUFA on the sphingomyelin (SM)-signalling cycle in human neutrophils. Treatment of neutrophils with eicosatetraenoic acid [arachidonic acid, 20:4(n-6)] caused a decrease in the mass of cellular SM and an increase in the level of ceramide. 20:4(n-6)-stimulated neutral sphingomyelinase (SMase) activity of the leucocytes in a time- and concentration-dependent manner. Other unsaturated fatty acids, docosahexaenoic [22:6(n-3)], eicosapentaenoic [20:5(n-3)], octadecenoic [oleic, 18:1(n-9)] and octadecadienoic [linoleic, 18:2(n-6)] acids also had the capacity to activate neutral SMase; however, certain 20:4(n-6) derivatives ¿20:4(n-6) methyl ester [20:4(n-6)ME], 15-hydroperoxyeicosatetraenoic (15-HPETE) and 15-hydroxyeicosatetraenoic (15-HETE) acids¿, very-long-chain PUFA ¿tetracosatetraenoic [24:4(n-6)] and octacosatetraenoic [28:4(n-6)] acids¿ and saturated fatty acids [octadecanoic (stearic, 18:0) and eicosanoic (arachidic, 20:0) acids] had no significant effect. Activation of neutral SMase by 20:4(n-6) appeared to involve metabolism via 20:4(n-6)CoA (arachidonoyl CoA) and was not dependent on prostaglandin and leukotriene synthesis. All of the fatty acids and derivatives tested failed to activate acidic SMase of neutrophils. Ceramide was found to inhibit 20:4(n-6)-induced superoxide generation by the cells. It is envisaged that the PUFA-induced ceramide production in neutrophils plays a role in the regulation of biological responses.     

Pasteurella haemolytica leukotoxin enhances production of leukotriene B4 and 5-hydroxyeicosatetraenoic acid by bovine polymorphonuclear leukocytes.

The influence of the leukotoxin of Pasteurella haemolytica on the generation of arachidonic acid metabolites by bovine polymorphonuclear leukocytes (PMNs) was investigated. PMNs released 5-, 12-, and 15-hydroxyeicosatetraenoic acids (5-, 12-, and 15-HETE) and leukotriene B4 (LTB4) upon stimulation with arachidonic acid. The leukotoxin preparations dose dependently enhanced the release of the 5-lipoxygenase products 5-HETE and LTB4 in arachidonic acid-stimulated PMNs, whereas the release of 12- and 15-HETE was not affected. The enhanced release of LTB4 and 5-HETE was not due to a decreased cellular retention of the 5-lipoxygenase products. In addition, leukotoxin preparations by themselves were also able to induce LTB4 and 5-HETE production in the absence of exogenous arachidonic acid. Generation of 5-lipoxygenase products by PMNs stimulated by leukotoxin may represent an important cellular event that occurs during infections with P. haemolytica.     

Arachidonate 12-lipoxygenase and cyclooxygenase: PGE isomerase are predominant pathways for oxygenation in bovine tracheal epithelial cells

The capacity of bovine tracheal epithelial cells to convert arachidonic acid to oxygenation products with potential biologic activity was studied in homogeneous preparations of isolated cells. Purified epithelial cell suspensions were incubated with radiolabeled arachidonic acid, and oxygenated metabolites were identified using high-pressure liquid chromatography and gas chromatography-mass spectrometry. The cells released predominantly two products during incubation with 0.3 to 150 microM arachidonic acid for 1 to 60 min at 37 degrees C: prostaglandin E2 (PGE2) and 12-hydroxyeicosatetraenoic acid (12-HETE). Concentration-response curves for the two products yielded half-maximal effects at 2 and 45 microM arachidonic acid, respectively. Stereochemical analysis by chiral-phase high-pressure liquid chromatography demonstrated that the epithelial 12-HETE consisted exclusively of the 12(S) isomer, providing supporting evidence that it was derived from an arachidonate 12-lipoxygenase. Epithelial cells prelabeled with arachidonic acid and incubated with 5 microM A23187 to stimulate endogenous arachidonic acid metabolism also released two predominant products with the chromatographic properties of PGE2 and 12-HETE. The findings demonstrate that bovine tracheal epithelial cells express both a cyclooxygenase:PGE isomerase and a 12-lipoxygenase pathway and therefore implicate this pathway as a new source of epithelial cell mediators.     

Single derivatization method for routine analysis of bacterial whole-cell fatty acid methyl esters, including hydroxy acids.

Analysis of fatty acid methyl esters prepared from whole-cell bacteria by sodium hydroxide hydrolysis and boron trichloride-catalyzed methylation showed degradation of hydroxy acid peaks after several injections on a fused silica capillary column. A simple base wash of the extracts before injection prevented the tailing of the hydroxy acid peaks even after extended use. This eliminates the need to form trifluoroacetic anhydride derivatives of the hydroxy acids.     

Virus-induced enhancement of arachidonate metabolism by bovine alveolar macrophages in vitro

Virus infection of alveolar macrophages both in vivo and in vitro has been associated with a variety of changes in cellular function. Some of these changes are identical to the effects that arachidonate-derived mediators, prostaglandins, leukotrienes, and hydroxyeicosatetraenoic acids, have on macrophage function. Virus infection of macrophages has been previously shown to increase the output of some arachidonate metabolites, most notably PGE2. However, the effect of virus infection on arachidonate metabolism in general has not been well described. In our experiments, primary cultures of alveolar macrophages obtained from normal cattle by bronchoalveolar lavage, were infected in vitro with parainfluenza type 3 virus. At days 0 to 4 post-infection (p.i.) these cells were labelled with 3H-arachidonic acid and stimulated with either serum-coated zymosan, the calcium ionophore A23187, or phorbol myristate acetate. The complete spectrum of arachidonate-derived metabolites was determined by reverse-phase high performance liquid chromatography with UV and on-line radiometric monitoring of column eluant. The total output of metabolites of arachidonic acid by virus-infected alveolar macrophages was increased over that of noninfected controls (with all stimuli tested) by day 4 p.i. (P less than or equal to 0.05). The production of metabolites by the cyclooxygenase, 12- and 5-lipoxygenase enzyme systems was significantly increased, as was the release of 3H-arachidonate. The lack of stimulus specificity and the increases in arachidonate release suggest that greater substrate availability, due either to increased phospholipase activity or direct virus-membrane interaction, may be responsible for the virus-induced enhancement of metabolite output.     

Second Messenger Function of Arachidonic Acid Lipoxygenation Products in Human Natural Killer Cell Lysis?

Human natural killer (NK) cells were tested in the presence of several fatty acid oxygenase inhibitors such as nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid, 3-amino-1-m-(trifluoromethyl)-phenyl-2-pyrazoline (BW 755C), and indomethacin. All drugs inhibited NK lysis at the post-target cell-binding level at concentrations that also suppressed lipoxygenation of arachidonic acid, suggesting that such reactivity may be required for effector cell triggering. NDGA gave a 50% NK cell inhibition in the range of 10-30 microM and also suppressed antibody-dependent and lectin-dependent systems. Further evidence of the involvement of arachidonic acid lipoxygenation was found as NK activity could be reconstituted to NDGA-suppressed effector cells with several metabolites such as LTB4, LTB4 analogues, and hydroxyeicosatetraenoic acids lipoxygenated at C-5, C-12, and C-15. Cyclic nucleotides such as cGMP and cAMP could also reconstitute activity with optimal effects at approximately 10(-8) M. The combined evidence is compatible with a model for triggering lysis in which lipoxygenation products have a second messenger function. Whether arachidonic acid lipoxygenation is necessary for effector cells at all different activation/differentiation stages and whether the lipoxygenation products activate guanylic cyclase, protein kinase C, or some other target molecule remain to be further investigated.     

Effect of dietary enrichment with eicosapentaenoic and docosahexaenoic acids on in vitro neutrophil and monocyte leukotriene generation and neutrophil function

The effects of dietary fish-oil fatty acids on the function of the 5-lipoxygenase pathway of peripheral-blood polymorphonuclear leukocytes and monocytes were determined in seven normal subjects who supplemented their usual diet for six weeks with daily doses of triglycerides containing 3.2 g of eicosapentaenoic acid and 2.2 g of docosahexaenoic acid. The diet increased the eicosapentaenoic acid content in neutrophils and monocytes more than sevenfold, without changing the quantities of arachidonic acid and docosahexaenoic acid. When the neutrophils were activated, the release of [3H]arachidonic acid and its labeled metabolites was reduced by a mean of 37 per cent, and the maximum generation of three products of the 5-lipoxygenase pathway was reduced by more than 48 per cent. The ionophore-induced release of [3H]arachidonic acid and its labeled metabolites from monocytes in monolayers was reduced by a mean of 39 per cent, and the generation of leukotriene B4 by 58 per cent. The adherence of neutrophils to bovine endothelial-cell monolayers pretreated with leukotriene B4 was inhibited completely, and their average chemotactic response to leukotriene B4 was inhibited by 70 per cent, as compared with values determined before the diet was begun and six weeks after its discontinuation. We conclude that diets enriched with fish-oil-derived fatty acids may have antiinflammatory effects by inhibiting the 5-lipoxygenase pathway in neutrophils and monocytes and inhibiting the leukotriene B4-mediated functions of neutrophils.     

Bacterial challenge stimulates formation of arachidonic acid metabolites by human keratinocytes and neutrophils in vitro.

Although the interactions of bacteria with keratinocytes induce the synthesis of various mediators, the capability of epithelial cells to form arachidonic acid mediators has not been studied, and therefore the first part of this study was initiated. The complex mixture of epithelium-derived mediators suggests that chemoattraction is not their only effect on neutrophils and that they may also affect neutrophil mediator synthesis. The effect of epithelium-derived mediators on neutrophil eicosanoide synthesis was evaluated in the second part of this study. We incubated human keratinocytes with human-pathogenic bacteria for 2 h and harvested the supernatants after 4, 6, 10, and 18 h of culture. Subsequently, the supernatants were coincubated for 5 min with human neutrophils with or without arachidonic acid. The formation of the arachidonic acid metabolites prostaglandin E(2) (PGE(2)), leukotriene B(4) (LTB(4)), 12-hydroxyeicosatetraenoic acid (12-HETE), and 15-HETE in keratinocytes and neutrophils was measured by reverse-phase high-pressure liquid chromatography. We demonstrated for the first time that keratinocytes produced significant amounts of LTB(4) and 12-HETE 4 to 6 h after bacterial challenge. Upon stimulation with epithelial supernatants, neutrophils produced significant amounts of PGE(2), LTB(4), 12-HETE, and 15-HETE throughout the observation period of 18 h, with a maximum synthesis by supernatants harvested 4 to 10 h after bacterial infection. The results of the study suggest that arachidonic acid mediator formation by epithelial cells following bacterial challenge may act as an early inflammatory signal for the initiation of the immune response. The epithelial supernatants were capable of inducing the formation of arachidonic acid mediators by neutrophils, which may have further regulatory effects on the immune response.     

Production of arachidonic and linoleic acid metabolites by guinea pig tracheal epithelial cells

Pulmonary epithelial cells may be responsible for regulating airway smooth muscle function, in part by release of fatty acid-derived mediators. Incubation of isolated guinea pig tracheal epithelial cells with radiolabeled arachidonic acid (AA) leads to the production of 5- and 15-hydroxyeicosatetraenoic acid (5- and 15-HETE) and smaller amounts of leukotriene (LT) B₄ and C₄ and 12-hydroxyheptadecatrienoic acid (HHT). Epithelial cells also are able to release linoleic acid (LA) metabolites. Incubation with radiolabeled linoleic acid leads to the formation of 9- and 13-hydroxyoctadecadienoic acid (9- and 13-HODE). The biological significance of these mediators produced by epithelial cells is discussed.     

Bacterial Challenge Stimulates Formation of Arachidonic Acid Metabolites by Human Keratinocytes and Neutrophils In Vitro

Although the interactions of bacteria with keratinocytes induce the synthesis of various mediators, the capability of epithelial cells to form arachidonic acid mediators has not been studied, and therefore the first part of this study was initiated. The complex mixture of epithelium-derived mediators suggests that chemoattraction is not their only effect on neutrophils and that they may also affect neutrophil mediator synthesis. The effect of epithelium-derived mediators on neutrophil eicosanoide synthesis was evaluated in the second part of this study. We incubated human keratinocytes with human-pathogenic bacteria for 2 h and harvested the supernatants after 4, 6, 10, and 18 h of culture. Subsequently, the supernatants were coincubated for 5 min with human neutrophils with or without arachidonic acid. The formation of the arachidonic acid metabolites prostaglandin E₂ (PGE₂), leukotriene B₄ (LTB₄), 12-hydroxyeicosatetraenoic acid (12-HETE), and 15-HETE in keratinocytes and neutrophils was measured by reverse-phase high-pressure liquid chromatography. We demonstrated for the first time that keratinocytes produced significant amounts of LTB₄ and 12-HETE 4 to 6 h after bacterial challenge. Upon stimulation with epithelial supernatants, neutrophils produced significant amounts of PGE₂, LTB₄, 12-HETE, and 15-HETE throughout the observation period of 18 h, with a maximum synthesis by supernatants harvested 4 to 10 h after bacterial infection. The results of the study suggest that arachidonic acid mediator formation by epithelial cells following bacterial challenge may act as an early inflammatory signal for the initiation of the immune response. The epithelial supernatants were capable of inducing the formation of arachidonic acid mediators by neutrophils, which may have further regulatory effects on the immune response.     

Metabolism of arachidonic acid in rabbit alveolar macrophages

Rabbit alveolar macrophages incorporated radioactive arachidonic acid primarily into phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and triglycerides. When these cells, so labelled, were washed and treated with polystyrene beads, there was a distinct and reproducible decrease in the radioactivity of phosphatidylcholine, phosphatidylethanolamine, and triglyceerides. This decrease was accounted for by the appearance of two major prostaglandins, PGE₂ and PGF_2a, and uncharacterized hydroxy fatty acids, which may be 12-hydroxy-5,8,10,14-eicosatetraenoic acid, 5-hydroxy-6,8,11, 14-eicosatetraenoic acid, and/or 8-hydroxy-9,11,14-eicosatrienoic acid.     

Production of 15-Hydroxyeicosatetraenoic Acid by Purified Human Eosinophils and Neutrophils

In the presence of high concentrations of exogenous arachidonic acid (greater than or equal to 10 microM), eosinophils produced 15-hydroxyeicosatetraenoic acid (15-HETE) in the absence of stimuli. The calcium ionophore A23187, as well as the chemotaxins used in this study--complement split product C5a, platelet-activating factor (PAF), and N-formyl-methionyl-leucyl-phenylalanine (FMLP)--failed to increase 15-HETE production, indicating that eosinophil 15-lipoxygenase is already active. Production of 15-HETE from eosinophils increased with increasing concentrations of arachidonic acid, exogenously added. Maximal 15-HETE production was observed to be 1111 +/- 380 ng per 10(6) eosinophils at the concentration of 100 microM of arachidonic acid. With low concentrations of exogenous arachidonic acid (below 2 microM), eosinophils were considered to incorporate exogenous arachidonic acid into their cell membrane, and did not produce 15-HETE. In contrast, 15-HETE formation in highly purified neutrophils (eosinophils less than 1%) was negligible compared with that in eosinophils (300-fold less), suggesting that 15-HETE-forming activity in granulocytes is derived from the eosinophil 15-lipoxygenase pathway and that neutrophils may lack 15-lipoxygenase activity.