Regulation of macrophage eicosanoid production by hydroperoxy-and hydroxy-eicosatetraenoic acids.

Resident mouse peritoneal macrophages when exposed to zymosan during the first day of cell culture synthesize and secrete large amounts of prostaglandin E2 (PGE2) and leukotriene C4 (LTC4), the respective products of cyclo-oxygenase- and 5-lipoxygenase-catalysed oxygenations of arachidonic acid. Under these conditions of cell stimulation only small amounts of hydroxyeicosatetraenoic acids (HETEs) are concomitantly produced. However, exogenously added arachidonic acid is metabolized to large amounts of 12- and 15-HETE and only relatively small amounts of PGE2. No LTC4 is formed under these conditions. In contrast, resident mouse peritoneal macrophages in cell culture for 4 days synthesized less PGE2 and LTC4 when exposed to zymosan. However, these macrophage populations continue to synthesize 12-HETE from exogenously added arachidonic acid. Zymosan induced the secretion of a lysosomal enzyme, N-acetyl-beta-glucosaminidase, equally in both 1- and 4-day cultures. Both 12- and 15-hydroperoxyeicosatetraenoic acids (HPETEs), the precursors of 12- and 15-HETE, were found to be irreversible inhibitors of the cyclo-oxygenase pathway and reversible inhibitors of the 5-lipoxygenase pathway in macrophages. 15-HETE were found to be reversible inhibitors of both pathways. Thus the oxidation of arachidonic oxidation of arachidonic acid to both prostaglandins and leukotrienes may be under intracellular regulation by products of 12- and 15-lipoxygenases.     

Differential effects of docosahexaenoic acid and eicosapentaenoic acid on suppression of lipoxygenase pathway in peritoneal macrophages

Docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) was facilely incorporated into phospholipids of mouse peritoneal macrophages following incubation with pure fatty acids complexed to bovine serum albumin. Following stimulation with calcium ionophore A23187, the DHA-enriched cells synthesized significantly smaller amounts of leukotriene C4 and leukotriene B4 compared to control or EPA-enriched cells. The EPA-enriched cells synthesized lower amounts of leukotriene C4 and leukotriene B4 compared to control cells. The stimulated macrophages utilized endogenously released arachidonic acid for leukotriene B4 and leukotriene C4 synthesis. Exogenous arachidonic acid increased the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE and macrophages enriched with DHA or EPA produced similar amounts of 12-HETE and 15-HETE compared to control cells. These studies demonstrated that the synthesis of leukotriene C4, leukotriene B4 and HETE in macrophages is differentially affected by DHA and EPA.     

Opposite effects of adrenalectomy on eicosanoid release in rat peritoneal macrophages and spleen

The effect of adrenalectomy on the formation of cyclo-oxygenase and lipoxygenase products by activated peritoneal rat macrophages was determined and compared with that of the spleen. After isolation, the cells and tissues were incubated with [1-14C] arachidonic acid and the Ca-ionophore A23187 and the metabolites isolated by HPLC chromatography. The main components formed in the macrophages of the controls are 6-keto-PGF1 alpha, TxB2 and 12-HETE. One peak represents 5, 12 di HETE. Smaller amounts of PGF2 alpha, PGE2, PGD2, LTB4 and 15-HETE are also present. After adrenalectomy, a considerable increase occurs in the amounts of LTB4, 15-HETE and 12-HETE. The increase in the PG is smaller. The compounds formed from endogenous arachidonic acid are also determined. In the cells of the controls, the formation of LTB4 is considerably increased after adrenalectomy. In the spleen, PGD2 and 12-HETE are decreased after adrenalectomy. The effect of the macrophages is most probably related to a diminished amount or inactivation of lipocortin, a glucocorticosteroid induced peptide with PlA2 inhibitory activity in adrenalectomized animals. In the decrease in formation in the spleen, the absence of the permissive effect of glucocorticosteroids on the hormone-induced lipolysis may play a role.     

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.     

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.     

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.     

The mitogenic effect of 15- and 12-hydroxyeicosatetraenoic acid on endothelial cells may be mediated via diacylglycerol kinase inhibition

15-Hydroxyeicosatetraenoic acid (15-HETE), a major lipoxygenase metabolite of arachidonic acid in fetal bovine aortic endothelial cells, was a mitogen for these cells, stimulating both cell proliferation and DNA synthesis in the presence of serum and serum-deprived cells. In [14C]arachidonic acid-labeled confluent endothelial cell monolayers, 15-HETE (30 microM) caused an elevation of [14C]diacylglycerol (DAG) with a concomitant decrease in cellular [14C]phosphatidylinositol (PI) in both unstimulated and stimulated cells. 1-Oleoyl-2-acetylglycerol, a synthetic DAG analog, stimulated endothelial cell DNA synthesis in a concentration-dependent manner. In [3H]inositol-labeled cells, 15-HETE also caused a decrease in cellular PI content under both basal and stimulated conditions. 15-HETE, however, had no effect on either isolated phospholipase C activity or phosphoinositide turnover in lithium chloride-treated cells. In intact cells, 15-HETE (30 microM) inhibited the synthesis of [3H]PI from [3H]inositol (80% inhibition, p less than 0.001). In human red cell membranes, the production of phosphatidic acid from endogenous DAG was inhibited by 15-HETE in a concentration-dependent manner with an IC50 of 41 microM. Although 12-HETE had effects similar to those of 15-HETE, the parent compound arachidonic acid did not affect DNA synthesis or DAG kinase activity. Our study thus demonstrates that the mitogenic activity of 15- and 12-HETE on endothelial cells may be mediated via DAG kinase inhibition with the concomitant accumulation of cellular DAG.     

15(S)-hydroxyeicosatetraenoic acid is the major arachidonic acid metabolite in human bronchi: association with airway epithelium

15(S)-Hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) was by far the most abundant metabolite of arachidonic acid in chopped human bronchi, as identified by reverse phase HPLC, uv spectrometry, and GC/MS. The quantitation of monohydroxyeicosatetraenoic acids (mono-HETEs) was performed by the use of 16(S)-hydroxy-9(Z),12(Z),14(E)-heneicosatrienoic acid as internal standard. Thus, significant amounts of 15-HETE were obtained in incubations of bronchi in buffer alone, but the addition of exogenous arachidonic acid (3-100 microM), dose-dependently increased the formation, with maximal levels reached at around 10 min. In contrast, challenge with ionophore A23187 or anti-human IgE did not stimulate the production of 15-HETE in the bronchi. Nordihydroguaiaretic acid inhibited the production of 15-HETE, whereas indomethacin did not. Small amounts of 8,15-diHETEs were detected in incubations with exogenous 15H(P)ETE. Lipoxins were however not detected under any of the incubation conditions used. Furthermore, removal of the airway epithelium substantially diminished the production of 15-HETE in the bronchi. Finally, bronchi were obtained from three patients with asthma, and the amounts of 15-HETE in these specimens were significantly higher than those found in tissues from nonasthmatics. Also, in peripheral lung parenchyma and pulmonary blood vessels 15-HETE was the major mono-HETE after stimulation with arachidonic acid but the levels were about 10 times lower than in the bronchi. As another difference, challenge of the parenchyma with the ionophore A23187 made 5-HETE the predominant mono-HETE. Taken together, airway epithelium appears to be the major source of 15-HETE in the human lung and the findings in specimens of asthmatics raise the possibility that 15-HETE somehow is involved in airway inflammation.     

Synthesis of hydroxyeicosatetraenoic (HETEs) and epoxyeicosatrienoic acids (EETs) by cultured bovine coronary artery endothelial cells

Endothelial cells release several factors which influence vascular tone, leukocyte function and platelet aggregation. Some of these factors are metabolites of arachidonic acid, most notably prostacyclin. However, many of the endothelial metabolites of arachidonic acid have not been positively identified. The purpose of these studies is to identify the arachidonic acid metabolites synthesized by bovine coronary endothelial cells. Cultured bovine coronary artery endothelial cells were incubated with [ ¹⁴C]arachidonic acid. The incubation media was extracted and the radioactive metabolites resolved by a combination of reverse phase- and normal phase-high pressure liquid chromatography (HPLC). The cells synthesized 6-keto prostaglandin (PG)F_1α, PGE₂, 12-hydroxyheptadecatrienoic acid (HHT), 12-, 15-, and 11- hydroxyeicosatetraenoic acids (HETE), and 14,15-, 11,12-, 8,9-, and 5,6-epoxyeicosatrienoic acids (EET). Several of the HETEs were further analyzed by chiral-phase HPLC. The cells synthesized predominately 12(S)-, 15(S)-, and 11(R)-HETE. The synthesis of the S optical isomers of 12- and 15-HETE suggested that the 12- and 15-lipoxygenases were present in these cells. 11(R)-HETE is probably derived from cyclooxygenase. They also synthesized smaller amounts of 9-, 8- and 5-HETEs. The structures of the HETEs and EETs were confirmed by mass spectrometry. The release of 6-keto PGF_1α and 15-HETE was measured by specific radioimmunoassays. Melittin, thrombin, arachidonic acid and A23187 stimulated the release of both eicosanoids in a concentration-related matter. Under all conditions, the release of 6-keto PGF_1α exceed the release of 15-HETE. Therefore, cultured bovine coronary artery endothelial cells synthesize cyclooxygenase, lipoxygenase and cytochrome P-450 metabolites of arachidonic acid.     

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.     

Binding of 13-HODE and 5-, 12- and 15-HETE to endothelial cells and subsequent platelet, neutrophil and tumor cell adhesion

Some studies report that endothelial cells preferentially take up the lipoxygenase-derived arachidonic acid metabolite, 5-hydroxyeicosatetraenoic acid (5-HETE), released from stimulated leukocytes (polymorphonuclear leukocytes, PMNs), whereas others report that endothelial cells preferentially take up 12-HETE released from platelets. The biological relevance of these observations, however, is unknown. Recently, we and others have found that, under basal conditions, endothelial cells, PMNs and tumor cells metabolize linoleic acid via the lipoxygenase enzyme to 13-hydroxyoctadecadienoic acid (13-HODE). We propose that endogenous levels of these metabolites regulate blood-vessel wall cell adhesion. In this study, we have measured (1) the relative binding of 5-, 12- and 15-HETE, and 13-HODE to endothelial cell monolayers, and (2) their effects on endothelial cell adhesivity with platelets, PMNs and tumor cells. There was a dose-related and specific binding of 5-[3H]HETE to endothelial cells but no binding of 12- or 15-HETE or 13-HODE. Platelet or PMN adhesion to endothelial cells was unaffected by the 5-HETE binding, but tumor cell adhesion was blocked by 40% (P less than 0.01). Interestingly, preincubation of endothelial cells with 13-HODE, 12-HETE or 15-HETE decreased platelet adhesion to endothelial cells (P less than 0.05), even though these metabolites did not bind to the endothelial cells. We conclude that 5-HETE preferentially binds to endothelial cells and interferes with a specific receptor for tumor cells, whereas the other metabolites neither bind to cells nor affect cell adhesion.     

Enhancement of endogenous production of 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid in aortic smooth muscle cells by platelet-derived growth factor

Platelet-derived growth factor (PDGF) has a chemotactic effect on smooth muscle cells, which is inhibited by lipoxygenase inhibitor caffeic acid. In order to study the role of endogenous lipoxygenase products of arachidonic acid on the chemotactic action of PDGF, effects of PDGF on the lipoxygenase pathway in smooth muscle cells were examined. Lipoxygenase products were analyzed by high-performance liquid chromatography. 15-, 5- and 12-lipoxygenase activities, in order of magnitude, were found in smooth muscle cell homogenate. However, when the lipoxygenase products were analyzed using intact cells prelabelled with [14C]arachidonic acid, only 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) was found to be produced endogenously. In addition, 12-HETE was not released into the medium. Treatment of the cells with PDGF increased the endogenous production of 12-HETE. The amounts of intracellular 12-HETE in PDGF-treated cells were 126, 132 and 146% at 1, 3, and 10 hr's after the initiation of PDGF treatment, respectively, when control value at each time point was considered as 100%. Caffeic acid (10(-4) M) completely inhibited the PDGF effect on 12-HETE production. However, PDGF treatment did not significantly alter the 12-lipoxygenase activity. These results suggest that the stimulatory effect of PDGF on 12-HETE production was not mediated by the activation of 12-lipoxygenase activity. Since 12-HETE itself is a potent chemoattractant for smooth muscle cells, the present dat strongly suggest that 12-HETE could be an important intracellular mediator of the chemotactic action of PDGF on aortic smooth muscle cells.     

Opposite effects of adrenalectomy on eicosanoid release in rat peritoneal macrophages and spleen

The effect of adrenalectomy on the formation of cyclo-oxygenase and lipoxygenase products by activated peritoneal rat macrophages was determined and compared with that of the spleen. After isolation, the cells and tissues were incubated with [1-¹⁴C] arachidonic acid and the Ca-ionophore A23187 and the metabolites isolated by HPLC chromatography. The main components formed in the macrophages of the controls are 6-keto-PGF_1α, TxB₂ and 12-HETE. One peak represents 5, 12 di HETE. Smaller amounts of PGF_2α, PGE₂, PGD₂, LTB₄ and 15-HETE are also present. After adrenalectomy, a considerable increase occurs in the amounts of LTB₄, 15-HETE and 12-HETE. The increase in the PG is smaller. The compounds formed from endogenous arachidonic acid are also determined. In the cells of the controls, the formation of LTB₄ is considerably increased after adrenalectomy. In the spleen, PGD₂ and 12-HETE are decreased after adrenalectomy.The effect of the macrophages is most probably related to a diminished amount or inactivation of lipocortin, a glucocorticosteroid induced peptide with PlA₂ inhibitory activity in adrenalectomized animals. In the decrease in formation in the spleen, the absence of the permissive effect of glucocorticosteroids on the hormone-induced lipolysis may play a role.     

The 650-kDa 12(S)-hydroxyeicosatetraenoic acid binding complex: occurrence in human platelets, identification of hsp90 as a constituent, and binding properties of its 50-kDa subunit.

A cytosolic 650-kDa complex which binds 12(S)-hydroxy-5,8,10, 14-eicosatetraenoic acid (12(S)-HETE) with high affinity and specificity has been found in various cell lines but not until now in platelet cytosol. After incubation of human platelets with 12(S)-[3H]HETE, a labeled cytosolic 650-kDa complex was isolated. As previously shown for the binding complex in Lewis lung carcinoma (LLC) cells, ATP treatment transformed the platelet complex into a 50-kDa ligand-binding subunit. These results are of interest for two reasons: (a) 12(S)-HETE is a major arachidonic acid metabolite in platelets, and (b) platelets contain large amounts of the cell adhesion molecule GpIIb/IIIa, the activation of which is regulated by 12(S)-HETE. Hsp90 was found to be a component of the 12(S)-HETE binding complex in Lewis lung carcinoma cells, and the 50-kDa ligand-binding subunit itself bound 12(S)-HETE with high affinity. Competition experiments showed that 12(R)-HETE, 15-deoxy-Delta12, 14-prostaglandin J2, and 5(S)-HETE had lower affinity for the 50-kDa subunit than 12(S)-HETE. The 12(S)-HETE binding protein appears to be distinct from known members of the steroid hormone receptor superfamily of nuclear receptors.     

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.     

Arachidonic acid metabolism in guinea pig Langerhans cells: studies on cyclooxygenase and lipoxygenase pathways

Epidermal Langerhans cells are macrophage-like la+ leukocytes that are critically involved in cutaneous immune reactions. Because macrophages exert their immunoregulatory activity in part by generation of oxygenated arachidonic acid metabolites, we systematically studied arachidonic acid transformations by purified guinea pig Langerhans cells and compared them with mixed epidermal cells and Langerhans cell-depleted keratinocytes. Products formed from arachidonic acid by cell homogenates were measured after thin-layer or reverse-phase high-pressure liquid chromatographic separation. In addition, leukotriene B4 and C4 formation was assessed in supernatants of Ca ionophore A23187-challenged intact cells by radioimmunoassay. Mixed epidermal cells converted arachidonic acid predominantly via cyclooxygenase and 12-lipoxygenase pathways. The main products were prostaglandin D2 (PGD2) and 12-hydroxyeicosatetraenoic acid (12-Hete), although significant amounts of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha were formed as well. PGD2 synthesis was dependent on the presence of reduced glutathione. The product spectrum formed by Langerhans cell-depleted keratinocytes was virtually indistinguishable from mixed epidermal cells. In contrast, Langerhans cells showed a markedly different metabolism of arachidonic acid. They exhibited an exceedingly high PGD2-generating capacity, whereas only minor amounts of 12-HETE and very low amounts of other prostaglandins were synthesized. The PGD2/12-HETE ratio was 1.22 for mixed epidermal cells and 4.37 for Langerhans cells. Leukotriene production from exogenous or endogenous arachidonic acid could not be demonstrated by either radioenzymatic or radioimmunologic detection methods. We conclude that guinea pig Langerhans cells transform arachidonic acid predominantly to PGD2, which might mediate significant immunoregulatory, inflammatory, and antitumoral activity in the skin.     

Uptake, release and novel species-dependent oxygenation of arachidonic acid in human and animal airway epithelial cells

To determine identities of mediators and mechanisms for their release from pulmonary airway epithelial cells, we examined the capacities of epithelial cells from human, dog and sheep airways to incorporate, release and oxygenate arachidonic acid. Purified cell suspensions were incubated with radiolabeled arachidonic acid and/or ionophore A23187; fatty acid esterification and hydrolysis were traced chromatographically, and oxygenated metabolites were identified using high-pressure liquid chromatography and mass-spectrometry. In each species, cellular uptake of 10 nM arachidonic acid was concentrated in the phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine fractions, and subsequent incubation with 5 microM A23187 caused release of 10-12% of the radiolabeled pool selectively from phosphatidylcholine and phosphatidylinositol. By contrast, the products of arachidonic acid oxygenation were species-dependent and in the case of human cells were also novel: A23187-stimulated human epithelial cells converted arachidonic acid predominantly to 15-hydroxyeicosatetraenoic acid (15-HETE) and two distinct 8,15-diols in addition to prostaglandin (PG) E2 and PGF2 alpha. Cell incubation with exogenous arachidonic acid (2.0-300 microM) led to progressively larger amounts of 15-HETE and the dihydroxy, epoxyhydroxy and keto acids characteristic of arachidonate 15-lipoxygenase. Both dog and sheep cells converted exogenous or endogenous arachidonic acid to low levels of 5-lipoxygenase products, including leukotriene B4 without significant 15-lipoxygenase activity. In the cyclooxygenase series, sheep cells selectively released PGE2, while dog cells generated predominantly PGD2. The findings demonstrate that stereotyped esterification and phospholipase activities are expressed at uniform levels among airway epithelial cells from these species, but pathways for oxygenating arachidonic acid allow mediator diversity depending greatly on species and little on arachidonic acid presentation.     

Lipoxygenase Metabolism of Arachidonic Acid in Brain

When blood-free mouse brain slices were incubated with exogenous radiolabeled arachidonic acid, gas chromatography/mass spectrometry confirmed that the major radioactive lipoxygenase enzyme product of arachidonic acid was 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), with lesser amounts of 5-hydroxy-5,6,8,11,14-eicosatetraenoic acid and 15-hydroxy-5,8,11,13-eicosatetraenoic acid. When 12-[2H]HETE was used to measure endogenous 12-HETE in brain tissue frozen with liquid nitrogen, the level of 12-HETE was 41 +/- 6 ng/g of wet weight tissue. This frozen tissue level was not due to the presence of blood. When brain slices were incubated in vitro for 20 min, the 12-HETE level increased to 964 +/- 35 ng/g of wet weight tissue. Elimination of residual intravascular blood before tissue incubation reduced the brain slice 12-HETE concentration by one-half.     

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.     

E. coli hemolysin-induced lipid mediator metabolism in alveolar macrophages: impact of eicosapentaenoic acid

Escherichia coli hemolysin (HlyA) is a prototype of a large family of pore-forming proteinaceous exotoxins that have been implicated in the pathogenetic sequelae of severe infection and sepsis, including development of acute lung injury. In the present study in rabbit alveolar macrophages (AMs), subcytolytic concentrations of purified HlyA evoked rapid synthesis of platelet-activating factor, with quantities approaching those in response to maximum calcium ionophore challenge. In parallel, large quantities of leukotriene (LT) B(4) and 5-, 8-, 9-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE) were liberated from HlyA-exposed AMs depending on exogenous arachidonic acid (AA) supply. Coadministration of eicosapentaenoic acid (EPA) dose dependently suppressed generation of the proinflammatory lipoxygenase products LTB(4) and 5-, 8-, 9-, and 12-HETE in parallel with the appearance of the corresponding EPA-derived metabolites LTB(5) and 5-, 8-, 9-, and 12-hydroxyeicosapentaenoic acid (HEPE). At equimolar concentrations, EPA turned out to be the preferred substrate over AA for these AM lipoxygenase pathways, with the sum of LTB(5) and 5-, 8-, 9-, and 12-HEPE surpassing the sum of LTB(4) and 5-, 8-, 9-, and 12-HETE by >80-fold. In contrast, coadminstration of EPA did not significantly reduce HlyA-elicited generation of the anti-inflammatory AA lipoxygenase product 15-HETE. We conclude that AMs are sensitive target cells for HlyA attack, resulting in marked proinflammatory lipid mediator synthesis. In the presence of EPA, lipoxygenase product formation is shifted from a pro- to an anti-inflammatory profile.