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.     

Lipoxygenase Pathway of Arachidonic Acid Metabolism in Growth Control of Tumor Cells of Different Type

The influence of inhibitors of different lipoxygenases (LOX) on the growth of human tumor cells with different profiles of synthesized eicosanoids was studied. The studied LOX inhibitors had virtually no influence on the growth of A549 cells actively synthesizing cyclooxygenase and lipoxygenase metabolites of arachidonic acid (AA). The inhibitor of 12-LOX, baicalein, significantly inhibited proliferation in cultures of A431 epidermoid carcinoma cells with a characteristic domination of the major lipoxygenase metabolite of AA, 12-hydroxyeicosatetraenoic acid (12-HETE), in the profile of synthesized eicosanoids and reduced to 70% the incorporation of [3H]thymidine into DNA. Treatment of these cultures with 12-HETE virtually restored the growth potential of the tumor cells. The findings suggest that the lipoxygenase metabolite of AA, 12-HETE, is a growth-limiting factor for tumor cells of definite type.     

A role for lipoxygenase metabolites of arachidonic acid in porcine ovulation

Prostaglandins, products of arachidonic acid via the cyclooxygenase pathway, are essential to the porcine ovulatory process in that inhibition of their synthesis results in ovulation failure. Studies in the rat have shown that ovulation is also preceded by a rise in three ovarian hydroxyeicosatetraenoic acids, products of the lipoxygenase pathway, and inhibition of this pathway also inhibits ovulation. Experiments were designed, using a pregnant mare serum gonadotropin/human chorionic gonadotropin (hCG)-treated prepuberal gilt model, to measure pre-ovulatory changes in follicular fluid concentrations of 15-hydroxyeicosatetraenoic acid (15-HETE), and to compare the effects of indomethacin and nordihydroguaiaretic acid (NDGA) on ovulation in the pig and on 15-HETE and prostaglandin F₂α synthesis both in vivo and in vitro. Follicular fluid concentrations of 15-HETE were elevated significantly just prior to the expected time of ovulation (40 h after hCG). When indomethacin (10 mg) was injected into the ovarian stalk at 24 h after hCG, follicular fluid concentrations of both 15-HETE and prostaglandin F₂α were lower (P<0.01) than controls at 40 h and ovulation rate was suppressed (P<0.01). When NDGA (5 mg) was administered in the same manner, ovulation rate was suppressed (P<0.01), but the levels of 15-HETE and prostaglandin F₂α were not altered. Synthesis of 15-HETE by cultured granulosa and theca interna cells was reduced by the presence of NDGA (1 mg/ml), whereas indomethacin (100 ng/ml) lowered 15-HETE production in theca interna cells only. These results clearly demonstrate that indomethacin can block the lipoxygenase as well as the cyclooxygenase pathways, depending on the dose used, and suggest that lipoxygenase metabolites of arachidonic acid are involved in the ovulatory process in the pig.     

Effects of lipoxygenase metabolites of arachidonic acid on the growth of human mononuclear marrow cells and marrow stromal cell cultures.

The effects of various lipoxygenase metabolites of arachidonic acid (AA) were investigated on the growth of freshly isolated human bone marrow mononuclear cells and marrow stromal cell cultures. LTB4, LXA4, LXB4, 12-HETE and 15-HETE (1 microM) decreased [3H]-thymidine incorporation on marrow stromal cell cultures without affecting cell number. Only 12-HETE showed a dose-response effect on [3H]-thymidine incorporation. While LTB4 (1 microM) decreased thymidine incorporation on marrow mononuclear cells, LTC4, LXA4, LXB4, 12-HETE and 15-HETE had no effect. The lipoxygenase inhibitor NDGA had no effect on both cell types suggesting no role of endogenous lipoxygenase metabolites on cell growth. These results suggest no important role of lipoxygenase metabolites of AA on the proliferation of human marrow mononuclear cells and marrow stromal cell cultures.     

Lipoxygenase- and cyclooxygenase-reaction products and incorporation into glycerolipids of radiolabeled arachidonic acid in the bovine retina

The metabolism of radiolabeled arachidonic acid (AA) by the intact bovine retina has been studied. Synthesis of prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs), and incorporation of AA into glycerolipids has been measured by reverse-phase and straight-phase high performance liquid chromatography with flow scintillation detection, and by thin-layer chromatography. AA was actively acylated into glycerolipids, particularly triglycerides, phosphatidylcholine and phosphatidylinositol. AA was also converted to the major PGs, PGF_2α, PGE₂, PGD₂, 6-keto-PGF_1α and TXB₂, and to the lipoxygenase reaction products, 12-HETE, 5-HETE, and other monohydroxy isomers. Approximately 6% of the radiolabeled AA was converted to eicosanoids. The synthesis of HETEs was inhibited in a concentration-dependent manner (IC₅₀ = 8.3 NM) by nordihydroguaiaretic acid (NDGA). PG synthesis was inhibited by aspirin (10 μM), indomethacin (1 μM) and NDGA (IC₅₀ = 380 nM). Metabolism of AA via lipoxygenase, cyclooxygenase and activation-acylation was inhibited by boiling retinal tissue prior to incubation. These studies demonstrate an active system for the uptake and utilization of AA in the bovine retina, and provide the first evidence of lipoxygenase-mediated metabolism of AA, resulting in the synthesis of mono-hydroxyeicosatetraenoic acids, in the retina.     

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.     

Arachidonic acid metabolim in rat pancreatic acinar cells: Calcium-mediated stimulation of the lipoxygenase system

Isolated rat pancreatic acini were employed to demonstrate that the exocrine pancreas can metabolize [¹⁴C]-arachidonic acid by way of the lipoxygenase pathway as well as the cyclooxygenase pathway. Analysis by high performance liquid chromtography delineated a monohydroxy acid, presumably 12-L-hydroxy-5,8–10,14-eicosatetraenoic acid (12-HETE) as the major lipoxygenase product. The formation of this hydroxy arachidonic derivative was stimulated by the calcium ionophore ionomycin. Stimulation of lipoxygenase pathway by ionomycin was confirmed by thin layer chromatography. In addition, 6-keto-PGF_1α, PGF_2α, and PGE₂ were identified; and ionomycin, carbamylcholine, and caerulein enhanced the formation of these metabolites of the cyclooxygenase pathway. Ionomycin induced stimulation of HETE formation was inhibited by ETYA and nordihydroguaiaretic acid, but spontaneous and evoked enzyme secretion was unaffected. Thus, although ionomycin, a pancreatic secretagogue, stimulates the lipoxygenase pathway, the precise role of these arachidonate metabolites in the physiology of the exocrine pancreas is still obscure.     

The effects of indomethacin, NDGA, allopurinol and superoxide dismutase on prostaglandin E2 and leukotriene C4 levels after mesenteric ischemia-reperfusion injury

In this study, the changes of arachidonic acid metabolites after an ischemia-reperfusion (I/R) period are investigated. The cyclooxygenase and lipoxygenase metabolites were found to be significantly increased after a 45 min period of ischemia followed by 5 min of reperfusion. Prostaglandin E₂ (PGE₂)- and leukotriene C₄ (LTC₄)-like activities did not change in the ischemic period, but they both increased after reperfusion. A cyclooxygenase inhibitor indomethacin and lipoxygenase inhibitor nordehydroguaretic acid (NDGA) decreased PGE₂- and LTC₄-like activities, respectively, while allopurinol and superoxide dismutase (SOD) decreased both activities.According to our results, it can be assumed that free oxygen radicals are responsible for the elevation of PGE₂- and LTC₄-like activities and both of these arachidonic acid metabolites and free oxygen radicals are the main necrotizing agents in ischemia-reperfusion induced damage.     

Prostaglandin production in cultured gastric mucosal cells: Role of cAMP on its modulation

The effect of cAMP on prostaglandin production may depend on cell types. To clarify the relationship between PG and cAMP, we examined arachidonate's effects on PG synthesis and intracellular cAMP accumulation in monolayers of rat gastric mucosal cells. These cells produced PGE₂, PGI₂ and thromboxaneA₂ (TXA₂) in amounts of 316±18, 100±7 and 30±5 pg per 10⁵ cells in 10 min, respectively, in response to 10μM arachidonic acid (AA). The production of these PG, however, leveled off subsequently. Cells initially exposed to AA responded poorly to a subsequent stimulation by AA. AA simultaneously stimulated intracellular cAMP accumulation; this stimulatory effect on cAMP production was abolished by the pretreatment with indomethacin. Nevertheless, the pretreatments with dibutyryl cAMP (0.1–5mM) did not alter the amount of subsequent AA-induced PGE₂ production. Furthermore, the preincubation with 1mM isobutyl methyl xanthine also failed to affect PGE₂ synthesis, while it increased intracellular cAMP accumulation. Our studies suggest (1) AA stimulates intracellular cAMP formation in cultured gastric mucosal cells, linked with conversion of AA to cyclooxygenase metabolites, (2) AA-induced PG production is limited in these cells, and (3) it seems, however, unlikely that intracellular cAMP modulates AA metabolism to PG.     

12-Hydroperoxyeicosatetraenoic acid (12-HPETE) and 15-HPETE stimulate melatonin synthesis in rat pineals

The role of arachidonic acid metabolites in norepinephrine (NE)-induced N-acetyltransferase (NAT) activity and melatonin release was examined from 6 h-incubations of rat pineal glands. A cyclooxygenase inhibitor, indomethacin (5×10⁻⁸ − 5×10⁻⁶ M) was ineffective on melatonin release, in the presence of absence of NE (5×10⁻⁶ M) while a lipoxygenase inhibitor, nordihydroguaiaretic acid (5×10⁻⁷ −5×10⁻⁵ M) had an inhibitory effect. Among the lipoxygenase metabolites, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 15-HPETE stimulated both NAT activity and melatonin release in a dose-dependent manner, with a maximal effect occuring at 10⁻⁶ M, while 5-HPETE or hydroxy derivatives of these compounds (12-HETE, 15-HETE and 5-HETE) were ineffective. These results indicate that 12-HPETE and 15-HPETE can be involved in NE-induced melatonin release.     

Signal transduction by bFGF, but not TGF beta 1, involves arachidonic acid metabolism in endothelial cells.

We investigated the stimulation of early cellular events resulting from the interaction of the growth factor basic FGF (bFGF) and of the growth inhibitor transforming growth factor beta-type 1 (TGFβ1), with their specific receptors on bovine endothelial cells. At mitogenic concentrations, bFGF stimulated the rapid release of arachidonic acid and its metabolites from (³H)-arachidonic acid labeled cells. When arachidonic acid metabolism was stimulated by addition of the calcium ionophore A23187, the effect of bFGF was amplified. Nordihydroguaïaretic acid, an inhibitor of the lipoxygenase pathway of arachidonic acid metabolism, decreased the mitogenic effect of bFGF, whereas indomethacin, an inhibitor of the cyclooxygenase pathway, was ineffective. These findings suggest that metabolism of arachidonic acid to lipoxygenase products may be necessary for the mitogenic effect of bFGF. Basic FGF did not stimulate the production of inositol phosphates from cells labelled with myo-(2-³H)-inositol nor did it induce calcium mobilization, as measured by fura-2 fluorescence, indicating that bFGF does not activate phosphoinositide specific phospholipase C in endothelial cells, but rather, that bFGF-induced arachidonic acid metabolism is mediated by another phospholipase. TGFβ1, which inhibits basal and bFGF-induced endothelial cell growth, had no effect on arachidonic acid matabolism and inositol phosphate formation and did not prevent bFGF-induced arachidonic acid metabolism. These results suggest that the inhibitory action of TGFβ1 on endothelial cell growth occurs through different mechanisms.     

Regulation of T-lymphocyte mitogenesis by the leukocyte product 15-hydroxy-eicosatetraenoic acid (15-HETE)

Synthesis of lipoxygenase metabolites of [¹⁴C]arachidonic acid by mouse spleen lymphocyte cultures was inhibited by the leukocyte product 15-hydroxy-eicosatetraenoic acid (15-HETE) in a dose-dependent manner. In parallel experiments, the influence of 15-HETE on mitogenesis in spleen lymphocyte cultures was examined. 15-HETE at concentrations similar to those which inhibited cellular lipoxygenases progressively inhibited mitogenesis induced by the T-cell mitogen PHA but had no significant effect on the mitogenic response to the B-cell mitogen LPS. The inhibitory response was maximal when 15-HETE was added within 8 hr of exposure to PHA. Several analogs of 15-HETE having progressively fewer double bonds were tested in the same systems. 15-OH,20:3 had approximately the same potency as 15-HETE in inhibiting both mitogenesis and formation of metabolites from [¹⁴C]arachidonic acid. 15-OH, 20:2 and 15-OH,20:0 were much less active in either assay. Mitogenesis, induced in spleen cell cultures by the tumor promoter phorbol myristate acetate, was also blocked by 15-HETE. These experiments indicate that lipoxygenase metabolites of arachidonic acid may play an important role in T-lymphocyte blastogenesis and suggest that 15-HETE, via its ability to selectively inhibit cellular lipoxygenases, may function as an endogenous regulator of T-lymphocyte responses.     

Metabolism of exogenous arachidonic acid by murine macrophage-like tumor cell lines

Murine macrophage-like cell lines, J774.2, P388D1, RAW264.7 and PU-5-1R, were incubated with exogenous arachidonic acid (AA). The major metabolites were identified by comigration with known standards in TLC and HPLC and by characteristic behavior following reduction. During a 30 min incubation J774.2 cells metabolized exogenous ¹⁴C-AA (10 μM) to PGE₂ (14.8%), 12-hydroxy-5,8,10-heptadecatrienoic acid (HTT)_ (13.0%), thromboxane B₂ (TXB₂) (7.4%), PGD₂ (4.4%) and PGF_2α (3.0%). The remainder was incorporated into phospholipids (39.0%), triglycerides (6.1%), and as yet unidentified metabolites (8.2%). No PGF_1α was found. Metabolism of exogenous AA was rapid, being >90% completed at 3.5 min. Metabolism of exogenous AA is not increased by the simultaneous addition of macrophage stimuli including the cation ionophore A-23187, particulate phagocytic stimuli and endotoxin. The synthesis of cyclooxygenase products was inhibited by low doses of indomethacin (ID₅₀=0.6 μM) while the synthesis of TXB₂ and HHT was selectively inhibited by benzylimidazole (ID₅₀=9.5 μM). Identification of a probable lipoxygenase product is being pursued. The synthesis of this product is not inhibited by indomethacin and migrates with an R_f value close to 5,12-diHETE in TLC. P388D1 and RAW264.7 cells metabolize exogenous AA to the same products as J774.2, in different proportions, while PU-5-1R does not produce cylooxygenase metabolites to any appreciable extent.     

Bio-synthesis of PGD2 and TXB2 by rabbit blood monocytes

A method for the preparation of a highly purified sample of rabbit blood monocytes is described. The metabolism of arachidonic acid (AA) in these cells was studied. Mononuclear cells were prepared by centrifugation on Ficoll-Paque gradients and the monocytes were obtained by further centrifugation and adherence onto plastic culture dishes. These procedures provided a preparation which contained 95% monocytes (non-specific esterase positive). Incubation of [1-¹⁴C]-AA with these cells produced four major metabolites which were separated by TLC; these corresponded to prostaglandin (PG) D₂, thromboxane (TX) B₂, 12-hydroxyheptadecatrienoic acid (HHT) and 12-/15- hydroxyeicosatetraenoic acid (HETE). A minor product which co-migrated with PGE₂ was also detected but neither 6-keto-PGF_1α nor PGF_2α were detected. Also, there was no evidence of the formation of 5-lipoxygenase products (5-HETE and LTB₄) by rabbit monocytes with or without calcium-ionophore A23187-stimulation. The production of PGD₂, TXB₂ and PGE₂ was further confirmed by analyzing [³H]-AA metabolites using high-performance liquid chromatography (HPLC) with tritiated standards as references. The biosynthesis of these compounds from endogenous substrate in A23187-stimulated monocytes was confirmed by specific radioimmunoassays with or without prior HPLC separation. The synthesis of immunoreactive LTB₄ and LTC₄ by A23187-stimulated cells was also monitored and found to be relatively low. The synthesis of PGD₂, TXB₂ and PGE₂ from both exogenous and endogenous substrate was suppressed by treatment of the monocytes with indomethacin (10⁻⁶ M).     

Gonadotropin releasing hormone activates the lipoxygenase pathway in cultured pituitary cells: role in gonadotropin secretion and evidence for a novel autocrine/paracrine loop.

The formation and role of arachidonic acid (AA) and its metabolites during gonadotropin releasing hormone- (GnRH-) induced gonadotropin secretion were investigated in primary cultures of rat pituitary cells. Prelabeled cells ([3H]AA) responded to GnRH challenge with increased formation (about 2-fold) of the leukotrienes LTC4, LTD4, and LTE4 as well as 5- and 15-eicosatetraenoic acids (5- and 15-HETE) as identified by HPLC. Formation of leukotrienes and 15-HETE was further verified by specific radioimmunoassays. No significant increase in the formation of 12-HETE or of the cyclooxygenase products prostaglandin E (PGE) and thromboxane A2 by GnRH was noticed. Addition of physiological concentrations of LTC4 enhanced basal LH release, while subphysiological concentrations of LTC4 (10(-15)-10(-12) M) inhibited GnRH-induced LH release by about 35% (p less than 0.02). Using specific lipoxygenase inhibitors L-656,224 and MK 886, we found inhibition of GnRH-induced LH release by about 40% at concentrations known to specifically inhibit the 5-lipoxygenase pathway. The peptidoleukotriene receptor antagonist ICI 198,615 inhibited LTC4- and LTE4-induced LH release and surprisingly also the effect of GnRH on LH release by 40%. The data strongly suggest a role for AA and its lipoxygenase metabolites in the on/off reactions of GnRH upon LH release. The data also present a novel amplification cycle in which newly formed leukotrienes become first messengers and establish an autocrine/paracrine loop.     

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.     

Endogenous prostaglandin E2 enhances polyclonal immunoglobulin production by tonically inhibiting T suppressor cell activity

The immunoregulatory effect of endogenous prostaglandin E₂ (PGE₂) on immunoglobulin production was studied in an in vitro culture system of human peripheral blood mononuclear cells, stimulated with pokeweed mitogen (PWM). Three different cyclooxygenase inhibitors (indomethacin, carprofen, and piroxicam) suppressed Ig synthesis by ~50%. This inhibitory effect could be reversed by adding low doses of exogenous PGE₂ (3 × 10^?9 to 3 × 10^?8M). These doses are endogenously produced in PWM-stimulated cultures, and a concentration of 2 × 10^?8M is reached after 48 hr of culture. When B cells were directly stimulated with helper factor, PGE₂ did not enhance Ig production and doses of 3 × 10^?7M to 3 × 10^?6M were inhibitory. The effects of indomethacin and PGE₂ were eliminated when T cells were irradiated or treated with mitomycin prior to culture. The enhancing effects of PGE₂ were substantially reduced after OKT8(+) T cells were removed from the system. PWM-stimulated cultures of lymphocytes from healthy subjects over age 70 were more sensitive to inhibition by indomethacin and to stimulation by PGE₂ than were cultures of lymphocytes from young controls. Thus the major role of endogenous PGE in polyclonal Ig production in vitro is to tonically inhibit a radiosensitive, OKT8(+) suppressor T cell. This tonic inhibition is increased in subjects over 70, which provides one explanation for decreased suppressor cell function in elderly subjects.     

Arachidonic acid metabolites and colchicine in Behçet's disease (BD)

Vasculitis is accepted to be the basis of Behçet's disease (BD) which is a multisystem disease, and the arachidonic acid(AA) metabolites acting as balancing mediators in the organism are accepted to be responsible for the vasculitis.In this study, we examined the prostaglandin E₂ (PGE₂) and leukotriene C₄ (LTC₄) levels of the patients with BD before and after colchicine therapy. We found a statistical decrease in the PGE₂ and LTC₄ levels after colchicine therapy compared to the previous levels, concluding that colchicine inhibits the inflammation and the polymorphonuclear leukocyte (PML) chemotaxis by inhibiting the cyclooxygenase and lipoxygenase patways.     

Interaction of human neutrophils with airway epithelial cells: Reduction of leukotriene B4 generation by epithelial cell derived prostaglandin E2

Airway epithelial cells (AEC) play an active role in the regulation of inflammatory airway disease. In the present study we analyzed the interaction of AEC with polymorphonuclear leukocytes (PMN) in coincubation with respect to their arachidonic acid (AA) metabolism using reversed phase-HPLC and post-HPLC-ELISA. Primary cultures of porcine AEC released predominantly PGE₂, PGF_2a, and 15-hydroxyeicosatetraenoic acid (15-HETE), whereas the major human PMN-derived AA metabolite was the chemotactic factor leukotriene B₄ (LTB₄). In AEC-PMN cocultures stimulated with the calcium ionophore A23187, PMN-related 5-lipoxygenase products were decreased by 45%. This reduction in LTB₄ formation in the presence of AEC was mainly due to PGE₂ generated by the epithelial cells, whereas 15-HETE made a minor contribution. Most of the effect was inhibited by AEC pretreatment with acetylsalicylic acid and restored by addition of equivalent amounts of exogenous PGE₂. LTB₄ degradation was not enhanced in PMN-AEC coincubations. Moreover, reduction of LTB₄ formation in this system did not require an intimate cell-to-cell contact as shown by studies involving filter membranes for PMN-AEC separation. Superoxide anion concentrations were also decreased in PMN-AEC coincubations; this effect, however, was unrelated to PGE₂ for quantitative reasons and was probably due to 2 is the major mediator in the coincubation of porcine AEC and human PMN that downregulates neutrophil responses by activating receptors on the neutrophil. A minor contributor in this course of PMN-AEC interaction may be the 15-HETE transcellular pathway. Overall, airway epithelium appears to play an antiinflammatory role by damping the proinflammatory potential of neutrophils. J. Cell. Physiol. 175:268–275, 1998. © 1998 Wiley-Liss, Inc.     

5-Hydroxyeicosatetraenoic acid and human parturition

5-Hydroxyeicosatetraenoic acid (5-HETE) is an arachidonic acid (AA) metabolite derived from the lipoxygenase pathway which is capable of inducing uterine contractions. The purpose of this study was to determine a). whether 5-HETE concentrations in amniotic fluid increase before or after the onset of labor and b). whether acetylsalicylic acid (ASA) could modulate the production of 5-HETE by human amnion cells. 5-HETE concentrations are increased in amniotic fluid before the onset of labor. Furthermore, ASA treatment as expected inhibited PGE₂, but also significantly increased 5-HETE production by amnion cells. 5-HETE concentrations on average increased by greater than 2.5 fold (p < 0.001) in amniotic fluid prior to spontaneous labor when compared with samples obtained from the same patients earlier in gestation and therefore may be important in mechanisms regulating the onset of labor. ASA provokes an increase in 5-HETE biosynthesis by amnion cells: control media 2.60 ± 1.5, ASA treatment alone 5.17 ± 0.20, IL-1β alone 6.39 ± 2.1, and ASA + IL-1β 8.95 ± 1.2 (mean ± SEM) picograms per microgram protein per 16 hours. These findings may explain in part why cyclooxygenase inhibitors are not always successful in treating women with preterm labor.