The contributions of aspirin and microbial oxygenase to the biosynthesis of anti-inflammatory resolvins: novel oxygenase products from omega-3 polyunsaturated fatty acids

Resolvins (Rvs) are oxygenated products derived from omega-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid that carry potent protective bioactions present in resolving inflammatory exudates. Resolvin E1 (RvE1) is biosynthesized in vivo from EPA via transcellular biosynthetic routes during cell-cell interactions, and thus RvE1 is formed in vivo during multicellular responses such as inflammation and microbial infections. RvE1 protects tissues from leukocyte-mediated injury and counterregulates proinflammatory gene expression. These newly identified Rvs may underlie the beneficial actions of omega-3 PUFAs especially in chronic disorders where unresolved inflammation is a key mechanism of pathogenesis. Here, we present an overview of the biosynthesis of RvE1, with a focus on the aspirin-triggered and microbial P450-initiated pathways. The generation of RvE1 and its actions appear to dampen acute leukocyte responses and facilitate the resolution of inflammation.     

Metabolic inactivation of resolvin E1 and stabilization of its anti-inflammatory actions

The resolvins (Rv) are lipid mediators derived from omega-3 polyunsaturated fatty acids that act within a local inflammatory milieu to stop leukocyte recruitment and promote resolution. Resolvin E1 (RvE1; (5S,12R,18R)-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) is an oxygenase product derived from omega-3 eicosapentaenoic acid that displays potent anti-inflammation/pro-resolution actions in vivo. Here, we determined whether oxidoreductase enzymes catalyze the conversion of RvE1 and assessed the biological activity of the RvE1 metabolite. With NAD+ as a cofactor, recombinant 15-hydroxyprostaglandin dehydrogenase acted as an 18-hydroxyl dehydrogenase to form 18-oxo-RvE1. In the murine lung, dehydrogenation of the hydroxyl group at carbon 18 position to form 18-oxo-RvE1 represented the major initial metabolic route for RvE1. At a concentration where RvE1 potently reduced polymorphonuclear leukocyte (PMN) recruitment in zymosan-induced peritonitis, 18-oxo-RvE1 was devoid of activity. In human neutrophils, carbon 20 hydroxylation of RvE1 was the main route of conversion. An RvE1 analog, i.e. 19-(p-fluorophenoxy)-RvE1, was synthesized that resisted rapid metabolic inactivation and proved to retain biological activity reducing PMN infiltration and pro-inflammatory cytokine/chemokine production in vivo. These results established the structure of a novel RvE1 initial metabolite, indicating that conversion of RvE1 to the oxo product represents a mode of RvE1 inactivation. Moreover, the designed RvE1 analog, which resisted further metabolism/inactivation, could be a useful tool to evaluate the actions of RvE1 in complex disease models.     

Resolvin E1 metabolome in local inactivation during inflammation-resolution

Resolvin E1 (RvE1; 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) is a potent anti-inflammatory and proresolving mediator derived from the omega-3 eicosapentaenoic acid. In this study, we report the RvE1 metabolome, namely, the metabolic products derived from RvE1. RvE1 was converted to several novel products by human polymorphonuclear leukocytes and whole blood as well as in murine inflammatory exudates, spleen, kidney, and liver. The potential activity of each of the newly identified products was directly compared with that of RvE1. The new RvE1 products elucidated included 19-hydroxy-RvE1, 20-carboxy-RvE1, and 10,11-dihydro-RvE1. Metabolomic profiles of RvE1 were species-, tissue-, and cell type-specific. Direct comparisons of the bioactions between isolated RvE1 metabolic products indicated that 10,11-dihydro-RvE1, 18-oxo-RvE1, and 20-carboxy-RvE1 displayed reduced bioactivity in vivo. At concentrations as low as 1 nM, RvE1 enhanced macrophage phagocytosis, a proresolving activity that was reduced by metabolic inactivation. These results document novel metabolic products of RvE1 that impact its actions and that both omega-1 hydroxylation and reduction of conjugated double bonds in RvE1 are new pathways of four main routes of RvE1 metabolism in mammalian tissues. Together, these findings indicate that, during inflammation and its controlled resolution, specific tissues inactivate proresolving signals, i.e., RvE1, to permit the coordinated return to homeostasis. Moreover, the RvE1 metabolome may serve as a biomarker of these processes.     

Resolvin E2 formation and impact in inflammation resolution

Acute inflammation and its resolution are essential processes for tissue protection and homeostasis. In this context, specialized proresolving mediators derived from polyunsaturated fatty acids are of interest. In this study, we report that resolvin E2 (RvE2) from eicosapentaenoic acid is endogenously produced during self-limited murine peritonitis in both the initiation and resolution phases. RvE2 (1-10 nM) carries potent leukocyte-directed actions that include: 1) regulating chemotaxis of human neutrophils; and 2) enhancing phagocytosis and anti-inflammatory cytokine production. These actions appear to be mediated by leukocyte G-protein-coupled receptors as preparation of labeled RvE2 gave direct evidence for specific binding of radiolabeled RvE2 to neutrophils (K(d) 24.7 ± 10.1 nM) and resolvin E1 activation of recombinant G-protein-coupled receptors was assessed. In addition to the murine inflammatory milieu, RvE2 was also identified in plasma from healthy human subjects. RvE2 rapidly downregulated surface expression of human leukocyte integrins in whole blood and dampened responses to platelet-activating factor. Together, these results indicate that RvE2 can stimulate host-protective actions throughout initiation and resolution in the innate inflammatory responses.     

Human lipoxygenase isoforms form complex patterns of double and triple oxygenated compounds from eicosapentaenoic acid

Lipoxygenases (ALOX) are lipid peroxidizing enzymes that catalyze the biosynthesis of pro- and anti-inflammatory lipid mediators and have been implicated in (patho-)physiological processes. In humans, six functional ALOX isoforms exist and their arachidonic acid oxygenation products have been characterized. Products include leukotrienes and lipoxins which are involved in the regulation of inflammation and resolution. Oxygenation of n3-polyunsaturated fatty acids gives rise to specialized pro-resolving mediators, e.g. resolvins. However, the catalytic activity of different ALOX isoforms can lead to a multitude of potentially bioactive products. Here, we characterized the patterns of oxygenation products formed by human recombinant ALOX5, ALOX15, ALOX15B and ALOX12 from eicosapentaenoic acid (EPA) and its 18-hydroxy derivative 18-HEPE with particular emphasis on double and triple oxygenation products. ALOX15 and ALOX5 formed a complex mixture of various double oxygenation products from EPA, which include 5,15-diHEPE and various 8,15-diHEPE isomers. Their biosynthetic mechanisms were explored using heavy oxygen isotopes (H₂¹⁸O, ¹⁸O₂ gas) and three catalytic activities contributed to product formation: i) fatty acid oxygenase activity, ii) leukotriene synthase activity, iii) lipohydroperoxidase activity. For ALOX15B and ALOX12 more specific product patterns were identified, which was also the case when these enzymes reacted in concert with ALOX5. Several double oxygenated compounds were formed from 18-HEPE by ALOX5, ALOX15B and ALOX12 including previously identified resolvins (RvE2, RvE3), while formation of triple oxygenation products, e.g. 5,17,18-triHEPE, required ALOX5. Taken together our data show that EPA can be converted by human ALOX isoforms to a large number of secondary oxygenation products, which might exhibit bioactivity.     

Resolvin E1 Receptor Activation Signals Phosphorylation and Phagocytosis

Resolvins are endogenous lipid mediators that actively regulate the resolution of acute inflammation. Resolvin E1 (RvE1; (5S,12R,18R)-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) is an endogenous anti-inflammatory and pro-resolving mediator derived from eicosapentaenoic acid that regulates leukocyte migration and enhances macrophage phagocytosis of apoptotic neutrophils to resolve inflammation. In the inflammatory milieu, RvE1 mediates counter-regulatory actions initiated via specific G protein-coupled receptors. Here, we have identified RvE1-specific signaling pathways initiated by the RvE1 receptor ChemR23. RvE1 stimulated phosphorylation of Akt that was both ligand- and receptor-dependent. RvE1 regulated Akt phosphorylation in a time (0–15 min)- and dose-dependent (0.01–100 nm) manner in human ChemR23-transfected Chinese hamster ovary cells. RvE1 stimulated phosphorylation of both Akt and a 30-kDa protein, a downstream target of Akt, identified using a phospho-Akt substrate antibody. The 30-kDa protein was identified as ribosomal protein S6, a translational regulator, and its phosphorylation was inhibited by a phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin) and an ERK inhibitor (PD98059) but not by a p38-MAPK inhibitor (SB203580). Ribosomal protein S6 is a downstream target of the PI3K/Akt signaling pathway as well as the Raf/ERK pathway. In ChemR23-expressing differentiated HL60 cells, RvE1 also stimulated the phosphorylation of ribosomal protein S6. In addition, RvE1 enhanced phagocytosis of zymosan A by human macrophages, which are inhibited by PD98059 and rapamycin (mTOR inhibitor). These results indicate that RvE1 initiates direct activation of ChemR23 and signals receptor-dependent phosphorylation. These phosphorylation-signaling pathways identified for RvE1 receptor-ligand interactions underscore the importance of endogenous pro-resolving agonists in resolving acute inflammation.     

Resolvins, docosatrienes, and neuroprotectins, novel omega-3-derived mediators, and their aspirin-triggered endogenous epimers: an overview of their protective roles in catabasis

The molecular basis for the beneficial impact of essential omega-3 fatty acids is of considerable interest. Recently, novel mediators generated from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) that displayed potent bioactions were first identified in resolving inflammatory exudates [J. Exp. Med. 192 (2000) 1197; J. Exp. Med. 196 (2002) 1025] and in tissues enriched with DHA [J. Exp. Med. 196 (2002) 1025; J. Biol. Chem. 278 (2003) 14677]. The trivial names Resolvin (resolution phase interaction products) and docosatrienes were introduced for the bioactive compounds belonging to these novel series because they demonstrate potent anti-inflammatory and immunoregulatory actions. The compounds derived from eicosapentaenoic acid carrying potent biological actions (i.e., 1-10 nM range) are designated E series, given their EPA precursor, and denoted as Resolvins of the E series (Resolvin E1 or RvE1), and those biosynthesized from the precursor docosahexaenoic acid are Resolvins of the D series (Resolvin D1 or RvD1). Bioactive members from DHA with conjugated triene structures are docosatrienes (DT) that are immunoregulatory [J. Exp. Med. 196 (2002) 1025; J. Biol. Chem. 278 (2003) 14677], and neuroprotective [J. Biol. Chem., 278 (2003) 43807; Proc. Natl. Acad. Sci. U.S.A. [submitted for publication]] and are termed neuroprotectins. The specific receptors for these novel bioactive products from omega-3 EPA and DHA are abbreviated Resolvin D receptors (i.e., ResoDR1), Resolvin E receptor (ResoER1; RER1), and neuroprotectin D receptors (NPDR), respectively, in recognition of their respective cognate ligands. Aspirin treatment impacts biosynthesis of these compounds and a related series by triggering endogenous formation of the 17R-D series Resolvins and docosatrienes. These novel epimers are denoted as aspirin-triggered (AT)-RvDs and -DTs, and possess potent anti-inflammatory actions in vivo essentially equivalent to their 17S series pathway products. Here, we provide a syntomy overview of the formation and actions of these newly uncovered pathways and products as well as highlight their role(s) as endogenous protective mediators generated in anti-inflammation and catabasis.     

Maresin Biosynthesis and Identification of Maresin 2, a New Anti-Inflammatory and Pro-Resolving Mediator from Human Macrophages

Maresins are a new family of anti-inflammatory and pro-resolving lipid mediators biosynthesized from docosahexaenoic acid (DHA) by macrophages. Here we identified a novel pro-resolving product, 13R,14S-dihydroxy-docosahexaenoic acid (13R,14S-diHDHA), produced by human macrophages. PCR mapping of 12-lipoxygenase (12-LOX) mRNA sequence in human macrophages and platelet showed that they are identical. This human 12-LOX mRNA and enzyme are expressed in monocyte-derived cell lineage, and enzyme expression levels increase with maturation to macrophages or dendritic cells. Recombinant human 12-LOX gave essentially equivalent catalytic efficiency (k_cat/K_M) with arachidonic acid (AA) and DHA as substrates. Lipid mediator metabololipidomics demonstrated that human macrophages produce a novel bioactive product 13,14-dihydroxy-docosahexaenoic acid in addition to maresin-1, 7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoic acid (MaR1). Co-incubations with human recombinant 12-LOX and soluble epoxide hydrolase (sEH) demonstrated that biosynthesis of 13,14-dihydroxy-docosahexaenoic acid (13,14-diHDHA) involves the 13S,14S-epoxy-maresin intermediate produced from DHA by 12-LOX, followed by conversion via soluble epoxide hydrolase (sEH). This new 13,14-diHDHA displayed potent anti-inflammatory and pro-resolving actions, and at 1 ng reduced neutrophil infiltration in mouse peritonitis by ∼40% and at 10 pM enhanced human macrophage phagocytosis of zymosan by ∼90%. However, MaR1 proved more potent than the 13R,14S-diHDHA at enhancing efferocytosis with human macrophages. Taken together, the present findings demonstrate that macrophages produced a novel bioactive product identified in the maresin metabolome as 13R,14S-dihydroxy-docosahexaenoic acid, from DHA via conversion by human 12-LOX followed by sEH. Given its potent bioactions, we coined 13R,14S-diHDHA maresin 2 (MaR2).     

Transformation of 15-hydroperoxyeicosapentaenoic acid to lipoxin A5 and B5, mono- and dihydroxyeicosapentaenoic acids by porcine leukocytes

15-Hydroperoxy[1-14C]eicosapentaenoic acid derived from eicosapentaenoic acid (EPA) was incubated with suspensions of porcine leukocytes. Incubation with porcine leukocytes resulted in the formation of seven dihydroxy compounds, one monohydroxy and one hydroxyepoxy compound. After separation by reverse-phase and straight-phase HPLC, GC/MS analysis revealed that these metabolites were four isomers of 8,15-diHEPEs, two isomers of 14,15-diHEPEs, one isomer of 5,15-diHEPE, 15-HEPE and an epoxyalcohol: 13-hydroxy-14,15-epoxyeicosatetraenoic acid. In addition to the above metabolites, two trihydroxytetraene derivatives were also isolated. GC/MS and ultraviolet spectroscopy identified the two trihydroxypentaene derivatives as 5,6,15-trihydroxy-7,9,11,13,17-eicosapentaenoic acid (lipoxin A5) and 5,14,15-trihydroxy-6,8,10,12,17-eicosapentaenoic acid (lipoxin B5). This study demonstrated that the 15-hydroperoxide of EPA can be actively converted to various hydroxylated products via the 5-, 12- and 15-lipoxygenase as well as epoxyisomerase pathways in the porcine leukocytes.     

Resolvin E1 selectively interacts with leukotriene B4 receptor BLT1 and ChemR23 to regulate inflammation

Resolvin E1 (RvE1) is a potent anti-inflammatory and proresolving mediator derived from omega-3 eicosapentaenoic acid generated during the resolution phase of inflammation. RvE1 possesses a unique structure and counterregulatory actions that stop human polymorphonuclear leukocyte (PMN) transendothelial migration and PMN infiltration in several murine inflammatory models. To examine the mechanism(s) underlying anti-inflammatory actions on PMNs, we prepared [(3)H]RvE1 and characterized its interactions with human PMN. Results with membrane fractions of human PMN demonstrated specific binding with a K(d) of 48.3 nM. [(3)H]RvE1 specific binding to human PMN was displaced by leukotriene B(4) (LTB(4)) and LTB(4) receptor 1 (BLT1) antagonist U-75302, but not by chemerin peptide, a ligand specific for another RvE1 receptor ChemR23. Recombinant human BLT1 gave specific binding with [(3)H]RvE1 with a K(d) of 45 nM. RvE1 selectively inhibited adenylate cyclase with BLT1, but not with BLT2. In human PBMC, RvE1 partially induced calcium mobilization, and blocked subsequent stimulation by LTB(4). RvE1 also attenuated LTB(4)-induced NF-kappaB activation in BLT1-transfected cells. In vivo anti-inflammatory actions of RvE1 were sharply reduced in BLT1 knockout mice when given at low doses (100 ng i.v.) in peritonitis. In contrast, RvE1 at higher doses (1.0 mug i.v.) significantly reduced PMN infiltration in a BLT1-independent manner. These results indicate that RvE1 binds to BLT1 as a partial agonist, potentially serving as a local damper of BLT1 signals on leukocytes along with other receptors (e.g., ChemR23-mediated counterregulatory actions) to mediate the resolution of inflammation.     

Protective effects of dietary EPA and DHA on ischemia–reperfusion-induced intestinal stress

The immunoregulatory effects of dietary omega-3 fatty acids are still not fully characterized. The aim of this study was to determine whether dietary eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) intake limits intestinal ischemia–reperfusion (IR) injury. To test this, rats were fed either control or EPA/DHA supplemented diet for 3 weeks following which they underwent either a sham or an IR surgical protocol. A significant reduction in mucosal damage was observed after EPA/DHA supplemented diet as reflected by maintenance of total protein content. To address the underlying mechanisms of protection, we measured parameters of oxidative stress, intestinal and serological cytokines and intestinal eicosanoids. Interestingly, EPA/DHA fed animals displayed a higher activity of oxidative stress enzyme machinery, i.e., superoxide dismutase and catalase in addition to a reduction in total nitrate/nitrite content. While no changes in cytokines were observed, eicosanoid analyses of intestinal tissue revealed an increase in metabolites of the 12-lipoxygenase pathway following IR. Further, IR in EPA/DHA fed animals was accompanied by a significant increase of 17,18-epoxyeicosatetraenoic acid, 8-Iso prostaglandin F_3α and thromboxane B₃, by more than 12-, 6-, 3-fold, respectively. Thus, the data indicate that EPA/DHA supplementation may be able to reduce early intestinal IR injury by anti-oxidative and anti-inflammatory mechanisms.     

Resolvin E1 dampens airway inflammation and hyperresponsiveness in a murine model of asthma

Resolvin E1 (RvE1; 5S, 12R, 18R-trihydroxyeicosapentaenoic acid) is an anti-inflammatory lipid mediator derived from the omega-3 fatty acid eicosapentaenoic acid (EPA). It has been recently shown that RvE1 is involved in the resolution of inflammation. However, it is not known whether RvE1 is involved in the resolution of asthmatic inflammation. To investigate the anti-inflammatory effect of RvE1 in asthma, a murine model of asthma was studied. After RvE1 was administered to mice intraperitoneally, there were decreases in: airway eosinophil and lymphocyte recruitment, specific Th2 cytokine, IL-13, ovalbumin-specific IgE, and airway hyperresponsiveness (AHR) to inhaled methacholine. Moreover, RvE1-treated mice had significantly lower mucus scores compared to vehicle-treated mice based on the number of goblet cells stained with periodic acid-schiff (PAS). These findings provide evidence that RvE1 is a pivotal counterregulatory signal in allergic inflammation and offer novel multi-pronged therapeutic approaches for human asthma.     

The biosynthetic pathway to abscisic acid via ionylideneethane in the fungus Botrytis cinerea

The biosynthetic pathway to abscisic acid (ABA) from isopentenyl diphosphate in the fungus, Botrytis cinerea, was investigated. Labeling experiments with (18)O2 and H2(18)O indicated that all oxygen atoms at C-1, -1, -1' and -4' of ABA were derived from molecular oxygen, and not from water. This finding was inconsistent not only with the known carotenoid pathway via oxidative cleavage of carotenoids, but also with the classical direct pathway via cyclization of farnesyl diphosphate. The fungus produced new C15-compounds, 2E,4E-alpha-ionylideneethane and 2Z,4E-alpha-ionylideneethane, along with 2E,4E,6E-allofarnesene and 2Z,4E,6E-allofarnesene, but did not apparently produce carotenoids except for a trace of phytoene. The C15-compounds labeled with 13C were converted to ABA by the fungus, and the incorporation ratio of 2Z,4E-alpha-ionylideneethane was higher than that of 2E,4E-alpha-ionylideneethane. From these results, it was concluded that farnesyl diphosphate was reduced at C-1, desaturated at C-4, and isomerized at C-2 to form 2Z,4E,6E-allofarnesene before being cyclized to 2Z,4E-alpha-ionylideneethane; the ionylideneethane was then oxidized to ABA with molecular oxygen. This direct pathway via ionylideneethane means that the biosynthetic pathway to fungal ABA, not only before but also after isopentenyl diphosphate, differs from that to ABA in plants, since plant ABA is biosynthesized using the non-mevalonate and carotenoid pathways.     

Protective effect of resolvin E1 on the development of asthmatic airway inflammation

Resolvin E1 (RvE1) is an anti-inflammatory lipid mediator derived from the omega-3 fatty acid eicosapentaenoic acid (EPA), and strongly acts in the resolution of inflammation. We previously reported that RvE1 dampens airway inflammation and hyperresponsiveness in a murine model of asthma. In the present study, to elucidate the effects of RvE1 on the development of asthmatic airway inflammation, we investigated whether RvE1 acts on different phases of an OVA-sensitized and -challenged mouse model of asthma. RvE1 treatments at the time of either OVA sensitization or at the time of OVA challenge were investigated and compared with RvE1 treatments at the time of both OVA sensitization and challenge. After RvE1 was administered to mice intraperitoneally at the time of both OVA sensitization and challenge, there were decreases in airway eosinophil and lymphocyte recruitment, as well as a reduction in Th2 cytokine and airway hyperresponsiveness. RvE1 treatment at the time of either OVA sensitization or challenge also improved AHR and airway inflammation. Our results suggest that RvE1 acts on several phases of asthmatic inflammation and may have anti-inflammatory effects on various cell types.     

Candida albicans modulates host defense by biosynthesizing the pro-resolving mediator resolvin E1

Candida albicans is an opportunistic fungal pathogen of humans that resides commensally on epithelial surfaces, but can cause inflammation when pathogenic. Resolvins are a class of anti-inflammatory lipids derived from omega-3 polyunsaturated fatty acids (PUFA) that attenuate neutrophil migration during the resolution phase of inflammation. In this report we demonstrate that C. albicans biosynthesizes resolvins that are chemically identical to those produced by human cells. In contrast to the trans-cellular biosynthesis of human Resolvin E1 (RvE1), RvE1 biosynthesis in C. albicans occurs in the absence of other cellular partners. C. albicans biosynthesis of RvE1 is sensitive to lipoxygenase and cytochrome P450 monoxygenase inhibitors. We show that 10nM RvE1 reduces neutrophil chemotaxis in response to IL-8; 1nM RvE1 enhanced phagocytosis of Candida by human neutrophils, as well as intracellular ROS generation and killing, while having no direct affect on neutrophil motility. In a mouse model of systemic candidiasis, RvE1 stimulated clearance of the fungus from circulating blood. These results reveal an inter-species chemical signaling system that modulates host immune functions and may play a role in balancing host carriage of commensal and pathogenic C. albicans.     

Anti-inflammatory actions of neuroprotectin D1/protectin D1 and its natural stereoisomers: assignments of dihydroxy-containing docosatrienes

Protectin D1, neuroprotectin D1 when generated by neural cells, is a member of a new family of bioactive products generated from docosahexaenoic acid. The complete stereochemistry of protectin D1 (10,17S-docosatriene), namely, chirality of the carbon-10 alcohol and geometry of the conjugated triene, required for bioactivity remained to be assigned. To this end, protectin D1/neuroprotectin D1 (PD1) generated by human neutrophils during murine peritonitis and by neural tissues was separated from natural isomers and subjected to liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. Comparisons with six 10,17-dihydroxydocosatrienes prepared by total organic and biogenic synthesis showed that PD1 from human cells carrying potent bioactivity is 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid. Additional isomers identified included trace amounts of Delta15-trans-PD1 (isomer III), 10S,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid (isomer IV), and a double dioxygenation product 10S,17S-dihydroxy-docosa-4Z,7Z,11E,13Z,15E,19Z-hexaenoic acid (isomer I), present in exudates. 18O2 labeling showed that 10S,17S-diHDHA (isomer I) carried 18O in the carbon-10 position alcohol, indicating sequential lipoxygenation, whereas PD1 formation proceeded via an epoxide. PD1 at 10 nM attenuated (approximately 50%) human neutrophil transmigration, whereas Delta15-trans-PD1 was essentially inactive. PD1 was a potent regulator of polymorphonuclear leukocyte (PMN) infiltration (approximately 40% at 1 ng/mouse) in peritonitis. The rank order at 1- to 10-ng dose was PD1 approximately PD1 methyl ester > Delta15-trans-PD1 > 10S,17S-diHDHA (isomer I). 10S,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid (isomer VI) proved > or = PD1 in blocking PMN infiltration, but was not a major product of leukocytes. PD1 also reduced PMN infiltration after initiation (2 h) of inflammation and was additive with resolvin E1. These results indicate that PD1 is a potent stereoselective anti-inflammatory molecule.     

15-lipoxygenase metabolites of gamma-linolenic acid/eicosapentaenoic acid suppress growth and arachidonic acid metabolism in human prostatic adenocarcinoma cells: possible implications of dietary fatty acids

Although gammalinolenic acid (GLA) and eicosapentaenoic acid (EPA) have independently been reported to suppress growth of cancer cells, their relative potencies are unknown. To determine the possible attenuating efficacies of dietary GLA or EPA on prostate carcinogenesis, we hereby report the in vitro effects of GLA, EPA and their 15-lipoxygenase (15-LOX) metabolites: 15(S)-HETrE and 15(S)-HEPE, respectively, on growth and arachidonic acid (AA) metabolism in human androgen-dependent (LNCaP) and androgen-independent (PC-3) prostatic cancer cells in culture. Specifically, both cells were preincubated respectively with the above PUFAs. Growth was determined by [3H]thymidine uptake and AA metabolism by HPLC analysis of the extracted metabolites. Our data revealed increased biosynthesis of prostaglandin E2 (PGE2) and 5-hydroxyeicosatetraenoic acid (5(S)-HETE) by both cells. Preincubation of the cells with 15(S)-HETrE or 15(S)-HEPE more markedly inhibited cellular growth and AA metabolism when compared to precursor PUFAs. Notably, 15(S)-HETrE exerted the greatest inhibitory effects. These findings therefore imply that dietary GLA rather than EPA should better attenuate prostate carcinogenesis via its in vivo generation of 15(S)-HETrE, thus warranting exploration.     

Enhancement of platelet 12-HETE production in the presence of polymorphonuclear leukocytes during calcium ionophore stimulation.

This study investigates the effect of platelet/neutrophil interactions on eicosanoid production. Human platelets and polymorphonuclear leukocytes (PMNs) were stimulated alone and in combination, with calcium ionophore A23187 and the resulting eicosanoids 12S-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid (12-HETE), 12S-heptadecatrienoic acid (HHT), 5S,12R-dihydroxy-(6Z,8E,10E,14Z)-eicosatetraenoi c acid (LTB4) and 5S-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE) were measured by HPLC. The addition of PMNs to platelet suspensions caused a 104% increase in 12-HETE, a product of 12-lipoxygenase activity, but had only a modest effect on the cyclooxygenase product HHT (increase of 18%). By using PMNs labelled with [14C]arachidonic acid it was shown that the increases in these platelet eicosanoids could be accounted for by translocation of released arachidonic acid from PMNs to platelets and its subsequent metabolism. The observation that 12-lipoxygenase was about five times more efficient than cyclooxygenase at utilising exogenous arachidonic acid during the platelet/PMN interactions was confirmed in experiments in which platelets were stimulated with A23187 in the presence of [14C]arachidonic acid. Stimulations of platelets with thrombin in the presence of PMNs resulted in a decrease in 12-HETE and HHT levels of 40% and 26%, respectively. The presence of platelets caused a small increase in neutrophil LTB4 output but resulted in a decrease in 5-HETE production of 43% during stimulation with A23187. This study demonstrates complex biochemical interactions between platelets and PMNs during eicosanoid production and provides evidence of a mechanism to explain the large enhancement in 12-HETE production.     

Human neutrophils convert the sebum-derived polyunsaturated fatty acid Sebaleic acid to a potent granulocyte chemoattractant

Sebaleic acid (5,8-octadecadienoic acid) is the major polyunsaturated fatty acid in human sebum and skin surface lipids. The objective of the present study was to investigate the metabolism of this fatty acid by human neutrophils and to determine whether its metabolites are biologically active. Neutrophils converted sebaleic acid to four major products, which were identified by their chromatographic properties, UV absorbance, and mass spectra as 5-hydroxy-(6E,8Z)-octadecadienoic acid (5-HODE), 5-oxo-(6E,8Z)-octadecadienoic acid (5-oxo-ODE), 5S,18-dihydroxy-(6E,8Z)-octadecadienoic acid, and 5-oxo-18-hydroxy-(6E,8Z)-octadecadienoic acid. The identities of these metabolites were confirmed by comparison of their properties with those of authentic chemically synthesized standards. Both neutrophils and human keratinocytes converted 5-HODE to 5-oxo-ODE. This reaction was stimulated in neutrophils by phorbol myristate acetate and in keratinocytes by oxidative stress (t-butyl-hydroperoxide). Both treatments dramatically elevated intracellular levels of NADP(+), the cofactor required by 5-hydroxyeicosanoid dehydrogenase. In keratinocytes, this was accompanied by a rapid increase in intracellular GSSG levels, consistent with the involvement of glutathione peroxidase. 5-Oxo-ODE stimulated calcium mobilization in human neutrophils and induced desensitization to 5-oxo-6,8,11,14-eicosatetraenoic acid but not leukotriene B(4), indicating that this effect was mediated by the OXE receptor. 5-Oxo-ODE and its 8-trans isomer were equipotent with 5-oxo-6,8,11,14-eicosatetraenoic acid in stimulating actin polymerization and chemotaxis in human neutrophils, whereas 5-HODE, 5-oxo-18-hydroxy-(6E,8Z)-octadecadienoic acid, and 5S,18-dihydroxy-(6E,8Z)-octadecadienoic acid were much less active. We conclude that neutrophil 5-lipoxygenase converts sebaleic acid to 5-HODE, which can be further metabolized to 5-oxo-ODE by 5-hydroxyeicosanoid dehydrogenase in neutrophils and keratinocytes. Because of its chemoattractant properties, sebum-derived 5-oxo-ODE could be involved in neutrophil infiltration in inflammatory skin diseases.     

Inhibition by eicosapentaenoic acid of IL-1beta-induced PGHS-2 expression in human microvascular endothelial cells: involvement of lipoxygenase-derived metabolites and p38 MAPK pathway

Prostaglandin H synthase 2 (PGHS-2), a highly inducible isoenzyme, is responsible for overproduction of the prostaglandins (PGs) in inflammatory sites.We established that among fish oil polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA), but not docosahexaenoic acid (DHA), greatly decreased interleukin-1beta (IL-1beta)-induced PGHS-2 expression in human pulmonary microvascular endothelial cells (HPMECs). Lipoxygenase products 12 (S)-hydroperoxyeicosapentaenoic acid ((S)-HpEPE), 15 (S)-HpEPE and leukotriene (LT) D5 reproduced similar inhibitory effect, suggesting that they may be the intermediate metabolites responsible for PGHS-2 down-regulation by EPA. Accordingly, the EPA effect is prevented by nordihydroguaiaretic acid (NDGA) and by REV 5901, nonspecific and specific 5-lipoxygenase inhibitors, respectively. Besides, inhibition of cyclooxygenase activity by ibuprofen, indomethacin or aspirin was not able to prevent this effect. Moreover, cyclooxygenase metabolites of EPA (PGs D3, E3 and I3) markedly potentiate IL-1beta-induced PGHS-2 expression, probably by increasing intracellular cAMP levels. Peroxisome proliferator-activated receptors (PPARs) are known to be activated by fatty acids (FAs) such as EPA. We found here that HPMECs express only weak amounts of PPARalpha and PPARgamma whose activation by synthetic agonists, Wy-14,643 and ciglitazone, does not cause any inhibition of IL-1beta-induced PGHS-2 expression. This finding ruled out the involvement of PPARs in the EPA inhibitory effect. In addition, we established that EPA, which failed to inhibit nuclear factor-kappaB (NF-kappaB) activation, suppressed p38 mitogen-activated protein kinase (MAPK) phosphorylation in stimulated HPMECs.Our data demonstrate that EPA, unlike DHA, down-regulates PGHS-2 expression in HPMECs probably through its 5-lipoxygenase-dependent metabolites and advocates a beneficial role for this FA in limiting inflammatory response.