Lipoxins and aspirin-triggered 15-epi-lipoxins are the first lipid mediators of endogenous anti-inflammation and resolution

Lipoxins (LXs) or the lipoxygenase interaction products are generated from arachidonic acid via sequential actions of lipoxygenases and subsequent reactions to give specific trihydroxytetraene-containing eicosanoids. These unique structures are formed during cell-cell interactions and appear to act at both temporal and spatially distinct sites from other eicosanoids produced during the course of inflammatory responses and to stimulate natural resolution. Lipoxin A4 (LXA4) and lipoxin B4 (LXB4) are positional isomers that each possesses potent cellular and in vivo actions. These LX structures are conserved across species. The results of numerous studies reviewed in this work now confirm that they are the first recognized eicosanoid chemical mediators that display both potent anti-inflammatory and pro-resolving actions in vivo in disease models that include rabbit, rat, and mouse systems. LXs act at specific GPCRs as agonists to regulate cellular responses of interest in inflammation and resolution. Aspirin has a direct impact in the LX circuit by triggering the biosynthesis of endogenous epimers of LX, termed the aspirin-triggered 15-epi-LX, that share the potent anti-inflammatory actions of LX. Stable analogs of LXA4, LXB4, and aspirin-triggered lipoxin were prepared, and several of these display potent actions in vitro and in vivo. The results reviewed herein implicate a role of LX and their analogs in many common human diseases including airway inflammation, asthma, arthritis, cardiovascular disorders, gastrointestinal disease, periodontal disease, kidney diseases and graft-vs.-host disease, as well as others where uncontrolled inflammation plays a key role in disease pathogenesis. Hence, the LX pathways and mechanisms reviewed to date in this work provide a basis for new approaches to treatment of many common human diseases that involve inflammation.     

Lipoxins and lipoxin analogs in asthma

The pathobiology of asthma is characterized by production of eicosanoids, a diverse family of bioactive fatty acids that play important roles in regulating airway inflammation and reactivity. Lipoxins (LXs) are products of arachidonic acid metabolism that are distinct from leukotrienes (LTs) and prostaglandins (PGs) in structure and function. Unlike the pro-inflammatory PGs and LTs, LXs display counter-regulatory actions. Cell-type specific biological actions have been uncovered for LXs and LX stable analogs that promote resolution of acute inflammatory responses. At least two classes of receptors, CysLT1 receptors and LXA4 receptors (named ALX), can interact with LXA4 and LXA4 analogs to mediate their biological actions. LXs are generated during asthma and LXA4 signaling blocks asthmatic responses in humans and experimental model systems. Of interest, respiratory diseases of increased severity, such as aspirin-intolerant asthma, cystic fibrosis and steroid-dependent, severe asthma, display defective generation of these protective lipid signals. Together, these findings indicate a pivotal role for LXs in mediating airway homeostasis.     

Cutting edge: lipoxins rapidly stimulate nonphlogistic phagocytosis of apoptotic neutrophils by monocyte-derived macrophages

Lipoxins (LX) are lipoxygenase-derived eicosanoids generated during inflammation. LX inhibit polymorphonuclear neutrophil (PMN) chemotaxis and adhesion and are putative braking signals for PMN-mediated tissue injury. In this study, we report that LXA4 promotes another important step in the resolution phase of inflammation, namely, phagocytosis of apoptotic PMN by monocyte-derived macrophages (Mphi). LXA4 triggered rapid, concentration-dependent uptake of apoptotic PMN. This bioactivity was shared by stable synthetic LXA4 analogues (picomolar concentrations) but not by other eicosanoids tested. LXA4-triggered phagocytosis did not provoke IL-8 or monocyte chemoattractant protein-1 release. LXA4-induced phagocytosis was attenuated by anti-CD36, alphavbeta3, and CD18 mAbs. LXA4-triggered PMN uptake was inhibited by pertussis toxin and by 8-bromo-cAMP and was mimicked by Rp-cAMP, a protein kinase A inhibitor. LXA4 attenuated PGE2-stimulated protein kinase A activation in Mphi. These results suggest that LXA4 is an endogenous stimulus for PMN clearance during inflammation and provide a novel rationale for using stable synthetic analogues as anti-inflammatory compounds in vivo.     

Endogenous anti-inflammatory mediators from arachidonate in human neutrophils.

Eicosanoids have been historically involved in the pathogenesis of various inflammatory diseases. Lipoxins (LXs) and epi-LXs show physiological effects relevant to inflammation regulation. In this study, we focused on LX precursors based on the hypothesis that their entrance and metabolism into the cell may facilitate their targeting at the inflammation site. Because compound chirality is of considerable importance in the efficacy of therapeutic agents, our aim was to study the anti-inflammatory effects of various epimers of LXA(4) precursors compared to LXA(4). Blood polymorphonuclear cells (PMNs) were incubated with 15(S)- or 15(R)-hydroxyeicosatetraenoic acid (HETE), 14(R)-,15(S)-, or 14(S),15(S)-diHETE, and LXA(4) and then stimulated with the calcium ionophore A23187. We found that 15(R)-HETE rather than 15(S)-HETE was preferentially metabolized and that 15-epi-LXs were produced in larger amounts than LXs. In contrast, when PMNs were incubated with the diastereoisomers of 14,15(S)-diHETE, 14-epi-LXB(4) was produced in lower amounts than LXB(4). Enantiomers of 15-HETE and diastereoisomers of 14,15-diHETE and LXA(4) were able to significantly decrease LTB(4) release by PMNs. These results suggest a potential resolution of the inflammatory process through endogenous anti-inflammatory mediators released by the way of trans-cellular metabolism.     

Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators

Active resolution of acute inflammation is a previously unrecognized interface between innate and adaptive immunity. Once thought to be a passive process, the resolution of inflammation is now shown to involve active biochemical programmes that enable inflamed tissues to return to homeostasis. This Review presents new cellular and molecular mechanisms for the resolution of inflammation, revealing key roles for eicosanoids, such as lipoxins, and recently discovered families of endogenous chemical mediators, termed resolvins and protectins. These mediators have anti-inflammatory and pro-resolution properties, thereby protecting organs from collateral damage, stimulating the clearance of inflammatory debris and promoting mucosal antimicrobial defence.     

Electrophilic eicosanoids: Signaling and targets

Electrophilic eicosanoids are reactive mediators that arise by non-enzymatic transformations of arachidonic acid or of its products and display varied biological actions. Various electrophilic eicosanoids have shown anti-proliferative and anti-inflammatory effects, which have elicited a great interest in their study as potential therapeutic agents. A key feature of these compounds is their ability to covalently modify proteins, thus altering their structure and function. The modification of several components of the NF-κB pathway contributes to the anti-inflammatory effects of electrophilic eicosanoids, whereas addition to redox-sensitive proteins plays a key role in the antioxidant response. However, electrophilic eicosanoids may also have a dark side, and accumulating evidence points towards their involvement in neurotoxicity and/or neurodegeneration. The ability of some electrophilic eicosanoids to induce protein oligomerization or aggregation through various mechanisms may contribute to these effects. Biochemical and proteomic studies have led to the identification of numerous protein targets for modification by electrophilic eicosanoids, the number of which continues to expand, revealing novel potential functions for these compounds and providing a basis for their pleiotropic effects. The ample number of targets identified, together with the non-enzymatic nature of the modification argue against the potential specificity or regulation of electrophilic eicosanoid action. However, protein modification displays selectivity depending on structural features of the proteins and of the electrophilic compounds as well as on context factors such as cell type and GSH availability. Understanding the factors which control the extent and selectivity of protein modification by electrophilic eicosanoids is therefore essential to elucidate their pathophysiological roles and therapeutic potential in specific settings.     

Lipoxin A(4) and aspirin-triggered 15-epi-lipoxin A(4) antagonize TNF-alpha-stimulated neutrophil-enterocyte interactions in vitro and attenuate TNF-alpha-induced chemokine release and colonocyte apoptosis in human intestinal mucosa ex vivo

Lipoxins (LXs) are lipoxygenase-derived eicosanoids and putative endogenous braking signals for inflammation in the gastrointestinal tract and other organs. Aspirin triggers the production of 15-epimers during cell-cell interaction in a cytokine-primed milieu, and aspirin-triggered 15-epi-5(S),6(R),15(S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid (15-epi-LXA(4)) may contribute to the bioactivity profile of this prototype nonsteroidal anti-inflammatory drug in vivo. We determined the effect of LXA(4), 15-(R/S)-methyl-11,12-dehydro-LXA(4) methyl ester (15-(R/S)-methyl-LXA(4)), and stable analogs of LXA(4) on TNF-alpha-stimulated neutrophil-enterocyte interaction in vitro and TNF-alpha-stimulated chemokine release, changes in mucosal architecture, and enterocyte apoptosis in cytokine-activated intact human colonic mucosa ex vivo. LXA(4), 15-(R/S)-epi-LXA(4), and 16-phenoxy-11,12-dehydro-17,18,19,20-tetranor-LXA(4) methyl ester (16-phenoxy-LXA(4)) inhibited TNF-alpha-stimulated neutrophil adherence to epithelial monolayers at nanomolar concentrations. In parallel experiments involving human colonic mucosa ex vivo, LXA(4)potently attenuated TNF-alpha-stimulated release of the C-X-C chemokine IL-8, and the C-C chemokines monocyte-chemoattractant protein-1 (MCP-1) and RANTES. Exposure of strips of normal human colonic mucosa to TNF-alpha induced disruption of mucosa architecture and enhanced colonocyte apoptosis via a caspase-3-independent mechanism. Prior exposure of the mucosa strips to 15-(R/S)-methyl-LXA(4) attenuated TNF-alpha-stimulated colonocyte apoptosis and protected the mucosa against TNF-alpha-induced mucosal damage. In aggregate, our data demonstrate that lipoxins and aspirin-triggered 15-epi-LXA(4) are potent antagonists of TNF-alpha-mediated neutrophil-enterocyte interactions in vitro, attenuate TNF-alpha-triggered chemokine release and colonocyte apoptosis, and are protective against TNF-alpha-induced morphological disruption in human colonic strips ex vivo. Our observations further expand the anti-inflammatory profile of these lipoxygenase-derived eicosanoids and suggest new therapeutic approaches for the treatment of inflammatory bowel disease.     

Reduced inflammation and tissue damage in transgenic rabbits overexpressing 15-lipoxygenase and endogenous anti-inflammatory lipid mediators

PGs and leukotrienes (LTs) mediate cardinal signs of inflammation; hence, their enzymes are targets of current anti-inflammatory therapies. Products of arachidonate 15-lipoxygenases (LO) types I and II display both beneficial roles, such as lipoxins (LXs) that stereoselectively signal counterregulation, as well as potential deleterious actions (i.e., nonspecific phospholipid degradation). In this study, we examined transgenic (TG) rabbits overexpressing 15-LO type I and their response to inflammatory challenge. Skin challenges with either LTB(4) or IL-8 showed that 15-LO TG rabbits give markedly reduced neutrophil (PMN) recruitment and plasma leakage at dermal sites with LTB(4). PMN from TG rabbits also exhibited a dramatic reduction in LTB(4)-stimulated granular mobilization that was not evident with peptide chemoattractants. Leukocytes from 15-LO TG rabbits gave enhanced LX production, underscoring differences in lipid mediator profiles compared with non-TG rabbits. Microbe-associated inflammation and leukocyte-mediated bone destruction were assessed by initiating acute periodontitis. 15-LO TG rabbits exhibited markedly reduced bone loss and local inflammation. Because enhanced LX production was associated with an increased anti-inflammatory status of 15-LO TG rabbits, a stable analog of 5S,6R,15S-trihydroxyeicosa-7E,9E,11Z,13E-tetraenoic acid (LXA(4)) was applied to the gingival crevice subject to periodontitis. Topical application with the 15-epi-16-phenoxy-para-fluoro-LXA(4) stable analog (ATLa) dramatically reduced leukocyte infiltration, ensuing bone loss as well as inflammation. These results indicate that overexpression of 15-LO type I and LXA(4) is associated with dampened PMN-mediated tissue degradation and bone loss, suggesting that enhanced anti-inflammation status is an active process. Moreover, they suggest that LXs can be targets for novel approaches to diseases, e.g., periodontitis and arthritis, where inflammation and bone destruction are features.     

COX-2 in inflammation and resolution

Aspirin and the other NSAIDs have popularized the notion of inhibiting prostaglandins as a common anti-inflammatory strategy based on the erroneous premise that all eicosanoids are, within the context of inflammation, generally detrimental. However, our fascination with aspirin and the emergence of COX-2 has shown a more affable side to lipid mediators based on our increasing interest in the endogenous control of acute inflammation and in factors that mediate its resolution. Epilipoxins, for instance, are produced from aspirin's acetylation of COX-2 and together with Resolvins and COX-2-derived prostaglandins of the D(2) and J(2) series represent an increasingly important family of immunoregulatory lipid mediators with strong implications for disease control and drug discovery.     

Modeling studies on phospholipase A2-inhibitor complexes

Phospholipase A2 (PLA2) is a ubiquitous enzyme that specifically catalyzes hydrolysis of membrane phospholipids to produce lysophospholipids and free fatty acid, namely arachidonic acid, which provides substrate for eicosanoids biosynthesis. Thus, the compounds inhibiting PLA2 have been implicated as potential therapeutic agents in treatment of inflammation related diseases. Plant and marine organisms serve as sources of compounds that act as potential therapeutic agents for treatment of various diseases. The present study reveals the relationship between the structure and function of the medicinally important herbal compounds (acalyphin, chlorogenic acid, stigmasterol, curcumin and tectoridin) and marine compounds (gracilin A and aplysulphurin A). To understand the binding mechanisms of these compounds, molecular modeling studies has been performed with Russell's viper and bovine pancreatic PLA2 as target molecules using molecular operating environment (MOE) software. These compounds show favorable interactions with the amino acid residues at the active site of Russell's viper and bovine pancreatic PLA2, thereby substantiating their proven efficacy as anti-inflammatory compounds and antidotes.     

Resolution of inflammation by N-arachidonoylglycine

N-arachidonoylglycine (NAgly) is an endogenous signaling lipid that is a member of the eicosanoid super family and is related to anandamide. It shows anti-inflammatory activity in vivo in the mouse peritonitis model where it reduces migration of inflammatory leukocytes following injection of pro-inflammatory agents into the peritoneal cavity. Using cell culture models, including GPR18 transfected HEK-293 cells, evidence is presented that the orphan receptor GPR18 is involved in this action. Increases in free arachidonic acid, and robust stimulation of anti-inflammatory eicosanoids were observed at low micromolar concentrations. These included 15-deoxy-delta-13,14-PGJ(2) and lipoxin A(4) both of which are believed to mediate the resolution stage of inflammation. It was further shown that NAgly might act via GPR18 activation in promoting the number of Trypan Blue stained cells, a possible indicator of programmed cell death. Thus, we hypothesize that NAgly induces the death of inflammatory cells, a process that is considered to be important for the resolution of inflammation.     

Regulation of inflammation in cancer by eicosanoids

Inflammation in the tumor microenvironment is now recognized as one of the hallmarks of cancer. Endogenously produced lipid autacoids, locally acting small molecule lipid mediators, play a central role in inflammation and tissue homeostasis, and have recently been implicated in cancer. A well-studied group of autacoid mediators that are the products of arachidonic acid metabolism include: the prostaglandins, leukotrienes, lipoxins and cytochrome P450 (CYP) derived bioactive products. These lipid mediators are collectively referred to as eicosanoids and are generated by distinct enzymatic systems initiated by cyclooxygenases (COX 1 and 2), lipoxygenases (5-LOX, 12-LOX, 15-LOXa, 15-LOXb), and cytochrome P450s, respectively. These pathways are the target of approved drugs for the treatment of inflammation, pain, asthma, allergies, and cardiovascular disorders. Beyond their potent anti-inflammatory and anti-cancer effects, non-steroidal anti-inflammatory drugs (NSAIDs) and COX-2 specific inhibitors have been evaluated in both preclinical tumor models and clinical trials. Eicosanoid biosynthesis and actions can also be directly influenced by nutrients in the diet, as evidenced by the emerging role of omega-3 fatty acids in cancer prevention and treatment. Most research dedicated to using eicosanoids to inhibit tumor-associated inflammation has focused on the COX and LOX pathways. Novel experimental approaches that demonstrate the anti-tumor effects of inhibiting cancer-associated inflammation currently include: eicosanoid receptor antagonism, overexpression of eicosanoid metabolizing enzymes, and the use of endogenous anti-inflammatory lipid mediators. Here we review the actions of eicosanoids on inflammation in the context of tumorigenesis. Eicosanoids may represent a missing link between inflammation and cancer and thus could serve as therapeutic target(s) for inhibiting tumor growth.     

Lipoxin A4 inhibits acute edema in mice: implications for the anti-edematogenic mechanism induced by aspirin

Lipoxin A4 (LXA4) is a lipid mediator that plays an important role in the resolution of inflammation. However, the role of LXA4 and aspirin (ASA)-triggered lipoxins (ATLs) in inflammatory edema formation remains unclear. Here, we investigated the inhibitory role played by LXA4 in the carrageenan-induced and other inflammatory mediator-induced edematogenic response in mice, and also assessed the role of ATLs in the anti-edematogenic action of aspirin. Our results showed that LXA4 (1-20 ng/paw or 5 microg/kg i.p.) was effective in inhibiting carrageenan-induced paw edema from 30 min to 2 h. LXA4 (10 ng/paw) was also able to acutely inhibit PAF-, histamine-, PGE2- or bradykinin-induced paw edema, as well as the PAF-induced myeloperoxidase activity increase in the paws. Likewise, LXA4 (10 ng/cavity) also inhibited the pleural edema triggered by histamine (1h), and this response was not followed by leukocyte accumulation. Of note, the lipoxin receptor (ALX-r) antagonist Boc2 (butoxycarbonyl-Phe-Leu-Phe-Leu-Phe, 200 ng/paw) significantly reverted the anti-edematogenic effect of ASA (300 mg/kg p.o.) against carrageenan, PAF, PGE2 and BK, without affecting the anti-edematogenic action caused by indomethacin (3 mg/kg i.p.) in the carrageenan-induced paw edema. Collectively, our results demonstrate for the first time that LXA4 displays an acute and rapid onset anti-edematogenic activity that does not discriminate among different pro-inflammatory stimuli, an effect that is most likely independent of its action on the leukocyte influx. Finally, the present study demonstrates that ATLs exert a very important role in the acute anti-edematogenic action of ASA.     

Cyclopentenone-containing oxidized phospholipids and their isoprostanes as pro-resolving mediators of inflammation

Inflammation represents a powerful innate immune response that defends tissue homeostasis. However, the appropriate termination of inflammatory processes is essential to prevent the development of chronic inflammatory disorders. The resolution of inflammation is actively induced by specialized pro-resolving lipid mediators, which include eicosanoids, resolvins, protectins and maresins. The responsible pro-resolution pathways have emerged as promising targets for anti-inflammatory therapies since they mitigate excessive inflammation without compromising the anti-microbial defenses of the host. We have recently shown that the lipid peroxidation of membrane phospholipids, which is associated with inflammatory conditions, generates oxidized phospholipid (OxPL) species with potent pro-resolving activities. These pro-resolving OxPLs contain a cyclopentenone as their common determinant, and are structurally and functionally related to endogenous pro-resolving prostaglandins. Here, we review the regulation of inflammatory responses by OxPLs with particular focus on the bioactivities and structural characteristics of cyclopentenone-OxPLs, and discuss the impact of the responsible signaling pathways on inflammatory diseases. This article is part of a Special Issue entitled: Lipid modification and lipid peroxidation products in innate immunity and inflammation edited by Christoph J. Binder.     

Stability and analysis of eicosanoids and docosanoids in tissue culture media

Bioactive lipids derived from polyunsaturated fatty acids constitute an important set of lipid mediators with diverse homeostatic and pathophysiological activities. The role of bioactive lipids in inflammation, active resolution of inflammation, reproduction, cardiovascular biology, chronic diseases such as cancer and metabolic disorders is an active area of research. Other than the few clinically used eicosanoids such as prostaglandin F(2)α, prostacyclin, and prostaglandin E(2), little is known about the stability of a majority of the bioactive lipids in aqueous solutions despite their extensive use under in vitro experimental conditions. The present study evaluated the stability of eicosanoids and docosanoids as well as their parent polyunsaturated fatty acids in tissue culture media with respect to incubation time, temperature, and composition of the media using multiple reaction monitoring (MRM) LC-MS. The results provide a comprehensive and valuable data set on the stability of these bioactive lipid mediators to guide in vitro experimentation.     

Red algae natural products for prevention of lipopolysaccharides (LPS)-induced liver and kidney inflammation and injuries

Background: The liver and kidney inflammation due to bacterial infection is one of the most common pathological problems leading to tissue damage or disease. In many liver and kidney disorders, which represent serious global health burden with a high economic cost, oxidative stress-related inflammation and apoptosis are important pathogenic components, finally resulting in acute liver and/or kidney failure. Erythropoietin and its analogues are well known to influence the interaction between apoptosis and inflammation in liver and kidney. Objective: The aim of the present study is to investigate and clarify the effect of Galaxaura oblongata (G. oblongata) red algae on lipopolysaccharides (LPS)-induced acute liver and kidney injury of mice with endotoxemia and associated molecular mechanism from inflammation, apoptosis and oxidative stress levels. Results: The current study cleared out that treatment of rats with the G. oblongata extract prior to LPS injection significantly lowered serum cytokines, including NF-κB, MPO and LPO, and improved liver apoptosis through suppressing protein tyrosine kinase signaling pathway, and that may be due to antibacterial activity as well antioxidant capacity of G. oblongata extract. Conclusion: The present study was cleared out the possibility of administration of G. oblongata red algae as a multi products source for biotechnological, medical, nutraceutical and pharmaceutical applications due to highly antioxidant and anti-inflammatory capacities even although more investigations are required for separating, purifying and characterizing these bioactive compounds.     

The role of marine omega-3 (n-3) fatty acids in inflammatory processes, atherosclerosis and plaque stability

Atherosclerosis has an important inflammatory component and acute cardiovascular events can be initiated by inflammatory processes occurring in advanced plaques. Fatty acids influence inflammation through a variety of mechanisms; many of these are mediated by, or associated with, the fatty acid composition of cell membranes. Human inflammatory cells are typically rich in the n-6 fatty acid arachidonic acid, but the contents of arachidonic acid and of the marine n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can be altered through oral administration of EPA and DHA. Eicosanoids produced from arachidonic acid have roles in inflammation. EPA also gives rise to eicosanoids and these are usually biologically weak. EPA and DHA give rise to resolvins which are anti-inflammatory and inflammation resolving. EPA and DHA also affect production of peptide mediators of inflammation (adhesion molecules, cytokines, etc.). Thus, the fatty acid composition of human inflammatory cells influences their function; the contents of arachidonic acid, EPA and DHA appear to be especially important. The anti-inflammatory effects of marine n-3 polyunsaturated fatty acids (PUFAs) may contribute to their protective actions towards atherosclerosis and plaque rupture.     

Aspirin-triggered lipoxins (15-epi-LX) are generated by the human lung adenocarcinoma cell line (A549)-neutrophil interactions and are potent inhibitors of cell proliferation.

BACKGROUND: The mechanism by which aspirin (ASA) acts to protect against human cancer is not yet known. We recently showed that ASA triggers the formation of a new series of potent bioactive eicosanoids, 15-epi-lipoxins (15-epi-LXs or ASA-triggered LX [ATL]), during interactions between prostaglandin endoperoxide synthase-2 (PGHS-2) in endothelial cells and 5-lipoxygenase (LO) in leukocytes. Here, we investigated the transcellular biosynthesis of these eicosanoids during costimulation of the human tumor A549 cell line (alveolar type II epithelial cells) and neutrophils, and evaluated their impact on cell proliferation. MATERIALS AND METHODS: A549 cells and isolated neutrophils were coincubated and mRNA expression levels of key enzymes in eicosanoid biosynthesis were measured. In addition, product formation was analysed by physical methods. The effect of LX on cell proliferation was determined by using a soluble microculture tetrazolium (MTT) assay and by measuring [3H]-thymidine incorporation. RESULTS: Interleukin-1 beta (IL-1 beta)-primed A549 cells showed selective elevation in the levels of PGHS-2 mRNA and generated 15-hydroxyeicosatetraenoic acid (15-HETE). ASA markedly increased 15-HETE formation by A549 cells, while treatment with an inhibitor of cytochrome P450 reduced by approximately 50%, implicating both PGHS-2- and cytochrome P450-initiated routes in 15-HETE biosynthesis in these cells. Maximal production of 15-HETE from endogenous sources occurred within 24 hr of cytokine (IL-1 beta) exposure and declined thereafter. Chiral analysis revealed that approximately 85% of ASA-triggered epithelial-derived 15-HETE carries its carbon 15 alcohol group in the R configuration. Costimulation of ASA-treated A549 cells and polymorphonuclear neutrophilic leukocytes (PMN) led to production of both LXA4 and LXB4, as well as 15-epi-LXA4 and 15-epi-LXB4 (9.5 +/- 0.5 ng LX/10(7) A549 cells). 15-epi-LXA4 accounted for approximately 88% of the total amount of LXA4 produced. In addition to LXs, stimulation of A549 cells and PMN also liberated substantial amounts (77.2 +/- 8.2 ng/10(7) A549 cells) of peptidoleukotrienes (pLTs), which were not generated by either cell type alone. Addition of ASA to these co-incubations led to an increase in the amounts of LXs generated that was paralleled by a decrease in pLTs. LXA4, LXB4, 15-epi-LXA4 and 15-epi-LXB4, as well as dexamethasone, inhibited cell proliferation at 100 nM range with a rank order of activity of 15-epi-LXB4 >>> LXB4 > dexamethasone > or = 15-epi-LXA4 > LXA4. CONCLUSIONS: These results indicate that ASA promotes the formation of antiproliferative 15-epi-LXs by epithelial cell-leukocyte interactions. Moreover, they suggest that these novel eicosanoids, when generated within the microenvironment of tissues, may contribute to ASA's therapeutic role in decreasing the risk of human cancer.