Profiling in resolving inflammatory exudates identifies novel anti-inflammatory and pro-resolving mediators and signals for termination.

A highly orchestrated inflammatory response and its completion, termed resolution, are essential for ongoing health. Thus, complete understanding of the cellular and molecular events that govern natural resolution is vital. Using an unbiased systems approach to profile self-limited inflammatory exudates, we identified a novel genus of specialized pro-resolving lipid mediators (SPMs) comprised of three new families coined the resolvins, protectins and most recently the maresins biosynthesized from omega-3 fatty acids. These join the lipoxin- and aspirin-triggered lipoxins as anti-inflammatory and pro-resolving lipid mediators formed from arachidonic acid with the genus. SPMs have proven stereoselective, and control both the duration and magnitude of inflammation. Mapping these endogenous resolution circuits provides new avenues to probe the molecular basis of many widely occurring diseases where uncontrolled inflammation is characteristic. The focus of this JIM review is to depict recent advances from studies by the authors and colleagues on the biosynthesis and actions of these novel anti-inflammatory, pro-resolving and protective lipid mediators. Together these findings indicate that defective mechanisms and pathways in resolution may underlie our current appreciation of the inflammatory phenotype(s) that characterize some prevalent human diseases.

Systems approach to inflammation resolution: identification of novel anti-inflammatory and pro-resolving mediators.

Using a systems approach to profile self-limited inflammatory exudates, we identified three novel families of lipid-derived mediators, coined the resolvins, protectins and most recently, the maresins that control both the magnitude and duration of inflammation. The mapping of these endogenous resolution circuits provides new avenues to probe the molecular basis of many widely occurring inflammatory diseases. This article focuses on our recent advances on the functional metabolomics of this novel genus of specialized pro-resolving mediators (SPM). SPM include resolvins, protectins and maresins and are biosynthesized from essential omega-3 fatty acid precursors. Each possesses potent multi-pronged actions that proved to be stereoselective with human cells and in animal disease models. Resolvins and protectins are also produced in bone marrow. Together, these findings suggest that defective resolution mechanism(s) may underlie some chronic inflammatory diseases. Moreover, identification of functional SPM biosynthesized during inflammation-resolution indicates that resolution is an active process.

Lipid mediator class switching during acute inflammation: signals in resolution.

Leukotrienes (LTs) and prostaglandins (PGs) amplify acute inflammation, whereas lipoxins (LXs) have unique anti-inflammatory actions. Temporal analyses of these eicosanoids in clinical and experimental exudates showed early coordinate appearance of LT and PG with polymorphonuclear neutrophil (PMN) recruitment. This was followed by LX biosynthesis, which was concurrent with spontaneous resolution. Human peripheral blood PMNs exposed to PGE2 (as in exudates) switched eicosanoid biosynthesis from predominantly LTB4 and 5-lipoxygenase (5-LO)-initiated pathways to LXA4, a 15-LO product that "stopped" PMN infiltration. These results indicate that first-phase eicosanoids promote a shift to anti-inflammatory lipids: functionally distinct lipid-mediator profiles switch during acute exudate formation to "reprogram" the exudate PMNs to promote resolution.

Novel functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3 fatty acids via cyclooxygenase 2-nonsteroidal antiinflammatory drugs and transcellular processing.

Aspirin therapy inhibits prostaglandin biosynthesis without directly acting on lipoxygenases, yet via acetylation of cyclooxygenase 2 (COX-2) it leads to bioactive lipoxins (LXs) epimeric at carbon 15 (15-epi-LX, also termed aspirin-triggered LX [ATL]). Here, we report that inflammatory exudates from mice treated with omega-3 polyunsaturated fatty acid and aspirin (ASA) generate a novel array of bioactive lipid signals. Human endothelial cells with upregulated COX-2 treated with ASA converted C20:5 omega-3 to 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-HEPE. Each was used by polymorphonuclear leukocytes to generate separate classes of novel trihydroxy-containing mediators, including 5-series 15R-LX(5) and 5,12,18R-triHEPE. These new compounds proved to be potent inhibitors of human polymorphonuclear leukocyte transendothelial migration and infiltration in vivo (ATL analogue > 5,12,18R-triHEPE > 18R-HEPE). Acetaminophen and indomethacin also permitted 18R-HEPE and 15R-HEPE generation with recombinant COX-2 as well as omega-5 and omega-9 oxygenations of other fatty acids that act on hematologic cells. These findings establish new transcellular routes for producing arrays of bioactive lipid mediators via COX-2-nonsteroidal antiinflammatory drug-dependent oxygenations and cell-cell interactions that impact microinflammation. The generation of these and related compounds provides a novel mechanism(s) for the therapeutic benefits of omega-3 dietary supplementation, which may be important in inflammation, neoplasia, and vascular diseases.

Anti-microinflammatory lipid signals generated from dietary N-3 fatty acids via cyclooxygenase-2 and transcellular processing: a novel mechanism for NSAID and N-3 PUFA therapeutic actions.

Aspirin therapy inhibits prostaglandin biosynthesis; yet via acetylation of cyclooxygenase 2 (COX-2) it leads to bioactive lipoxins epimeric at carbon 15 (15-epi-LX, also termed aspirin-triggered lipoxin or ATL). Here, we review our findings indicating that inflammatory exudates from mice treated with omega-3 PUFA and aspirin (ASA) generate a novel array of bioactive lipid signals. Also, human endothelial cells, both HUVEC and microvascular, with upregulated COX-2 and treated with ASA converted C20:5 omega-3 to 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-HEPE. Human PMN activated with serum treated zymosan (STZ) utilized each of these R-HEPEs to generate novel classes of trihydroxy-containing mediators including 5-series 15R-LX and 5,12,18R-triHEPE. The novel products were potent inhibitors of human PMN transendothelial migration and infiltration of PMN in dorsal air pouches in vivo. In addition to ASA, both acetaminophen and indomethacin also permitted 18R-HEPE and 15R-HEPE generation with recombinant human COX-2 as well as omega-5 and omega-9 oxygenations of other fatty acids that act on leukocytes, platelets and endothelial cells. These findings establish new transcellular routes for producing arrays of lipid mediators via COX-2-NSAIDs and cell-cell interactions that impact microinflammation. Moreover, they provide novel mechanism(s) that could underlie the many reported therapeutic benefits of omega-3 dietary supplementation of interest in inflammation, cancer, and vascular disorders.

Identification of a human cDNA encoding a functional high affinity lipoxin A4 receptor.

Lipoxin A4 (LXA4) triggers selective responses with human neutrophils that are pertussis toxin sensitive and binds to high affinity receptors (Kd = 0.5 +/- 0.3 nM) that are modulated by stable analogues of guanosine 5'-triphosphate (GTP). Here, we characterized [11,12-(3)]LXA4 specific binding with neutrophil granule and plasma membranes, which each display high affinity binding sites (Kd = 0.7 +/- 0.1 nM) that were regulated by GTP gamma S. Since functional LXA4 receptors are inducible in HL-60 cells, we tested orphan cDNAs encoding 7-transmembrane region receptors cloned from these cells for their ability to bind and signal with LXA4. Chinese hamster ovary (CHO) cells transfected with the orphan receptor cDNA (pINF114) displayed specific 3H-LXA4 high affinity binding (1.7 nM). When displacement of LXA4 binding with pINF114-transfected CHO cells was tested with other eicosanoids, including LXB4, leukotriene D4 (LTD4), LTB4, or prostaglandin E2, only LTD4 competed with LXA4, giving a Ki of 80 nM. In transfected CHO cells, LXA4 also stimulated GTPase activity and provoked the release of esterified arachidonate, which proved to be pertussis toxin sensitive. These results indicate that pINF114 cDNA encodes a 7-transmembrane region-containing protein that displays high affinity for 3H-LXA4 and transmits LXA4-induced signals. Together, they suggest that the encoded protein is a candidate for a LXA4 receptor in myeloid cells.

Lipoxin synthase activity of human platelet 12-lipoxygenase.

Human platelets and megacaryocytes generate lipoxins from exogenous leukotriene A4 (LTA4). We examined the role of human 12-lipoxygenase (12-LO) in lipoxin generation with recombinant histidine-tagged human platelet enzyme (6His-12-LO), partially purified 12-LO from human platelets (HPL 12-LO) and, for the purposes of direct comparison, permeabilized platelets. Recombinant and HPL 12-LO catalysed the conversion of intact LTA4 into both lipoxin A4 (LXA4) and lipoxin B4 (LXB4). In contrast, only negligible quantities of LXA4 were generated when recombinant 12-LO was incubated with the non-enzymic hydrolysis products of LTA4.6His-12-LO also converted a non-allylic epoxide, 5(6)-epoxy-(8Z,11Z,14Z)-eicosatrienoic acid. The apparent Km and Vmax. for lipoxin synthase activity of 6His-12-LO were estimated to be 7.9 +/- 0.8 microM and 24.5 +/- 2.5 nmol/min per mg respectively, and the LXB4 synthase activity of this enzyme was selectively regulated by suicide inactivation. Aspirin gave a 2-fold increase in lipoxin formation by platelets but did not enhance the conversion of LTA4 by the recombinant 12-LO. These results provide direct evidence for LXA4 and LXB4 synthase activity of human platelet 12-LO. Moreover, they suggest that 12-LO is a dual-function enzyme that carries both oxygenase and lipoxin synthase activity.