SummaryLipoxin 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 [11,12-3H]LXA4 specific binding with neutrophil granule and plasma membranes, which each display high affinity binding sites (Kd = 0.7 _+ 0.1 riM) that were regulated by GTP3,S. Since functional LXAa 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 eDNA (plNF114) displayed specific 3H-LXA4 high affinity binding (1.7 nM). When displacement of LXA4 binding with plNFl14-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 plNF114 eDNA 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.poxygenase (LO)l-derived eicosanoids are important lipid mediators (1). The 5-LO-derived products include leukotriene B4 (LTB4), a potent stimulus for phagocytic cells, and peptido-leukotrienes C4, D4, and E4, which are potent bronchoconstrictors and are associated with the pathogenesis of asthma (1). Lipoxins (LX) are a newer class of bioactive LO-derived products that are generated by the interactions of either 5-and 12-LO and/or 15-and 5-LO followed by subsequent reactions (for a review see reference 2). The LXs are functionally distinct from leukotrienes and other eicosanoids and are primarily generated in human tissues during cell-cell interactions that are exemplified by leukocyte-platelet interactions (2). LXA4 displays intriguing biological re-1 Abbreuiations used in this paper: AMP-PNP, 5'-adenylylimidodiphosphate; CHO, Chinese hamster ovary cells; DPBS/PBS, Dulbecco's PBS; PBS 2-without divalent cations; FPR, formyl peptide receptor; GTPyS, guanosine 5'-O-(3-thiotriphosphate); leukotriene B4 (LTB4), 5S,12R-dihydroxy-6,14-cis-8,10-trans-dihydroxyeicosatetraenoic acid; leukotriene D4 (LTD4), 5S-hydroxy-6R- lipoxin A4 (LXA4), 5S,6R,9, lipoxin B4 (LXB4), 5S,14R,10,-ciseicosatetraenoic acid; LO, lipoxygenase; PGE2, 9-oxy-11o~,15S-dihydroxy-5-cis-13-trans-prostadienoic acid; PT, Pertussis toxin holotoxin.sponses in several tissues (2), and with neutrophils they involve G protein-mediated signal transduction events (3-5). A LXA4 receptor is induced in HL-60 cells upon differentiation, and it activates phospholipase D (5). LXA4-induced lipid remodeling events are similar to those of other leuko...
Lipoxins are bioactive eicosanoids that are immunomodulators. In human myeloid cells, lipoxin (LX) A4 actions are mediated by interaction with a G protein–coupled receptor. To explore functions of LXA4 and aspirin-triggered 5(S),6(R),15(R)-trihydroxy-7,9,13-trans-11-cis–eicosatetraenoic acid (15-epi-LXA4) in vivo, we cloned and characterized a mouse LXA4 receptor (LXA4R). When expressed in Chinese hamster ovary cells, the mouse LXA4R showed specific binding to [3H]LXA4 (K d ≈ 1.5 nM), and with LXA4 activated GTP hydrolysis. Mouse LXA4R mRNA was most abundant in neutrophils. In addition to LXA4 and 15-epi-LXA4, bioactive LX stable analogues competed with both [3H]LXA4 and [3H]leukotriene D4 (LTD4)– specific binding in vitro to neutrophils and endothelial cells, respectively. Topical application of LXA4 analogues and novel aspirin-triggered 15-epi-LXA4 stable analogues to mouse ears markedly inhibited neutrophil infiltration in vivo as assessed by both light microscopy and reduced myeloperoxidase activity in skin biopsies. The 15(R)-16-phenoxy-17,18, 19,20-tetranorLXA4 methyl ester (15-epi-16-phenoxy-LXA4), an analogue of aspirin triggered 15-epi-LXA4, and 15(S)-16-phenoxy-17,18,19,20-tetranor-LXA4 methyl ester (16-phenoxy-LXA4) were each as potent as equimolar applications of the anti-inflammatory, dexamethasone. Thus, we identified murine LXA4R, which is highly expressed on murine neutrophils, and showed that both LXA4 and 15-epi-LXA4 stable analogues inhibit neutrophil infiltration in the mouse ear model of inflammation. These findings provide direct in vivo evidence for an anti-inflammatory action for both aspirin-triggered LXA4 and LXA4 stable analogues and their site of action in vivo.
Lipoxins (LX) are bioactive eicosanoids that carry a tetraene structure and serve as regulators of inflammation, in part by inhibiting neutrophil migration and adhesion. Lipoxin A4 is rapidly regulated by conversion to inactive LX metabolites via local metabolism that involves dehydrogenation as the predominant route. Here, several LXA4 analogs were designed that resisted rapid conversion by both differentiated HL-60 cells and recombinant 15-hydroxyprostaglandin dehydrogenase, systems where native LXA4 is degraded within minutes. The rank order of conversion by recombinant dehydrogenase was LXA4 methyl ester > PGE2 approximately PGE2 methyl ester > LXA4 >>> the novel LXA4 analogs. In addition, 15(R/S)-methyl-LXA4, 15-cyclohexyl-LXA4, and 16-phenoxy-LXA4 proved to retain LXA4 bioactivity and inhibited neutrophil transmigration across polarized epithelial cell monolayers as well as adhesion to vascular endothelial cells. These results indicate that LXA4 analogs can be designed using these criteria to resist rapid transformation and to retain biological actions of native LXA4. Moreover, the results suggest that LXA4 stable analogs can be useful tools both in vitro and in vivo to evaluate LXA4 actions and therapeutic potential.
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