Aspirin-Triggered 15-epi-Lipoxin A 4 (ATL) Generation by Human Leukocytes and Murine Peritonitis Exudates: Development of a Specific 15-epi-LXA 4 ELISA 1 NAN CHIANG, TOMOKO TAKANO, CLARY B. CLISH, NICOS A. PETASIS 2 , HSIN-HSIUNG TAI 3 , and CHARLES N. SERHAN Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesia, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts (N.C., T.T., C.B.C., C.N.S.), Department of Chemistry, University of Southern California, Los Angeles, California (N.A.P), and Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington, Kentucky (H.-H.T.) Accepted for publication June 1, 1998 This paper is available online at http://www.jpet.org ABSTRACT Aspirin (ASA) triggers the formation of 15-epi-lipoxins (15-epi- LXs or ATL [ASA-triggered LX]), which are potent bioactive eicosanoids that may contribute to the therapeutic impact of ASA. To elucidate the role of these new compounds in vivo, it is essential to establish quick and sensitive detection methods. To this end, we prepared an enzyme-linked immunosorbent assay specific for 15-epi-LXA 4 that proved to be highly sensi- tive (IC 50  50 pg, minimum detection  3.5 pg) and stereo- selective. The amounts of 15-epi-LXA 4 generated by human neutrophils from peripheral blood of healthy volunteers using this enzyme-linked immunosorbent assay were in agreement with those values obtained by liquid chromatography. Forma- tion of 15-epi-LXA 4 was cell ratio-dependent during THP-1 (a monocytic leukemia cell line)-neutrophil interactions with ASA-treated cells, and 15-epi-LXA 4 was not detected with ei- ther cell type alone. Generation of 15-epi-LXA 4 was also exam- ined in murine peritonitis with ASA administration. Exudates from ASA-treated mice showed increased production of 15- epi-LXA 4 that was diminished by indomethacin, a blocker of ASA-dependent acetylation of prostaglandin G/H synthase. A cytochrome P450 inhibitor administered in the presence of ASA did not prevent 15-epi-LXA 4 formation, which suggests that P450 does not significantly contribute to formation of 15-epi- LXA 4 in this murine model. These results indicate that the new enzyme-linked immunosorbent assay is both sensitive and se- lective for 15-epi-LXA 4 and that 15-epi-LXA 4 is produced by human leukocyte-leukocyte interactions. In addition, 15-epi- LXA 4 is generated by inflammatory exudates when ASA is administered during murine peritonitis and when prostaglandin G/H synthase is upregulated and acetylated. This assay should provide rapid means to investigate 15-epi-LXA 4 generation in both cellular and animal models. Lipoxins belong to the eicosanoid family of bioactive lipid mediators that carry trihydroxytetraene structures as distin- guishing features. They are generated in mammals predom- inantly by transcellular biosynthetic routes during cell-cell interactions, which is now recognized as an important means of both amplifying and generating new lipid-derived media- tors (reviewed in Serhan, 1997). Two major transcellular routes of LX biosynthesis by LO interactions in human cell types are established. LXs generated by these two pathways carry their C-15 hydroxyl group mainly in the 15S-configu- ration, which is inserted by lipoxygenase-based mechanisms. LXs not only are formed in vitro in isolated cells but also are generated in humans and in experimental animals. Situa- Received for publication January 8, 1998. 1 This work was supported in part by grants no. GM38765 and DK50305 (C.N.S.) from the National Institutes of Health. 2 Present address: Nephrology Division, Royal Victoria Hospital, 3775 Uni- versity Street, Montreal, Quebec, Canada H3A 2B4. ABBREVIATIONS: AA, arachidonic acid; ASA, aspirin (acetylsalicylic acid); ATL, aspirin-triggered lipoxins; ELISA, enzyme-linked immunosorbent assay; GM-CSF, granulocyte monocyte colony-stimulating factor; HETE, hydroxy eicosatetraenoic acid; 5S-HETE, (5S)-5-hydroxy-8,11,14-cis- 6-trans-eicosatetraenoic acid; 12S-HETE, (12S)-12-hydroxy-5,8,14-cis-10-trans-eicosatetraenoic acid; 15S-HETE, (15S)-15-hydroxy-5,8,11-cis- 13-trans-eicosatetraenoic acid; 15R-HETE, (15R)-15-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid; HRP, horseradish peroxidase; LC/MS/ MS, liquid chromatography tandem mass spectrometry-mass spectrometry; LO, lipoxygenase; LPS, lipopolysaccharide; LTB 4 (leukotriene B 4 ), 5S, 12R-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid; LX, lipoxin; 15-epi-LXA 4 ,5S, 6R, 15R-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid; 15-epi-LXB 4 ,5S, 14R, 15R-trihydroxy-6,10,12-trans-8-cis-eicosatetraenoic acid; 15-R/S-methyl-LXA 4 ,5S, 6R, 15(R/S)-trihydroxy-methyl- 7,9,13-trans-11-cis-eicosatetraenoic acid; LXA 4 ,5S, 6R, 15S-trihydroxyl-7,9,13-trans-11-cis-eicosatetraenoic acid; LXB 4 ,5S, 14R, 15S-trihy- droxyl-6,10,12-trans-8-cis-eicosatetraenoic acid; ODYA, 17-octadecynoic acid; PGHS, prostaglandin G/H synthase; PMN, neutrophil(s); RP- HPLC, reverse-phase high-pressure liquid chromatography; KLH, keyhole limpets hemocyanin; THP-1, human acute monocytic leukemia cell line. PVDF, polyvinylidine difluoride; SDS-Page, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 0022-3565/98/2872-0779$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 287, No. 2 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 287:779 –790, 1998 779 at ASPET Journals on June 1, 2016 jpet.aspetjournals.org Downloaded from