A Stable Aspirin-Triggered Lipoxin A 4 Analog Blocks Phosphorylation of Leukocyte-Specific Protein 1 in Human Neutrophils 1 Taisuke Ohira,* † Gerard Bannenberg,* Makoto Arita,* Minoru Takahashi,* Qingyuan Ge, ‡ Thomas E. Van Dyke, † Gregory L. Stahl,* Charles N. Serhan,* and John A. Badwey 2 * Lipoxins and their aspirin-triggered 15-epimers are endogenous anti-inflammatory agents that block neutrophil chemotaxis in vitro and inhibit neutrophil influx in several models of acute inflammation. In this study, we examined the effects of 15-epi-16- (p-fluoro)-phenoxy-lipoxin A 4 methyl ester, an aspirin-triggered lipoxin A 4 -stable analog (ATLa), on the protein phosphorylation pattern of human neutrophils. Neutrophils stimulated with the chemoattractant fMLP were found to exhibit intense phosphor- ylation of a 55-kDa protein that was blocked by ATLa (10 –50 nM). This 55-kDa protein was identified as leukocyte-specific protein 1, a downstream component of the p38-MAPK cascade in neutrophils, by mass spectrometry, Western blotting, and immuno- precipitation experiments. ATLa (50 nM) also reduced phosphorylation/activation of several components of the p38-MAPK pathway in these cells (MAPK kinase 3/MAPK kinase 6, p38-MAPK, MAPK-activated protein kinase-2). These results indicate that ATLa exerts its anti-inflammatory effects, at least in part, by blocking activation of the p38-MAPK cascade in neutrophils, which is known to promote chemotaxis and other proinflammatory responses by these cells. The Journal of Immunology, 2004, 173: 2091–2098. L ipoxins are endogenous anti-inflammatory lipid mediators that promote the resolution of inflammation. Biosynthesis of these lipid mediators in humans requires cell-cell in- teractions and is highly influenced by the presence of certain cy- tokines and aspirin (reviewed in Refs. 1–3). Monocytes, eosino- phils, and airway-epithelial cells can convert arachidonate into 15S-hydroxyeicosatetraenoic acid (15S-HETE) 3 by a 15-lipoxy- genase catalyzed reaction. 15S-HETE is rapidly taken up by neu- trophils and converted to lipoxin A 4 (LXA 4 ) by a 5-lipoxygenase catalyzed reaction. Of interest, acetylation of cyclooxygenase 2 by aspirin alters the activity of this enzyme to catalyze conversion of arachidonate to 15R-HETE (1). 15R-HETE can also be converted by neutrophils and other cells to 15-epi-LXA 4 . LXA 4 and 15-epi LXA 4 are rapidly generated in response to specific signals, act locally, and then are quickly inactivated by conversion to 15-oxo- containing metabolites in monocytes (1–3). A series of metaboli- cally resistant LXA 4 analogues (e.g., 15-epi-16-( p-fluoro)- phenoxy-LXA 4 -methyl ester or aspirin-triggered LXA 4 -stable analog (ATLa)) were designed and synthesized to prevent this rapid inactivation (4). Lipoxins and their stable analogues potently block many of the functional responses of neutrophils in vitro (chemo- taxis, transmigration, degranulation, superoxide production, IL-1 release) (4 – 8) and prevent leukocyte influx/recruitment in acute inflammation (8 –12). For example, topical application of ATLa dramatically blocks leukocyte infiltration and tissue destruction in a rabbit model of periodontal disease (9). Specific G-protein coupled receptors for lipoxins have been identified on a variety of cells (13–15), and some of the biochem- ical pathways/second messenger systems that become activated when these receptors are occupied have been described (16 –20). Protein phosphorylation reactions are critically involved in most, if not all, of the functional responses of stimulated neutrophils (21). At this juncture, little is known about how lipoxins might impact protein phosphorylation in human neutrophils. In this manuscript, we report that ATLa blocks phosphorylation of leukocyte-specific protein 1 (LSP1) and alters phosphorylation/activation of compo- nents of the p38-MAPK cascade in chemoattractant-stimulated hu- man neutrophils. The significance of the p38-MAPK cascade as a “target” for lipoxins is consistent with the proinflammatory nature of this pathway and the anti-inflammatory actions of ATLa. Materials and Methods Materials Affinity-purified rabbit phosphospecific Abs (pAbs) that recognize phos- pho-Ser/Thr in proteins preceded by two or more Lys/Arg residues at the -2 to -5 positions (pAkt(S) Ab), MAPK-activated protein kinase-2 (MAPKAP-K2) when phosphorylated at Thr 334 (pMAPKAP-K2(Thr 334 ) Ab), MAPK kinase 3/6 (MKK3/MKK6) when phosphorylated at Ser 189 and Ser 207 , respectively (pMKK3/MKK6(Ser 189/207 ) Ab), p44/p42-MAPK (ERK1/2) when phosphorylated at both Thr 202 and Tyr 204 (p44/42(Thr 202 and Tyr 204 ) Ab), and p38-MAPK when phosphorylated at both Thr 180 and *Center for Experimental Therapeutics and Reperfusion Injury, Department of An- esthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital and Harvard University Medical School, Boston, MA 02115; † Boston University Gold- man School of Dental Medicine, Boston, MA 02118; and ‡ Cell Signaling Technology, Beverly, MA 01915 Received for publication March 11, 2004. Accepted for publication May 18, 2004. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grants PO1 DE-13499 (to C.N.S.), AI-23323 (to J.A.B.), and HL-56086 (to G.L.S.). G.B. was supported by the Arthritis Foundation. 2 Address correspondence and reprint requests to Dr. John A. Badwey, Center for Ex- perimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Periop- erative and Pain Medicine, Brigham and Women’s Hospital, Thorn Building, Room 703, 75 Francis Street, Boston, MA 02115. E-mail address: Badwey@zeus.bwh.harvard.edu 3 Abbreviations used in this paper: 15S-HETE, 15S-hydroxyeicosatetraenoic acid; LXA 4 , lipoxin A 4 ; Bim I, bisindolylmaleimide I; ATLa, aspirin-triggered LXA 4 - stable analog; LSP1, leukocyte-specific protein 1; pAb, phosphospecific Ab; MAPKAP-K2, MAPK-activated protein kinase-2; MKK, MAPK kinase; Hsp27, heat shock protein 27; LC, liquid chromatography; MS, mass spectrometry; m/z, mass/ charge; PKC, protein kinase C; GST-fl-LSP1, GST fusion protein containing full- length LSP1; GST-N-LSP1, GST fusion protein containing the N-terminal region of LSP1; PLD, phospholipase D. The Journal of Immunology Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00