NADPH Oxidase-dependent Generation of Lysophosphatidylserine Enhances Clearance of Activated and Dying Neutrophils via G2A
2008; Elsevier BV; Volume: 283; Issue: 48 Linguagem: Inglês
10.1074/jbc.m807047200
ISSN1083-351X
AutoresS. Courtney Frasch, Karin Zemski Berry, Ruby Fernandez-Boyanapalli, Hyun-Sun Jin, Christina C. Leslie, Peter M. Henson, Robert C. Murphy, Donna L. Bratton,
Tópico(s)Neutrophil, Myeloperoxidase and Oxidative Mechanisms
ResumoExofacial phosphatidylserine (PS) is an important ligand mediating apoptotic cell clearance by phagocytes. Oxidation of PS fatty acyl groups (oxPS) during apoptosis reportedly mediates recognition through scavenger receptors. Given the oxidative capacity of the neutrophil NADPH oxidase, we sought to identify oxPS signaling species in stimulated neutrophils. Using mass spectrometry analysis, only trace amounts of previously characterized oxPS species were found. Conversely, 18:1 and 18:0 lysophosphatidylserine (lyso-PS), known bioactive signaling phospholipids, were identified as abundant modified PS species following activation of the neutrophil oxidase. NADPH oxidase inhibitors blocked the production of lyso-PS in vitro, and accordingly, its generation in vivo by activated, murine neutrophils during zymosan-induced peritonitis was absent in mice lacking a functional NADPH oxidase (gp91phox-/-). Treatment of macrophages with lyso-PS enhanced the uptake of apoptotic cells in vitro, an effect that was dependent on signaling via the macrophage G2A receptor. Similarly, endogenously produced lyso-PS also enhanced the G2A-mediated uptake of activated PS-exposing (but non-apoptotic) neutrophils, raising the possibility of non-apoptotic mechanisms for removal of inflammatory cells during resolution. Finally, antibody blockade of G2A signaling in vivo prolonged zymosan-induced neutrophilia in wild-type mice, whereas having no effect in gp91phox-/- mice where lyso-PS are not generated. Taken together, we show that lyso-PS are modified PS species generated following activation of the NADPH oxidase and lyso-PS signaling through the macrophage G2A functions to enhance existing receptor/ligand systems for optimal resolution of neutrophilic inflammation. Exofacial phosphatidylserine (PS) is an important ligand mediating apoptotic cell clearance by phagocytes. Oxidation of PS fatty acyl groups (oxPS) during apoptosis reportedly mediates recognition through scavenger receptors. Given the oxidative capacity of the neutrophil NADPH oxidase, we sought to identify oxPS signaling species in stimulated neutrophils. Using mass spectrometry analysis, only trace amounts of previously characterized oxPS species were found. Conversely, 18:1 and 18:0 lysophosphatidylserine (lyso-PS), known bioactive signaling phospholipids, were identified as abundant modified PS species following activation of the neutrophil oxidase. NADPH oxidase inhibitors blocked the production of lyso-PS in vitro, and accordingly, its generation in vivo by activated, murine neutrophils during zymosan-induced peritonitis was absent in mice lacking a functional NADPH oxidase (gp91phox-/-). Treatment of macrophages with lyso-PS enhanced the uptake of apoptotic cells in vitro, an effect that was dependent on signaling via the macrophage G2A receptor. Similarly, endogenously produced lyso-PS also enhanced the G2A-mediated uptake of activated PS-exposing (but non-apoptotic) neutrophils, raising the possibility of non-apoptotic mechanisms for removal of inflammatory cells during resolution. Finally, antibody blockade of G2A signaling in vivo prolonged zymosan-induced neutrophilia in wild-type mice, whereas having no effect in gp91phox-/- mice where lyso-PS are not generated. Taken together, we show that lyso-PS are modified PS species generated following activation of the NADPH oxidase and lyso-PS signaling through the macrophage G2A functions to enhance existing receptor/ligand systems for optimal resolution of neutrophilic inflammation. Neutrophils are often robustly recruited early in inflammation. Within hours of their activation in tissues, they are removed by phagocytes, an event required for resolution of inflammation and the return to normalcy of tissue function. It is known that neutrophils undergoing apoptosis drive the production of anti-inflammatory mediators such as transforming growth factor-β that actively suppress production of inflammatory cytokines, chemokines, eicosanoids, and nitric oxide (1Freire-de-Lima C.G. Xiao Y.Q. Gardai S.J. Bratton D.L. Schiemann W.P. Henson P.M. J. Biol. Chem. 2006; 281: 38376-38384Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 2Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y. Henson P.M. J. Clin. Investig. 1998; 101: 890-898Crossref PubMed Scopus (2577) Google Scholar). Indeed, enhanced induction of neutrophil apoptosis in vivo is potently anti-inflammatory (3Rossi A.G. Hallett J.M. Sawatzky D.A. Teixeira M.M. Haslett C. Biochem. Soc. Trans. 2007; 35: 288-291Crossref PubMed Scopus (47) Google Scholar, 4Hallett J.M. Leitch A.E. Riley N.A. Duffin R. Haslett C. Rossi A.G. Trends Pharmacol. Sci. 2008; 29: 250-257Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). However, if recognition and clearance fail, activated and dying neutrophils ultimately disintegrate releasing injurious intracellular constituents (e.g. serine proteases) (5Fadok V.A. Bratton D.L. Guthrie L. Henson P.M. J. Immunol. 2001; 166: 6847-6854Crossref PubMed Scopus (312) Google Scholar). Failure of timely cell clearance is associated with both autoimmunity and enhanced inflammation (6Mevorach D. Mascarenhas J.O. Gershov D. Elkon K.B. J. Exp. Med. 1998; 188: 2313-2320Crossref PubMed Scopus (576) Google Scholar, 7Taylor P.R. Carugati A. Fadok V.A. Cook H.T. Andrews M. Carroll M.C. Savill J.S. Henson P.M. Botto M. Walport M.J. J. Exp. Med. 2000; 192: 359-366Crossref PubMed Scopus (625) Google Scholar). Phosphatidylserine (PS) 2The abbreviations used are: PS, phosphatidylserine; lysophosphatidylserine(s); oxPS, oxidized phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; COOH, carboxylate-modified beads; MRM, multiple reaction monitoring; MOX, methoxylamine hydrochloride; MPO, myeloperoxidase; OPZ, opsonized zymosan; DPI, diphenyleneiodonium; PI, phagocytic index; RPMΦ, resident peritoneal macrophage; TGMΦ, thioglycollate-elicited peritoneal macrophages; 18:0/9-al-PS, 1-stearoyl-2-(9′-oxo-nonanoyl)-glycerophosphoserine; 18:0/5-al-PS, 1-stearoyl-2-(5′-oxovaleroyl)-glycerophosphoserine; PLA2, phospholipase A2; PMA, phorbol 12-myristate 13-acetate; PBS, phosphate-buffered saline; fMLP, formylmethionylleucylphenylalanine; RP-HPLC, reverse phase-high performance liquid chromatography. exposed in the plasma membrane outer leaflet of apoptotic cells has long been known as a key ligand important for their recognition and removal. Interaction with various PS receptors, including the recently identified TIM4 (8Miyanishi M. Tada K. Koike M. Uchiyama Y. Kitamura T. Nagata S. Nature. 2007; 450: 435-439Crossref PubMed Scopus (850) Google Scholar, 9Kobayashi N. Karisola P. Pena-Cruz V. Dorfman D.M. Jinushi M. Umetsu S.E. Butte M.J. Nagumo H. Chernova I. Zhu B. Sharpe A.H. Ito S. Dranoff G. Kaplan G.G. Casasnovas J.M. Umetsu D.T. Dekruyff R.H. Freeman G.J. Immunity. 2007; 27: 927-940Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar), BAI1 (10Park D. Tosello-Trampont A.C. Elliott M.R. Lu M. Haney L.B. Ma Z. Klibanov A.L. Mandell J.W. Ravichandran K.S. Nature. 2007; 450: 430-434Crossref PubMed Scopus (607) Google Scholar), and stabilin 2 (11Park S.Y. Jung M.Y. Kim H.J. Lee S.J. Kim S.Y. Lee B.H. Kwon T.H. Park R.W. Kim I.S. Cell Death Differ. 2008; 15: 192-201Crossref PubMed Scopus (341) Google Scholar) or PS-recognizing bridge molecule-receptor combinations (e.g. MFG-E8 and αv integrins or Gas6 and Mer (12Hanayama R. Tanaka M. Miwa K. Shinohara A. Iwamatsu A. Nagata S. Nature. 2002; 417: 182-187Crossref PubMed Scopus (1057) Google Scholar)), have been demonstrated. In many, but not all cases, these interactions have been shown to have stereospecificity for the l-phosphoserine moiety, and not the d-isomer (13Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216Crossref PubMed Google Scholar, 14Fadok V.A. de Cathelineau A. Daleke D.L. Henson P.M. Bratton D.L. J. Biol. Chem. 2001; 276: 1071-1077Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar, 15Hoffmann P.R. Kench J.A. Vondracek A. Kruk E. Daleke D.L. Jordan M. Marrack P. Henson P.M. Fadok V.A. J. Immunol. 2005; 174: 1393-1404Crossref PubMed Scopus (169) Google Scholar). Recently it has been demonstrated that oxidation of the sn-2 fatty acyl chain of PS (oxPS) makes it a more potent stimulus for the clearance of apoptotic cells involving scavenger receptors, particularly CD36 (16Arroyo A. Modriansky M. Serinkan F.B. Bello R.I. Matsura T. Jiang J. Tyurin V.A. Tyurina Y.Y. Fadeel B. Kagan V.E. J. Biol. Chem. 2002; 277: 49965-49975Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 17Kagan V.E. Gleiss B. Tyurina Y.Y. Tyurin V.A. Elenstrom-Magnusson C. Liu S.X. Serinkan F.B. Arroyo A. Chandra J. Orrenius S. Fadeel B. J. Immunol. 2002; 169: 487-499Crossref PubMed Scopus (209) Google Scholar, 18Greenberg M.E. Sun M. Zhang R. Febbraio M. Silverstein R. Hazen S.L. J. Exp. Med. 2006; 203: 2613-2625Crossref PubMed Scopus (322) Google Scholar). In contrast to PS-derived signals that promote recognition and removal of apoptotic cells, other lipid-derived mediators released from cells, such as lipoxins, resolvins, and protectins, signaling through their cognate receptors, have been shown to play a role in orchestrating resolution of inflammation by enhancing these existing mechanisms for engulfment of apoptotic cells and tissue homeostasis (19Schwab J.M. Chiang N. Arita M. Serhan C.N. Nature. 2007; 447: 869-874Crossref PubMed Scopus (969) Google Scholar). The exact PS species signaling for the recognition and removal of infiltrating neutrophils have not been fully elucidated. Given that recruited neutrophils robustly activate their NADPH oxidase, we reasoned that activated neutrophils would be an excellent source for the identification of additional oxPS species signaling for apoptotic cell engulfment and specifically for the resolution of neutrophilic inflammation. Here, we show that neutrophils generate trace amounts of previously identified oxPS species, but more striking was the production of nanogram quantities of lyso-PS species both in vitro and in vivo following activation of the NADPH oxidase. Lyso-PS species have been shown to be biologically active, signaling via G protein-coupled receptors (e.g. GPR34 on mast cells and via G2A on neutrophils (20Frasch S.C. Zemski-Berry K. Murphy R.C. Borregaard N. Henson P.M. Bratton D.L. J. Immunol. 2007; 178: 6540-6548Crossref PubMed Scopus (77) Google Scholar, 21Sugo T. Tachimoto H. Chikatsu T. Murakami Y. Kikukawa Y. Sato S. Kikuchi K. Nagi T. Harada M. Ogi K. Ebisawa M. Mori M. Biochem. Biophys. Res. Commun. 2006; 341: 1078-1087Crossref PubMed Scopus (120) Google Scholar)). Our studies demonstrate that lyso-PS are modified PS species that signal via the macrophage G2A receptor to enhance existing receptor/ligand systems for the engulfment of PS exposing activated and apoptotic cells in vitro and in vivo. These data support inclusion of lyso-PS/G2A in the growing repertoire of lipid-derived signaling molecules and signaling receptors demonstrated to play a role in anti-inflammatory signaling and resolution of inflammation. Materials—All lipids were purchased from Avanti Polar Lipids (Alabaster, AL) unless otherwise noted. Solvents, glucose, glucose oxidase, NaNO2, fMLP, phorbol myristate acetate (PMA), methoxylamine hydrochloride (MOX), cytochrome c, and zymosan were from Sigma. Myeloperoxidase (MPO), diphenyleneiodonium (DPI), cPLA2α inhibitor, and bromoenol lactone were from EMD Biosciences (Gibbstown, NJ). Aminopropyl Sep-Pak (NH2-SPE) columns were from Supelco (subsidiary of Sigma). Flash red carboxylate-modified beads (5 μm) were from Bangs Labs (Fishers, IN). ONO-RS-082 was from Biomol International (Plymouth Meeting, PA). HLB reverse solid phase columns were from Phenomenex (Torrance, CA). Anti-G2A M-20 was from Santa Cruz Biotechnology (Santa Cruz, CA). Annexin V, Alexa 488, and anti-goat IgG Alexa 488 were from Molecular Probes (Eugene, OR). Lyso-PS internal standard (17:1/OH-PS) was a generous gift from Dr. Walter Shaw at Avanti Polar Lipids (Alabaster, AL). Animals—Male and female C57BL/6 and gp91phox-/- mice were purchased from Jackson Laboratories (Bar Harbor, ME) and also used from a breeding colony at National Jewish Health (Denver, Colorado). All animals received care in accordance with the guidelines of the Institutional Animal Care and Use Committee and were maintained on food and water ad libitum. Mice between the ages of 8 and 16 weeks provided a source of resident peritoneal and thioglycollate-elicited MΦ and were age and gender matched for all experiments. Isolation and Culture of Murine Peritoneal MΦ—Thioglycollate (TG)-elicited peritoneal MΦ were obtained according to previously established methods (13Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216Crossref PubMed Google Scholar). Briefly, mice were injected intraperitoneally with 1.5 ml of a 4% sterile and aged (3 month) solution of Brewer thioglycollate medium (Difco Laboratories, Detroit, MI). At 3 days post injection, mice were euthanized with CO2, and the peritoneal cavity lavaged with 5 ml of sterile Hanks' balanced salt solution (Cellgro, Kansas City, MO). Peritoneal cells were collected, centrifuged at 1,000 × g for 10 min at 4 °C, and plated at 2.5 × 105 cells/well in a 24-well tissue culture plate in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA), 2 mm l-glutamine, 100 μg/ml streptomycin, and 100 units/ml penicillin. Macrophages were allowed to adhere for 2 h at 37 °C in a 10% CO2 humidified incubator at which time non-adherent cells were removed and macrophages were cultured for an additional 48 h before use in phagocytosis assays. Resident peritoneal (RP) MΦ were isolated from mice using 5 ml of sterile Hanks' balanced salt solution to lavage the peritoneum following euthanization with CO2. Resident peritoneal cells were collected, centrifuged at 1,000 rpm for 10 min at 4 °C, and plated at 4 × 105 cells/well and cultured as described for thioglycollate-elicited macrophages. Murine macrophage RAW264.7 cell line (from ATCC, Manassas, VA) was cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mm l-glutamine, 100 μg/ml streptomycin, and 100 units/ml penicillin at 37 °C in a 5% CO2 humidified incubator. Cells were plated at 4 × 104 cells per well in a 24-well tissue culture plate for 48 h prior to phagocytosis assays. Induction of Sterile Peritonitis—Mice were injected intraperitoneal with 1 mg of zymosan (in 1 ml of PBS) and peritoneal cells were harvested by lavage with sterile Hanks' balanced salt solution supplemented with 1 mm EDTA and 10 mm HEPES (pH 7.2) at the times indicated. Cell counts and cytospins were done to determine cell differentials and absolute numbers. In some cases, peritoneal cells were separated into neutrophils and mononuclear cell fractions by Percoll density gradient centrifugation as described by Suratt et al. (22Suratt B.T. Young S.K. Lieber J. Nick J.A. Henson P.M. Worthen G.S. Am. J. Physiol. 2001; 281: L913-L921Crossref PubMed Google Scholar). Peritoneal cells were suspended in 6 ml of PBS and peritoneal lavage fluid (6 ml) was subjected to lipid extraction as described below. To test the role of lyso-PS signaling via G2A during resolution of inflammation, mice were injected intraperitoneally (100 μg/mouse) with either goat IgG or anti-G2A antibody (dialyzed against PBS) 24 h post-zymosan injection. Peritoneal cells were collected by lavage at the times indicated. Total cells counts and differentials were determined as described above. Lipid Extraction and Derivatization—Lipids were extracted by the method of Folch et al. (23Folch J. Lees M. Sloane Stanley G.H. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). Briefly, peritoneal cells or human neutrophils (2-5 × 106/ml) in 6 ml of PBS, 6 ml of culture supernatant or peritoneal lavage fluid were extracted in a separatory funnel with CHCl3/MeOH/dH2O, 20:10:6 (v/v/v), supplemented with 70 mm NaCl. Fifty ng of 17:1/OH-PS, 1 μg of 19:0/OH-PC, and 1 μg of 14:0/OH-PE were added to each sample as internal standards and in some samples 10 μg of 1,2-dimyristoyl-sn-glycero-3-phosphoserine was also added. The extractions were allowed to sit overnight at room temperature. The organic layer was collected and brought to dryness under a stream of nitrogen. Reactive aldehyde or ketone groups were derivatized in the gas phase with MOX as described (24Zemski Berry K.A. Murphy R.C. Antioxid. Redox Signal. 2005; 7: 157-169Crossref PubMed Scopus (30) Google Scholar). In brief, sodium hydroxide (1 ml) and MOX (50 mg) were added together and attached to an enclosed glass apparatus. During a 60-min incubation at 60 °C, the liberated CH3ONH2 gas derivatized the ketone or aldehyde groups present on the oxidized phospholipids. Following derivatization, the dried lipids were suspended in 200 μl of CHCl3 and applied to a hexane-conditioned aminopropyl Sep-Pak (NH2-SPE) column to separate lipids by class (25Kim H.Y. Salem Jr., N. J. Lipid Res. 1990; 31: 2285-2289Abstract Full Text PDF PubMed Google Scholar, 26Bodennec J. Pelled D. Futerman A.H. J. Lipid Res. 2003; 44: 218-226Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The neutral lipids were eluted with 4 ml of CHCl3, isopropyl alcohol, 2:1 (v/v). Polar lipids (phosphatidylcholine and phosphatidylethanolamine) were collected with 4 ml of MeOH and the acidic lipids (phosphatidylserine, phosphatidic acid, and phosphatidylglycerol) were collected with 4 ml of MeOH, CHCl3, 3.6 m ammonium acetate, 60:30:8 (v/v/v). The acidic lipid fraction from the NH2-SPE, which contained PS was dried under nitrogen until 200 μl of liquid remained. An equal volume of MeOH was added to suspend the lipids and water was added to reduce the MeOH content to less than 10% of final volume. The ammonium acetate present in this NH2-SPE fraction was removed by introducing the acidic lipid fraction onto a MeOH-conditioned and rinsed HLB reverse phase column. Bound phospholipids were washed with 4-5 volumes of dH2O and eluted with 2 ml of MeOH/CHCl3, 2:1 (v/v), dried under a stream of nitrogen and suspended in 65 μl of reverse phase buffer A. During the course of phospholipid extraction using known quantities of a standard lyso-PS, it was determined that the recovery efficiency for lyso-PS species reached 50%. Reverse Phase Chromatography and Electrospray Ionization Tandem Mass Spectrometry—RP-HPLC/MS/MS analysis of PS present in the methanol, chloroform, 3.6 m ammonium acetate, 60:30:8 (v/v/v), elution of the NH2-SPE was performed using an Eclipse Plus 3.5 μmC18 (2.1 × 50 mm) column (Agilent, Santa Clara, CA) with mass spectrometric detection using a Sciex API 3000 triple quadrupole mass spectrometer (PE Sciex, Toronto, Canada). The HPLC was operated at a flow rate of 0.2 ml/min with a mobile phase of methanol/acetonitrile/water, 60:20:20 (v/v/v), with 1 mm ammonium acetate (solvent A) and 1 mm methanolic ammonium acetate (solvent B). The gradient for the PS analysis was 0% solvent B to 100% solvent B in 20 min and then an isocratic hold at 100% B for 10 min. In some cases, the PS species were detected in the negative ion mode by monitoring for the neutral loss of serine (neutral loss of 87 atomic mass unit) with a collision energy of -30 V (27Pulfer M. Murphy R.C. Mass Spectrom. Rev. 2003; 22: 332-364Crossref PubMed Scopus (735) Google Scholar). The mass range scanned for neutral loss of the 87 atomic mass unit scan was m/z 400-900 at a rate of 3 s/scan. In other cases, multiple reaction monitoring (MRM) in the negative ion mode of m/z 508.5 → 421.5 for 17:1/OH-PS (internal standard), m/z 522.5 → 435.5 for 18:1/OH-PS, m/z 524.5 → 437.5 for 18:0/OH-PS, and m/z 707.6 → 620.5 for MOX derivatized 18:0/9al-PS was used to detect the major oxPS species eluting from the RP-HPLC column. In addition, minor oxPS species observed previously (16Arroyo A. Modriansky M. Serinkan F.B. Bello R.I. Matsura T. Jiang J. Tyurin V.A. Tyurina Y.Y. Fadeel B. Kagan V.E. J. Biol. Chem. 2002; 277: 49965-49975Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 17Kagan V.E. Gleiss B. Tyurina Y.Y. Tyurin V.A. Elenstrom-Magnusson C. Liu S.X. Serinkan F.B. Arroyo A. Chandra J. Orrenius S. Fadeel B. J. Immunol. 2002; 169: 487-499Crossref PubMed Scopus (209) Google Scholar, 18Greenberg M.E. Sun M. Zhang R. Febbraio M. Silverstein R. Hazen S.L. J. Exp. Med. 2006; 203: 2613-2625Crossref PubMed Scopus (322) Google Scholar) were monitored in the negative ion mode with their specific MRM transitions (see supplemental Table S1). For PS analysis in the negative ion mode, the electrospray voltage was -4000 V, the focusing potential was -200 V, and the declustering potential was -45 V. In addition PC and PE were also analyzed using the same chromatography conditions above and using a precursor of m/z 184 and neutral loss of 141 atomic mass units in the positive ion mode to specifically detect PC and PE lipids, respectively (27Pulfer M. Murphy R.C. Mass Spectrom. Rev. 2003; 22: 332-364Crossref PubMed Scopus (735) Google Scholar). Quantitation—The quantitation of 18:1/OH-PS and 18:0/OH-PS in the samples was performed using a standard isotope dilution curve as previously described (28Hall L.M. Murphy R.C. J. Am. Soc. Mass Spectrom. 1998; 9: 527-532Crossref PubMed Scopus (103) Google Scholar). The internal standard used for this quantitative analysis was 17:1/OH-PS. Because small amounts of lyso-PS were generated during the MOX derivatization process, quantitation of all lyso-PS was carried out on duplicate samples in the absence of MOX derivatization. Vesicle Preparation and Modification—1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine small unilamellar vesicles (SUVs), some containing either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine or 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoserine (lyso-PS) (at the indicated molar ratios) at a final concentration of 100 mm were prepared by evaporating the lipid to dryness under nitrogen in a glass tube. Dried lipids were suspended in PBS or media without serum or protein supplementation by vigorous vortexing, and small unilamellar vesicles were created by sonication in a water bath sonicator (29Fadok V.A. Savill J.S. Haslett C. Bratton D.L. Doherty D.E. Campbell P.A. Henson P.M. J. Immunol. 1992; 149: 4029-4035PubMed Google Scholar). Small unilamellar vesicles were stored on ice and used within 1 h of preparation. For phagocytosis assays, 100 μl (100 nmol of total lipid) were used per well. For in vitro oxidation, 100 μg/ml synthetic 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphoserine or 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine suspended in PBS was oxidized using the MPO/H2O2/NO2− generating system according to the methods of Greenburg et al. (18Greenberg M.E. Sun M. Zhang R. Febbraio M. Silverstein R. Hazen S.L. J. Exp. Med. 2006; 203: 2613-2625Crossref PubMed Scopus (322) Google Scholar). Oxidation of lipids was also performed using 2,2′-azobis(2-methylpropionamidine)-dihydrochloride (Sigma) at a final concentration of 10 mm at 37 °C for 2.5 h. To detect oxidized species containing reactive aldehydes or ketones, lipids were derivatized in the gas phase with MOX as stated above. Following derivatization, lipids were processed as stated above and suspended at 250 μg/ml in CHCl3/MeOH, 2:1. For LC/MS/MS, 5 μg of the lipid was dried under nitrogen and suspended in 65 μl of reverse phase buffer A. Surface G2A Staining—Murine resident or thioglycollate-elicited peritoneal macrophages or the RAW264.7 murine macrophage cell line were incubated with 5 μg/ml anti-G2A or isotype control for 1 h at 4 °C. Cells were washed 2 times with ice-cold PBS and incubated with donkey anti-goat IgG Alexa 488 for 30 min at 4 °C. Cells were washed 2 times with ice-cold PBS and analyzed by flow cytometry. Stimulation and Preparation of Neutrophils for Lipid Extraction and Engulfment Assays—Human neutrophils were obtained from normal, healthy donors in accordance with a protocol reviewed and approved by the Institutional Review Board. Using endotoxin-free reagents and plasticware, human neutrophils were isolated by the plasma Percoll method as described previously (30Haslett C. Guthrie L.A. Kopaniak M.M. Johnston Jr., R.B. Henson P.M. Am. J. Pathol. 1985; 119: 101-110PubMed Google Scholar). For in vitro studies, human neutrophils were suspended at 5 × 106/ml in a HEPES buffer (137 mm NaCl, 2.7 mm KCl, 2 mm MgCl2, 5 mm glucose, 1 mm CaCl2, 10 mm HEPES (pH 7.4)) supplemented with 0.05% fatty acid-free bovine serum albumin. For stimulation, zymosan was opsonized with pooled human serum as described previously (31Yamamori T. Inanami O. Sumimoto H. Akasaki T. Nagahata H. Kuwabara M. Biochem. Biophys. Res. Commun. 2002; 293: 1571-1578Crossref PubMed Scopus (38) Google Scholar) and added to neutrophils at a concentration of 200 μg/ml. Phorbol myristate acetate (PMA) was used at a final concentration of 20 ng/ml and fMLP at 100 nm. Cells were stimulated for the times indicated at 37 °C and subjected to lipid extraction and LC/MS/MS analysis as described above. For phagocytosis assays, human neutrophils were stimulated with 20 ng/ml PMA for 30 min at 37 °C or UV irradiated for 5 min on a trans-illuminator followed by incubation at 37 °C for 2 h. Surface PS exposure was detected by Annexin V binding and propidium iodide staining (as a test for permeability) and flow cytometry according to the manufacturers instructions. Under these conditions UV-irradiated neutrophils were greater than 50% apoptotic as determined by nuclear morphology and were 63.4 ± 5.8% Annexin V positive, and >97% propidium iodide negative as determined by flow cytometry. PMA-stimulated neutrophils were less than 5% apoptotic as determined by nuclear morphology and were 43.5 ± 6.5% Annexin V positive, and >96% propidium iodide negative as determined by flow cytometry. In some experiments human neutrophils were preincubated with liposomes according to the methods described by Fadok et al. (14Fadok V.A. de Cathelineau A. Daleke D.L. Henson P.M. Bratton D.L. J. Biol. Chem. 2001; 276: 1071-1077Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar) with minor modifications. Briefly, unstimulated viable neutrophils or neutrophils UV irradiated to induce apoptosis (as described above) were suspended at 2 × 107 cells/ml in PBS supplemented with 0.01% fatty acid-free bovine serum albumin. An equal volume of liposomes containing the indicated lipids in PBS (as described above) was added to the cells at a final concentration of 1 μmol of total lipid per 1 × 107 cells (final concentration of 0.005% bovine serum albumin). Cells were incubated at 37 °C for 30 min and washed twice with PBS and used in phagocytosis assays as described below. Following stimulation or induction of apoptosis, neutrophils were washed once and suspended at 2 × 107 cells/ml in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum for use in the phagocytosis assays (see below). Where indicated, unstimulated viable neutrophils were opsonized with mouse anti-human CD45. Briefly, cells at 5 × 106/ml were incubated with 10 μg/ml anti-CD45 for 30 min at 4 °C. Binding of anti-CD45 was confirmed by incubating the cells with anti-mouse IgG Alexa 488 (1:100) for 30 min on ice and analysis by flow cytometry (data not shown). Anti-CD45-opsonized neutrophils were washed as stated above for use in phagocytosis assays. Where the phagocytic index (PI) is represented as percent of control, the PI for control resident peritoneal macrophages with UV-irradiated neutrophils was 20.1 ± 2.72, and with COOH beads was 44.4 ± 6.4. The PI for thioglycollate-elicited macrophages with UV neutrophils was 8.5 ± 1.7, and for RAW264.7 with COOH beads was 49.0 ± 4.5. Superoxide Measurement—Release of O2.¯ was determined by cytochrome c reduction as described previously (32Guthrie L.A. McPhail L.C. Henson P.M. Johnston Jr., R.B. J. Exp. Med. 1984; 160: 1656-1671Crossref PubMed Scopus (466) Google Scholar). Phagocytosis Assays—Phagocytosis assays were performed as previously described (14Fadok V.A. de Cathelineau A. Daleke D.L. Henson P.M. Bratton D.L. J. Biol. Chem. 2001; 276: 1071-1077Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). For antibody blocking experiments, 10 μg/ml anti-G2A or isotype control antibody was added for 30 min before liposomes or target cells were added. Liposomes (100 nmol in 100 μl) were added as indicted, either 1 h prior to or simultaneously with target cells. Target cells (either 2 × 106 UV-irradiated or PMA-stimulated neutrophils) or 2.5 × 105 COOH 5-μm beads in 100 μl were added per well. The macrophages/target cells were cocultured for 60 min at 37 °C in 10% CO2, washed three times with PBS, and stained with a modified Wright Giemsa stain (Fisher Scientific, Pittsburgh, PA). The phagocytic index (PI) was calculated by multiplying the percentage of MΦ that have phagocytosed by the average number of engulfed cells per MΦ (13Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216Crossref PubMed Google Scholar). A minimum of 200 mΦ were counted blindly. Each condition was tested in duplicate using at least four mice per experiment and repeated 3-10 times as indicated. Statistical Analysis—Statistical analyses and p value calculations were conducted using analysis of variance (JMP statistical program (SAS Institute, Cary, NC)). The Dunnett and Tukey-Kramer tests were used for single and multiple comparisons, respectively. Lyso-PS Are Abundant Modified PS Species Produced in Neutrophils following Act
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