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Nitrosative Stress Inhibits the Aminophospholipid Translocase Resulting in Phosphatidylserine Externalization and Macrophage Engulfment

2007; Elsevier BV; Volume: 282; Issue: 11 Linguagem: Inglês

10.1074/jbc.m606950200

ISSN

1083-351X

Autores

Yulia Y. Tyurina, Liana Basova, Nagarjun V. Konduru, Vladimir A. Tyurin, Ala I. Potapovich, Peter Y. Cai, Huölya Bayir, Detcho A. Stoyanovsky, Bruce R. Pitt, Anna A. Shvedova, Bengt Fadeel, Valerian E. Kagan,

Tópico(s)

Sphingolipid Metabolism and Signaling

Resumo

Macrophage recognition of apoptotic cells depends on externalization of phosphatidylserine (PS), which is normally maintained within the cytosolic leaflet of the plasma membrane by aminophospholipid translocase (APLT). APLT is sensitive to redox modifications of its -SH groups. Because activated macrophages produce reactive oxygen and nitrogen species, we hypothesized that macrophages can directly participate in apoptotic cell clearance by S-nitrosylation/oxidation and inhibition of APLT causing PS externalization. Here we report that exposure of target HL-60 cells to nitrosative stress inhibited APLT, induced PS externalization, and enhanced recognition and elimination of "nitrosatively" modified cells by RAW 264.7 macrophages. Using S-nitroso-l-cysteine-ethyl ester (SNCEE) and S-nitrosoglutathione (GSNO) that cause intracellular and extracellular trans-nitrosylation of proteins, respectively, we found that SNCEE (but not GSNO) caused significant S-nitrosylation/oxidation of thiols in HL-60 cells. SNCEE also strongly inhibited APLT, activated scramblase, and caused PS externalization. However, SNCEE did not induce caspase activation or nuclear condensation/fragmentation suggesting that PS externalization was dissociated from the common apoptotic pathway. Dithiothreitol reversed SNCEE-induced S-nitrosylation, APLT inhibition, and PS externalization. SNCEE but not GSNO stimulated phagocytosis of HL-60 cells. Moreover, phagocytosis of target cells by lipopolysaccharide-stimulated macrophages was significantly suppressed by an NO. scavenger, DAF-2. Thus, macrophage-induced nitrosylation/oxidation plays an important role in cell clearance, and hence in the resolution of inflammation. Macrophage recognition of apoptotic cells depends on externalization of phosphatidylserine (PS), which is normally maintained within the cytosolic leaflet of the plasma membrane by aminophospholipid translocase (APLT). APLT is sensitive to redox modifications of its -SH groups. Because activated macrophages produce reactive oxygen and nitrogen species, we hypothesized that macrophages can directly participate in apoptotic cell clearance by S-nitrosylation/oxidation and inhibition of APLT causing PS externalization. Here we report that exposure of target HL-60 cells to nitrosative stress inhibited APLT, induced PS externalization, and enhanced recognition and elimination of "nitrosatively" modified cells by RAW 264.7 macrophages. Using S-nitroso-l-cysteine-ethyl ester (SNCEE) and S-nitrosoglutathione (GSNO) that cause intracellular and extracellular trans-nitrosylation of proteins, respectively, we found that SNCEE (but not GSNO) caused significant S-nitrosylation/oxidation of thiols in HL-60 cells. SNCEE also strongly inhibited APLT, activated scramblase, and caused PS externalization. However, SNCEE did not induce caspase activation or nuclear condensation/fragmentation suggesting that PS externalization was dissociated from the common apoptotic pathway. Dithiothreitol reversed SNCEE-induced S-nitrosylation, APLT inhibition, and PS externalization. SNCEE but not GSNO stimulated phagocytosis of HL-60 cells. Moreover, phagocytosis of target cells by lipopolysaccharide-stimulated macrophages was significantly suppressed by an NO. scavenger, DAF-2. Thus, macrophage-induced nitrosylation/oxidation plays an important role in cell clearance, and hence in the resolution of inflammation. Programmed cell death (apoptosis), aimed at harmless elimination of irreparably damaged or unwanted cells, and subsequent removal of apoptotic cells are strongly coupled with the regulation of normal tissue function and structure, both in the developing and adult organism (1Voll R.E. Herrmann M. Roth E.A. Stach C. Kalden J.R. Girkontaite I. Nature. 1997; 390: 350-351Crossref PubMed Scopus (1494) Google Scholar, 2Huynh M.L. Fadok V.A. Henson P.M. J. Clin. Investig. 2002; 109: 41-50Crossref PubMed Scopus (1018) Google Scholar, 3Huynh M.L. Malcolm K.C. Kotaru C. Tilstra J.A. Westcott J.Y. Fadok V.A. Wenzel S.E. Am. J. Respir. Crit. Care Med. 2005; 172: 972-979Crossref PubMed Scopus (120) Google Scholar, 4Henson P.M. Hume D.A. Trends Immunol. 2006; 27: 244-250Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Moreover, recognition of apoptotic cells and their clearance by macrophages play an active role in the resolution of inflammation, through production of anti-inflammatory cytokines, down-regulation of proinflammatory mediators (1Voll R.E. Herrmann M. Roth E.A. Stach C. Kalden J.R. Girkontaite I. Nature. 1997; 390: 350-351Crossref PubMed Scopus (1494) Google Scholar, 2Huynh M.L. Fadok V.A. Henson P.M. J. Clin. Investig. 2002; 109: 41-50Crossref PubMed Scopus (1018) Google Scholar), and macrophage generation and release of reactive oxygen and nitrogen species (5Serinkan B. Gambelli F. Potapovich A.I. Babu H. Di Giuseppe M. Ortiz L.A. Fabisiak J.P. Kagan V.E. Cell Death Differ. 2005; 12: 1141-1144Crossref PubMed Scopus (21) Google Scholar). Dysregulation and ineffective clearance of apoptotic cells result in postapoptotic cytolysis (6Gardai S.J. Bratton D.L. Ogden C.A. Henson P.M. J. Leukocyte Biol. 2006; 79: 896-903Crossref PubMed Scopus (184) Google Scholar) and cause pro-inflammatory conditions that are associated with a number of autoimmune and chronic inflammatory diseases such as systemic lupus erythematosus (7Gaipl U.S. Kuhn A. Sheriff A. Munoz L.E. Franz S. Voll R.E. Kalden J.R. Herrmann M. Curr. Dir. Autoimmun. 2006; 9: 173-187PubMed Google Scholar), chronic obstructive pulmonary disease (8Demedts I.K. Demoor T. Bracke K.R. Joos G.F. Brusselle G.G. Respir. Res. 2006; 7: 53Crossref PubMed Scopus (369) Google Scholar), chronic granulomatous disease (9Fadeel B. Aåhlin A. Henter J-I. Orrenius S. Hampton M. Blood. 1998; 92: 4808-4818Crossref PubMed Google Scholar), and asthma (3Huynh M.L. Malcolm K.C. Kotaru C. Tilstra J.A. Westcott J.Y. Fadok V.A. Wenzel S.E. Am. J. Respir. Crit. Care Med. 2005; 172: 972-979Crossref PubMed Scopus (120) Google Scholar).Phagocytic recognition of apoptotic cells requires a remarkable rearrangement of their membrane surface. Most notably, collapse of plasma membrane phospholipid asymmetry, appearance of phosphatidylserine (PS), 2The abbreviations used are: PS, phosphatidylserine; NBD-PS, 1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phospho-l-serine; NBD-PC, 1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine; PC, phosphatidylcholine; DTT, dithiothreitol; GSNO, S-nitrosoglutathione; SNCEE, S-nitroso-l-cysteine ethyl ester; LPS, lipopolysaccharide; DAF-2, 4,5-diaminofluorescein; DAF-2DA, 4,5-diaminofluorescein diacetate; DHE, dihydroethidium; O¯·2, superoxide; NO·, nitric oxide; ONOO-, peroxynitrite; SWCNT, single walled carbon nanotubes; APLT, aminophospholipid translocase; ANOVA, analysis of variance; HPTLC, high performance thin layer chromatography; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone. 2The abbreviations used are: PS, phosphatidylserine; NBD-PS, 1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phospho-l-serine; NBD-PC, 1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine; PC, phosphatidylcholine; DTT, dithiothreitol; GSNO, S-nitrosoglutathione; SNCEE, S-nitroso-l-cysteine ethyl ester; LPS, lipopolysaccharide; DAF-2, 4,5-diaminofluorescein; DAF-2DA, 4,5-diaminofluorescein diacetate; DHE, dihydroethidium; O¯·2, superoxide; NO·, nitric oxide; ONOO-, peroxynitrite; SWCNT, single walled carbon nanotubes; APLT, aminophospholipid translocase; ANOVA, analysis of variance; HPTLC, high performance thin layer chromatography; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone. an essential "eat-me" signal, on the cell surface (10Savill J. Fadok V. Nature. 2000; 407: 784-788Crossref PubMed Scopus (1268) Google Scholar), and its interactions with specialized binding proteins (11Bottcher A. Gaipl U.S. Furnrohr B.G. Herrmann M. Girkontaite I. Kalden J.R. Voll R.E. Arthritis Rheum. 2006; 54: 927-938Crossref PubMed Scopus (81) Google Scholar, 12Hanayama R. Miyasaka K. Nakaya M. Nagata S. Curr. Dir. Autoimmun. 2006; 9: 162-172PubMed Google Scholar) are involved in recognition, tethering, and engulfment of apoptotic cells by phagocytes (13Kagan V.E. Borisenko G.G. Serinkan B.F. Tyurina Y.Y. Tyurin V.A. Jiang J. Liu S.X. Shvedova A.A. Fabisiak J.P. Uthaisang W. Fadeel B. Am. J. Physiol. 2003; 285: 1-17Crossref PubMed Scopus (33) Google Scholar). Moreover, externalized PS is essential for triggering of antiinflammatory responses in macrophages (2Huynh M.L. Fadok V.A. Henson P.M. J. Clin. Investig. 2002; 109: 41-50Crossref PubMed Scopus (1018) Google Scholar, 4Henson P.M. Hume D.A. Trends Immunol. 2006; 27: 244-250Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 7Gaipl U.S. Kuhn A. Sheriff A. Munoz L.E. Franz S. Voll R.E. Kalden J.R. Herrmann M. Curr. Dir. Autoimmun. 2006; 9: 173-187PubMed Google Scholar). Therefore, failure to externalize PS may disrupt the clearance of apoptotic cells in vivo and contribute to perpetuating a calamitous proinflammatory environment (14Fadeel B. Cell. Mol. Life Sci. 2003; 60: 2575-2585Crossref PubMed Scopus (85) Google Scholar).Normally PS is confined exclusively to the cytoplasmic surface of the plasma membrane (15Devaux P.F. Lopez-Montero I. Bryde S. Chem. Phys. Lipids. 2006; 141: 119-132Crossref PubMed Scopus (61) Google Scholar); maintenance of this asymmetric PS distribution is because of energy (ATP)-dependent aminophospholipid translocase (APLT) responsible for the inward translocation of amino phospholipids (15Devaux P.F. Lopez-Montero I. Bryde S. Chem. Phys. Lipids. 2006; 141: 119-132Crossref PubMed Scopus (61) Google Scholar, 16Daleke D.L. Lyles J.V. Biochim. Biophys. Acta. 2000; 1486: 108-127Crossref PubMed Scopus (178) Google Scholar). During apoptosis, APLT is inactivated causing egress of PS from the inner to the outer leaflet of the plasma membrane (17Fabisiak J.P. Tyurin V.A. Tyurina Y.Y. Sedlov A. Lazo J.S. Kagan V.E. Biochemistry. 2000; 39: 127-138Crossref PubMed Scopus (30) Google Scholar, 18Tyurina Y.Y. Serinkan F.B. Tyurin V.A. Kini V. Yalowich J.C. Schroit A.J. Fadeel B. Kagan V.E. J. Biol. Chem. 2004; 279: 6056-6064Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 19Gleiss B. Gogvadze V. Orrenius S. Fadeel B. FEBS Lett. 2002; 519: 153-158Crossref PubMed Scopus (60) Google Scholar). The mechanisms of apoptotic APLT inactivation remain to be elucidated. Reportedly, APLT is not a substrate for apoptosis-activated caspases suggesting that alternative mechanisms may be involved (20Verhoven B. Krahling S. Schlegel R.A. Williamson P. Cell Death Differ. 1999; 6: 262-270Crossref PubMed Scopus (114) Google Scholar). In line with this, PS externalization can be divorced from the common caspase-initiated pathway of apoptosis (21Zhuang J. Ren Y. Snowden R.T. Zhu H. Gogvadze V. Savill J.S. Cohen G.M. J. Biol. Chem. 1998; 273: 15628-15632Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 22Uthaisang W. Nutt L.K. Orrenius S. Fadeel B. FEBS Lett. 2003; 545: 110-114Crossref PubMed Scopus (21) Google Scholar). Connor and Schroit (23Connor J. Schroit A.J. Biochemistry. 1990; 29: 37-43Crossref PubMed Scopus (58) Google Scholar) demonstrated that reversible PS externalization can be induced by -SH reagents in non-apoptotic cells. Conversely, certain cell types can undergo apoptosis without PS externalization (24Fadeel B. Gleiss B. Hoögstrand K. Chandra J. Wiedmer T. Sims P.J. Henter J.I. Orrenius S. Samali A. Biochem. Biophys. Res. Commun. 1999; 266: 504-511Crossref PubMed Scopus (127) Google Scholar, 25Forsberg A.J. Kagan V.E. Schroit A.J. Antioxid. Redox. Signal. 2004; 6: 203-208Crossref PubMed Scopus (10) Google Scholar), but the resistance to PS externalization in these cells can be completely reversed by treatment with thiol reagents (23Connor J. Schroit A.J. Biochemistry. 1990; 29: 37-43Crossref PubMed Scopus (58) Google Scholar, 25Forsberg A.J. Kagan V.E. Schroit A.J. Antioxid. Redox. Signal. 2004; 6: 203-208Crossref PubMed Scopus (10) Google Scholar). Apparently, the sensitivity of catalytically competent cysteines of APLT to oxidation/alkylation may be important in the regulation of the enzyme activity during apoptosis (5Serinkan B. Gambelli F. Potapovich A.I. Babu H. Di Giuseppe M. Ortiz L.A. Fabisiak J.P. Kagan V.E. Cell Death Differ. 2005; 12: 1141-1144Crossref PubMed Scopus (21) Google Scholar, 16Daleke D.L. Lyles J.V. Biochim. Biophys. Acta. 2000; 1486: 108-127Crossref PubMed Scopus (178) Google Scholar, 17Fabisiak J.P. Tyurin V.A. Tyurina Y.Y. Sedlov A. Lazo J.S. Kagan V.E. Biochemistry. 2000; 39: 127-138Crossref PubMed Scopus (30) Google Scholar, 18Tyurina Y.Y. Serinkan F.B. Tyurin V.A. Kini V. Yalowich J.C. Schroit A.J. Fadeel B. Kagan V.E. J. Biol. Chem. 2004; 279: 6056-6064Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 26de Jong K. Geldwerth D. Kuypers F.A. Biochemistry. 1997; 36: 6768-6776Crossref PubMed Scopus (79) Google Scholar).S-Nitrosylation is another redox-related mechanism participating in the modification of protein cysteines (27Foster M.W. McMahon T.J. Stamler J.S. Trends Mol. Med. 2003; 9: 160-168Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar). A variety of enzymes contain essential cysteines whose S-nitrosylation results in changed catalytic activity (28Hess D.T. Matsumoto A. Kim S.O. Marshall H.E. Stamler J.S. Nat. Rev. Mol. Cell Biol. 2005; 6: 150-166Crossref PubMed Scopus (1706) Google Scholar). The two major pathways for protein S-nitrosylation are as follows: 1) trans-nitrosylation resulting from the transfer of the nitrosyl functionality from a donor S-nitrosothiol (commonly S-nitrosoglutathione or other low molecular weight thiol) to a recipient cysteine, and 2) direct nitrosylation by peroxynitrite (ONOO-) (formed as a product of the reaction between radical species, nitric oxide (NO.) and superoxide (O2.)) (29Crow J.P. Beckman J.S. Adv. Pharmacol. 1995; 34: 17-43Crossref PubMed Scopus (290) Google Scholar). Importantly, during inflammation, NADPH oxidase and inducible nitric-oxide synthase in activated macrophages massively produce O2. and NO., respectively, to yield ONOO- (30Radi R. Denicola A. Álvarez B. Ferrer-Sueta G. Rubbo H. Nitric Oxide: Biology and Pathobiology. 2000; (Ignarro, L. J., ed) pp. , Academic Press, San Diego, CA: 57-82Crossref Google Scholar, 31Forman H.J. Torres M. Mol. Aspects Med. 2001; 22: 189-216Crossref PubMed Scopus (432) Google Scholar). The latter can readily S-nitrosylate low molecular thiols (32van der Vliet A. Hoen P.A. Wong P.S. Bast A. Cross C.E. J. Biol. Chem. 1998; 273: 30255-30262Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) and hence generate pools of trans-nitrosylating species ready to attack protein cysteines (27Foster M.W. McMahon T.J. Stamler J.S. Trends Mol. Med. 2003; 9: 160-168Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar).We hypothesized that activated macrophages can directly participate in the clearance of target (apoptotic) cells and bystander cells by causing S-nitrosylation/oxidation and inhibition of APLT and inducing PS externalization independently of the execution of the common caspase-dependent pathway of apoptosis. In this study, we demonstrate that exposure of target HL-60 cells to nitrosative stress indeed inhibits APLT, induces PS externalization, and enhances recognition and elimination of "nitosatively" modified cells by RAW 264.7 macrophages. We suggest that this novel mechanism of macrophage-induced nitrosative stress contributes to effective clearance of apoptotic cells and regulates switching of the acute inflammatory reaction to an anti-inflammatory phase of the response.EXPERIMENTAL PROCEDURESReagents—16:0-12:0 NBD-PS, 1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phospho-l-serine (ammonium salt), and 16:0-12:0 NBD-PC, 1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino] dodecanoyl]-sn-glycero-3-Phospho-choline (ammonium salt), were from Avanti Polar Lipids Inc. (Alabaster, AL). Dithiothreitol (DTT), S-nitrosoglutathione (GSNO), dithionite, HEPES, glucose, MgCl2, CaCl2, KCl, NaCl, acetonitrile, phenylmethylsulfonyl fluoride, lipopolysaccharide (LPS), and Hoechst 33342 were from Sigma. S-Nitroso-l-cysteine-ethyl ester (SNCEE) was synthesized as described previously (33Clancy R. Cederbaum A.I. Stoyanovsky D.A. J. Med. Chem. 2001; 44: 2035-2038Crossref PubMed Scopus (49) Google Scholar). DAF-2, DAF-2DA, and ThioGlo™-1 were purchased from Calbiochem. DHE-DA and Cell Tracker Orange were from Molecular Probes (Eugene, OR).Cell Culture—HL-60 human promyelocytic leukemia cells (American Type Culture Collection) were grown in RPMI 1640 with phenol red supplemented with 12.5% heat-inactivated fetal bovine serum at 37 °C in a humidified atmosphere (5% CO2 plus 95% air). Cells from passages 45-50 were used for the experiments. The density of the cells at collection time was 0.5 × 106 cell/ml. RAW 264.7 macrophages (American Type Culture Collection) were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 100 units/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere (5% CO2 plus 95% air) at 37 °C.Measurement of Thiol Contents—Low molecular weight thiols and protein thiol contents in the cells were determined fluorometrically using ThioGlo™-1 as described previously (18Tyurina Y.Y. Serinkan F.B. Tyurin V.A. Kini V. Yalowich J.C. Schroit A.J. Fadeel B. Kagan V.E. J. Biol. Chem. 2004; 279: 6056-6064Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Briefly, cells treated with either SNCEE (50-300 μm) or GSNO (50-300 μm) for 30 min at 37 °C were collected by centrifugation, washed, and resuspended in PBS. GSH was measured in cell lysates prepared by freezing and thawing cells. Immediately after addition of ThioGlo™-1 to the cell lysates, fluorescence was measured in a Packard Fusion™ Multifunctional Plate Reader (PerkinElmer Life Sciences) using excitation 390 ± 15 nm and emission 515 ± 30 nm. Protein sulfhydryls were determined as an additional increase in fluorescence response after addition of SDS (4 mm) to cell lysates.Measurement of S-Nitrosothiols—The content of S-nitrosothiols was determined by fluorescence HPLC using DAF-2 that specifically reacts with NO. (but not with other NOx, ONOO-, NO¯2, or NO¯3), yielding fluorescent DAF-2 triazole (34Kojima H. Nakatsubo N. Kikuchi K. Kawahara S. Kirino Y. Nagoshi H. Hirata Y.T. Naganno T. Anal. Chem. 1998; 70: 2446-2453Crossref PubMed Scopus (1175) Google Scholar). Briefly, samples (50 μl) were mixed with DAF-2 (5 μm) in 2.5 ml of PBS and exposed to UV irradiation (15 min at room temperature) using an Oriel UV light source (model 66002) (Oriel Instruments, Stratford, CT) and cut-off filter (Balzers, >330 nm) (35Tyurin V.A. Tyurina Y.Y. Liu S.X. Bayir H. Hubel C.A. Kagan V.E. Methods Enzymol. 2002; 352: 347-360Crossref PubMed Scopus (19) Google Scholar). After UV irradiation, chloroform (200 μl) was added to precipitate proteins, and samples were centrifuged at 14,000 × g for 5 min. Supernatant was used for the measurements. Fluorescence HPLC (Eclipse XDB-C18 column, 5 μm, 150 × 4.6 mm; mobile phase was composed of 25 mm disodium phosphate buffer, pH 7.0, acetonitrile (94:6 v/v), Ex = 495 nm, Em = 515 nm) was performed on a Shimadzu LC-100AT HPLC system equipped with fluorescence detector (RF-10Axl) and autosampler (SIL-10AD). The data obtained were exported and processed using Shimadzu RF-5301 PC personal software. A standard curve was established using GSNO as the standard.APLT and Scramblase Activity Assays—APLT and scramblase activity were measured using NBD-PS and NBD-PC, respectively, as described previously (36McIntyre J.C. Sleight R.G. Biochemistry. 1991; 30: 11819-11827Crossref PubMed Scopus (431) Google Scholar, 37Connor J. Pak C.H. Zwaal R.F. Schroit A.J. J. Biol. Chem. 1992; 267: 19412-19417Abstract Full Text PDF PubMed Google Scholar). HL-60 cells (5 × 106) were exposed to SNCEE (50-300 μm) or GSNO (50-300 μm) for 30 min at 37 °C in serum-free RPMI 1640 medium without phenol red. At the end of incubation, the cells were washed twice and suspended in ice-cold incubation buffer (136 mm NaCl, 2.7 mm KCl, 2 mm MgCl2, 5 mm glucose, 500 μm phenylmethylsulfonyl fluoride, 10 mm HEPES, pH 7.5) and incubated with either NBD-PS or NBD-PC (10 μm) for 10 min at 4 °C. Then the cells were pelleted by centrifugation, resuspended in the same buffer (5 × 106 cells/ml), and placed in a water bath (at 28 °C) to initiate internalization of NBD-PS. For measurements of scramblase activity, 2 mm CaCl2 was added to the incubation medium. A Shimadzu RF-5301PC spectrofluorophotometer was employed for these measurements using the following instrumental conditions: Ex = 470 nm, Em = 540 nm; slits 5 and 10 nm, respectively. We normalized the differences in fluorescence intensities recorded immediately after removal of unbound of NBD-PS by centrifugation and the responses obtained from residual noninternalized NBD-PS (after allowing its internalization for given periods of time) in the presence and absence of a reducing agent, dithionite, to the fluorescence intensity of bound NBD-PS before initiation of its internalization (at 28 °C). Specifically, the percent of internalized NBD-PS was calculated using the following equation: % internalized NBD-PS = (FLt - FL0)/(FLtotal - FL0) × 100, where FLt is the fluorescence intensity measured in the presence of sodium dithionite (10 mm) at a given time point; FLtotal is the fluorescence response from bound plus internalized NBD-PS measured in the absence of dithionite; FL0 is the fluorescence intensity from the cells with NBD-lipid before the initiation of internalization but measured in the presence of dithionite.To estimate possible effects of endocytosis on the uptake of NBD-PS, HL-60 cells were incubated (30 min at 37 °C) in the presence of a mixture containing several commonly used inhibitors of endocytosis (38Ogretmen B. Pettus B.J. Rossi M.J. Wood R. Usta J. Szulc Z. Bielawska A. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2002; 277: 12960-12969Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) as follows: nystatin (25 μg/ml), genistein (200 μm), chlorpromazine (6 μg/ml), and brefeldin A (10 μg/ml). The cells were then washed with serum-free RPMI 1640 medium and incubated in the presence and in the absence of SCNEE (300 μm for 30 min at 37 °C). At the end of incubation, cells were washed with PBS, and the activity of APLT was determined as described above.Two-dimensional High Performance Thin Layer Chromatography (HPTLC)—HL-60 cells with integrated NBD-PS (1.7 nmol × 106 cells for 10 min at 4 °C) were incubated at 28 °C for 20 min. At the end of incubation, lipids were extracted using the Folch procedure (39Folch J. Lees M. Sloane Stanley G.H. J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar) and separated by two-dimensional HPTLC on Silica G plates (Whatman and Schleicher & Schuell). The plates were first developed with a solvent system consisting of chloroform, methanol, 28% ammonium hydroxide (65:25:5, v/v). After drying the plates with a forced air blower to remove the solvent, the plates were developed in the second dimension with a solvent system consisting of chloroform:acetone:methanol:glacial acetic acid:water (50:20:10:10:5, v/v). NBD-PS was localized by exposure of HPTLC plates to UV light by using a Fluor-S™ Multimager (Bio-Rad). The phospholipids were visualized by exposure to iodine vapors. The identified spots were scraped, and the silica was transferred to tubes. Lipid phosphorus was determined by the submicromethod as described by Bottcher et al. (40Bottcher C.J.F. Van Gent C.M. Pries C. Anal. Chim. Acta. 1961; 24: 203-204Crossref Scopus (845) Google Scholar). In addition, TLC plates were scanned, and fluorescence of NBD-PS on plates was estimated using Fluor-S™ Multimager.PS Exposure on Cell Surface—PS exposure on the surface of cells was determined by flow cytometric detection of annexin V staining using a protocol outlined in the annexin V-fluorescein isothiocyanate apoptosis detection kit (BioVision Research Products, Mountain View, CA). Briefly, HL-60 cells (0.5 × 106) exposed to either SNCEE (50-300 μm) or GSNO (50-300 μm) for 30 min at 37 °C in serum-free RPMI 1640 medium without phenol red, washed once with PBS, and resuspended in binding buffer were stained with annexin V (0.5 μg/ml) and propidium iodide (0.6 μg/ml) for 5 min at room temperature. After staining, cells were immediately analyzed using a FACScan flow cytometer (BD Biosciences) with simultaneous monitoring of green fluorescence (530 nm, 30 nm bandpass filter) for annexin V-fluorescein isothiocyanate and red fluorescence (long pass emission filter that transmits light >650 nm) associated with propidium iodide. Ten thousand events were collected and analyzed using the LYSIS™ II software (BD Biosciences).Caspase-3/7 Activity Assay—Caspase-3/7 activity was measured using a luminescence Caspase-Glo® 3/7 assay kit (Promega, Madison, WI). HL-60 cells (4 × 105) were incubated in serum-free RPMI 1640 medium without phenol red in the presence of SNCEE (300 μm) or GSNO (300 μm) for 30 min at 37 °C. At the end of the incubation, cells were washed twice, and caspase-3/7 activity was measured. Luminescence was measured using a plate reading chemiluminometer ML1000 (Dynatech Laboratories). Activity of caspase-3/7 was expressed as luminescence arbitrary units per mg of protein.Nuclear Fragmentation—Nuclear condensation/fragmentation of Hoechst 33342 (1 μg/ml)-labeled cells was evaluated as described previously (22Uthaisang W. Nutt L.K. Orrenius S. Fadeel B. FEBS Lett. 2003; 545: 110-114Crossref PubMed Scopus (21) Google Scholar). Cells were scored using a fluorescence microscope (Nikon ECLIPSE TE 200, Tokyo, Japan) equipped with a digital Hamamatsu CCD camera (C4742-95-12NBR).Phagocytosis of HL-60 Cells by RAW 264.7 Macrophages—RAW 264.7 macrophages were seeded into an 8-well chamber slide (5 × 104 cells/well) and cultured overnight in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Naiöve or nitrosatively modified HL-60 cells (i.e. treated in the presence of SNCEE (50-300 μm) as described above) were loaded with Cell Tracker Orange™ (10 μm) for 10 min at 37 °C. Cells were then washed twice and co-cultured with macrophages for 1 h at 37 °C. To inhibit caspase activation, HL-60 cells were pretreated with a pan-caspase inhibitor, Z-VAD-fmk (10 μm, for 30 min at 37 °C). To prevent oxidative damage leading to nonspecific changes in membrane biophysical properties, HL-60 cells pretreated with Z-VAD-fmk were exposed to a potent lipid antioxidant, etoposide (50 μm, for 1 h at 37 °C). In the same experiments macrophages were stimulated by lipopolysaccharide (LPS, 0.1 μg/ml for 6 h at 37 °C) and zymosan (0.25 mg/ml for 1 h at 37 °C). After incubation, unbound target cells were washed three times with RPMI 1640 medium and three times with PBS and fixed with solution of 2% formaldehyde in PBS containing Hoechst 33342 (1 μg/ml) for 1 h at 4 °C. The cells were examined under a Nikon ECLIPSE TE 200 fluorescence microscope (Tokyo, Japan) equipped with a digital Hamamatsu CCD camera (C4742-95-12NBR) and analyzed using the MetaImaging Series™ software version 4.6 (Universal Imaging Corp., Downingtown, PA). A minimum of 300 macrophages was analyzed per experimental condition. Results are expressed as the percentage of phagocytosis-positive macrophages.Superoxide Production—Intracellular production of O2. was assessed using the dihydroethidium (DHE) oxidation assay (41Zhao H. Kalivendi S. Zhang H. Joseph J. Nithipatikom K. Vasquez-Vivar J. Kalyanaraman B. Free Radic. Biol. Med. 2003; 34: 1359-1368Crossref PubMed Scopus (642) Google Scholar). DHE has been specifically recommended for assessments of superoxide production in cells because of its relatively high specificity (41Zhao H. Kalivendi S. Zhang H. Joseph J. Nithipatikom K. Vasquez-Vivar J. Kalyanaraman B. Free Radic. Biol. Med. 2003; 34: 1359-1368Crossref PubMed Scopus (642) Google Scholar). Macrophages (0.3 × 106/well) were preincubated with DHE-DA (10 μm for 10 min at 37 °C) and then stimulated by zymosan (0.25 mg/ml) for 1 h at 37 °C. At the end of incubation, cells were washed three times with PBS, fixed with a solution of 2% formaldehyde in PBS. Treated cells were then examined under a Nikon ECLIPSE TE 200 fluorescence microscope (Tokyo, Japan) equipped with a digital Hamamatsu charge-coupled device camera (C4742-95-12NBR) and analyzed using the MetaImaging Series™ software version 4.6 (Universal Imaging Corp.).Nitric Oxide Production—Intracellular production of NO. was assessed using the DAF-2DA oxidation assay (42Jourd'heuil D. Free Radic. Biol. Med. 2002; 33: 676-684Crossref PubMed Scopus (107) Google Scholar). DAF-2DA has been extensively used for analysis of NO production in cells. In contrast to other NO scavengers, DAF-2 does not readily react with NO. metabolites (NO¯2 and NO¯3) and reactive nitrogen and oxygen species (O¯·2, H2O2, or ONOO-) (34Kojima H. Nakatsubo N. Kikuchi K. Kawahara S. Kirino Y. Nagoshi H. Hirata Y.T. Naganno T. Anal. Chem. 1998; 70: 2446-2453Crossref PubMed Scopus (1175) Google Scholar). Acetylated DAF-2DA readily penetrates into cells and, after hydrolysis, acts as a sensitive and one of relatively specific fluorogenic reagents for NO.. Naiöve and LPS-stimulated (0.1 μg/ml, for 6 h at 37 °C) macrophages (0.3 × 106/well) were i

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