Phospholipid Flip-Flop and Phospholipid Scramblase 1 (PLSCR1) Co-localize to Uropod Rafts in Formylated Met-Leu-Phe-stimulated Neutrophils
2004; Elsevier BV; Volume: 279; Issue: 17 Linguagem: Inglês
10.1074/jbc.m313414200
ISSN1083-351X
AutoresS. Courtney Frasch, Peter M. Henson, Kaz Nagaosa, Michael B. Fessler, Niels Borregaard, Donna L. Bratton,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoMovement of phosphatidylserine (PS) to the plasma membrane outer leaflet is a nearly universal marker of apoptosis and occurs during activation of many cells. Neutrophils stimulated with the chemotactic peptide formylated Met-Leu-Phe (fMLP) demonstrated transient PS exposure. Stimulated outward movement of PS was accompanied by enhanced inward movement of several phosphorylcholine lipid probes and was associated with enhanced FM 1-43 staining indicative of phospholipid packing changes. Unlike apoptosis, inward movement of exogenously added fluorescent PS did not decline, and DNA was not cleaved during fMLP stimulation. Movement of phospholipids occurred within minutes following stimulation, was independent of endocytosis/pinocytosis, and was consistent with bidirectional, transbilayer phospholipid flip-flop. While the role of phospholipid scramblase 1 (PLSCR1) is controversial in flip-flop, we sought evidence for its role in enhanced phospholipid movements during fMLP stimulation. Using antibodies to the carboxyl-terminal domain of PLSCR1, its presence in the plasma membranes of non-permeabilized neutrophils was confirmed by flow cytometry. Additionally subcellular fractionation demonstrated that PLSCR1 was also located in secretory vesicles and tertiary and secondary granules. Activation of neutrophils with fMLP, however, did not significantly alter surface labeling suggesting that stimulated phospholipid flip-flop does not require additional mobilization of PLSCR1 to the plasma membrane. As expected for palmitoylated proteins, PLSCR1 was enriched in detergent-insoluble membranes and co-localized with raft markers at the neutrophil uropod after stimulation. Of note, PS exposure, phospholipid uptake, and FM 1-43 staining also localized to the uropod following stimulation demonstrating that both PLSCR1 and phospholipid flip-flop characterize this specialized domain of polarized neutrophils. Movement of phosphatidylserine (PS) to the plasma membrane outer leaflet is a nearly universal marker of apoptosis and occurs during activation of many cells. Neutrophils stimulated with the chemotactic peptide formylated Met-Leu-Phe (fMLP) demonstrated transient PS exposure. Stimulated outward movement of PS was accompanied by enhanced inward movement of several phosphorylcholine lipid probes and was associated with enhanced FM 1-43 staining indicative of phospholipid packing changes. Unlike apoptosis, inward movement of exogenously added fluorescent PS did not decline, and DNA was not cleaved during fMLP stimulation. Movement of phospholipids occurred within minutes following stimulation, was independent of endocytosis/pinocytosis, and was consistent with bidirectional, transbilayer phospholipid flip-flop. While the role of phospholipid scramblase 1 (PLSCR1) is controversial in flip-flop, we sought evidence for its role in enhanced phospholipid movements during fMLP stimulation. Using antibodies to the carboxyl-terminal domain of PLSCR1, its presence in the plasma membranes of non-permeabilized neutrophils was confirmed by flow cytometry. Additionally subcellular fractionation demonstrated that PLSCR1 was also located in secretory vesicles and tertiary and secondary granules. Activation of neutrophils with fMLP, however, did not significantly alter surface labeling suggesting that stimulated phospholipid flip-flop does not require additional mobilization of PLSCR1 to the plasma membrane. As expected for palmitoylated proteins, PLSCR1 was enriched in detergent-insoluble membranes and co-localized with raft markers at the neutrophil uropod after stimulation. Of note, PS exposure, phospholipid uptake, and FM 1-43 staining also localized to the uropod following stimulation demonstrating that both PLSCR1 and phospholipid flip-flop characterize this specialized domain of polarized neutrophils. Most resting cells appear to demonstrate marked asymmetry in the transbilayer distribution of phospholipids composing the plasma membrane with the majority of sphingomyelin and phosphatidylcholine (PC) 1The abbreviations used are: PC, phosphatidylcholine; PS, phosphatidylserine; AEBSF, 4-(2-aminoethyl)benzenesulfonylfluoride, HCl; BSA, bovine serum albumin; PAF, platelet-activating factor; cPAF, 1-O-[hexadecyl-1′-,2′-3H(N)]2-N-methylcarbamyl-platelet activating factor; CT-B, cholera toxin B; fMLP, formylated Met-Leu-Phe; FVa, activated Factor V; GM1, Galβ1,3GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1-ceramide, NH4; GM3, Neu5Acα2,3Galβ1,4Glcβ1,1-ceramide, NH4; KRPD, Krebs-Ringer phosphate-dextrose, MES, 2-(N-morpholino)ethanesulfonic acid; NBD, (7-nitro-2,1,3-benzoxadiazol-4-yl)amino; PBS, phosphate-buffered saline; PLSCR1, phospholipid scramblase 1; ABC, ATP-binding cassette; CHO, Chinese hamster ovary. localizing to the membrane outer leaflet and most phosphatidylethanolamine and nearly all phosphatidylserine (PS) localizing to the membrane inner leaflet (1Daleke D.L. J. Lipid Res. 2003; 44: 233-242Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar, 2Zwaal R.F.A. Schroit A.J. Blood. 1997; 89: 1121-1132Crossref PubMed Google Scholar). This phospholipid asymmetry is thought to be maintained by both an aminophospholipid translocase activity that flips (inward) PS, and to a lesser extent phosphatidylethanolamine, from the outer leaflet back to the inner leaflet and a low spontaneous rate of phospholipid flip and flop (outward) across the plasma membrane bilayer (1Daleke D.L. J. Lipid Res. 2003; 44: 233-242Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar, 3Tang X. Halleck M.S. Schlegel R.A. Williamson P. Science. 1996; 272: 1495-1497Crossref PubMed Scopus (424) Google Scholar, 4Schlegel R.A. Williamson P. Cell Death Differ. 2001; 8: 551-563Crossref PubMed Scopus (298) Google Scholar, 5Halleck M.S. Lawler J.J. Blackshaw S. Gao L. Nagarajan P. Hacker C. Pyle S. Newman J.T. Nakanishi Y. Ando H. Weinstock D. Williamson P. 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Where it has been investigated, PS exposure is thought to result, at least in part, from loss of PS flip due to declining aminophospholipid translocase activity (14Bratton D.L. Fadok V.A. Richter D.A. Kailey J.M. Guthrie L.A. Henson P.M. J. Biol. Chem. 1997; 272: 26159-26165Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 17Verhoven B. Schlegel R.A. Williamson P. J. Exp. Med. 1995; 182: 1597-1601Crossref PubMed Scopus (615) Google Scholar). The mechanisms of PS exposure during cellular activation have received much less attention. Where it has been studied, PS exposure in activation, like apoptosis, is accompanied by enhanced staining with lipophilic dyes that indicate changes in membrane lipid packing and enhanced nonspecific uptake of phospholipids bearing various head groups including PC species (12Williamson P. Bevers E.M. Smeets E.F. Comfurius P. Schlegel R.A. Zwaal R.F. 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Notably the mechanisms of phospholipid movement and the proteins involved in either outward movement of PS or inward movement of PC have not been unequivocally identified (see "Discussion"). Members of the P-type ATPase family have been proposed as candidate proteins mediating flip of PS from the outer leaflet to the inner leaflet and are credited with establishing and maintaining the basal asymmetric distribution of PS, but reverse function resulting in PS exposure has not been noted (1Daleke D.L. J. Lipid Res. 2003; 44: 233-242Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar, 3Tang X. Halleck M.S. Schlegel R.A. Williamson P. Science. 1996; 272: 1495-1497Crossref PubMed Scopus (424) Google Scholar, 4Schlegel R.A. Williamson P. Cell Death Differ. 2001; 8: 551-563Crossref PubMed Scopus (298) Google Scholar, 5Halleck M.S. Lawler J.J. Blackshaw S. Gao L. Nagarajan P. Hacker C. Pyle S. Newman J.T. Nakanishi Y. Ando H. Weinstock D. Williamson P. Schlegel R.A. Physiol. Genomics. 1999; 1: 139-150Crossref PubMed Scopus (70) Google Scholar). Other proteins of the ABC family have been proposed as mediating flop of phospholipids from inner leaflet to outer leaflet (e.g. ABCA1, ABCB1, and ABCB4) (for reviews, see Refs. 1Daleke D.L. J. Lipid Res. 2003; 44: 233-242Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar and 22Pomorski T. Hrafnsdottir S. Devaux P.F. van Meer G. Semin. Cell Dev. Biol. 2001; 12: 139-148Crossref PubMed Scopus (126) Google Scholar) but have not been found to enhance inward flip of phospholipids from outer leaflet to inner leaflet. Also these proteins appear to be constitutively active with little evidence that they are activated within minutes of stimulation as has been described for PS exposure in inflammatory cells. Although controversial, the PLSCR1 is hypothesized to mediate calcium-activated, bidirectional, nonspecific (with regard to head group) phospholipid movement that has been shown to accompany PS exposure in apoptosis and cellular activation (23Yu A. McMaster C.R. Byers D.M. Ridgway N.D. Cook H.W. J. Biol. Chem. 2003; 278: 9706-9714Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 24Frasch S.C. Henson P.M. Kailey J.M. Richter D.A. Janes M.S. Fadok V.A. Bratton D.L. J. Biol. Chem. 2000; 275: 23065-23073Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 25Zhao J. Zhou Q. Wiedmer T. Sims P.J. J. Biol. Chem. 1998; 273: 6603-6606Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 26Murakami M. Kambe T. Shimbara S. Higashino K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) (see "Discussion"). However, where phospholipid movement has been associated with PLSCR1 expression, the precise mechanisms of movement have not been elucidated. Movement of phospholipids could involve either transbilayer flip-flop across the plasma membrane (27Zhou Q. Zhao J. Stout J.G. Luhm R.A. Wiedmer T. Sims P.J. J. Biol. Chem. 1997; 272: 18240-18244Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar) and/or, given PLSCR1 localization to recycling endosomes in epithelial cells (28Sun J. Nanjundan M. Pike L.J. Wiedmer T. Sims P.J. Biochemistry. 2002; 41: 6338-6345Crossref PubMed Scopus (80) Google Scholar), could involve bilayer mixing during vesicle-plasma membrane fission and fusion. Here we have shown that stimulated neutrophils transiently expose PS during fMLP activation in the absence of evidence for apoptosis. PS exposure was accompanied by altered plasma membrane phospholipid packing and enhanced inward movement of both alkyl- and acyl-linked choline-containing phospholipids. As these phospholipid movements were not accompanied by evidence of either actin-dependent or actin-independent endocytosis/pinocytosis, the data are strongly supportive of phospholipid movement by flip-flop across the plasma membrane. Furthermore we present evidence that phospholipid flip-flop and membrane packing changes occurred in "raft" membranes at the uropod of polarized neutrophils, and that plasma membrane PLSCR1 co-localized to these same domains. Reagents—Cholera toxin B (CT-B)-Alexa 555, FM 1-43, annexin V-Alexa 488, fluorescein isothiocyanate-phalloidin, 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosyl phosphocholine (NBD-sphingomyelin), and Lucifer Yellow were from Molecular Probes (Eugene, OR). 1-Palmitoyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoyl[-sn-glycero-3-phosphoserine] (NBD-PS) and 1-palmitoyl-2-[12-[(7-nitro-2–1,3-benzoxadiazol-4-yl)amino]hexanoyl[-sn-glycero-3-phosphocholine] (NBD-PC) were from Avanti Polar Lipids (Alabaster, AL). Radiolabeled 1-O-[hexadecyl-1′,2′-3H(N)]2-N-methylcarbamyl-platelet activating factor (cPAF) was synthesized by PerkinElmer Life Sciences. Anti-CD55 was from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-CD11b was from Dako Corp. (Carpenteria, CA). Anti-CD45 clone BRA-55, fatty acid-free BSA, and fMLP were from Sigma. AEBSF, aprotinin, and leupeptin were from Calbiochem. Factor Va and anti-Factor Va were from Hematologic Technologies, Inc. (Essex Junction, VT). Rabbit PLSCR1 antiserum was raised against the 14 carboxyl-terminal amino acids of PLSCR1 (sequence CESTGSQEQKSGVW). Cell Isolation and Culture—Using endotoxin-free reagents and plasticware, human neutrophils were isolated by the plasma Percoll method as described previously (29Haslett C. Guthrie L.A. Kopaniak M.M. Johnston Jr., R.B. Henson P.M. Am. J. Pathol. 1985; 119: 101-110PubMed Google Scholar). Unless otherwise noted, all incubations were done in NBD buffer (137 mm NaCl, 2.7 mm KCl, 2 mm MgCl2,5mm glucose, 10 mm HEPES (pH 7.4)) or Krebs-Ringer phosphate-dextrose (KRPD) buffer (0.9% saline, 4.8 mm KCl, 0.93 mm CaCl2, 1.2 mm MgSO4, 3.1 mm NaH2PO4, 12.5 mm Na2HPO4, 5% dextrose) with designated amounts of fatty acid-free BSA. T-Rex-293 cells (Invitrogen) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, l-glutamine, 1% penicillin, 1% streptomycin, 1 mm sodium pyruvate at 37 °C in a 10% CO2 humidified incubator. cPAF Uptake—Uptake of radiolabeled cPAF was carried out as described previously (24Frasch S.C. Henson P.M. Kailey J.M. Richter D.A. Janes M.S. Fadok V.A. Bratton D.L. J. Biol. Chem. 2000; 275: 23065-23073Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) with the following modifications. Uptake of [3H]cPAF took place over the last 5 min of stimulation with fMLP at 37 °C. Samples were diluted with an equal volume of ice-cold 10% BSA in KRPD buffer and washed two more times with 5% BSA in KRPD buffer at 4 °C to remove outer leaflet lipid. The cell pellets were resuspended in 500 μl of 1% Triton X-100. Uptake of labeled lipid was determined by scintillation counting. NBD-PS, NBD-PC, NBD-sphingomyelin, Annexin V, and Factor Va Staining—NBD-PC (2.5 μg), NBD-PS (2.5 μg), or NBD-sphingomyelin (5.0 μg) was dried under nitrogen and resuspended in 20 μl of NBD buffer and 180 μl of propidium iodide (50 μg/ml stock). Neutrophils were stimulated as described at a concentration of 5 × 106/ml in NBD buffer with 1 mm CaCl2 and 0.25% BSA. Following stimulation, a final concentration of 1.25 μg/ml NBD-PC or NBD-PS or 2.5 μg/ml NBD-sphingomyelin was added to 100 μl of cells at 37 °C for 5 min. Outer leaflet lipid was removed by diluting cells with an equal volume of ice-cold 10% BSA in NBD buffer followed by two additional washes at 4 °C with 5% BSA in NBD buffer. Cells were resuspended in 500 μl of ice-cold NBD buffer and analyzed by flow cytometry. For annexin V staining, 1 × 106 cells in 100 μl of NBD buffer with 2.5 mm CaCl2, annexin V-Alexa 488 (1:50), and 5 μg/ml propidium iodide were incubated for 15 min at room temperature, diluted with 400 μl of ice-cold NBD buffer with 2.5 mm CaCl2, and analyzed by flow cytometry. For activated Factor V (FVa) staining, 1 × 106 neutrophils in 100 μl NBD buffer with 1% BSA and 1 mm CaCl2 were transferred to 100 μlof FVa and propidium iodide (10 μg/ml and 5 μg/ml, respectively) or propidium iodide alone for the last 10 min of stimulation. Cells were then fixed for 10 min at room temperature with 3% paraformaldehyde, 3% sucrose in PBS, washed once, and incubated with 100 μl of anti-FVa antibody (final concentration of 10 μg/ml) for 10 min at room temperature. Cells were washed once and incubated with 100 μl of anti-mouse IgG F(ab′)2 (1:100 in NBD buffer plus 1 mm CaCl2 and 0.1% BSA) for 30 min on ice, washed once, and analyzed by flow cytometry. For fluorescence microscopy, FVa staining was performed as for flow cytometry except in the absence of propidium iodide and incubated for 15 min at room temperature with Cy3 goat anti-mouse IgG F(ab′)2 diluted 1:100. Cells were mounted on coverslips with OPDA (20 mg/ml o-phenylenediamine dihydrochloride in 1 m Tris base (pH 8.5) diluted 1:1 with glycerol) and viewed with a Lieca DMRXA fluorescence microscope using a 63× oil (numerical aperture 1.32) Plan-Apo objective. Images were analyzed using Slidebook software (Intelligent Imaging Innovations, Inc., Denver, CO). FM 1-43 and Lucifer Yellow Staining—Neutrophils at 10 × 106 cells/ml in NBD buffer with 0.05% BSA and 1 mm CaCl2 were stimulated with fMLP for the times indicated. For the last minute of stimulation, 5 × 105 cells were transferred to 450 μl of NBD buffer and FM 1-43 (final concentration of 2.0 μm) and allowed to incubate for an additional 1 min at 37 °C. Cells were centrifuged and resuspended in 500 μl of ice-cold NBD buffer and analyzed by flow cytometry. Lucifer Yellow staining was carried out as described previously (30Fittschen C. Henson P.M. J. Clin. Investig. 1994; 93: 247-255Crossref PubMed Scopus (38) Google Scholar). Raft Membrane Isolation—Neutrophils (300 × 106)at20 × 106/ml in KRPD buffer with 0.25% BSA were stimulated with fMLP for 15 min. Following stimulation, cells were centrifuged and resuspended in 1 ml of ice-cold lysis buffer (25 mm MES (pH 6.5), 150 mm NaCl, 1% Triton X-100, 1 mm AEBSF, 1 mm NaF, 5 μg/ml leupeptin, 5 μg/ml aprotinin) and sonicated for 1 min on ice. Unbroken cells, nuclei, and debris were removed by centrifugation at 1000 × g for 10 min at 4 °C. The supernatant was added to 2 ml of 80% sucrose in MES-buffered saline (25 mm MES (pH 6.5), 150 mm NaCl), and the lysates were centrifuged through a 10–40% continuous sucrose gradient for 18–24 h at 37,000 rpm at 4 °C. Following centrifugation, 1-ml fractions were harvested from the bottom by puncturing the tube with an 18-gauge needle. Protein concentrations in each fraction were determined by the Bradford protein assay (31Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (223660) Google Scholar), and 10 μg of protein were run on 10% SDS-PAGE, transferred to nitrocellulose, and probed for PLSCR1 or CD55. Alkaline Phosphatase Assay—Sucrose density fractions were measured for alkaline phosphatase activity to identify rafts. Fraction samples (20 μl) were added to a 96-well flat bottom plate and mixed with 200 μl of reaction buffer (5 mm p-nitrophenyl phosphate in 100 mm 2-amino-2-methyl-1-propanol (pH 10.0)). Reactions were incubated at 37 °C for at least 15 min, and the absorbance was read in a microplate reader at 405 nm. Cloning and Transfection of PLSCR1—RNA isolated from Jurkat cells was synthesized to cDNA using a reverse transcriptase for PCR kit (Clontech Inc.) according to the manufacturer's instructions. cDNA was used as a template to amplify full-length PLSCR1 using the forward primer 5′-AAGAATTCGGCAGCCAGAGAACTGTTTTAA-3′ and the reverse primer 5′-AAGAATTCGCAGTTTTTCAAAGGAAGTTTCA-3′, both of which have engineered an EcoRI restriction enzyme cloning site on the 5′ ends. The PCR product of the correct size was purified and cloned directly into the EcoRI sites of pcDNA4/TO (Invitrogen), which allows the expression of the cloned gene in the presence of doxycycline. T-Rex-293 cells, expressing the tetracycline repressor, were transfected with PLSCR1 in the absence or presence of doxycycline to induce expression of PLSCR1 using FuGENE transfection reagent (Roche Applied Science) according to the manufacturer's instructions. Cells were harvested 24 h after transfection into lysis buffer (50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1 mm EGTA, 0.25% deoxycholate, 1% Triton X-100). Protein concentrations were determined by the Bradford protein assay (31Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (223660) Google Scholar), and 10 μg of protein were run on 10% SDS-PAGE, blotted to nitrocellulose, and probed for PLSCR1. PLSCR1, CD45, and Cholera Toxin B Staining—For PLSCR1 staining, neutrophils (2 × 106)at20 × 106/ml were blocked for 15 min on ice with 1% BSA in KRPD buffer and then incubated with either PLSCR1 serum (1:50) or preimmune serum (1:50) from the same rabbit on ice for an additional 30 min. Primary antibody was removed, and neutrophils (in KRPD buffer with 0.25% BSA) were stimulated with fMLP for 10 min at 37 °C, fixed in 3% paraformaldehyde, 3% sucrose in PBS at room temperature for 10 min, washed twice with PBS, and incubated with Cy3 goat anti-Rabbit F(ab′)2 diluted 1:100 for 30 min on ice. Cells were washed twice more with PBS and analyzed by flow cytometry. For CD45, neutrophils were preincubated as above except with 10 μg/ml mouse anti-human CD45 or isotype control antibody, and Cy3 anti-mouse F(ab′)2 diluted 1:100 was used. For fluorescence microscopy, cells were pretreated with antibody as above or with CT-B-Alexa 555 diluted 1:200 and either settled for 20 min at room temperature on poly-l-lysine-coated coverslips (pretreated with 100 μg/ml in PBS at 4 °C overnight) followed by fMLP stimulation at 37 °C or stimulated in suspension. Following stimulation, cells were fixed as above and incubated for 15 min at room temperature with secondary antibody, washed three times with PBS, and permeabilized with 0.2% Triton X-100 for 10 min at room temperature. Cells were washed three times with PBS, stained with fluorescein isothiocyanate-phalloidin (1:250) and 5 μg/ml Hoechst in PBS for 15 min at room temperature, washed with PBS, mounted with OPDA, and viewed with a Zeiss fluorescence microscope using a 63× oil (numerical aperture 1.4) Axiovert 200M objective. Images were analyzed using Slidebook software. Live Cell Imaging—Neutrophils (7.5 × 106 cells at 5 × 106/ml) in NBD buffer with 0.01% BSA, 1 mm EDTA, 300 μm EGTA, and either FM 1-43 or NBD-PC at a final concentration of 16 μm or 5 μg/ml, respectively, were placed in a BSA (0.01%)-coated Delta T dish (Bioptechs, Inc.) that was preheated to 37 °C. Neutrophils were stimulated with fMLP, and images were taken over 5 min with an inverted Olympus IX70 microscope equipped with a Sensicam camera connected to TILL-vision software (T.I.L.L. Photonics GmbH) every 3 s with a 500-ms exposure time for FM 1-43 and every 5 s with a 3-s exposure time for NBD-PC. Subcellular Fractionation—Neutrophil granules were isolated by Percoll density gradients from control neutrophils as described previously (32Kjeldsen L. Sengelov H. Borregaard N. J. Immunol. Methods. 1999; 232: 131-143Crossref PubMed Scopus (111) Google Scholar). Plasma membrane and secretory vesicles from control neutrophils were isolated by free flow electrophoresis as described previously (33Sengelov H. Borregaard N. J. Immunol. Methods. 1999; 232: 145-152Crossref PubMed Scopus (23) Google Scholar). PS Exposure on fMLP-stimulated Neutrophils—Using FVa as a sensitive probe to detect outer leaflet PS (34Zhai X. Srivastava A. Drummond D.C. Daleke D. Lentz B.R. Biochemistry. 2002; 41: 5675-5684Crossref PubMed Scopus (33) Google Scholar), we were able to demonstrate that fMLP stimulation resulted in transient PS exposure that was evident as early as 10 min and returned to base-line levels by 45 min (Fig. 1A). In contrast, this transient PS exposure was minimally detectable by the less sensitive annexin V binding (data not shown). PS exposure during fMLP stimulation was not accompanied by loss of aminophospholipid translocase activity as was seen during UV-induced apoptosis (Fig. 1B); indeed PS uptake during fMLP stimulation appeared to be enhanced. Nor was activation of caspase 3 or DNA degradation (data not shown) seen following stimulation with fMLP. These data demonstrate that transient PS exposure during neutrophil activation was not associated with apoptosis. PS Exposure Was Accompanied by Enhanced Uptake of Other Glycerophospholipids—To test whether the outward flop of endogenous PS was both specific for PS and unidirectional or alternatively was bidirectional as suggested by enhanced PS uptake observed in Fig. 1B, we tested whether exogenous phosphocholine-containing diradyl lipid probes presented to the activated neutrophil would be internalized. Using a non-hydrolyzable, alkyl-linked phosphocholine probe, c-PAF, fMLP stimulation resulted in enhanced phospholipid uptake that was rapid in onset, occurred as early as 5 min, and peaked at 15 min following stimulation (Fig. 1C). Enhanced phospholipid uptake did not result from stimulation of the PAF receptor by c-PAF as it occurred in the presence of the PAF receptor inhibitor WEB 2086 (data not shown) and was identical for the fluorescently labeled NBD-PC, an ester-linked phosphatidylcholine probe that does not stimulate the PAF receptor (Fig. 1D). As a positive control, neutrophils stimulated to undergo apoptosis with UV irradiation also showed increased phospholipid uptake of choline-containing probes (14Bratton D.L. Fadok V.A. Richter D.A. Kailey J.M. Guthrie L.A. Henson P.M. J. Biol. Chem. 1997; 272: 26159-26165Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 17Verhoven B. Schlegel R.A. Williamson P. J. Exp. Med. 1995; 182: 1597-1601Crossref PubMed Scopus (615) Google Scholar, 24Frasch S.C. Henson P.M. Kailey J.M. Richter D.A. Janes M.S. Fadok V.A. Bratton D.L. J. Biol. Chem. 2000; 275: 23065-23073Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). While phospholipid uptake during apoptosis was inhibited by the caspase 3 inhibitor DEVD phospholipid uptake during fMLP stimulation was not inhibited, again dissociating it from the process of apoptosis (data not shown) (24Frasch S.C. Henson P.M. Kailey J.M. Richter D.A. Janes M.S. Fadok V.A. Bratton D.L. J. Biol. Chem. 2000; 275: 23065-23073Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Phospholipid Uptake Was Independent of Endocytosis and Pinocytosis—In contrast to the enhanced uptake of choline-containing glycerophospholipids, internalization of NBD-sphingomyelin, which has been used as a lipid marker of endocytosis (35Hao M. Maxfield F.R. J. Biol. Chem. 2000; 275: 15279-15286Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), was not enhanced during either stimulation or apoptosis (data not shown); this was despite equivalent staining for both NBD-sphingomyelin and NBD-PC of both control and unwashed samples. To further investigate whether PC uptake was attributable to an endocytic/pinocytic pathway and whether PS exposure was the consequence of bilayer mixing during vesicle-plasma membrane budding and fusion during endosome recycling, the following experiment
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