Anaphylatoxin Signaling in Human Neutrophils
2004; Elsevier BV; Volume: 279; Issue: 43 Linguagem: Inglês
10.1074/jbc.m403977200
ISSN1083-351X
AutoresFarazeela Bte Mohd Ibrahim, See Jay Pang, Alirio J. Melendez,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoAnaphylatoxins activate immune cells to trigger the release of proinflammatory mediators that can lead to the pathology of several immune-inflammatory diseases. However, the intracellular signaling pathways triggered by anaphylatoxins are not well understood. Here we report for the first time that sphingosine kinase (SPHK) plays a key role in C5a-triggered signaling, leading to physiological responses of human neutrophils. We demonstrate that C5a rapidly stimulates SPHK activity in neutrophils and differentiated HL-60 cells. Using the SPHK inhibitor N,N-dimethylsphingosine (DMS), we show that inhibition of SPHK abolishes the Ca2+ release from internal stores without inhibiting phospholipase C or protein kinase C activation triggered by C5a but has no effect on calcium signals triggered by other stimuli (FcγRII). We also show that DMS inhibits degranulation, activation of the NADPH oxidase, and chemotaxis triggered by C5a. Moreover, an antisense oligonucleotide against SPHK1, in neutrophil-differentiated HL-60 cells, had similar inhibitory properties as DMS, suggesting that the SPHK utilized by C5a is SPHK1. Our data indicate that C5a stimulation decreases cellular sphingosine levels and increases the formation of sphingosine-1-phosphate. Exogenously added sphingosine has a dual effect on C5a-stimulated oxidative burst: it has a priming effect at lower concentrations but a dose-dependent inhibitory effect at higher concentrations; however, C5a-triggered protein kinase C activity was only reduced at high concentration of sphingosine. In contrast, C5a-triggered Ca2+ signals, chemotaxis, and degranulation were not affected by sphingosine at all. Exogenous sphingosine-1-phosphate, by itself, did not induce degranulation or chemotaxis, but it did marginally induce Ca2+ signals and oxidative burst and had a priming effect, enhancing all the C5a-triggered responses. Taken together, these results suggest that SPHK plays an important role in the immune-inflammatory pathologies triggered by anaphylatoxins in human neutrophils and point out SPHK as a potential therapeutic target for the treatment of diseases associated with neutrophil hyperactivation. Anaphylatoxins activate immune cells to trigger the release of proinflammatory mediators that can lead to the pathology of several immune-inflammatory diseases. However, the intracellular signaling pathways triggered by anaphylatoxins are not well understood. Here we report for the first time that sphingosine kinase (SPHK) plays a key role in C5a-triggered signaling, leading to physiological responses of human neutrophils. We demonstrate that C5a rapidly stimulates SPHK activity in neutrophils and differentiated HL-60 cells. Using the SPHK inhibitor N,N-dimethylsphingosine (DMS), we show that inhibition of SPHK abolishes the Ca2+ release from internal stores without inhibiting phospholipase C or protein kinase C activation triggered by C5a but has no effect on calcium signals triggered by other stimuli (FcγRII). We also show that DMS inhibits degranulation, activation of the NADPH oxidase, and chemotaxis triggered by C5a. Moreover, an antisense oligonucleotide against SPHK1, in neutrophil-differentiated HL-60 cells, had similar inhibitory properties as DMS, suggesting that the SPHK utilized by C5a is SPHK1. Our data indicate that C5a stimulation decreases cellular sphingosine levels and increases the formation of sphingosine-1-phosphate. Exogenously added sphingosine has a dual effect on C5a-stimulated oxidative burst: it has a priming effect at lower concentrations but a dose-dependent inhibitory effect at higher concentrations; however, C5a-triggered protein kinase C activity was only reduced at high concentration of sphingosine. In contrast, C5a-triggered Ca2+ signals, chemotaxis, and degranulation were not affected by sphingosine at all. Exogenous sphingosine-1-phosphate, by itself, did not induce degranulation or chemotaxis, but it did marginally induce Ca2+ signals and oxidative burst and had a priming effect, enhancing all the C5a-triggered responses. Taken together, these results suggest that SPHK plays an important role in the immune-inflammatory pathologies triggered by anaphylatoxins in human neutrophils and point out SPHK as a potential therapeutic target for the treatment of diseases associated with neutrophil hyperactivation. Activation of the complement cascade plays a key role in host defense. However, anaphylatoxins produced after the activation of the complement system are associated with a variety of pathologies, including septic shock, adult respiratory distress syndrome, and immune complex-dependent diseases such as rheumatoid arthritis (1Smedegard G. Cui L.X. Hugli T.E. Am. J. Pathol. 1989; 135: 489-497PubMed Google Scholar, 2Stevens J.H. O'Hanley P. Shapiro J.M. Mihm F.G. Satoh P.S. Collins J.A. Raffin T.A. J. Clin. Investig. 1986; 77: 1812-1816Crossref PubMed Scopus (189) Google Scholar, 3Wang Y. Rollins S.A. Madri J.A. Matis L.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8955-8959Crossref PubMed Scopus (320) Google Scholar). Recently, the anaphylatoxin C5a has been shown to have an immune-regulatory role able to stimulate mediators of both acute and chronic inflammation (4Boyden S.V. J. Exp. Med. 1962; 115: 453-466Crossref PubMed Scopus (2072) Google Scholar, 5Okusawa S. Yancey K.B. van der Meer J.W. Endres S. Lonnemann G. Hefter K. Frank M.M. Burke J.F. 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Siever S. Gotze O. Burchardi H. Eur. J. Clin. Investig. 1998; 28: 227-234Crossref PubMed Scopus (32) Google Scholar, 11Short A. Wong A.K. Finch A.M. Haaima G. Shiels I.A. Fairlie D.P. Taylor S.M. Br. J. Pharmacol. 1999; 126: 551-554Crossref PubMed Scopus (65) Google Scholar, 12Czermak B.J. Sarma V. Pierson C.L. Warner R.L. Huber-Lang M. Bless N.M. Schmal H. Friedl H.P. Ward P.A. Nat. Med. 1999; 5: 788-792Crossref PubMed Scopus (346) Google Scholar, 13Heller T. Hennecke M. Baumann U. Gessner J.E. zu Vilsendorf A.M. Baensch M. Boulay F. Kola F. Klos A. Bautsch W. Kohl J. J. Immunol. 2000; 163: 985-994Google Scholar). Most of these studies used blocking antibodies raised against C5a (10Mohr M. Hopken U. Oppermann M. Mathes C. Goldmann K. Siever S. Gotze O. Burchardi H. Eur. J. Clin. Investig. 1998; 28: 227-234Crossref PubMed Scopus (32) Google Scholar, 12Czermak B.J. Sarma V. Pierson C.L. Warner R.L. Huber-Lang M. Bless N.M. Schmal H. Friedl H.P. Ward P.A. Nat. Med. 1999; 5: 788-792Crossref PubMed Scopus (346) Google Scholar) or recombinant proteins that are receptor antagonists or analogues of C5a (9Pellas T.C. Boyar W. van Oostrum J. Wasvary J. Fryer L.R. Pastor G. Sills M. Braunwalder A. Yarwood D.R. Kramer R. Kimble E. Hadala J. Haston W. Moreira-Ludewig R. Uziel-Fusi S. Peters P. Bill K. Wennogle L.P. J. Immunol. 1998; 160: 5616-5621PubMed Google Scholar, 13Heller T. Hennecke M. Baumann U. Gessner J.E. zu Vilsendorf A.M. Baensch M. Boulay F. Kola F. Klos A. Bautsch W. Kohl J. J. Immunol. 2000; 163: 985-994Google Scholar). However, there are many problems associated with the use of such proteins to treat human patients. Immunogenicity is a problem, and proteins are expensive to manufacture, very susceptible to degradation by proteases in serum or gastrointestinal tract, and generally display poor pharmacokinetic properties. More recently, attempts have been made to produce smaller molecules that are more stable, cheaper to make, have better bioavailability, and are more attractive as drug candidates for treating human diseases mediated by C5a (14Wong A.K. Finch A.M. Pierens G.K. Craik D.J. Taylor S.M. Fairlie P. J. Med. Chem. 1998; 41: 3417-3425Crossref PubMed Scopus (72) Google Scholar, 15Finch A.M. Wong A.K. Paczkowski N.J. Wadi S.K. Craik D.J. Fairlie D.P. Taylor S.M. J. Med. Chem. 1999; 42: 1965-1974Crossref PubMed Scopus (219) Google Scholar). However, very little is known about the intracellular signaling pathways activated by C5a in immune effector cells. During the last few years, it has become clear that sphingo-lipids, in addition to being structural constituents of cell membranes, are sources of important signaling molecules. In particular, the sphingolipid metabolites, ceramide and sphingosine-1-phosphate (SPP), 1The abbreviations used are: SPP, sphingosine-1-phosphate; SPHK, sphingosine kinase; IP3, inositol-1,4,5-trisphosphate; DMS, N,N-dimethylsphingosine; fMLP, formylmethionylleucylphenylalanine; PKC, protein kinase C. have emerged as a new class of potent bioactive molecules implicated in a variety of cellular processes such as cell differentiation, apoptosis, and proliferation (16Hannun Y.A. J. Biol. Chem. 1994; 269: 3125-3128Abstract Full Text PDF PubMed Google Scholar, 17Heller R.A. Krönke M. J. Cell Biol. 1994; 126: 5-9Crossref PubMed Scopus (404) Google Scholar, 18Kolesnik R. Golde D.W. Cell. 1994; 77: 325-328Abstract Full Text PDF PubMed Scopus (920) Google Scholar, 19Spiegel S. Milstien S. J. Membr. Biol. 1995; 146: 225-237Crossref PubMed Scopus (226) Google Scholar). Interest in SPP focused recently on two distinct cellular actions of this lipid, namely, the function of SPP as an extracellular ligand activating specific G protein-coupled receptors and the role of SPP as an intracellular second messenger (20Meyer zu Heringdorf D. van Koppen C.J. Jakobs K.H. FEBS Lett. 1997; 410: 34-38Crossref PubMed Scopus (121) Google Scholar). Several findings enforced the notion of SPP as an important intracellular second messenger. First, activation of various plasma membrane receptors, such as the platelet-derived growth factor receptor (21Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (827) Google Scholar, 22Bornfeldt K.E. Graves L.M. Raines E.W. Igarashi Y. Wayman G. Yamamura S. Yatomi Y. Sidhu J.S. Krebs E.G. Hakomori S. Ross R. J. Cell Biol. 1995; 130: 193-206Crossref PubMed Scopus (265) Google Scholar), FcϵRI and FcγRI antigen receptors (23Choi O.H. Kim J.H. Kinet J.P. Nature. 1996; 18: 634-636Crossref Scopus (386) Google Scholar, 24Melendez A.J. Khaw A.K. J. Biol. Chem. 2002; 277: 17255-17262Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 25Melendez A. Floto R.A. Gillooly D.J. Harnett M.M. Allen J.M. J. Biol. Chem. 1998; 273: 9393-9402Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), and fMLP receptor (26Alemany R. zu Henringdorf D.M. van Koppen C.J. Jacobs K.H. J. Biol. Chem. 1999; 274: 3994-3999Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), was found to rapidly increase intracellular SPP production through stimulation of the sphingosine kinase. Second, inhibition of sphingosine kinase stimulation strongly reduced or even prevented cellular events triggered by these receptors, such as receptor-stimulated DNA synthesis, Ca2+ mobilization, and vesicular trafficking (21Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (827) Google Scholar, 22Bornfeldt K.E. Graves L.M. Raines E.W. Igarashi Y. Wayman G. Yamamura S. Yatomi Y. Sidhu J.S. Krebs E.G. Hakomori S. Ross R. J. Cell Biol. 1995; 130: 193-206Crossref PubMed Scopus (265) Google Scholar, 23Choi O.H. Kim J.H. Kinet J.P. Nature. 1996; 18: 634-636Crossref Scopus (386) Google Scholar, 24Melendez A.J. Khaw A.K. J. Biol. Chem. 2002; 277: 17255-17262Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 25Melendez A. Floto R.A. Gillooly D.J. Harnett M.M. Allen J.M. J. Biol. Chem. 1998; 273: 9393-9402Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 26Alemany R. zu Henringdorf D.M. van Koppen C.J. Jacobs K.H. J. Biol. Chem. 1999; 274: 3994-3999Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Our goal is to investigate the intracellular signaling pathways triggered by anaphylatoxins. We have used primary human neutrophils and differentiated HL-60 cells (a human neutrophil cell model) to better understand the intracellular molecular mechanisms responsible for C5a-triggered physiological events and to identify key molecules as candidates for novel therapeutic intervention. Here we report for the first time that the anaphylatoxin C5a activates the intracellular signaling molecule sphingosine kinase and present data that support the role for sphingosine kinase in the physiological responses triggered by C5a in human neutrophils, showing that inhibition of this enzyme has potential anti-inflammatory properties. We demonstrate that C5a receptor activation rapidly stimulates sphingosine kinase activity in primary human neutrophils and neutrophil-differentiated HL-60 cells. Moreover, inhibition of sphingosine kinase by N,N-dimethylsphingosine (DMS) does not affect phospholipase C stimulation or protein kinase C (PKC) activation triggered by C5a, but it largely inhibits C5a-stimulated Ca2+ mobilization, enzyme release, chemotaxis, and NADPH activation from both primary neutrophils and differentiated HL-60 cells. Furthermore, an antisense oligonucleotide specific for sphingosine kinase (SPHK) 1, transfected to the HL-60 cells, also inhibited the responses triggered by C5a. Interestingly, DMS had no effect calcium signals triggered by another stimulus (FcγRII). We also show here that C5a stimulation decreases cellular sphingosine levels and increases the formation of SPP; this may suggest a role for SPHK in removing a negative regulator (sphingosine) and generating a positive regulator (SPP), as has been suggested for mast cell activation (27Prieshl E.E. Csonga R. Novotny V. Kikuchi G.E. Baumruker T. J. Exp. Med. 1999; 190: 1-8Crossref PubMed Scopus (148) Google Scholar). Thus, we studied the effects of exogenously added sphingosine and SPP; we found that sphingosine has a dual effect on C5a-stimulated NADPH oxidase activation: it has a priming effect at lower concentrations but has a dose-dependent inhibitory effect at higher concentrations. On the other hand, C5a-triggered PKC activity was only reduced at high concentrations of sphingosine. C5a-triggered Ca2+ signals, chemotaxis, and degranulation were not affected by sphingosine at all. SPP, by itself, did not induce degranulation or chemotaxis, but it did marginally induce Ca2+ signals and the oxidative burst. However, SPP showed a priming effect, enhancing all C5a-triggered responses. Thus, our data contribute not only to the understanding of the intracellular molecular mechanisms utilized by C5a, suggesting that SPHK plays a key role in anaphylatoxin-triggered physiological functions, but also point out SPHK as a novel candidate for therapeutic intervention to treat inflammatory diseases. All materials, unless stated otherwise, were purchased from Sigma-Aldrich (Singapore). Isolation of Primary Human Neutrophils—Neutrophils were purified from healthy donors as described previously (28Smith W.B. Gamble J.R. Clark-Lewis I. Vadas M.A. Immunology. 1991; 72: 65-72PubMed Google Scholar). Briefly, this was done using dextran sedimentation, followed by density gradient centrifugation with Lymphoprep (Nycomed) and hypotonic lysis of erythrocytes. Cells were resuspended in assay medium (RPMI 1640 medium with 10 mm HEPES and 2.5% fetal calf serum) before use. Cytological examination of stained centrifuged preparations showed that 95% of the cells were neutrophils. Trypan blue staining confirmed that >98% of these cells were viable. After purification, the cells (2 × 106 cells/ml) were resuspended in RPMI 1640 medium supplemented with 2.5% fetal calf serum and allowed to recover for 30 min at 37 °C in a 5% CO2 atmosphere. For experiments, the cells were left untreated or pretreated with DMS (10 μm) for 20 min before stimulation. HL-60 Cell Culture and Differentiation to Neutrophil-like Cells—HL-60 cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 150 units/ml penicillin, and 150 μg/ml streptomycin at 37 °C in 5% CO2. Differentiation into neutrophil-like cells was induced by culturing HL-60 cells for 48 h in the presence of 0.5 mm dibutyril cyclic AMP, followed by an additional 48 h of differentiation in the presence or absence of antisense oligonucleotides and 0.5 mm dibutyril cyclic AMP. C5a Stimulation—Cells (2 × 106 cells/ml) resuspended in RPMI 1640 medium supplemented with 2.5% fetal calf serum, untreated or pretreated with DMS (10 μm), sphingosine (at the concentrations indicated in Fig. 9), or SPP (10 μm) for 20 min or antisense oligonucleotides for 48 h, were stimulated by the addition of 1 μm C5a and incubated at 37 °C for the times indicated in each figure. Sphingosine-1-phosphate Generation in Whole Cells—SPP generation was measured by assaying the amount of intracellular SPP generation after receptor activation as described previously (25Melendez A. Floto R.A. Gillooly D.J. Harnett M.M. Allen J.M. J. Biol. Chem. 1998; 273: 9393-9402Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Briefly, cells were preincubated overnight in media containing [3H]serine (2 μCi/ml) to label cellular sphingolipids and free sphingosine pools. After labeling, the cells were stimulated by the addition of C5a and warming to 37 °C, and the reactions were terminated at specified times. Lipids were extracted and analyzed by TLC on Silica Gel G60. Standard sphingosine 1-phosphate was applied with the samples, and the lipids were visualized using iodine vapors. Bands corresponding to sphingosine-1-phosphate were excised from the plate and counted by liquid scintillation spectrometry. Results were calculated as a percentage of the total radioactivity incorporated in the lipids. Sphingosine Kinase Activity—After C5a stimulation, activation of sphingosine kinase was measured as described previously (24Melendez A.J. Khaw A.K. J. Biol. Chem. 2002; 277: 17255-17262Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 29Melendez A. Floto R.A. Cameron A.J. Gillooly D.J. Harnett M.M. Allen J.M. Curr. Biol. 1998; 8: 210-221Abstract Full Text Full Text PDF PubMed Google Scholar) in total cell lysates. Briefly, cells were resuspended in ice-cold 0.1 m phosphate buffer (pH 7.4) containing 20% glycerol, 1 mm mercaptoethanol, 1 mm EDTA, phosphatase inhibitors (20 mm ZnCl2, 1 mm sodium orthovanadate, and 15 mm sodium fluoride), protease inhibitors (10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mm phenylmethylsulfonyl fluoride), and 0.5 mm 4-deoxypyridoxine and disrupted by freeze-thawing. Lysates were assayed for sphingosine kinase activity, based on the SPHK-catalyzed transfer of the γ-phosphate group of ATP (using a mixture of cold ATP and [γ32P]ATP (1 μCi/sample)) to a specific substrate, and the products were separated by TLC on Silica Gel G60 (Whatman) and visualized by autoradiography. The radioactive spots corresponding to sphingosine phosphate were scraped and counted in a scintillation counter. Phospholipase C Activity—After stimulating the cells with C5a for the indicated times, inositol-1,4,5-trisphosphate (IP3) was measured as described previously (24Melendez A.J. Khaw A.K. J. Biol. Chem. 2002; 277: 17255-17262Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), using the BIOTRAK TRK 1000 kit (Amersham Biosciences). Briefly, the system is based on the competitive binding of cellularly formed IP3 and a known amount of radiolabeled IP3 to the IP3 receptor. Cytosolic Calcium Measurement—Cytosolic calcium was measured as described previously (24Melendez A.J. Khaw A.K. J. Biol. Chem. 2002; 277: 17255-17262Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Briefly, cells were loaded with 1 μg/ml Fura-2/AM (Molecular Probes) in PBS, 1.5 mm Ca2+, and 1% bovine serum albumin. After removal of excess reagents by dilution and centrifugation, the cells were resuspended in 1.5 mm Ca2+-supplemented PBS and warmed to 37 °C in the cuvette; after the basal line was obtained, the cells were stimulated by the addition of C5a. Fluorescence was measured at 340 and 380 nm, and the background-corrected 340: 380 ratio was calibrated as described previously (24Melendez A.J. Khaw A.K. J. Biol. Chem. 2002; 277: 17255-17262Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Degranulation/Enzyme Release—After C5a stimulation, β-glucorinidase release from neutrophils and neutrophil differentiated HL-60 cells was measured as described previously (26Alemany R. zu Henringdorf D.M. van Koppen C.J. Jacobs K.H. J. Biol. Chem. 1999; 274: 3994-3999Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Enzyme release is measured as a percentage of the total cellular enzyme content. Chemotaxis Assay—Chemotaxis was assayed using the Chemicin QCM™ Chemotaxis 3 μm 96-well Cell Migration Assay kit (catalog no. ECM 515) following the manufacturer's instructions. Briefly, the assay is based on the 3-μm pore size of Boyden chambers. Cells are placed in the upper chamber, and 5 nm C5a is placed in the lower chamber, and cells are incubated at 37 °C for 2 h. Migratory cells found on the lower chamber are collected, and migratory cells attached on the bottom of the insert membrane are dissociated from the membranes with the Cell Detachment Buffer provided. These cells are subsequently lysed and detected using the CyQuant GR dye (Molecular Probes) provided in the kit. This green fluorescent dye exhibits strong fluorescence enhancement when bound to cellular nucleic acids. The number of migratory cells is determined by running a fluorescent cell dose curve, in which a known number of cells are lysed and detected using the CyQuant GR dye to generate a standard curve. NADPH Oxidative Burst Assays—Whole cell superoxide production after C5a stimulation was measured in primary neutrophils and differentiated HL-60 cells. Cells were assayed using an enhanced luminol-based substrate (DIOGENES, National Diagnostics) added to the cells at the same time as C5a (1 μm), and luminescence was measured using a luminometer (Wallac 1420 Multilabel counter). Western Blots—Unless otherwise stated, 40 μg of lysate for each sample was resolved on 12% polyacrylamide gels (SDS-PAGE) under denaturing conditions and then transferred to 0.45 μm nitrocellulose membranes. After blocking overnight at 4 °C with 5% nonfat milk in Tris-buffered saline and 0.1% Tween 20 and washing, the membranes were incubated with the relevant antibodies for 4 h at room temperature. The membranes were washed extensively in Tris-buffered saline/0.1% Tween 20 (washing buffer). The blots were probed using specific anti-SPHK1 polyclonal (made in house as described previously (21Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (827) Google Scholar)) and monoclonal anti-Arf1 (Santa Cruz Biotechnologies) primary antibodies. Bands were visualized using anti-rabbit horseradish peroxidase-conjugated and anti-mouse horseradish peroxidase-conjugated secondary antibodies and the ECL Western blotting detection system (Amersham Biosciences). Fluorescence Microscopy—After C5a stimulation, suspended cells were fixed in 4% paraformaldehyde and deposited on microscope slides in a cytospin centrifuge and then permeabilized for 5 min in 0.1% Triton X-100 in PBS. Fluorescence labeling was performed using the anti-SPHK1 polyclonal antibody made in house as described previously (21Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (827) Google Scholar) as primary antibody and an anti-rabbit, fluorescein isothiocyanate-conjugated secondary antibody. Staining was analyzed by fluorescence microscopy using a Leica DM IRB microscope, and images were captured using a Leica DC 300F camera. Antisense Knock-down of Sphingosine Kinase 1—The antisense down-regulation of SPHK1 was carried out as described previously (24Melendez A.J. Khaw A.K. J. Biol. Chem. 2002; 277: 17255-17262Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Antisense oligonucleotides were purchased from Oswell DNA Services; 20-mers were synthesized, capped at either end by the phosphorothioate linkages (the first two and the last two linkages), and corresponded to the reverse complement of the first 20 coding nucleotides for SPHK1; a scrambled oligonucleotide was used as a control. The sequences of the oligonucleotides were 5′-CCCGCAGGATCCATAACCTC-3′ (antisense) for SPHK1 and 5′-CTGGTGGAAGAAGAGGACGT-3′ (scrambled antisense) for control. Flow Cytometry for Cell Viability—Cell viability was detected by propidium iodide staining, which stains dead or dying cells. 100 μl of propidium iodide (catalog no. 1 348 639; Roche Diagnostics) was added to a 1-ml cell suspension containing 106 cells (final concentration of propidium iodide, 50 μg/ml) just before analysis using a Coulter EPICS-XL flow cytometer. PKC Activity—PKC enzyme activity was measured using the Biotrak Protein Kinase C enzyme assay system (Amersham Biosciences). Briefly, the system is based on the PKC-catalyzed transfer of the γ-phosphate group of ATP (using a mixture of cold ATP and [γ-32P]ATP (1 μCi/sample)) to a peptide substrate specific for PKC. After receptor stimulation, PKC assays were carried out. Results are expressed as phosphorylation rate per pmol of protein per minute. Sphingosine Levels—Sphingosine levels were analyzed as follows after C5a stimulation in cells pretreated or not pretreated with the antisense oligonucleotide to SPHK1: cells were preincubated overnight in media containing [3H]serine (2 μCi/ml) to label cellular sphingolipids and free sphingosine pools. After labeling, the cells were stimulated by the addition of C5a and warming to 37 °C, and the reactions were terminated at the specified times. Lipids were extracted and analyzed by TLC on Silica Gel G60 as described previously (25Melendez A. Floto R.A. Gillooly D.J. Harnett M.M. Allen J.M. J. Biol. Chem. 1998; 273: 9393-9402Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). The sphingosine standard was applied to the samples to act as carrier and aid visualization of the lipid after TLC, and the lipids were visualized using iodine vapors. Bands corresponding to sphingosine were excised from the plate and counted by liquid scintillation spectrometry. Results were expressed as cpm per 2 × 106 cells. FcγRII (CD32) Aggregation/Stimulation.—FcγRII aggregation was carried out as described previously (29Melendez A. Floto R.A. Cameron A.J. Gillooly D.J. Harnett M.M. Allen J.M. Curr. Biol. 1998; 8: 210-221Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly, cells were incubated at 5 °C, rocked for 45 min with a specific mouse monoclonal anti-CD32 antibody, and then washed to remove excess antibody. After that, anti-mouse IgG was added to the cells, which were incubated at 37 °C, and reactions were stopped at the indicated times. C5a Stimulates Sphingosine Kinase Activity in Human Neutrophils—We first investigated whether the C5a receptor would stimulate sphingosine kinase activity in primary human neutrophils and differentiated HL-60 cells (a human neutrophil model). Direct measurement of sphingosine kinase activity showed that the enzyme is activated after C5a receptor engagement in both the primary neutrophils and the HL-60 neutrophil model (Fig. 1A) and that this activation was inhibited in cells pretreated with DMS (Fig. 1A). After that, direct measurement of cellular SPP generation showed that SPP is generated after C5a receptor engagement in both the primary neutrophils and the HL-60 neutrophil model (Fig 1B) and that this SPP generation was inhibited in cells pretreated with DMS (Fig. 1B); the kinetics of SPP generation correlate with the kinetics for SPHK activity. These data show that the C5a receptor is capable of stimulating sphingosine kinase activity and the generation of intracellular SPP in both the primary neutrophils and the HL-60 neutrophil model. Role of Sphingosine Kinase on C5a-triggered Ca2+ Signals—Previous studies have demonstrated that sphingosine kinase mediates Ca2+ signals for several plasma membrane receptors (20Meyer zu Heringdorf D. van Koppen C.J. Jakobs K.H. FEBS Lett. 1997; 410: 34-38Crossref PubMed Scopus (121) Google Scholar, 21Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (827) Google Scholar, 22Bornfeldt K.E. Graves L.M. Raines E.W. Igarashi Y. Wayman G. Yamamura S. Yatomi Y. Sidhu J.S. Krebs E.G. Hakomori S. Ross R. J. Cell Biol. 1995; 130: 193-206Crossref PubMed Scopus (265) Google Scholar, 23Choi O.H. Kim J.H. Kinet J.P. Nature. 1996; 18: 634-636Crossref Scopus (386) Google Scholar), and because our results show that C5a triggers sphingosine kinase activity in human neutrophils, we decided to investigate the role of sphingosine kinase in C5a-triggered Ca2+ mobilization in these cells. C5a stimulation of primary neutrophils rapidly and transiently triggered calcium release from internal stores (Fig. 2A). However, in cells pretreated with DMS, these Ca2+ signals were inhibited (Fig. 2A). Similar results were obtained after C5a stimulation in differentiated HL-60 cells (Fig. 2B). The inhibitory action of DMS on Ca2+ signals was not due to inhibition of phosph
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