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Originally published In Press as doi:10.1074/jbc.M403977200 on August 9, 2004

J. Biol. Chem., Vol. 279, Issue 43, 44802-44811, October 22, 2004
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Anaphylatoxin Signaling in Human Neutrophils

A KEY ROLE FOR SPHINGOSINE KINASE*

Farazeela Bte Mohd Ibrahim, See Jay Pang, and Alirio J. Melendez{ddagger}

From the Department of Physiology, National University of Singapore, Singapore 117597

Received for publication, April 9, 2004 , and in revised form, July 14, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (Fc{gamma}RII). 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (1–3).

Recently, the anaphylatoxin C5a has been shown to have an immune-regulatory role able to stimulate mediators of both acute and chronic inflammation (4–8). The significance of C5a in several inflammatory diseases is demonstrated by the fact that agents that blocked the action of C5a also suppressed inflammation in several animal models (9–13). Most of these studies used blocking antibodies raised against C5a (10, 12) or recombinant proteins that are receptor antagonists or analogues of C5a (9, 13). 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 (14, 15). 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),1 have emerged as a new class of potent bioactive molecules implicated in a variety of cellular processes such as cell differentiation, apoptosis, and proliferation (16–19). 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 (20). 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 (21, 22), Fc{epsilon}RI and Fc{gamma}RI antigen receptors (23–25), and fMLP receptor (26), 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 (21–26).

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 (Fc{gamma}RII). 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 (27). 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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 (28). 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 x 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 x 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.



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  		FIG. 9.
Role of exogenous added sphingosine or SPP on neutrophil functions. A, oxidative burst triggered by C5a on human neutrophils pretreated with increasing concentrations of sphingosine. B, protein kinase C activity triggered by C5a on human neutrophils pretreated with increasing concentrations of sphingosine. C, calcium release from internal stores triggered by C5a on untreated human neutrophils (Control) or human neutrophils treated with 10 µM sphingosine (+Sphingosine (10 µM)). D, {beta}-glucuronidase release from untreated resting neutrophils (Basal) and untreated neutrophils triggered by C5a (C5a), unstimulated neutrophils treated with 10 µM sphingosine (Basal, Sphingosine 10 µM), and neutrophils pretreated with 10 µM sphingosine triggered by C5a (C5a, Sphingosine 10 µM). E, number of migratory cells: untreated resting neutrophils (Basal), untreated neutrophils using C5a as chemoattractant (C5a), resting neutrophils pretreated with 10 µM sphingosine (Basal, Sphingosine 10 µM), and neutrophils pretreated with 10 µM sphingosine, using C5a as chemoattractant (C5a, Sphingosine 10 µM). F, number of migratory cells: untreated resting neutrophils (Basal), untreated neutrophils using C5a as chemoattractant (C5a), resting neutrophils pretreated with 10 µM SPP (Basal, SPP 10 µM), and neutrophils pretreated with 10 µM SPP, using C5a as chemoattractant (C5a, SPP 10 µM). G, {beta}-glucuronidase release from untreated resting neutrophils (Basal), C5a-triggered {beta}-glucuronidase release from untreated neutrophils (C5a), {beta}-glucuronidase release from unstimulated neutrophils treated with 10 µM SPP (Basal, SPP 10 µM), and C5a-triggered {beta}-glucuronidase release from neutrophils pretreated with 10 µM SPP (C5a, SPP 10 µM). H, calcium release from internal stores in untreated resting neutrophils (Control) and from neutrophils triggered with 10 µM SPP (SPP 10 µM). I, oxidative burst from untreated resting neutrophils (Basal), C5a-triggered oxidative burst from untreated neutrophils (C5a); oxidative burst from resting neutrophils pretreated with 10 µM SPP (Basal, SPP 10 µM), and C5a-triggered oxidative burst from neutrophils pretreated with 10 µM SPP (C5a, SPP 10 µM). J, C5a-triggered calcium release from internal stores in untreated neutrophils (Control) and C5a-triggered calcium release from neutrophils pretreated with 10 µM SPP (+SPP 10 µM).

 
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 (25). 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 (24, 29) 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 {gamma}-phosphate group of ATP (using a mixture of cold ATP and [{gamma}32P]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 (24), 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 (24). 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 (24).

Degranulation/Enzyme Release—After C5a stimulation, {beta}-glucorinidase release from neutrophils and neutrophil differentiated HL-60 cells was measured as described previously (26).

Enzyme release is measured as a percentage of the total cellular enzyme content.

Chemotaxis Assay—Chemotaxis was assayed using the Chemicin QCMTM 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 (21)) 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 (21) 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 (24). 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 {gamma}-phosphate group of ATP (using a mixture of cold ATP and [{gamma}-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 (25). 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 x 106 cells.

Fc{gamma}RII (CD32) Aggregation/Stimulation.—Fc{gamma}RII aggregation was carried out as described previously (29). 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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.



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  		FIG. 1.
C5a triggers SPHK activity and SPP generation in primary human neutrophils and differentiated HL-60 cells (neutrophil model). A, basal SPHK activity in primary neutrophils (Neutrophil basal), SPHK activity after C5a stimulation in primary neutrophils (Neutrophil + C5a), basal SPHK activity in differentiated HL-60 cells (HL-60 basal), SPHK activity after C5a stimulation in differentiated HL-60 cells (HL-60 + C5a), SPHK activity in primary neutrophils triggered by C5a in cells pretreated with the SPHK inhibitor DMS (Neutrophil + DMS + C5a), and SPHK activity triggered by C5a in differentiated HL-60 cells pretreated with the SPHK inhibitor DMS (HL-60 + DMS + C5a). Results shown are the mean ± S.D. of triplicate measurements and of three separate experiments. B, basal SPP in primary neutrophils (Neutrophil basal), SPP generation after C5a stimulation in primary neutrophils (Neutrophil + C5a), basal SPP in differentiated HL-60 cells (HL-60 basal), SPP generation after C5a stimulation in differentiated HL-60 cells (HL-60 + C5a), SPP generation triggered by C5a in primary neutrophils pretreated with the SPHK inhibitor DMS (Neutrophil + DMS + C5a), and SPP generation triggered by C5a in differentiated HL-60 cells pretreated with the SPHK inhibitor DMS (HL-60 + DMS + C5a). Results shown are the mean ± S.D. of triplicate measurements and of three separate experiments.

 
Role of Sphingosine Kinase on C5a-triggered Ca2+ Signals—Previous studies have demonstrated that sphingosine kinase mediates Ca2+ signals for several plasma membrane receptors (20–23), 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).



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  		FIG. 2.
C5a-triggered cytosolic Ca2+ signals are inhibited by DMS: role for SPHK. A, cytosolic Ca2+ triggered by C5a stimulation in primary neutrophils (Neutrophils + C5a) and cytosolic Ca2+ triggered by C5a in primary neutrophils pretreated with the SPHK inhibitor DMS (Neutrophils + DMS + C5a). Results shown are representative of three separate experiments. B, cytosolic Ca2+ triggered by C5a stimulation in differentiated HL-60 cells (HL-60 + C5a) and cytosolic Ca2+ triggered by C5a in differentiated HL-60 cells pretreated with the SPHK inhibitor DMS (HL-60 + DMS + C5a). Results shown are representative of three separate experiments. C, C5a-triggered phospholipase C activity is not inhibited by DMS. IP3 generation in differentiated HL-60 cells (basal control; Basal HL60), IP3 generation after C5a stimulation in differentiated HL-60 cells (C5a HL60), basal IP3 generation in the neutrophil model pretreated with the SPHK inhibitor DMS (Basal HL60+DMS), IP3 generation after C5a stimulation in differentiated HL-60 cells pretreated with the SPHK inhibitor DMS (C5a HL-60+DMS), IP3 generation in primary neutrophils (basal control; Basal Neutrophils), IP3 generation after C5a stimulation in primary neutrophils (C5a Neutrophils), basal IP3 generation in primary neutrophils pretreated with the SPHK inhibitor DMS (Basal Neutrophils +DMS), and IP3 generation after C5a stimulation in primary neutrophils pretreated with the SPHK inhibitor DMS (C5a Neutrophils +DMS). Results shown are the mean ± S.D. of triplicate measurements and of three separate experiments.

 
The inhibitory action of DMS on Ca2+ signals was not due to inhibition of phospholipase C stimulation. Preincubation of cells with DMS did not alter basal or inhibit C5a-triggered generation of IP3 (Fig. 2C).

Role of Sphingosine Kinase on C5a-triggered Physiological Responses—Following the role of sphingosine kinase on triggering Ca2+ signals, we investigated the role of sphingosine kinase in other well-characterized functional responses of neutrophils after cellular activation by C5a: degranulation, chemotaxis, and NADPH oxidative burst.

Role of Sphingosine Kinase on C5a-triggered Degranulation—Enzyme release (degranulation) is an important effector function of phagocytic cells and has been shown to require Ca2+ signals. Therefore we investigated whether SPHK activity plays any role in C5a-triggered degranulation. In contrast to phospholipase C activity, C5a-stimulated degranulation was strongly inhibited by the sphingosine kinase inhibitor DMS (Fig. 3). C5a very rapidly triggered the release of {beta}-glucoronidase in primary neutrophils (Fig. 3A) and in the neutrophil model (Fig. 3B). Pretreatment of cells with DMS substantially inhibited the C5a-triggered enzyme release (Fig. 3). These data suggest that SPHK plays a role in neutrophil degranulation.



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  		FIG. 3.
Degranulation triggered by C5a is dependent on SPHK activity. A, {beta}-glucuronidase release form primary neutrophils. {beta}-Glucuronidase release from resting cells (control; Basal Control), {beta}-glucuronidase release after C5a stimulation control (C5a Control), {beta}-glucuronidase release from resting cells pretreated with the SPHK inhibitor DMS (Basal + DMS), and {beta}-glucuronidase release after C5a stimulation in cells pretreated with DMS (C5a + DMS). Results shown are the mean ± S.D. of triplicate measurements and of three separate experiments. B, {beta}-glucuronidase release from differentiated HL-60 cells. {beta}-Glucuronidase release from resting cells (control; Basal Control), {beta}-glucuronidase release after C5a stimulation control (C5a Control), {beta}-glucuronidase release from resting cells pretreated with the SPHK inhibitor DMS (Basal + DMS), and {beta}-glucuronidase release after C5a stimulation in cells pretreated with DMS (C5a + DMS). Results shown are the mean ± S.D. of triplicate measurements and of three separate experiments.

 
Role of Sphingosine Kinase in C5a-triggered Chemotaxis—One of the most important functions of anaphylatoxins is their role as chemoattractants; therefore, we decided to find out whether sphingosine kinase plays any role in chemotaxis. First, we worked out the number of cells passing through the 3.0 µM pore membrane in response to a concentration range of 0.1–10 nM C5a; a concentration of 5 nM C5a was shown to give the maximum linear chemotactic response induced by C5a (data not shown) and chosen as the concentration to be used for the chemotaxis experiments. C5a triggered a chemotactic response for both the primary neutrophils and the neutrophil-like HL-60 cells (Fig. 4). However, pretreatment of the primary neutrophils or differentiated HL-60 cells with DMS substantially inhibited C5a-triggered chemotaxis for both cell types (Fig. 4), showing that SPHK activity does indeed play a role in C5a-triggered chemotaxis on human neutrophils.



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  		FIG. 4.
C5a-induced chemotaxis is inhibited by the SPHK inhibitor. Number of migratory differentiated HL-60 cells. Migration of unstimulated cells (basal; HL-60 basal), migration of resting cells pretreated with the SPHK inhibitor DMS (HL-60 basal +DMS), migration of untreated cells toward the C5a-containing chamber (HL60+C5a), and migration of cells pretreated with the SPHK inhibitor DMS toward the C5a-containing chamber (HL60+C5a+DMS). Number of migratory primary neutrophils. Migration of unstimulated cells (basal; Neutrophil basal), migration of resting cells pretreated with the SPHK inhibitor DMS (Neutrophil basal +DMS), migration of untreated cells toward the C5a-containing chamber (Neutrophil +C5a), and migration of cells pretreated with the SPHK inhibitor DMS toward the C5a-containing chamber (Neutrophil +C5a+DMS). Results shown are the mean ± S.D. of triplicate measurements and of three separate experiments.

 
Role of Sphingosine Kinase in C5a-triggered NADPH Oxidative Burst—Another important role for anaphylatoxins is to promote the release of oxygen radicals. Here we show that C5a rapidly stimulates the NADPH oxidase activity of primary human neutrophils and differentiated HL-60 cells (Fig. 5). However, in cells pretreated with DMS, C5a-triggered NADPH oxidase activity was inhibited. These results show that inhibition of sphingosine kinase completely blocks the C5a-triggered oxidative burst.



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  		FIG. 5.
C5a-induced NADPH oxidase activity is inhibited by DMS. NADPH oxidative burst triggered by C5a in primary neutrophils (Neutrophil + C5a), NADPH oxidative burst triggered by C5a in differentiated HL-60 cells (HL-60 + C5a), NADPH oxidative burst triggered by C5a in primary neutrophils pretreated with the SPHK inhibitor DMS (Neutrophil + DMS + C5a), and NADPH oxidative burst triggered by C5a in differentiated HL-60 cells pretreated with the SPHK inhibitor DMS (HL-60 + DMS + C5a). The oxidative burst is measured in relative luminescence units (RLU). Results shown are representative of three separate experiments.

 
SPHK1 Is the Enzyme Activated by C5a Antisense Knock-down of SPHK1—The experiments described above pointed out a potential important role for SPHK in several cellular responses triggered by C5a in human neutrophils. However, the use of pharmacological inhibitors sometimes gives rise to false results; therefore, we decided to investigate which specific SPHK was involved in the C5a-triggered physiological responses, using a knock-down approach for SPHK1, and thus determine whether this was the enzyme activated in the C5a-triggered signals. Fluorescence microscopy confirmed the presence of SPHK1 and showed that SPHK1 is primarily cytosolic in resting cells, but after C5a receptor engagement, it rapidly translocates to the cell periphery (Fig. 6A).



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  		FIG. 6.
SPHK1 expression, subcellular localization, and antisense knock-down. A, fluorescence microscopy of differentiated HL-60 cells and primary neutrophils, immunostained for SPHK1. Resting differentiated HL-60 cells (HL-60 resting), differentiated HL-60 cells after stimulation with C5a for 2 min (HL-60 + C5a), resting primary neutrophils (Neutrophil resting), and primary neutrophils after stimulation with C5a for 2 min (Neutrophils + C5a). Results shown are representative of three separate experiments. B, Western blot analysis of SPHK1 expression before and after antisense treatment in differentiated HL-60 cells. SPHK1 expression level of control (HL-60 control), SPHK1 expression levels in cells pretreated for 48 h with the antisense oligonucleotide against SPHK1 (HL-60 a.s. SPHK1); and SPHK1 expression levels in the neutrophil cell model pretreated for 48 h with the scrambled antisense oligonucleotide as a control (HL-60 a.s. control). The blot was simultaneously probed with an anti-Arf1 antibody to show equal loading for all lanes. Results shown are representative of three separate experiments.

 
We pretreated the HL-60 cells with an antisense oligonucleotide specific for SPHK1 and found that the antisense oligonucleotide knocks down SPHK1 levels by ~80%, as shown by Western blot analysis (Fig. 6B); an antisense control (scrambled oligonucleotide) had no effect on the SPHK1 levels (Fig. 6B) on these cells. These results suggested that SPHK1 was responsible for the SPHK activity observed in these cells after C5a stimulation.

SPHK1 Mediates C5a-triggered Ca2+ Release, Degranulation, Chemotaxis, and NADPH Oxidase Activity—To clarify whether SPHK1 was indeed involved in the responses triggered by C5a (such as Ca2+ signals, degranulation, chemotaxis, and the oxidative burst), we next used the antisense oligonucleotides against human SPHK1 to investigate these functions.

We found that C5a-triggered functions were substantially inhibited in cells pretreated with the antisense oligonucleotide specific for SPHK1; that is, a substantial reduction in the peak of Ca2+ release from intracellular stores was seen (Fig. 7A). Similarly, levels of enzyme release (degranulation) were also substantially inhibited for the human neutrophil model (Fig. 7B). Furthermore, chemotaxis was also significantly reduced in cells pretreated with the antisense oligonucleotide against SPHK1 (Fig. 7C). Moreover, the C5a-triggered oxidative burst was also inhibited in cells pretreated with the anti-SPHK1 antisense oligonucleotides (Fig. 7D).



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  		FIG. 7.
SPHK1 mediates the physiological responses triggered by C5a in differentiated HL-60 cells. A, C5a-triggered cytosolic Ca2+ signals via SPHK1. Cytosolic Ca2+ triggered by C5a stimulation time course control (HL-60 + C5a), cytosolic Ca2+ triggered by C5a in cells pretreated with the antisense oligonucleotide against SPHK1 (HL-60 + a.s.SPHK1 + C5a), and cytosolic Ca2+ triggered by C5a in cells pretreated with the scrambled antisense oligonucleotide (HL60 + a.s.scrambled + C5a). Results shown are representative of three separate experiments. B, degranulation triggered by C5a is dependent of SPHK1. {beta}-Glucuronidase release from resting cells (HL-60 Basal), {beta}-glucuronidase release after C5a stimulation (HL-60+C5a), {beta}-glucuronidase release from resting cells pretreated with the antisense oligonucleotide against SPHK1 (HL-60 Basal+a.sSPHK1), {beta}-glucuronidase release after C5a stimulation in cells pretreated with the antisense oligonucleotide against SPHK1 (HL-60+C5a+a.s.SPHK1), {beta}-glucuronidase release after C5a stimulation in cells pretreated with the scrambled antisense oligonucleotide (HL-60+C5a+a.s.scrambled). Results shown are the mean ± S.D. of triplicate measurements and of three separate experiments. C, C5a-induced chemotaxis in differentiated HL-60 cells is inhibited by the antisense oligonucleotide against SPHK1. Migration of resting cells (control; HL-60 Basal), migration of resting cells pretreated with the antisense oligonucleotide against SPHK1 (HL-60 Basal+a.s.SPHK1), migration of cells toward the C5a-containing chamber (HL60+C5a), migration of cells toward the C5a-containing chamber of cells pretreated with antisense oligonucleotide against SPHK1 (HL60+C5a+a.s.SPHK1), and migration of cells toward the C5a-containing chamber of cells pretreated with the scrambled antisense oligonucleotide (HL60+C5a+a.s.scrambled). Results shown are the mean ± S.D. of triplicate measurements and of three separate experiments. D, NADPH oxidative burst triggered by C5a in differentiated HL-60 cells is dependent on SPHK1. NADPH oxidative burst triggered by C5a in differentiated HL-60 cells (HL-60 + C5a), NADPH oxidative burst triggered by C5a in differentiated HL-60 cells pretreated with the antisense oligonucleotide against SPHK1 (HL-60 + a.s.SPHK1 + C5a), and NADPH oxidative burst triggered by C5a in differentiated HL-60 cells pretreated with the scrambled antisense oligonucleotide (HL-60 + a.s.scrambled + C5a). The oxidative burst is measured in relative luminescence units (RLU). Results shown are representative of three separate experiments.

 
It has been suggested that DMS and sphingosine can induce apoptosis of neutrophils and/or inhibit PKC activity (30–36); therefore, we decided to investigate this in our experimental setup. Here we demonstrate that treatment with 10 µM DMS (the concentration of DMS used in our experimental setup) and the antisense oligonucleotide against SPHK1 did not induce cell death (Fig. 8A, 1–5). However, the much higher DMS concentration (100 µM) previously reported to induce neutrophil apoptosis (30–33) did indeed induce the death of our experimental cells (Fig. 8A, 6 and 7). Protein kinase C activity triggered by C5a was not inhibited by DMS in our experimental conditions (Fig. 8B). Furthermore, pretreatment of neutrophils with DMS had no effect on calcium signals triggered by the IgG receptor present on neutrophil Fc{gamma}RII (Fig. 8C). We also show here that the antisense oligonucleotide against SPHK1 inhibits the C5a-triggered generation of SPP (Fig. 8D) and that the cellular levels of sphingosine are reduced after C5a stimulation. On the other hand, when SPHK activity is inhibited, sphingosine levels remain unchanged (Fig. 8E), suggesting that SPHK acts on a preexisting pool of sphingosine.



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  		FIG. 8.
Role of DMS and/or SPHK1-antisense oligonucleotides on cell viability, PKC activity, calcium signals, SPP generation, and sphingosine levels in neutrophils. A, cytotoxicity assay. 1, control neutrophils; 2, neutrophils treated with DMS (10 µM)for2h; 3, control HL-60 cells; 4, HL-60 cells treated with DMS (10 µM) for 2 h; 5, HL-60 cells pretreated with the antisense oligonucleotide against SPHK1; 6, neutrophils treated with DMS (100 µM) for 2 h; 7, HL-60 cells treated with DMS (100 µM) for 2 h. Results shown are representative of three separate experiments. B, PKC activity assay. PKC activity triggered by C5a in HL-60 cells (HL-60+C5a), PKC activity triggered by C5a in HL-60 cells pretreated with DMS (HL-60+DMS+C5a), PKC activity triggered by C5a in primary neutrophils (Neutrophil+C5a), PKC activity triggered by C5a in primary neutrophils pretreated with DMS (Neutrophil+DMS+C5a), PKC activity triggered by C5a in primary HL-60 cells pretreated with the PKC inhibitor bisindolylmaleimide-I (HL-60+Bis+C5a), and PKC activity triggered by C5a in primary neutrophils pretreated with the PKC inhibitor bisindolylmaleimide-I (Neutrophil+Bis+C5a). Results shown are the mean ± S.D. of triplicate measurements and of three separate experiments. C, DMS does not inhibit Fc{gamma}RII-triggered calcium release from human neutrophils. Intracellular calcium triggered by Fc{gamma}RIIa aggregation, XL Fc{gamma}RIIa; intracellular calcium triggered by Fc{gamma}RIIa aggregation in neutrophils pretreated with DMS, XL Fc{gamma}RIIa + DMS. XL and arrow indicate the moment when the cross-linking antibody was added. Results shown are representative of three separate experiments. D, role of antisense oligonucleotide against SPHK1 on SPP production in HL-60 cells. Basal levels of SPP in unstimulated HL-60 cells (Basal), C5a-triggered SPP generation in HL-60 cells (+C5a), C5a-triggered SPP generation in HL-60 cells pretreated with the antisense oligonucleotide against SPHK1 (a.s.SPHK1 +C5a), and C5a-triggered SPP generation in HL-60 cells pretreated with the scrambled antisense control (a.s.scrambled +C5a). Results shown are the mean ± S.D. of triplicate measurements and of three separate experiments. E, role of antisense oligonucleotide against SPHK1 on sphingosine levels in HL-60 cells. Basal levels of sphingosine in unstimulated HL-60 cells (Basal), sphingosine levels in HL-60 cells after C5a stimulation (+C5a), sphingosine levels after C5a stimulation in HL-60 cells pretreated with the antisense oligonucleotide against SPHK1, a.s.SPHK1 +C5a; sphingosine levels after C5a stimulation in HL-60 cells pretreated with the scrambled antisense control, a.s.scrambled +C5a. Results shown are the mean ± S.D. of triplicate measurements and of three separate experiments.

 
Effects of Sphingosine and SPP on Signaling—The data shown in Fig. 8E may suggest a role for SPHK in removing a negative regulator (sphingosine) and generating a positive regulator (SPP), similar to the rheostat model proposed for mast cell activation (27). Thus, we studied the effects of exogenously added sphingosine and SPP on C5a-triggered responses. In agreement with a previous study on the role of these lipids on superoxide production triggered by fMLP on neutrophils (37), we found that sphingosine has a dual effect on C5a-stimulated oxidative burst: a priming effect is seen at concentrations between 0.2 and 1 µM, but a dose-dependent inhibitory effect is seen at higher concentrations of 2–10 µM (Fig. 9A). C5a-triggered PKC activity was only partially reduced in cells pretreated with 10 µM sphingosine (Fig. 9B). On the other hand, the C5a-triggered Ca2+ signals were not affected by sphingosine (Fig. 9C); similarly, no effect of sphingosine on chemotaxis or degranulation was observed (Fig. 9, D and E). SPP (10 µM), by itself, did not induce degranulation or chemotaxis (Fig. 9, F and G, left), but it did marginally induce Ca2+ signals (Fig. 9H) and superoxide production (Fig. 9I, left). However, SPP showed a priming effect enhancing C5a-triggered Ca2+ signals (Fig. 9J), stimulation of the oxidative burst, chemotaxis, and degranulation (Fig. 9, F, G, and I, right).

Taken together, these results suggest that SPHK plays an important role in the immune-inflammatory pathologies triggered by anaphylatoxins and that SPHK1 is a key enzyme for C5a to trigger physiological responses in human neutrophils.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
At the onset of an inflammatory response, there is an acute elevation of circulating C5a that causes a number of rapid physiological responses, including up-regulation of cell adhesion molecules resulting in a the rapid adhesion of neutrophils to the vascular endothelium, cytokine and enzyme release, and extravasation (38, 39).

Sphingolipids (in particular, SPP) have been shown to play an important role in immune cell activation, including cytoskeletal changes and degranulation. Here we report for the first time that sphingosine kinase is rapidly activated by C5a in primary human neutrophils and in a human neutrophil model (differentiated HL-60 cells). Furthermore, by using the sphingosine kinase inhibitor DMS, we provide evidence suggesting that sphingosine kinase plays an important role in the physiological responses triggered by C5a. Our data show that the sphingosine kinase pathway is involved in C5a receptor-mediated Ca2+ mobilization in human neutrophils. Pretreatment of cells with the sphingosine kinase inhibitor DMS substantially inhibited the C5a-triggered calcium release from internal stores in a manner that was independent of the phospholipase C/IP3 pathway because the inhibitor did not have any effect on C5a-triggered IP3 generation. Previous studies have shown that DMS inhibits PKC activity (34, 36). Of interest, DMS did not inhibit PKC activation triggered by C5a and did not have any effect on calcium signals triggered by other stimuli (i.e., Fc{gamma}RII). We further investigated other physiological roles of the sphingosine kinase in the C5a signaling pathways; we studied the effects of the sphingosine kinase inhibitor on degranulation, chemotaxis, and the NADPH oxidative burst. There is a tight correlation between calcium increase and degranulation (enzyme release) in immune effector cells (23, 24, 26). Pretreatment of primary neutrophils or differentiated HL-60 with DMS strongly inhibited the C5a-triggered release of {beta}-glucoronidase from these cells. Of interest, the inhibition of C5a-triggered degranulation and calcium increase by DMS showed similar concentration dependence, strongly suggesting that inhibition of degranulation is due, to a major extent, to blockade of calcium mobilization. To confirm this, we pretreated the cells with the intracellular calcium chelator BAPTA-AM and showed that in cells pretreated with the calcium chelator, C5a could not trigger degranulation (data not shown).

Inflammatory responses are characterized by the accumulation of PMNs at an inflammatory site. Where local inflammation is triggered by infection, trauma, or immune complex deposition, C5a is likely to be an important chemokine. C5a has been shown to trigger chemotaxis in cell suspensions (40), and C5a-triggered cell migration has been used as a sensitive test for measuring the activation of the cell's internal motile apparatus (41). In the present study, DMS potently inhibited the C5a-induced chemotaxis of both primary neutrophils and differentiated HL-60 cells. Moreover, activation of the neutrophil NADPH oxidative burst, which is another important effector function of activated neutrophils, was also inhibited in cells pretreated with DMS. It has been suggested that DMS induces apoptosis in neutrophils (30–33). However, in our experimental setup, the concentration of DMS (10 µM) we used did not induce apoptosis in primary neutrophils or HL-60 differentiated cells.

To further dissect the role of sphingosine kinase(s) in the above-mentioned C5a-triggered events in phagocytic cells, we decided to investigate which particular SPHK isoform was playing a role in these events. We found that SPHK1 was involved in the above-mentioned effector function triggered by anaphylatoxin in the human neutrophil model. Thus, C5a stimulation resulted in rapid translocation of SPHK1 from the cytosol to the periphery of the cells. To clarify whether SPHK1 was indeed involved in the responses triggered by C5a (such as Ca2+ signals, degranulation, chemotaxis, and NADPH oxidase activity), we next used an antisense oligonucleotide against human SPHK1. We found that antisense knock-down of SPHK1 levels did indeed substantially inhibit the rise in Ca2+ release from intracellular stores, degranulation, chemotaxis, and the oxidative burst triggered by C5a; because antisense treatment cannot be carried out on primary neutrophils, due to their very short life in culture, all the antisense experiments could only be performed on the human neutrophil model (differentiated HL-60 cells).

Several studies have reported that sphingosine acts as an endogenous regulator of neutrophil functions (30–37). Sphingosine has been shown to inhibit the neutrophil oxidative burst and phagocytosis (36, 37) and to induce apoptosis in neutrophils and other cells (30–33). One interesting observation in this study is the fact that C5a stimulation decreases sphingosine levels and increases the formation of SPP (Fig. 8E); this would suggest a role for SPHK in removing a negative regulator (sphingosine) and generating a positive regulator (SPP), similar to the rheostat models proposed for mast cell activation (27). Thus, we studied the effects of exogenously added sphingosine and SPP on C5a-triggered responses. In agreement with a previous study on the role of these lipids in superoxide production triggered by fMLP on neutrophils (37), we found that sphingosine has a dual effect on C5a-stimulated NADPH oxidase activation (a priming effect at lower concentrations but a dose-dependent inhibitory effect at higher concentrations). C5a-triggered PKC activity was only reduced at a high concentration of sphingosine. However, C5a-triggered Ca2+ signals were not affected by sphingosine at all; similarly, no effect of sphingosine on chemotaxis or degranulation was observed. SPP, by itself, did not induce degranulation or chemotaxis, but it did marginally induce Ca2+ release from internal stores and the NADPH oxidative burst. On the other hand, SPP showed a priming effect enhancing the C5a-triggered responses.

Taken together, the data presented in this study demonstrate that sphingosine kinase plays a major role in C5a-triggered reactions and that blocking sphingosine kinase activity inhibits the proinflammatory activities triggered by C5a in human phagocytic cells.

Phagocytic cell infiltration and the release of proinflammatory mediators by neutrophils are universal components of a wide range of disease states including immune complex-mediated conditions such as nephritis (42), arthritis (43), and acute graft rejection (44). Agents that can inhibit neutrophil infiltration and/or the release of proinflammatory mediators from neutrophils will have wide therapeutic applications in the prevention and treatment of inflammatory diseases. The application of a sphingosine kinase inhibitor in disease states involving anaphylatoxin activation and the production of proinflammatory agents by phagocytic cells need to be further investigated as both preventive and therapeutic agents. The findings presented in this study open new ways to better understand the intracellular signaling cascades triggered by anaphylatoxins and point out new ways for the development and validation of novel targets as potential therapeutic candidates for treating inflammatory diseases.


   FOOTNOTES
 
* This work was supported by National Medical Research Council Grant R-185-000-052-213. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Physiology, Faculty of Medicine, National University of Singapore, 2 Medical Dr., MD9 #01-05, Singapore 117597. Tel.: 65-6874-1697; Fax: 65-6778-8161; E-mail: phsmraj{at}nus.edu.sg.

1 The 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. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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