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Antisense Knockdown of Sphingosine Kinase 1 in Human Macrophages Inhibits C5a Receptor-Dependent Signal Transduction, Ca2+ Signals, Enzyme This information is current as Release, Cytokine Production, and of September 25, 2021. Chemotaxis Alirio J. Melendez and Farazeela Bte Mohd Ibrahim

J Immunol 2004; 173:1596-1603; ; Downloaded from doi: 10.4049/jimmunol.173.3.1596 http://www.jimmunol.org/content/173/3/1596

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Antisense Knockdown of Sphingosine Kinase 1 in Human Macrophages Inhibits C5a Receptor-Dependent Signal Transduction, Ca2؉ Signals, Enzyme Release, Cytokine Production, and Chemotaxis1

Alirio J. Melendez2 and Farazeela Bte Mohd Ibrahim

The anaphylatoxin C5a is produced following the activation of the complement system and is associated with a variety of pathologies, including septic shock and adult respiratory distress syndrome, and with immune complex-dependent diseases such as rheumatoid arthritis. C5a has been shown to regulate inflammatory functions by interacting with its receptor, C5aR, which belong to the rhodopsin family of seven-transmembrane GPCRs. However, the intracellular signaling pathways triggered by C5aR on immune-effector cells are not well understood. In this report we present data showing that, in human monocyte-derived macro- Downloaded from phages, C5aR uses the intracellular signaling molecule sphingosine kinase (SPHK)1 to trigger various physiological responses. Our data show that C5a rapidly stimulates the generation of sphingosine-1-phosphate, SPHK activity, and membrane translocation of SPHK1. Using an antisense oligonucleotide against SPHK1, we show that knockdown of SPHK1 abolishes the C5a-triggered 2؉ intracellular Ca signals, degranulation, cytokine generation, and chemotaxis. Our study shows for the first time that SPHK1 not g pj only plays a key role in the generation and release of proinflammatory mediators triggered by anaphylatoxins from human macrophages but is also involved in the process of immune cell motility, thus pointing out SPHK1 as a potential therapeutic target http://www.jimmunol.org/ for the treatment of inflammatory and autoimmune diseases. The Journal of Immunology, 2004, 173: 1596–1603.

ctivation of the complement cascade plays a key role in toxins in human immune-effector cells are still poorly understood. host defense. However, anaphylatoxins produced fol- The aim of our study was to dissect the intracellular signaling A lowing the activation of the complement system are as- pathways triggered by C5aR in human macrophages to better un- sociated with a variety of pathologies, including septic shock and derstand the C5a triggered responses in human macrophages. adult respiratory distress syndrome, and with immune complex- Recently, it has become clear that sphingolipids are sources of

dependent diseases such as rheumatoid arthritis (1–3). Anaphyla- important signaling molecules. Particularly, the sphingolipid me- by guest on September 25, 2021 toxins are generated by activation-induced cleavage of the third tabolites, ceramide and sphingosine-1-phosphate (S1P), have and fifth components of complement. C5a and, to a lesser extent, emerged as a new class of potent bioactive molecules, implicated C3a are mediators of proinflammatory and immunoregulatory ac- in a variety of cellular processes such as cell differentiation, apo- tivities (4). C5a is a 74 aa peptide shown to regulate inflammatory ptosis, and proliferation (16–19). Interest in S1P focused recently

functions by interacting with its receptor, C5aR, which belongs to on two distinct cellular actions of this lipid, namely its function y as gy the rhodopsin family of seven-transmembrane, G protein-coupled an extracellular ligand activating specific GPCRs and its role as an receptors (GPCRs)3 (5, 6). Anaphylatoxin receptors are present on intracellular second messenger (20). Several findings enforced the myeloid and nonmyeloid leukocyte populations, including granu- notion of S1P as an important intracellular second messenger. locytes, and monocytes and macrophages (7, 8), lymphocytes (9, First, activation of various plasma membrane receptors, such as the 10), and dendritic cells (DCs) (11, 12). One of the main functions platelet-derived growth factor receptor (21, 22), the Fc⑀RI and of anaphylatoxins is the recruitment of leukocytes to the sites of Fc␥RI Ag receptors (23–25), as well as the fMLP receptor (26), infection, inflammation, and trauma. It is well recorded that C5a is was found to rapidly increase intracellular S1P production through a potent chemoattractant for all C5aR-expressing cell types (13– the stimulation of the SPHK. Second, inhibition of SPHK stimu- 15), including the responses of macrophages to C5a (14, 15). How- lation strongly reduced or even prevented cellular events triggered ever, the intracellular signaling cascades triggered by anaphyla- by these receptors, such as receptor-stimulated DNA synthesis, Ca2ϩ mobilization, and vesicular trafficking (21–26). To our best knowledge this is the first study showing that C5a Department of Physiology, National University of Singapore, Singapore uses the intracellular signaling molecule SPHK1 as a key player in Received for publication March 19, 2004. Accepted for publication May 20, 2004. the physiological responses triggered by C5a in human macro- The costs of publication of this article were defrayed in part by the payment of page phages. In this study we show that C5aR activation rapidly stim- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ulates the production of S1P and SPHK activity. Moreover, knock- 1 This work was supported by a grant from the National Medical Research Council of ing down the expression levels of SPHK1, with a specific antisense Singapore (R-185-000-052-213). oligonucleotide, a number of the macrophage responses to C5a are 2ϩ 2 Address correspondence and reprint requests to Dr. Alirio J. Melendez, Department inhibited, such as the C5a-stimulated intracellular Ca signals, of Physiology, National University of Singapore, 2 Medical Drive, MD9 Room 01-05, degranulation, cytokine production as well as cell migration. Thus, 117597 Singapore. E-mail address: [email protected] our data suggest that SPHK1 is indeed a key player in the proin- 3 Abbreviations used in this paper: GPCR, G protein-coupled receptor; SPHK, sphingosine kinase; DC, dendritic cell; S1P, sphingosine-1-phosphate; PLC, phospholipase flammatory responses triggered by human monocyte-derived mac- C; PKC, protein kinase C; IP3, inositol 1,4,5-triphosphate; BisI, bisindolylmaleimide I. rophages stimulated by C5a.

Materials and Methods All chemicals and reagents unless stated otherwise were bought from Sig- ma-Aldrich (Singapore). Isolation of human primary monocytes and differentiation to macrophages Mononuclear cells were isolated from heparinized fasting venous blood by Ficoll-Hypaque centrifugation as previously described (27). The 20 ml of blood (anticoagulated with 10 U/ml heparin) was layered carefully on 15 ml of Ficoll-Hypaque gradient and centrifuged at 500 ϫ g, without brakes at room temperature for 30 min. The mixed mononuclear band was aspi- rated and the cells were washed three times in phenol red RPMI 1640 medium containing 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 2 mM glutamine and suspended in a known volume. Leukocyte count was performed on a Coulter counter and then cells were plated (5–7 ϫ 106 cells) in six-well culture plates in RPMI 1640 medium. Incubation was conducted at 37°C for2hin5%CO2/95% air, after which nonadherent cells were removed by washing the wells twice with RPMI 1640, and the remaining adherent cells were grown in the culture medium supplemented with 10% FCS and 2 mM glutamine. The medium was replaced every 2–3 days. The cells were used after 8 days of culture. Cell viability, determined by trypan blue exclusion, was Ͼ94% in all experiments. Downloaded from SPHK activity in whole macrophages and generation of S1P SPHK activity in intact cells was measured by assaying the amount of intracellular S1P generation following receptor activation as previously described (25, 28). Brieﬂy, cells were preincubated overnight in medium containing [3H]serine (2 ␮Ci/ml) to label cellular sphingolipids and free sphingosine pools. Following labeling, 2 ϫ 106 cells/sample were stimu- http://www.jimmunol.org/ lated by the addition of 5 nM 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. Standard S1P was applied with the samples, and the lipids were visualized using iodine vapors. Bands corresponding to S1P were excised from the plate and counted by liquid scintillation spectrom- etry. Results were calculated as a percentage of the total radioactivity in- corporated in the lipids. SPHK activity in cell extracts

Human macrophages, 2 ϫ 106 cells/sample, were stimulated by the addi- FIGURE 1. C5a triggers S1P generation and SPHK activity in human by guest on September 25, 2021 tion of 5 nM C5a. Following C5a stimulation the cells are lysed and cell macrophages. A, Cells were labeled with [3H]serine. Basal S1P generation extracts are assayed for SPHK activity. SPHK activity was measured as was 1.2 pmols S1P/2 ϫ 106 macrophages, expressed as 100% (macrophage previously described (24, 29). Briefly, the system is based upon the SPHK basal). S1P generation in the macrophages following C5a stimulation ␥ catalyzed transfer of the -phosphate group of ATP (using a mixture of (macrophageϩC5a) and S1P generation after C5a stimulation in macro- cold ATP and [␥-32P]ATP (1 ␮Ci/sample) to a specific substrate, the prod- phages pretreated with the SPHK inhibitor N,N-dimethylsphingosine, DMS ucts were separated by TLC on silica gel G60 (Whatman, Maidstone, U.K.) ϩ ϩ Ϯ and visualized by autoradiography. The radioactive spots corresponding to (macrophage C5a DMS) are shown. Results are the mean SD of trip- S1P were scraped and counted in a scintillation counter. licate measurements and from three separate experiments. B, SPHK activity in human macrophages. Basal SPHK activity from human macrophages Phospholipase C (PLC) activity (macrophage basal), SPHK activity from the macrophages following C5a stimulation (macrophageϩC5a), and SPHK activity following C5a stimu- Human macrophages, 2 ϫ 106 cells/sample, were stimulated by the addition of 5 nM C5a. Following stimulation, inositol 1,4,5-triphosphate (IP ) lation from macrophages pretreated with the SPHK inhibitor N,N-dimeth- 3 ϩ ϩ was measured as previously described (24), using the BIOTRAK TRK ylsphingosine (macrophage C5a DMS) are shown. Results are the 1000 kit (Amersham Biosciences, Buckinghamshire, U.K.). mean Ϯ SD of triplicate measurements and from three separate experiments. Cytosolic calcium measurement Cytosolic calcium was measured as previously described (24). Briefly, 2 ϫ 6 10 cells/ml were loaded with 1 ␮g/ml fura 2-AM (Molecular Probes, Degranulation and enzyme release Leiden, The Netherlands) in PBS, 1.5 mM Ca2ϩ and 1% BSA. After re- moval of excess reagents by dilution and centrifugation, the cells were ␤-Hexosaminidase is released from macrophages. Degranulation was mea- resuspended in 1.5 mM Ca2ϩ supplemented PBS and warmed to 37°C in sured using a previously described colorimetric assay to assess the release the cuvette. A total of 2 ϫ 105 cells/sample were stimulated by the addition of ␤-hexosaminidase (24). Briefly, 2 ϫ 105 cells/sample were stimulated of 5 nM C5a. Fluorescence was measured at 340 and 380 nm and the by the addition of 5 nM C5a for 30 min. Following stimulation, 50 ␮lof background-corrected 340:380 ratio was calibrated as previously the sample supernatant was incubated with 200 ␮lof1mMp-nitrophenyl described (24). N-acetyl-␤-D-glucosaminide for1hat37°C.Thetotal ␤-hexosaminidase concentration was determined by a 1/1 extraction of the remaining buffer Protein kinase C (PKC) activity and cells with 1% Triton X-100; a 50 ␮l aliquot was removed and analyzed as described. The enzyme concentration was determined by measuring the PKC enzyme activity was measured as previously stated (30, 31), using the OD at 400 nm. Biotrak Protein Kinase C enzyme assay system (Amersham Biosciences). Briefly, the system is based upon the PKC catalyzed transfer of the ␥-phos- Cytokine detection phate group of ATP (using a mixture of cold ATP and [␥-32P]ATP; 1 ␮Ci/sample) to a peptide substrate specific for PKC. A total of 2 ϫ 106 A total of 2 ϫ 106 cells/sample, pretreated or not with antisense oligos, cells/sample were stimulated by the addition of 5 nM C5a for 15 min. were stimulated by the addition of 5 nM C5a for 24 h. Following stimu- Following stimulation, PKC assays were conducted. Results are expressed lation, the supernatants were collected at the indicated time points and as phosphorylation rate per picomolar of protein per minute. stored at Ϫ20°C until use. TNF-␣, IL-6, and IL-8 levels in the supernatants 1598 ROLE OF SPHK1 IN C5AR SIGNALING Downloaded from jg pj http://www.jimmunol.org/

FIGURE 2. SPHK1 expression, subcellular localization, and antisense (a.s.) knockdown in the human monocyte-derived macrophages. A, RT-PCR by guest on September 25, 2021 analysis of mRNA expression levels for both SPHK1 and SPHK2 in the human monocyte-derived macrophages (Macrophage), and in a positive control for both isoforms, the human neuronal cell line SKNMC. Results are representative of at least three separate experiments. B, Fluorescent microscopy of cells immune-stained for SPHK1. Resting cells (Macrophage resting) and cells after stimulation with C5a for 2 min (MacrophageϩC5a) are shown. Results are representative of three separate experiments. C, Western blot analysis of SPHK1 expression before and after antisense treatment are represented by SPHK1 expression levels in untreated monocyte-derived macrophages (Macrophage control), SPHK1 expression levels in cells pretreated for 48 h with the antisense against SPHK1 (Macrophage a.s.SPHK1), and SPHK1 expression levels in cells pretreated for 48 h with the scrambled antisense (Macrophage y gy a.s.scrambled). The blot was simultaneously probed with an anti-Arf1 Ab to show equal loading for all the lanes. Results are representative of three separate experiments. D, Effect of the SPHK1 antisense on S1P generation following C5a stimulation. Cells were labeled with [3H]serine. Basal S1P generation was 1.2 pmols S1P/2 ϫ 106. S1P generation in untreated macrophages following C5a stimulation (MacrophageϩC5a), S1P generation after C5a stimulation in macrophages pretreated with the SPHK1 antisense (MacrophageϩC5aϩa.s.SPHK1), and S1P generation after C5a stimulation in macrophages pretreated with the scrambled antisense (MacrophageϩC5aϩa.s.scrambled) are shown. Results are the mean Ϯ SD of triplicate measurements and from three separate experiments. E, Effects of the SPHK1 antisense on the C5a stimulated SPHK activity in the macrophages. SPHK activity from untreated macrophages following C5a stimulation (MacrophageϩC5a), SPHK activity following C5a stimulation from macrophages pretreated with the SPHK1 antisense (MacrophageϩC5aϩa.s.SPHK1), and SPHK activity following C5a stimulation from macrophages pretreated with the scrambled antisense (MacrophageϩC5aϩa.s.scrambled) are shown. Results are the mean Ϯ SD of triplicate measurements and from three separate experiments.

were evaluated using ELISA (R&D Systems, Minneapolis, MN) following CGGGCAGTGG CAGTCCTCGG-3Ј) primers were designed for the hu- the manufacturer’s instructions. man SPHK2 to yield a 356 bp fragment. Chemotaxis assay Western blots ␮ Chemotaxis was assayed using the Chemicon QCM Chemotaxis 3 m ␮ 96-well Cell Migration Assay kit (catalogue no. ECM 515; Temecula, CA) Unless stated other ways, 40 g of lysate for each sample was resolved on 12% polyacrylamide gels (SDS-PAGE) under denaturing conditions and following manufacturers instructions. A total of 2 ϫ 105 cells were used then transferred to 0.45-␮m nitrocellulose membranes. After blocking per sample and the concentration of C5a used for the migration assays was overnight at 4°C with 5% nonfat milk in TBS, 0.1% Tween 20 and wash- 5 nM. ing, the membranes were incubated with the relevant Abs for4hatroom RT-PCR analysis temperature. The membranes were washed extensively in TBS/0.1% Tween 20 (washing buffer). The blots were probed using specific, anti- mRNA from BMMC was isolated using the Qiagen midi kit for mRNA SPHK1 polyclonal (made in house as previously described (21)), and a extraction. Specific forward (5Ј-TGAACCCGCGCGGCAAGGGC-3Ј) and monoclonal anti-Arf1 (Santa Cruz Biotechnologies, Santa Cruz, CA), pri- reverse (5Ј-GGTCAGCCGGCGCCATCCACG-3Ј) primers were designed mary Abs. Bands were visualized using anti-rabbit HRP-conjugated, or for the human SPHK1 to yield a 570 bp fragment, and specific forward anti-mouse HRP-conjugated, secondary Abs, and the ECL Western Blot- (5Ј-CCATGGCGAG TTTGGCTCCT-3Ј) and reverse (5Ј-CATCCC ting Detection System (Amersham Biosciences). The Journal of Immunology 1599

Fluorescent microscopy engagement it rapidly translocates to the cell periphery (Fig. 2B, top After stimulation with 5 nM C5a, cells were fixed in 4% paraformaldehyde ). and deposited on microscope slides in a cytospin centrifuge, then perme- We pretreated the cells with an antisense oligonucleotides spe- abilized for 5 min in 0.1% Triton X-100 in PBS. Fluorescent labeling was cific for SPHK1, and found that the antisense knocks down SPHK1 performed using an anti-SPHK1 polyclonal Ab made by us as previously levels by ϳ80% as shown by Western blot analysis (Fig. 2C), described (21). Staining was analyzed by fluorescence microscopy using a whereas an antisense control (scrambled oligo) had no effect on the Leica DM IRB microscope, and images captured using a Leica DC 300F camera (Leica Microsystems, Bannockburn, IL). SPHK1 levels (Fig. 2C). In cells pretreated with the antisense oligo against SPHK1, C5a- Antisense knockdown of SPHK 1 stimulated formation of S1P was substantially inhibited (Fig. 2D). The antisense down-regulation of SPHK1 was conducted as previously Direct measurement of SPHK activity was also inhibited in cells described (24). Antisense oligonucleotides were purchased from Oswell pretreated with the antisense against SPHK1 (Fig. 2E). However, DNA Services (Southampton, U.K.); 20-mers were synthesized, capped at pretreatment of the cells with a scrambled antisense oligonucleo- either end by the phosphorothiorate linkages (first two and last two link- tide had no effect on S1P generation (Fig. 2D), nor on SPHK ages), and corresponded to the reverse complement of the first 20 coding nucleotides for SPHK1, and a scrambled oligo for control. The sequences enzyme activity (Fig. 2E). of the oligonucleotides were: 5Ј-CCCGCAGGATCCATAACCTC-3Ј an- These data show that the C5aR is capable of stimulating SPHK tisense for SPHK1; 5Ј-CTGGTGGAAGAAGAGGACGT-3Ј scrambled activity and S1P generation by specifically activating SPHK1 antisense for control. Cells were cultured in the presence of antisense for 48 h. Role of SPHK1 on C5a-triggered Ca2ϩ signals 2ϩ Results Previous studies have demonstrated that SPHK mediates Ca sig- Downloaded from nals for several plasma membrane receptors (20–23), and because C5a stimulates SPHK activity our results showed that C5a triggers SPHK activity in human mac- We first investigated whether the C5aR would generate S1P and rophages (Figs. 1 and 2), we decided to investigate the role of activate SPHK activity in the human macrophages. For this model, SPHK1 in the C5a-triggered Ca2ϩ mobilization in these cells. C5a formation of [3H]S1P from [3H]sphingosine was first determined. stimulation rapidly and transiently triggered calcium release from Activation of the C5aR induced a rapid and transient increase in internal stores (Fig. 3A). However, in cells pretreated with the http://www.jimmunol.org/ [3H]S1P generation (Fig. 1A), reaching its maximal [3H]S1P gen- antisense against SPHK1, these Ca2ϩ signals were inhibited (Fig. eration at 2 min. Parallel to the generation of S1P, direct measure- 3A). Pretreatment of the cells with the scrambled antisense oligo- ment of SPHK activity showed that the enzyme is activated fol- nucleotide had no effect on the C5a-triggered Ca2ϩ signals (Fig. 3A). lowing C5aR engagement (Fig. 1B). The inhibitory action of antisense on Ca2ϩ signals was not due to inhibition of PLC stimulation. Preincubation of cells with the SPHK1 is the enzyme activated by C5a antisense knockdown of antisense against SPHK1 neither altered basal nor inhibited the SPHK1 C5a triggered generation of IP3 (Fig. 3B). Two human SPHKs have recently been cloned and characterized, namely SPHK1 (32) and SPHK2 (33). First, the mRNA presence Role of SPHK1 on C5a-triggered physiological responses by guest on September 25, 2021 of specific SPHK isozymes present in the cells was examined; only Following the role of SPHK1 on triggering Ca2ϩ signals, we in- the messenger for SPHK1 was found by RT-PCR in the human vestigated the role of SPHK1 in other well-characterized func- macrophages, and SKNMC were used as control (Fig. 2A). Fluo- tional responses of macrophages following cellular activation by rescent microscopy confirmed the presence of SPHK1, and showed C5a, namely PKC activity, degranulation, cytokine release, and that in resting cells SPHK1 is primarily cytosolic, but after C5aR chemotaxis.

FIGURE 3. C5a-triggered cytosolic Ca2ϩ signals are inhibited by N,N-dimethylsphingosine showing a role for SPHK. A, Cytosolic Ca2ϩ triggered by C5a stimulation in the monocyte-derived macrophages, time course control (Cells untreated); cytosolic Ca2ϩ triggered by C5a in cells pretreated with the SPHK1 antisense (Cellsϩa.s.SPHK1); and cytosolic Ca2ϩ triggered by C5a in cells pretreated with the scrambled antisense (Cellsϩa.s.scrambled) are shown. Results are representative of three separate experiments. B, C5a-triggered PLC activity is not inhibited by the SPHK1 antisense. Basal IP3 generation ϩ in untreated macrophages (Cells basal), IP3 generation after C5a stimulation in the untreated macrophages (Cells C5a), IP3 generation after C5a stimulation ϩ ϩ in macrophages pretreated with the SPHK1 antisense (Cells C5a a.s.SPHK1), and IP3 generation after C5a stimulation in macrophages pretreated with the scrambled antisense (CellsϩC5aϩa.s.scrambled) are shown. Results are the mean Ϯ SD of triplicate measurements and of three separate experiments. 1600 ROLE OF SPHK1 IN C5AR SIGNALING

C5a-triggered PKC activity. PKC activity is important for many cellular responses, and most receptor trigger PKC activity. C5a triggered a fast increase of PKC enzyme activity in the macrophages (Fig. 4). Pretreatment with the antisense oligo against SPHK1 did not affect the PKC activity triggered by C5a, however, pretreatment with the PKC inhibitor, bisindolylmaleimide (Bis)I, indeed inhibited the PKC activity triggered by C5a (Fig. 4). This shows that SPHK1 does not play a role on the C5a-triggered activation of PKCs. C5a-triggered degranulation. Enzyme release (degranulation) is an important effector function of phagocytic cells and has been shown to require Ca2ϩ signals. Therefore we investigated whether SPHK1 plays any role on the C5a-triggered degranulation. In contrast to PLC and PKC activities, C5a-stimulated degranulation was strongly inhibited in cells pretreated with the SPHK1 antisense oligo. C5a very rapidly triggered the release of ␤-hexosaminidase from the human macrophages (Fig. 5). Pretreatment of cells with the antisense oligo against SPHK1, substantially inhibited the C5a triggered enzyme release, whereas pretreatment with the scram- Downloaded from bled antisense oligonucleotide had no effect on degranulation (Fig. 5). These results suggest a role for SPHK1 in regulating C5a- triggered degranulation. C5a-triggered chemotaxis. One of the most important functions FIGURE 5. Degranulation triggered by C5a is dependent on SPHK activity. ␤-Hexosaminidase release from macrophages is shown as ␤-hex- of anaphylatoxins is their role as chemoattractants, therefore, we osaminidase release from resting cells, control (Basal Control); ␤-hex- http://www.jimmunol.org/ decided to ﬁnd whether SPHK1 plays any role in chemotaxis. C5a osaminidase release after C5a stimulation control (C5a Control); (5 nM) triggered a strong chemotactic response on the human mac- ␤-hexosaminidase release from resting cells pretreated with the SPHK1 rophages (Fig. 6). However, pretreatment of cells with the anti- antisense (Basalϩa.s.SPHK1); ␤-hexosaminidase release after C5a stimu- sense SPHK1 oligo substantially inhibited the C5a-triggered che- lation in cells pretreated with the SPHK1 antisense (C5aϩa.s.SPHK1); and motaxis, whereas pretreatment of the cells with the scrambled ␤-hexosaminidase release after C5a stimulation in cells pretreated with the antisense oligonucleotides did not altered the response (Fig. 6). scrambled antisense (C5aϩa.s.scrambled). Results are the mean Ϯ SD of This suggests a speciﬁc role for SPHK1 in the C5a-triggered mac- triplicate measurements and of three separate experiments. rophage migration.

C5a-triggered cytokine production. Another important role for and IL-8 (Fig. 7C) was inhibited. In contrast pretreatment with the by guest on September 25, 2021 anaphylatoxins is to promote the release of proinﬂammatory cy- scramble antisense had no inhibitory effect on cytokine production, tokines and chemokines. In this model we show that C5a stimu- suggesting that SPHK1 mediates the C5a-triggered cytokine pro- ␣ lates TNF- and IL6 and IL8 release from the human macrophages duction on human macrophages. (Fig. 7). However, in cells pretreated with the ansisense SPHK1 Taking these results together, our data indicates that SPHK1 ␣ oligo, C5a-triggered release of TNF- (Fig. 7A), IL-6 (Fig. 7B), plays a key role on the C5a-triggered proinﬂammatory responses of human macrophages.

Discussion Activation of C5aR triggers a number of rapid proinflammatory responses, including up-regulation of cell adhesion molecules, cytokine and enzyme release, and leukocyte migration (34, 35). Re- cently, C5a has been shown to have an immunoregulatory role able to stimulate mediators of both acute and chronic inflammation (36–39). 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 (40– 44). Most of these studies used blocking Abs raised against C5a (41, 43) or recombinant proteins that are receptor antagonists or analogues of C5a (40, 44). 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 man- FIGURE 4. C5a-triggered PKC activity in the monocyte-derived mac- ufacture, very susceptible to degradation by proteases in serum or rophages is not inhibited by the SPHK1 antisense (a.s.). Basal PKC activity the gastrointestinal track, and generally display poor pharmacoki- u in untreated cells (Cells control, ); PKC activity after C5a stimulation in netic properties. More recently attempts have been made to make untreated cells (Cells control, f); basal PKC activity in the cells pretreated smaller molecules that are more stable, cheaper to make, have with the SPHK1 antisense (Cellsϩa.s.SPHK1, u); PKC activity after C5a stimulation cells pretreated with the SPHK1 antisense (Cellsϩa.s.SPHK1, better bioavailability, and are more attractive as drug candidates f); basal PKC activity in cells pretreated with the PKC inhibitor BisI for treating human diseases mediated by C5a (45, 46). However, (CellsϩBisI, u); and PKC activity after C5a stimulation in cells pretreated very little is known about the intracellular signaling pathways with the PKC inhibitor BisI (CellsϩBisI, f) are shown. Results are the activated by C5a in immune-effector cells, such as human mean Ϯ SD of triplicate measurements and of three separate experiments. macrophages. The Journal of Immunology 1601

FIGURE 6. C5a-induced chemotaxis is inhibited in macrophages pretreated with the SPHK1 antisense (a.s.). Migratory number of cells shown Downloaded from include basal migration of untreated cells (Basal control); migration of cells pretreated with the SPHK1 antisense (Basalϩa.s.SPHK1); migration of untreated cells toward the C5a containing chamber (C5a control); migration toward the C5a containing chamber of cells pretreated with the SPHK1 antisense (C5aϩa.s.SPHK1); and migration toward the C5a containing chamber of cells pretreated with the scrambled antisense ϩ Ϯ (C5a a.s.scrambled). Results are the mean the SD of triplicate mea- http://www.jimmunol.org/ surements and of three separate experiments.

S1P has been shown to play an important role in immune cell activation including cytoskeletal changes and degranulation. We FIGURE 7. TNF-␣, IL-6, and IL-8 release triggered by C5a is inhibited report for the ﬁrst time that SPHK1 is rapidly activated by C5a in in macrophages pretreated with the SPHK1 antisense (a.s.). A, Basal the human macrophages. Our data shows that SPHK1 is involved TNF-␣ release from untreated cells (Basal); TNF-␣ release from untreated ϩ in the C5aR-mediated Ca2 mobilization in human macrophages. cells after C5a stimulation (C5a control); TNF-␣ release after C5a stimu-

Furthermore, knocking down SPHK1 levels, with the specific lation in cells pretreated with the SPHK1 antisense (C5aϩa.s.SPHK1); and by guest on September 25, 2021 SPHK1 antisense oligonucleotides, substantially inhibited the TNF-␣ release after C5a stimulation in cells pretreated with the scrambled ϩ Ϯ C5a-triggered calcium release from internal stores, in a manner antisense (C5a a.s.SPHK1) are shown. Results are the mean SD of that was independent of the PLC/IP pathway because the anti- triplicate measurements and of three separate experiments. B, Basal IL-6 3 release from untreated cells (Basal); IL-6 release from untreated cells after sense did not have any effect on the C5a-triggered IP generation. 3 C5a stimulation (C5a control); IL-6 release after C5a stimulation in cells To further investigate the physiological roles of the SPHK1 in pretreated with the SPHK1 antisense (C5aϩa.s.SPHK1); and IL-6 release the C5a signaling pathways, we studied the effects of the antisense after C5a stimulation in cells pretreated with the scrambled antisense knockdown of SPHK1 on PKC enzyme activity, degranulation, (C5aϩa.s.SPHK1). Results are the mean Ϯ SD of triplicate measurements cytokine production, and chemotaxis. Many physiological re- and of three separate experiments. C, Basal IL-8 release from untreated sponses triggered in phagocytic cells are dependent on the activa- cells (Basal); IL-8 release from untreated cells after C5a stimulation (C5a tion of PKC isoforms. Treatment of the macrophages with the control); IL-8 release after C5a stimulation in cells pretreated with the antisense oligonucleotides against SPHK1, which caused a marked SPHK1 antisense (C5aϩa.s.SPHK1); and IL-8 release after C5a stimula- ϩ ϩ inhibition on the C5a-induced intracellular Ca2 concentration tion in cells pretreated with the scrambled antisense (C5a a.s.SPHK1) are Ϯ signals, had no effect on the C5a-triggered stimulation of protein shown. Results are the mean SD of triplicate measurements and of three separate experiments. kinase activity, whereas the PKC inhibitor BisI did indeed inhibit PKC activation. These data suggest that SPHK is not involved in the signaling pathway to PKC activation. However, there is a tight correlation between increase of intracellular calcium and degran- an inflammatory site. Where local inflammation is triggered by ulation (23, 24, 26). Pretreatment of human macrophages with the infection, trauma, or immune-complex deposition, C5a is likely to antisense against SPHK1 strongly inhibited the C5a-triggered re- be an important chemokine. C5a has been shown to trigger che- lease of ␤-hexosaminidase from the macrophages, indicating the motaxis in cell suspensions (48), and C5a-triggered cell migration requirement for SPHK1 in this response. has been used as a sensitive test for measuring the activation of the It is well established that C5a can trigger proinflammatory cy- cell’s internal motile apparatus (49). In the present study we show tokines and chemokines, such as TNF-␣, IL-6, and IL-8 production that C5a is capable of triggering macrophage migration, and that (36–39). These molecules share many activities, including the pretreatment of cells with the antisense oligonucleotides against ability to induce fever and shock syndrome in animal models (47). SPHK1 strongly inhibited the C5a-induced chemotaxis. In this report we show that C5a triggered the generation of TNF-␣, SPHK activity also plays a potential role in the physiological IL-6, and IL-8 to different levels. Moreover, pretreatment of cells responses triggered on other immune-effector cells. Activated neu- with antisense against SPHK1 substantially inhibited the produc- trophils generate a broad and a vigorous set of alterations in gene tion of TNF-␣, IL-6, and IL-8 triggered by C5a. Inflammatory expression shaping the inflammatory response (50). It has been responses are characterized by the accumulation of macrophages at proposed that SPHK regulates neutrophil priming to provide an 1602 ROLE OF SPHK1 IN C5AR SIGNALING essential defense against infections (51), and in neutrophil inflam- 8. Zwirner, J., O. Go¨tze, G. Begemann, A. Kapp, K. Kirchhoff, and T. Werfel. 1999. matory responses (26, 51). The recruitment of circulating neutro- Evaluation of C3a receptor expression on human leucocytes by the use of novel monoclonal antibodies. Immunology 97:166. phils to damaged tissue plays a critical role in a number of phys- 9. Nataf, S., N. Davoust, R. S. Ames, and S. R. Barnum. 1999. Human T cells iological and pathophysiological events: circulating neutrophils express the C5a receptor and are chemoattracted to C5a. J. Immunol. 162:4018. attach to the activated endothelial cells in blood vessels through 10. Werfel, T., K. Kirchhoff, M. Wittmann, G. Begemann, A. Kapp, F. Heidenreich, O. Gotze, and J. Zwirner. 2000. Activated human T lymphocytes express a func- various adhesion molecules and then migrate through the endothe- tional C3a receptor. J. Immunol. 165:6599. lial cell layer into the injured sites; and SPHK activity has also 11. Kirchhoff, K., O. Weinmann, J. Zwirner, G. Begemann, O. Go¨tze, A. Kapp, and ϩ been shown to modulate the expression of vascular cell adhesion T. 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antagonists of the receptor for the complement factor C5a. J. M. Crawford, and J. L. Ferrara. 1999. Differential roles of IL-1 and TNF- on http://www.jimmunol.org/ 42:1965. graft-versus-host disease and graft versus leukemia. J. Clin. Invest. 104:459. by guest on September 25, 2021 The Journal of Immunology

Letter of Retraction

The editors hereby retract the article titled “Antisense Knockdown of Sphingosine Kinase 1 in Human Macrophages Inhibits C5a Receptor-Dependent Signal Transduction, Ca21 Signals, Enzyme Release, Cytokine Production, and Chemotaxis” by Alirio J. Melendez and Farazeela Bte Mohd Ibrahim, The Journal of Immunology, 2004, 173: 1596–1603. An investigation by the National University of Singapore concluded that Fig. 2A and 2C display irregular backgrounds that suggest the bands were improperly inserted. The investigation also concluded that Fig. 2A was duplicated in Zhi et al., Journal of Cellular Physiology, 2006, 208: 109–115, which has been retracted by Dr. Gary S. Stein, the Editor-in-Chief of that journal. The Investigative Committee of the National University of Singapore concluded that Dr. Melendez committed serious scientiﬁc misconduct. The committee found no evidence indicating that other coauthors were involved in the scientiﬁc misconduct.

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