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 References This article cites 56 articles, 25 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/173/3/1596.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 25, 2021 • No Triage! 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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 pa- thologies, 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 pj g 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 as gy y 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, sphin- gosine 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. Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 1597 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)