1 Is a Negative Regulator of CD4 + Th1 Cells Jianfei Yang, Brian E. Castle, Adedayo Hanidu, Lisa Stevens, Yang Yu, Xiang Li, Carol Stearns, Vladimir Papov, This information is current as Daniel Rajotte and Jun Li of September 25, 2021. J Immunol 2005; 175:6580-6588; ; doi: 10.4049/jimmunol.175.10.6580 http://www.jimmunol.org/content/175/10/6580 Downloaded from

References This article cites 47 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/175/10/6580.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

by guest on September 25, 2021 *average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Sphingosine Kinase 1 Is a Negative Regulator of CD4؉ Th1 Cells

Jianfei Yang,1 Brian E. Castle, Adedayo Hanidu, Lisa Stevens, Yang Yu, Xiang Li, Carol Stearns, Vladimir Papov, Daniel Rajotte, and Jun Li

CD4؉ Th1 cells produce IFN-␥, TNF-␣, and IL-2. These Th1 cytokines play critical roles in both protective immunity and inflammatory responses. In this study we report that 1 (SPHK1), but not SPHK2, is highly expressed in DO11.10 Th1 cells. The expression of SPHK1 in Th1 cells requires TCR signaling and new protein synthesis. SPHK1 phosphor- ylates sphingosine to form sphingosine-1-phosphate. Sphingosine-1-phosphate plays important roles in inhibition of apoptosis, promotion of cell proliferation, cell migration, calcium mobilization, and activation of ERK1/2. When SPHK1 expression was knocked down by SPHK1 short interfering RNA, the production of IL-2, TNF-␣, and IFN-␥ by Th1 cells in response to TCR stimulation was enhanced. Consistently, overexpression of dominant-negative SPHK1 increased the production of IL-2, TNF-␣, and IFN-␥ in Th1 cells. Furthermore, overexpression of SPHK1 in Th1 and Th0 cells decreased the expression of IL-2, TNF-␣, Downloaded from and IFN-␥. Several chemokines, including Th2 chemokines CCL17 and CCL22, were up-regulated by SPHK1 short interfering RNA and down-regulated by overexpression of SPHK1. We also showed that Th2 cells themselves express CCL17 and CCL22. Finally, we conclude that SPHK1 negatively regulates the inflammatory responses of Th1 cells by inhibiting the production of proinflammatory cytokines and chemokines. The Journal of Immunology, 2005, 175: 6580–6588.

aive CD4ϩ T cells differentiate into two distinct subsets, receptors (8). Intracellular S1P regulates several important pathways, http://www.jimmunol.org/ Th1 and Th2, depending on their environment and the as- such as inhibition of apoptosis, promotion of cell proliferation, cal- N sociated differentiation signals. Th1 cells are characterized cium mobilization, and activation of ERK1/2 (8, 9). IgE Ag stimula- by their ability to produce IFN-␥, but not IL-4, whereas Th2 cells tion of human bone marrow-derived mast cells triggers SPHK1-me- produce IL-4, but not IFN-␥ (1, 2). Th1 cells also produce more diated fast and transient Ca2ϩ release from intracellular stores, which TNF-␣ and IL-2 than Th2 cells. Th1 cells play critical roles in cell- depends primarily on the activation of both phospholipase D1 and mediated immune response, whereas Th2 cells are involved in hu- SPHK1 (10). In L929 cells, TNF-␣-induced cyclooxygenase-2 and ϩ moral immunity. Overactivation of CD4 Th1 and Th2 cells may PGE2 expression is significantly reduced by SPHK1 short-interfering

induce autoimmune and inflammatory diseases. For example, Th1 RNA (siRNA) (11). Although SPHK2 is known to associate with the by guest on September 25, 2021 cells are involved in inflammatory bowel disease, arthritis, type I di- mouse IL-12R␤1 cytoplasmic region and augment IL-12-induced abetes, and myocarditis. Crohn’s disease, one of the inflammatory STAT4-mediated transcriptional activation (12), the function of bowel diseases, is driven by the production of IL-12 and IFN-␥ (3, 4). SPHK1 in CD4ϩ T cells is still poorly understood. Mice lacking IFN-␥Rs were highly susceptible to collagen-induced The immunomodulatory drug FTY720 is phosphorylated by arthritis (5). TNF-␣ is the major cytokine in chronic destructive ar- SPHKs (13). The phosphorylated FTY720 is a partially selective thritis. Anti-TNF-␣ treatment shows great efficacy in rheumatoid ar- S1P analog (14). It binds and activates four of five S1P receptors thritis patients (6). TNF-␣ is up-regulated in the mucosa of patients (15, 16). FTY720 inhibits lymphocyte emigration from lymphoid with inflammatory disease, and anti-TNF-␣ Abs have shown marked organs (16). Graeler et al. (17) showed that S1P enhanced chemo- clinical benefit in the majority of patients with Crohn’s disease (7). taxis of CD4ϩ T cells to CCL-21 and CCL-5 by up to 2.5-fold at Thus, inhibition of the above cytokines is critical for the treatment of 10 nM to 0.1 ␮M, whereas 0.3–3 ␮M S1P inhibits this chemotaxis autoimmune and inflammatory diseases. by up to 70%. They also showed that S1P decreased CD4ϩ T cell There are two known sphingosine (SPHK)2: SPHK1 and generation of IFN-␥ and IL-4, without affecting IL-2. Meanwhile, SPHK2. SPHK phosphorylates sphingosine to form sphingosine-1- S1P inhibited chemotaxis to CCL-5 and CCL-21 (18). Thus, un- phosphate (S1P). S1P is a bioactive lipid mediator that can function as derstanding the regulation of S1P production and downstream ac- an intracellular second messenger as well as a ligand of cell surface tivity in CD4ϩ T cells is crucial. In this study we show that SPHK1 is highly expressed in Th1 cells. Overexpression of SPHK1 in these cells leads to decreased R&D Center, Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT 06877 expression of IL-2, TNF-␣, IFN-␥, CCL17, and CCL22, whereas Received for publication December 29, 2004. Accepted for publication September knocking down of SPHK1 by SPHK1 siRNA shows increased in- 7, 2005. duction of IL-2, TNF-␣, IFN-␥, CCL17, and CCL22 by TCR stim- 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 ulation. Thus, SPHK1 plays an inhibitory function in the proin- with 18 U.S.C. Section 1734 solely to indicate this fact. flammatory responses of Th1 cells. 1 Address correspondence and reprint requests to Dr. Jianfei Yang, Department of Immunology and Inflammation, Boehringer Ingelheim Pharmaceuticals, 900 Ridgebury Road, Ridgefield, CT 06877. E-mail address: [email protected] Materials and Methods ingelheim.com Mice, reagents, cytokines, and Abs 2 Abbreviations used in this paper: SPHK, sphingosine kinase; CBA, cytometric bead ␣␤ array; m, murine; GFP-RV, GFP retrovirus; siRNA, short-interfering RNA; S1P, DO11.10 -TCR transgenic mice have been described previously (19); sphingosine-1-phosphate; MGB, minor groove binder; GADD, growth arrest and they were maintained in our pathogen-free facility. Female BALB/c mice DNA damage-inducible. were bought from The Jackson Laboratory. Murine IL-4 and IL-12 were

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 6581 purchased from Calbiochem. Murine IL-2 and monoclonal anti-IL-4 expression of murine CD4 and GFP. Cells were then restimulated with (11B11) were obtained from eBioscience. Polyclonal anti-mouse IL-12 OVA and irradiated BALB/c splenocytes (2000 rad; 5 ϫ 106 cells/ml) for was purchased from Cell Sciences. Monoclonal anti-mouse CD3⑀ (500A2) another week. Finally, cells were counted, washed, suspended with 1 ϫ 106 and anti-mouse CD28 (37.51) were purchased from BD Biosciences. OVA, cells/ml, and then restimulated with plate-bound anti-CD3 for 2–18 h. cyclosporin A, PMA, ionomycin, and Histopaque-1119 were all obtained from Sigma-Aldrich. Cycloheximide, genistein, AG9 (tyrphostin A1), and Transfection of siRNA AG126 (tyrphostin AG 126) were purchased from Calbiochem. Human SPHK1-specific 21-nucleotide siRNA oligo (Dharmacon Research; Cell culture GAGCUGCAAGGCCUUGCCCdTdT and GGGCAAGGCCUUGCAG CUCdTdT) targets 70 nt downstream of the start codon as described pre- Differentiation of DO11.10 T cells to Th1 and Th2 phenotypes and infec- viously (23–25). These previous studies also showed that the above SPHK1 tion by retrovirus were conducted as previously described (20, 21). Briefly, siRNA specifically inhibits SPHK1 mRNA expression. Bioinformatics splenic T cells were activated by OVA (500 ␮g/ml) in 10% FBS/IMDM in study shows that the siRNA sequence is unique to SPHK1 mRNA. The the presence of IL-2 (2 ng/ml). IL-12 (5 ng/ml) and anti-IL-4 (11B11; 2 nonspecific control GL2 siRNA (5Ј-CGUACGCGGAAUACUUCGATT ␮g/ml) were added to the culture for Th1 development, whereas IL-4 (20 and TTGCAUGCGCCUUAUGAAGCU-5Ј) were described previously ng/ml) and anti-IL12 (1 ␮g/ml) were used for Th2 development. For the (26). T cells were harvested by centrifugation and resuspended at 3 ϫ 107 generation of Th0 cells, no additional cytokines or Abs were added to the cells/ml in Human T Cell kit buffer (VPA-1002; Amaxa), and 100 ␮l was culture. The T cells were passaged on a weekly basis by activation with dispensed to electroporation cuvettes containing 6 ␮l of siRNA and 1 ␮lof OVA and irradiated BALB/c splenocytes (2000 rad). For anti-CD3 stim- FITC-control siRNA (Invitrogen Life Technologies). The final concentra- ulation, plates were coated overnight at 4°C with 0.63–10 ␮g/ml anti-CD3 tion of siRNA was 100 nM. Cells were electroporated using the specific Ab (500A2) diluted in PBS. T cells were applied at 1 ϫ 106 cells/ml as program T23 by the Nucleofector device (Amaxa) and were incubated for indicated in the figure legends. For GeneChip experiments, T cells were 72 h in IMDM with hIL-2. Cells were then counted and placed into wells briefly spun down in anti-CD3-coated plates and stimulated with anti-CD3 at a concentration of 1 ϫ 106 cells/ml. Finally, the T cells were stimulated Downloaded from for2h. with anti-CD3 for 18 h before supernatants and cell pellets were harvested Human T cells were purified from peripheral blood donated by healthy for additional analysis. donors. RBC were separated by Ficoll-Paque centrifugation. The remain- ing cells were incubated according to label directions with StemSep Hu- Cytometric bead array (CBA) man Naive CD4ϩ T Cell Enrichment Cocktail (Stem Cell Technologies), ϩ ϩ Ϫ Ϫ The cytokine titers in the supernatants were analyzed by CBA (BD Bio- which purifies T cells that are CD4 , CD45RA , CD45RO , and CD8 by negative selection. The T cells were cultured in 10% FBS/IMDM. These sciences). Mouse or hTh1/Th2 cytokine CBA kits were used (BD Bio- sciences). Briefly, 25 ␮l of each sample or standard dilution was mixed naive T cells were then stimulated with plate-bound anti-CD3 and anti- ␮ ␮ http://www.jimmunol.org/ CD28 in the presence of 2.5 ng/ml human (h) IL-2 (BD Biosciences), 4 with 25 l of mixed capture beads and 25 l of the mouse Th1/Th2 PE ng/ml hIL-12 (Calbiochem), and 200 ng/ml anti-hIL-4 (eBioscience) at a detection reagent. After the samples were incubated at room temperature for2hinthedark, they were washed once and resuspended in 200 ␮lof concentration of 2 ϫ 106 cells/ml for 7 days. The cells were restimulated for another week before being transfected with siRNA. wash buffer before acquisition on a FACScan. Data were analyzed using the CBA software. The concentration of each cytokine in the supernatants Immunoblotting was calculated with the corresponding standard curve. Th1 and Th2 cells were stimulated with anti-CD3 for 18 h before being RNA extraction, TaqMan quantitative RT-PCR, and GeneChip harvested. Cells then were lysed by SDS loading buffer (187.5 mM Tris- analysis HCl (pH 6.8), 6% (w/v) SDS, 30% glycerol, 0.03% (w/v) bromphenol blue, Total RNA was extracted using RNeasy Mini kit (Qiagen). The possible and 42 mM DTT; New England Biolabs). One hundred microliters of the by guest on September 25, 2021 SDS loading buffer was used to lyse 1 ϫ 107 cells. The lysates were then remaining DNA was digested with the RNase-free DNase I (Qiagen) dur- boiled at 100°C for 5 min, centrifuged at 13,000 rpm, resolved by SDS- ing the extraction of total RNA. cDNA synthesis, TaqMan quantitative PAGE, and transferred to nitrocellulose membrane. The membrane was RT-PCR, and GeneChip analysis were described previously (27, 28). Taq- probed with rabbit polyclonal anti-SPHK1 (Exalpha Biologicals) and de- Man real-time RT-PCRs were performed using ABI 7700 to measure the veloped using an anti-rabbit HRP-conjugated Ab and SuperSignal West mRNA expression level. TaqMan signals were calculated as mRNA copy Pico Substrate (Pierce). The membrane was then striped and reprobed with numbers per sample input generated using a standard curve and normalized mouse anti-p38 MAPK (5F11) mAb (Cell Signaling) and developed using to endogenous GAPDH (28). The TaqMan signals provide relative quan- an anti-mouse-HRP-conjugated Ab. titation of expression within each experiment. TaqMan primers and probes are as follows: mSPHK1: mSPHK1-539 forward, CCATCCA Retroviral constructs, transfection, and infection GAAACCCCTGTGTA; mSPHK1-615 reverse, CCCAGCATAGTGGT TCACAGAA; and mSPHK1-563T-FAM minor groove binder (MGB) We obtained a mouse full-length SPHK1 cDNA clone from an established probe, TCCCTGGAGGCTCCGGCAATG; hSPHK: hSPHK1 forward, sequence tag IMAGE clone from OpenBiosystems. The SPHK1 cDNA CTTGCAGCTCTTC insert was generated by PCR using the IMAGE clone as a template. The CGGAGTC; SPHK1 reverse, GCTCAGTGAGCATCAGCGTG; and sequences of the primers for PCR are as follows: murine (m) SPHK1-1, SPHK1-FAM MGB probe, CCCTTTTGGCTGAGGCTGAAATCTCC; 5Ј-TAAGAAGCTGAACGCAGGAG; and mSPHK1-3, 5Ј-AAAGGCA mGAPDH: mGAPDH-864 forward, GCTACACTGAGGACCAGGTT CAGAGTTATGGTTC. The PCR products were then ligated into the GTCT; mGAPDH-980 reverse, ACCAGGAAATGAGCTTGACAAAGT; blunted BglII/XhoI site of internal ribosome entry site GFP retrovirus and mGAPDH-898T-FAM MGB probe, CAACAGCAACTCCCACTCT (GFP-RV) as described previously (20, 21). The final SPHK1-RV was TCCACCTTC; hGAPDH: hGAPDH forward, CCAGGTGGTCTCCTCT confirmed by sequencing. Changing hSPHK1 glycine 26 or glycine 80 to GACTTC; hGAPDH reverse, GTGGTCGTTGAGGGCAATG; and aspartic acid 26 or aspartic acid 80 by point mutation results in hGAPDH-FAM MGB probe, AGCGACACCCACTCCTCCACCTTTG. an catalytically inactive kinase (22). We designed the following oligonu- Human and mouse CCL17 and CCL22 and hSPHK1 TaqMan primers and cleotides to generate SPHK1 G26D- and G80D-RV: mSPHK1 G26D-F, probes were purchased from Applied Biosystems. 5Ј-GCTGCTGAACCCCCAGGGTGACAAGGGCAAGGCTCTGCAGC; mSPHK1G26D-R, 5Ј-GCTGCAGAGCCTTGCCCTTGTCACCCTGGGG Statistical analysis GTTCAGCAG; mSPHK1G80D-F, 5Ј-CGCCCTGGCAGTCATGTCCG ATGATGGTCTGATGCATGAGG; and mSPHK1G80D-R, 5Ј-CCTCATG For TaqMan and cytokine assays, statistical analysis was performed using CATCAGACCATCATCGGACATGACTGCCAGGGCG. The SPHK1-RV Student’s t test by Microsoft Excel. A value of p Ͻ 0.05 was considered was used as a template to generate SPHK1 G26D-RV and SPHK1 significant. G80D-RV using the QuikChange Site-Directed Mutagenesis kit (Strat- For Affymetrix GeneChip studies, levels of were quan- agene). Phoenix-Eco packaging cell line (licensed from Dr. G. Nolan, Stan- titated from the hybridization intensities of 11 pairs of perfectly matched ford University, CA) was transfected to generate the retroviral supernatants and mismatched control probes using the Affymetrix GeneChip Operating according to Dr. Nolan’s protocol, as described previously (21). T cells Software (GCOS) software. The software computes how the expression were activated as described above and infected 48 h later. The viral su- level of each transcript has changed between the baseline and experimental pernatant and 6 ␮g/ml polybrene (Sigma-Aldrich) were added to the T cells samples (change call). A change call describes whether a transcript in an in six-well plates and centrifuged at 1800 rpm for 30 min at room tem- experimental array has changed compared with a baseline array. Wilcox- perature. Cells were then cultured for 24 h before a 1:3 split. Infected T on’s signed rank is used to generate a change p value using hybridization cells were purified by cell sorting on day 7 after primary activation by the signals from all 22 probes for two comparison experiments. A change call 6582 SPHK1 IS A NEGATIVE REGULATOR OF Th1 CELLS is assigned based on analysis parameters. Change p values between 0.00 very low levels of IL-4 mRNA, whereas Th2 cells expressed high and 0.0025 are given an increase call. Change p values between 0.0025 and levels of IL-4 and very low levels of IFN-␥ mRNA (data not 0.003 are given a marginal increase call. Change p values between 0.997 and 0.998 are given a marginal decrease call. Change p values between shown). Many other were also differentially induced in Th1 0.998 and 1.00 are given a decrease call (Affymetrix User Manual). and Th2 cells. Among them, we identified that SPHK1 was spe- cifically induced in Th1 cells by anti-CD3, but not IL-12/IL-18, Results stimulation (Fig. 1A). Anti-CD3 stimulation had much less effect SPHK1 is highly induced in Th1 cells by TCR stimulation on SPHK1 mRNA expression in Th2 cells (Fig. 1A). In contrast, To identify genes that are specifically induced in Th1 or Th2 cells, SPHK2 showed similar expression levels between Th1 and Th2 we performed DNA microarray studies using Affymetrix U74Av2 cells (Fig. 1B). To confirm the GeneChip results, we designed chips. DO11.10 splenocytes were differentiated under Th1 or Th2 TaqMan probes and primers which have no overlap with the conditions for 7–14 days, and the cells were restimulated with SPHK1 oligos printed on the microarray chip. Real-time TaqMan anti-CD3, IL-12, and/or IL-18 for 2 h. RNA was extracted from PCR analysis showed that the SPHK1 was highly induced in Th1 these cells and subjected to GeneChip analysis. We first analyzed cells by anti-CD3 stimulation (Fig. 1C). There was an ϳ10-fold Th1/Th2 cytokine mRNA levels from the GeneChip data. As ex- induction after Th1 cells were stimulated with anti-CD3. This find- pected, the Th1 cells expressed high levels of IFN-␥ mRNA and ing is consistent with the GeneChip result. Meanwhile, day 2 Th1 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. SPHK1 is highly induced in Th1 cells. A, DO11.10 T cells were activated under Th1 or Th2 conditions for 7–14 days, then cells were harvested, washed, counted, and restimulated with anti-CD3, IL-12, and/or IL-18 for 2 h. Cells were harvested for RNA extraction and Affymetrix GeneChip analysis. Data show the absolute signal of SPHK1. Anti-CD3 stimulation of both Th1 and Th2 produces an increase call (p ϭ 0.000002; see Materials and Methods). Results are representative of two GeneChip experiments. B, Level of SPHK2 signal from the same GeneChip experiment as that in A. Based on the change call p values, all stimulation conditions in B yield no change call for SPHK2 expression compared with unstimulated controls. C, DO11.10 T cells were activated under Th1 or Th2 conditions for 48 h or 7 days, and restimulated with anti-CD3 for 2 h. Cells were harvested for RNA extraction and TaqMan real-time quantitative RT-PCR. Results shown are the average signals of SPHK1 after normalization with GAPDH. D, Th1 and Th2 cells were stimulated with anti-CD3 for 18 h before being harvested for immunoblotting analysis. The membrane was first probed with anti-SPHK1 Ab, then striped and reprobed with anti-p38 MAPK Ab. The Journal of Immunology 6583

cells were added to an anti-CD3-coated plate. Cells were then briefly spun down and cultured for 2 h. As shown in Fig. 2, genistein and cyclosporin A almost completely inhibited SPHK1 expression, whereas the inactive inhibitor tyrphostin A1 and the p42MAPK inhibitor tyrphostin AG 126 had no effect. Genistein is a potent inhibitor of tyrosine phosphorylation, whereas cyclosporin A inhibits calcineurin and the downstream NF-AT pathway (29). It is well known that TCR signaling induces both tyrosine phosphor- ylation and the activation of calcineurin and NF-AT. Taken to- gether, these data strongly suggest that SPHK1 mRNA expression is induced by TCR signaling. We also asked whether the expres- sion of SPHK1 is the result of direct TCR signaling or requires de novo protein synthesis. When cells were treated with cyclohexi- mide to block new protein synthesis after TCR stimulation, the FIGURE 2. SPHK1 mRNA expression requires TCR signaling and new induction of SPHK1 mRNA expression was suppressed (Fig. 2). protein synthesis. DO11.10 T cells were differentiated to Th1 cells for 7 This finding suggests that the SPHK1 promoter is not activated by days. Cells were washed, counted, and resuspended before being cultured direct signaling of the TCR, but requires de novo protein synthesis. with various inhibitors for 30 min. Cells were then stimulated with anti- CD3 for 2 h before being harvested for RNA extraction and TaqMan RT- SPHK1 inhibits the expression of IL-2, TNF-␣, and IFN-␥ Downloaded from PCR. Results shown are the percentage of the mRNA level compared with p Ͻ 0.05 vs DMSO control. Because SPHK1 is highly expressed in Th1 cells, we asked ,ء .noninhibitor-treated sample whether knocking down or overexpression of SPHK1 in Th1 cells would affect Th1 cytokine expression. RNA interference by spe- and Th2 cells did not express high levels of SPHK1 (Fig. 1C), cific siRNA oligos is now a common tool used to specifically suggesting that SPHK1 is highly expressed in differentiated Th1 knock down a gene of interest in mammalian cells (26, 30). How- http://www.jimmunol.org/ cells. We also detected the protein level of SPHK1 in Th1 and Th2 ever, there is no effective way to efficiently transfect siRNA oligos cells. Fig. 1D shows that Th1 cells expressed higher levels of into mouse primary T cells. Thus, we used human primary CD4ϩ SPHK1 protein than Th2 cells after cells were stimulated with T cells, because siRNA oligos can be efficiently transfected into anti-CD3 for 18 h. We used p38 MAPK levels as a loading control. these cells using the electroporation technology developed by Because SPHK1 phosphorylates sphingosine to form S1P, we de- Amaxa (31). We purified human naive CD4ϩCD45RAϩ termined the level of S1P in Th1 and Th2 cells by mass spectro- CD45ROϪ T cells and then stimulated the cells under Th1 condi- metric analysis. We found that Th1 cells produce ϳ7-fold more tions. After 2 wk of differentiation, Th1 cells were transiently intracellular S1P than Th2 cells (data not shown). Finally, Th0 transfected with SPHK1 siRNA or control siRNA (GL2). The cells expressed moderate levels of SPHK1 mRNA (data not transfection efficiency was ϳ95% as detected by FITC-siRNA by guest on September 25, 2021 shown). (data not shown). Cells were cultured for an additional 3 days after Because SPHK1 is highly induced in Th1 cells by anti-CD3 siRNA transfection and then restimulated with anti-CD3 for 18 h. stimulation, we sought to confirm the requirement for a TCR-me- The cells were harvested for RT-PCR analysis, and the superna- diated signal for the expression of SPHK1. Different inhibitors of tants were used to determine the cytokine titers by BD CBA. TCR signaling were first cultured with T cells for 30 min before SPHK1 siRNA knocked down ϳ70% of the SPHK1 mRNA level

FIGURE 3. SPHK1 siRNA treatment decreases SPHK1 mRNA expression and enhances IL-2, TNF-␣, and IFN-␥ production in Th1 cells. Human Th1 cells were transiently transfected with SPHK1 siRNA, con- trol siRNA GL2, or SPHK2 siRNA using the Amaxa method. Three days after transfection, cells were counted, resuspended at 1 ϫ 106/ml, and restimulated with anti-CD3 (10 ␮g/ml) for 18 h. Cells and superna- tants were then harvested for RNA extraction and CBA assay. A, SPHK1 and SPHK2 mRNA levels were deter- mined by TaqMan real-time PCR. The PCR signals were normalized to GAPDH. B, Cytokine production was detected by CBA after the siRNA-treated Th1 cells p Ͻ 0.05 vs ,ء .were stimulated with anti-CD3 for 18 h other siRNA samples. 6584 SPHK1 IS A NEGATIVE REGULATOR OF Th1 CELLS Downloaded from http://www.jimmunol.org/ FIGURE 4. Overexpression of SPHK1 reduces IL-2, TNF-␣, IFN-␥, and IL-5 production in Th0 cells. DO11.10 T cells were stimulated with OVA under Th0 conditions. Cells were infected with SPHK1-RV or control GFP-RV on day 2. The GFP- and CD4-positive cells were sorted on day 7 and stimulated with OVA and BALB/c APC for another week. Cells were then harvested, counted, and restimulated with anti-CD3 for 18 h. A, Cells were harvested for Western blot for SPHK1. p38 was used as a loading control. B, The supernatants were used to determine the titers of Th1/Th2 cytokines by CBA. Data are representative of four different experiments. p Ͻ 0.05, comparing GFP-RV- with SPHK1-RV-infected cells stimulated with anti-CD3 at 1.25–5 ␮g/ml. as determined by TaqMan real-time RT-PCR (Fig. 3A). We then natants were analyzed using the CBA Th1/Th2 cytokine kit. Fig. 4 measured cytokine titers of Th1 cytokines in the supernatants shows that SPHK1-RV-infected Th0 cells produced less IL-2, treated with SPHK1 siRNA or GL2 siRNA. Surprisingly, SPHK1 TNF-␣, IFN-␥, and IL-5 than the GFP control. However, no sig- by guest on September 25, 2021 siRNA-treated cells produced higher titers of IL-2, TNF-␣, and nificant reduction of IL-4 was observed (Fig. 4B). Table I shows a IFN-␥ than the control cells (Fig. 3B). SPHK1 siRNA did not similar pattern of expression when TaqMan analysis was used to inhibit SPHK2 mRNA expression (Fig. 3A). A subsequent SPHK2 examine changes in the mRNA levels of these four cytokines. Ad- siRNA study showed that this siRNA specifically inhibited SPHK2 ditionally, an increase in IL-10 was observed in Th0 cells infected mRNA expression, but not cytokine expression (Fig. 3, A and B). with SPHK1-RV compared with the control (Table I). As ex- This result indicates that SPHK1 is a negative regulator of cyto- pected, a decrease in cytokine expression of IL-2, TNF-␣, and kine production in these cells. IFN-␥ was also seen by CBA analysis of Th1 cells infected with To study this role, we asked whether overexpression of SPHK1 SPHK1-RV (Fig. 5A). Although a slight inhibition was observed could inhibit Th1 cytokine expression. To do this, we generated a (ϳ20–30% inhibition), it should be noted that the result was repro- mouse SPHK1 retroviral vector (SPHK1-RV) that also expressed ducible in four separate experiments. Because endogenous SPHK1 is GFP. The vectors were transiently transfected into a Phoenix E already highly expressed in activated T cells, additional overexpres- packaging cell line to produce retroviral supernatants, which were sion may not show a dramatic effect on the cytokine production. then used to infect mouse DO11.10 Th0 or Th1 cells. On day 7, Next, we asked whether the inhibitory effect was due to the sorting of GFP- and CD4-positive cells resulted in Ͼ95% GFP- enzymatic activity of SPHK1. Mutation of human SPHK1 glycine positive cells (data not shown). The sorted cells were also stimu- 26 or glycine 80 to aspartic acid 26 or aspartic acid 80 results in a lated with OVA and irradiated BALB/C spleen cells for another catalytically inactive sphingosine kinase (22). Therefore, we gen- week before restimulation with different concentrations of anti- erated SPHK1 G26D and SPHK1 G80D retroviral vectors. Th1 CD3 for 18 h. SPHK1 mRNA increased ϳ10-fold in SPHK1-RV- cells infected with SPHK1 G26D-RV and SPHK1 G80D-RV pro- infected T cells (data not shown). Western blot analysis confirmed duced more IL-2, TNF-␣, and IFN-␥ than cells infected with that SPHK1 protein expression was enhanced in SPHK1-RV-in- GFP-RV (Fig. 5B). Furthermore, SPHK1 G26D-RV and SPHK1 fected Th0 cells (Fig. 4A). Cytokine titers of the resulting super- G80D-RV produced much more IL-2, TNF-␣, and IFN-␥ than

Table I. Overexpression of SPHK1 in Th0 cells with inhibited TNF-␣, IFN-␥, IL-2, and IL-5 mRNA expression

IL-2 TNF-␣ IFN-␥ IL-4 IL-5 IL-10

GFP 100a 100 100 100 100 100 SPHK1 69 69 78 96 57 122

a The percentage of mRNA level compared with cells infected with SPHK1-RV. The Journal of Immunology 6585

FIGURE 5. SPHK1 reduces the production of IL-2, TNF-␣, and IFN-␥ in Th1 cells. DO11.10 T cells were stimulated with OVA under Th1 conditions. Cells were infected, sorted, and restimulated as described in Fig. 4. The supernatants were used to determine the titers of Th1 cyto- kines by CBA. A, Th1 cells overex- pressed with SPHK1 were stimulated with anti-CD3 for 18 h. Data are rep- resentative of four different experi- ments. B, Th1 cells overexpressed with dominant-negative SPHK1 (SPHK1 G26D or SPHK1 G80D) Downloaded from were stimulated with anti-CD3 for 18 h. Data are representative of three p Ͻ 0.05 vs ,ء .different experiments GFP-RV, SPHK1 G26D-RV, or SPHK1 G80D-RV. http://www.jimmunol.org/

cells infected with SPHK1-RV (Fig. 5B). The repeated observa- tions that IL-2, TNF-␣, and IFN-␥ were down-regulated by SPHK1 overexpression and up-regulated by SPHK1 G26D-RV, and SPHK1 G80D-RV are consistent with the siRNA data, indi- by guest on September 25, 2021 cating that SPHK1 negatively controls the expression of these Th1 cytokines.

CCL17 and CCL22 are highly expressed in Th2 cells and are inhibited by SPHK1 To obtain a broader view of SPHK1-controlled gene expression in T cells, we performed Affymetrix GeneChip microarray analysis of T cells overexpressing SPHK1. Th0 cells infected with SPHK1 GFP-RV or control GFP-RV were stimulated with anti-CD3 for 2 h, and the total RNA was extracted for the GeneChip analysis. As expected, we found that IL-2, TNF-␣, and IFN-␥ were inhibited in Th0 cells infected with SPHK1-RV (data not shown). We also discovered that the mRNA levels for several chemokines were decreased in SPHK1 RV-infected Th0 cells. Specifically, the mRNA level of chemokines CCL17 and CCL22 in SPHK1 RV-infected cells was significantly lower than that in control GFP- RV-infected cells (data not shown). CCL17 (also known as thymus- and activation-regulated che- mokine (TARC)) and CCL22 (also known as macrophage-derived chemokine), mainly produced by dendritic cells and epithelial cells (32–34), are known as Th2 chemokines that attract Th2 cells. However, there is no report showing that Th1 and Th2 cells them- selves express both chemokines. From our GeneChip data, we found that both CCL17 and CCL22 were expressed in Th1 and Th2 cells (Fig. 6A). Th2 cells expressed higher mRNA levels of CCL17 and CCL22 than Th1 cells. Anti-CD3 stimulation slightly FIGURE 6. CCL17 and CCL22 are highly expressed in Th2 cells. A, enhanced their expression. A similar observation was made for Affymetrix GeneChip analyses were performed as described in Fig. 1A. Th1 and Th2 cells from the C57BL/6 background (data not Data are representative of two different GeneChip experiments. B, Samples shown). We also performed TaqMan real-time PCR analysis to and TaqMan real-time PCR are shown in Fig. 1D. Data are representative detect the expression of CCL17 and CCL22 in Th1 and Th2 cells. of two separate experiments. 6586 SPHK1 IS A NEGATIVE REGULATOR OF Th1 CELLS

cells, suggesting that it is a conserved mechanism. However, it is unclear how SPHK1 regulates the cytokine and chemokine expression. The molecular mechanism of the inhibitory role of SPHK1 in cytokine production is not clear. SPHK1 activity may alter the half-life of transiently expressed messages or regulate cytokine mRNA expression at the transcription level. Yoshimoto et al. (12) showed that SPHK2 associates with the mouse IL-12R␤1 and aug- ments IL-12-induced STAT4-mediated transcriptional activation. In T cells overexpressing SPHK1, we observed no change in the mRNA level for the IL-12R ␤1- and ␤2-chains, IL-18R1, and IL- 18R accessory protein (GeneChip results; data not shown). The levels of the transcription factors GATA3 and T-bet were not af- fected (data not shown). IL-12/IL-18-induced IFN-␥, growth arrest and DNA damage-inducible (GADD) 45␤, and GADD45␥ were also not significantly impaired (data not shown). Thus, SPHK1 does not inhibit the process of Th1 differentiation. SPHK1 phosphorylates sphingosine to form S1P. The function

of S1P is through its binding and activation of S1P receptors. Downloaded from FIGURE 7. SPHK1 inhibits CCL17 and CCL22 expression. A, Rosen et al. (14) reported that S1P receptor agonists S1P and DO11.10 Th0 cells were prepared as described in Fig. 4. Cells were stim- FTY720 induced emptying of lymphoid sinuses by retention of ulated with anti-CD3 for 2 h before total RNA was extracted. TaqMan lymphocytes on the abluminal side of the sinus-lining endothelium RT-PCR for mouse CCL17 and CCL22 was performed. B, Human Th1 and inhibition of egress into lymph. They also showed that cells were treated as described in Fig. 3. TaqMan RT-PCR for human FTY720 induces immunosuppression through inhibition of both

CCL17 and CCL22 was performed. Data are representative of two different the recirculation of naive T cells and the release of Ag-activated T http://www.jimmunol.org/ experiments. cells from the draining lymph node (36). Matloubian et al. (16) showed that S1PR1 is essential for lymphocyte recirculation and that it regulates egress from both thymus and peripheral lymphoid Fig. 6B shows that Th2 cells expressed higher levels of CCL17 and organs. Mature T cells are unable to exit the thymus in mice whose CCL22 than Th1 cells after cells were differentiated for 48 h or 1 hemopoietic cells lack a single S1PR (16). They also discovered wk. Higher levels of CCL17 and CCL22 mRNA were observed that S1PR1 promotes B cell localization in splenic marginal zone after cells were differentiated for 1 wk. Anti-CD3 stimulation also (37). Furthermore, S1P (0.3–3 ␮M) inhibits chemotaxis of CD4ϩ slightly increased their expression. The TaqMan real-time PCR T cells to CCL-21 and CCL-5 (17, 18). These discoveries show the results were consistent with the GeneChip data. close relationship between S1P receptors and the migration of lym- by guest on September 25, 2021 Finally, we asked whether CCL17 and CCL22 mRNA were in- phocytes. We found that chemokine CCL17 and CCL22 are highly deed inhibited by SPHK1. As expected, TaqMan analysis showed expressed in Th2 cells, whereas Th1 cells express moderate level that CCL17 and CCL22 mRNA were decreased in SPHK1-RV- of CCL17 and CCL22 (Fig. 6). CCR4, which is expressed in Th2 infected T cells (Fig. 7A). In addition, increased expression of and T regulatory cells, is the receptor of CCL17 and CCL22 (38– CCL17 and CCL22 mRNA was observed in human Th1 cells 40). Both CCL17 and CCL22 could chemoattract Th2 and T reg- when SPHK1 was knocked down by SPHK1 siRNA (Fig. 7B). ulatory cells via CCR4 (38, 41, 42). The low levels of CCL17 and These results strongly support the idea that SPHK1 is a negative CCL22 in Th1 cells may be due to the inhibitory role of SPHK1. regulator of the expression of these chemokines. Thus, SPHK1 may play an important role in preventing Th2 and T regulatory cells from migrating into the inflammatory sites of Th1- Discussion related autoimmune and inflammatory diseases as well as Th1- In this report we showed that SPHK1, but not SPHK2, is selec- related protective immune reaction. tively expressed in activated Th1 cells. The expression of SPHK1 The inhibition of Th1 cytokines by SPHK1 could be associated in Th1 cells requires TCR signaling and new protein synthesis. with S1P production. Indeed, Graeler et al. (17) and Dorsam et al. When human SPHK1 expression was knocked down by SPHK1- (18) reported that S1P decreased CD4ϩ T cell production of IFN-␥ specific siRNA, the production of IL-2, TNF-␣, and IFN-␥ from and IL-4. Idzko et al. (43) showed that S1P reduces IL-12 and human Th1 cells in response to TCR stimulation was enhanced. TNF-␣ production and augments IL-10 release in maturating den- Consistently, overexpression of -dead, dominant-negative dritic cells. Furthermore, S1P in maturating dendritic cells inhibits SPHK1 mutants increased the production of IL-2, TNF-␣, and their capacity to induce Th1 immune response, resulting in a Th2 IFN-␥ in Th1 cells. Furthermore, overexpression of wide-type response. Although their finding was in dendritic cells, it is con- SPHK1 in Th1 and Th0 cells decreased the expression of IL-2, sistent with our observations in T cells. We showed SPHK1 re- TNF-␣, and IFN-␥. Moreover, overexpression of SPHK1 also in- duced the expression of Th1 cytokine mRNA and increased IL-10 hibited IL-5 and increased IL-10 mRNA expression. To our mRNA expression (Table I). The increased IL-10 expression was knowledge, this is the first report showing that SPHK1 is highly consistent with the report by Idzko et al. (43). However, Jin et al. induced in Th1 cells and that it is a negative regulator of cytokine (44) reported that S1P enhanced IL-2 and IFN-␥ production by T production in these cells. In addition, we report that the Th2 che- cells stimulated with anti-CD3 and anti-CD28, although T cell mokines CCL17 and CCL22 are highly expressed in Th2 cells. proliferation was inhibited by S1P. This apparent contradictory Indeed, Zhang et al. (35) first reported that CCL22 is differentially report may be due to the different cell types used. Jin et al. (44) expressed in Th2 cells. The expression of CCL17 and CCL22 was used whole human T cells, a mixture of naive and memory CD4ϩ also inhibited by SPHK1. We observed that SPHK1 inhibits cy- T cells and CD8ϩ T cells, for their experiments. Graeler et al. (17, tokine and chemokine production in both human and mouse T 18) used mouse CD4ϩ T cells for their S1P experiments. In our The Journal of Immunology 6587

ϩ experiments we used human naive CD4 T cells and mouse 8. Spiegel, S., and S. Milstien. 2003. Sphingosine-1-phosphate: an enigmatic sig- DO11.10 TCR-transgenic CD4ϩ T cells. In addition, we differen- nalling lipid. Nat. Rev. Mol. Cell Biol. 4: 397–407. ϩ 9. Pitson, S. M., P. A. Moretti, J. R. Zebol, H. E. Lynn, P. Xia, M. A. Vadas, and tiated the CD4 T cells into Th1 cells. An additional explanation B. W. Wattenberg. 2003. Activation of sphingosine kinase 1 by ERK1/2-medi- could be the difference between intracellular S1P and extracellular ated phosphorylation. EMBO J. 22: 5491–5500. 10. Melendez, A. J., and A. K. Khaw. 2002. Dichotomy of Ca2ϩ signals triggered by S1P. Other intracellular functions of SPHK1 may also be consid- different phospholipid pathways in antigen stimulation of human mast cells. ered. It has been recently shown that SPHK1 is an intracellular J. Biol. Chem. 277: 17255–17262. effector of phosphatidic acid (45). In addition to sphingosine and 11. Pettus, B. J., J. Bielawski, A. M. Porcelli, D. L. Reames, K. R. Johnson, J. Morrow, C. E. Chalfant, L. M. Obeid, and Y. A. Hannun. 2003. The sphin- S1P, SPHK1 expressed in Th1 cells could alter the intracellular gosine kinase 1/sphingosine-1-phosphate pathway mediates COX-2 induction balance of other sphingolipid metabolites, including ceramide and and PGE2 production in response to TNF-␣. FASEB J. 17: 1411–1421. sphingomyelin, which also play important roles in T cell signaling. 12. Yoshimoto, T., M. Furuhata, S. Kamiya, M. Hisada, H. Miyaji, Y. Magami, K. Yamamoto, H. Fujiwara, and J. Mizuguchi. 2003. Positive modulation of Furthermore, SPHK1 could have another substrate in addition to IL-12 signaling by sphingosine kinase 2 associating with the IL-12 receptor ␤1 sphingosine. Indeed, Gijsbers et al. (46) showed that 1-O-hexade- cytoplasmic region. J. Immunol. 171: 1352–1359. cyl-2-desoxy-2-amino-sn-glycerol is a substrate for human SPHK. 13. Billich, A., F. Bornancin, P. Devay, D. Mechtcheriakova, N. Urtz, and T. Baumruker. 2003. Phosphorylation of the immunomodulatory drug FTY720 During our submission of this manuscript, Allende et al. (47) re- by sphingosine kinases. J. Biol. Chem. 278: 47408–47415. ported that lymphocyte distribution is unaffected in lymphoid or- 14. Rosen, H., and J. Liao. 2003. Sphingosine 1-phosphate pathway therapeutics: a lipid ligand-receptor paradigm. Curr. Opin. Chem. Biol. 7: 461–468. gans of SPHK1-deficient mice, although S1P signaling regulated 15. Mandala, S., R. Hajdu, J. Bergstrom, E. Quackenbush, J. Xie, J. Milligan, lymphocyte trafficking. They also showed that the S1P level in R. Thornton, G. J. Shei, D. Card, C. Keohane, et al. 2002. Alteration of lym- most tissues from SPHK1-deficient mice is not markedly de- phocyte trafficking by sphingosine-1-phosphate receptor agonists. Science 296: 346–349. Downloaded from creased. Thus, SPHK1 is not essential for the generation of S1P, 16. Matloubian, M., C. G. Lo, G. Cinamon, M. J. Lesneski, Y. Xu, V. Brinkmann, which meditates T lymphocyte trafficking through S1P1R. In M. L. Allende, R. L. Proia, and J. G. Cyster. 2004. Lymphocyte egress from SPHK1Ϫ/Ϫ mice, SPHK2 or another enzyme(s) is able to phos- thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature Ϫ/Ϫ 427: 355–360. phorylate sphingosine to produce S1P. The SPHK1 study in- 17. Graeler, M., G. Shankar, and E. J. Goetzl. 2002. Cutting edge: suppression of T dicates that the suppressive function of SPHK1 might occur be- cell chemotaxis by sphingosine 1-phosphate. J. Immunol. 169: 4084–4087. cause of its alteration of the intracellular function of S1P and other 18. Dorsam, G., M. H. Graeler, C. Seroogy, Y. Kong, J. K. Voice, and E. J. Goetzl. 2003. Transduction of multiple effects of sphingosine 1-phosphate (S1P) on T cell

sphingosine metabolites. It also indicates the possibility that the functions by the S1P1 G protein-coupled receptor. J. Immunol. 171: 3500–3507. http://www.jimmunol.org/ suppression is independent of S1P. 19. Murphy, K. M., A. B. Heimberger, and D. Y. Loh. 1990. Induction by antigen of ϩ ϩ lo Our findings suggest that SPHK1 is a negative regulator of cy- intrathymic apoptosis of CD4 CD8 TCR thymocytes in vivo. Science 250: ϩ 1720–1723. tokine production in CD4 Th1 cells. The present work implies 20. Yang, J., H. Zhu, T. L. Murphy, W. Ouyang, and K. M. Murphy. 2001. IL-18- that SPHK1 plays a role in the balance of cytokine and chemokine stimulated GADD45␤ required in cytokine-induced, but not TCR-induced, IFN-␥ production. Nat. Immunol. 2: 157–164. expression levels in Th1 cells. It is well known that overexpression 21. Ouyang, W., S. H. Ranganath, K. Weindel, D. Bhattacharya, T. L. Murphy, of Th1 cytokines induces inflammation. The production of SPHK1 W. C. Sha, and K. M. Murphy. 1998. Inhibition of Th1 development mediated by after T cell activation may serve as a negative feedback to limit the GATA-3 through an IL-4-independent mechanism. Immunity 9: 745–755. 22. Pitson, S. M., P. A. Moretti, J. R. Zebol, R. Zareie, C. K. Derian, A. L. Darrow, overexpression of Th1 cytokines. It also may play an important J. Qi, R. J. D’Andrea, C. J. Bagley, M. A. Vadas, et al. 2002. The nucleotide- role in preventing the chemoattraction of Th2 and T regulatory of human sphingosine kinase 1. J. Biol. Chem. 277: 49545–49553. by guest on September 25, 2021 cells by inhibiting CCL17 and CCL22. Our current work indicates 23. Ancellin, N., C. Colmont, J. Su, Q. Li, N. Mittereder, S. S. Chae, S. Stefansson, G. Liau, and T. Hla. 2002. Extracellular export of sphingosine kinase-1 enzyme: that inhibition of SPHK1 might induce inflammatory and autoim- sphingosine 1-phosphate generation and the induction of angiogenic vascular mune diseases by overproduction of cytokines and chemokines. It maturation. J. Biol. Chem. 277: 6667–6675. 24. Chen, X. L., J. Y. Grey, S. Thomas, F. H. Qiu, R. M. Medford, M. A. Wasserman, remains unclear how SPHK1 inhibits the expression of these and C. Kunsch. 2004. Sphingosine kinase-1 mediates TNF-␣-induced MCP-1 proinflammatory cytokines and chemokines. Additional studies gene expression in endothelial cells: upregulation by oscillatory flow. will be required to understand this inhibitory mechanism. Am. J. Physiol. 287: H1452–H1458. 25. Bektas, M., P. S. Jolly, C. Muller, J. Eberle, S. Spiegel, and C. C. Geilen. 2005. Sphingosine kinase activity counteracts ceramide-mediated cell death in human Acknowledgments melanoma cells: role of Bcl-2 expression. Oncogene 24: 178–187. We thank Drs. Jeanne Magram, Sheenah Mische, and Mark Labadia for 26. Elbashir, S. M., J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, and T. Tuschl. their discussion of this manuscript. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494–498. 27. Li, X., P. E. Massa, A. Hanidu, G. W. Peet, P. Aro, A. Savitt, S. Mische, J. Li, Disclosures and K. B. Marcu. 2002. IKK␣, IKK␤, and NEMO/IKK␥ are each required for the ␬ The authors have no financial conflict of interest. NF- B-mediated inflammatory response program. J. Biol. Chem. 277: 45129–45140. 28. Li, X., and X. Wang. 2000. Application of real-time chain reaction References for the quantitation of interleukin-1␤ mRNA upregulation in brain ischemic tol- 1. Mosmann, T. R., H. Cherwinski, M. W. Bond, M. A. Giedlin, and R. L. Coffman. erance. Brain Res. Brain Res. Protoc. 5: 211–217. 1986. Two types of murine helper T cell clone. I. Definition according to profiles 29. Crabtree, G. R. 1999. Generic signals and specific outcomes: signaling through ϩ of lymphokine activities and secreted proteins. J. Immunol. 136: 2348–2357. Ca2 , calcineurin, and NF-AT. Cell 96: 611–614. 2. Murphy, K. M., W. Ouyang, J. D. Farrar, J. Yang, S. Ranganath, H. Asnagli, 30. Brummelkamp, T. R., R. Bernards, and R. Agami. 2002. A system for stable M. Afkarian, and T. L. Murphy. 2000. Signaling and transcription in T helper expression of short interfering RNAs in mammalian cells. Science 296: 550–553. development. Annu. Rev. Immunol. 18: 451–494. 31. Bidere, N., H. K. Lorenzo, S. Carmona, M. Laforge, F. Harper, C. Dumont, and 3. Fuss, I. J., M. Neurath, M. Boirivant, J. S. Klein, M. C. de la, S. A. Strong, A. Senik. 2003. Cathepsin D triggers Bax activation, resulting in selective ap- C. Fiocchi, and W. Strober. 1996. Disparate CD4ϩ lamina propria (LP) lympho- optosis-inducing factor (AIF) relocation in T lymphocytes entering the early com- kine secretion profiles in inflammatory bowel disease: Crohn’s disease LP cells mitment phase to apoptosis. J. Biol. Chem. 278: 31401–31411. manifest increased secretion of IFN-␥, whereas ulcerative colitis LP cells man- 32. Soumelis, V., P. A. Reche, H. Kanzler, W. Yuan, G. Edward, B. Homey, ifest increased secretion of IL-5. J. Immunol. 157: 1261–1270. M. Gilliet, S. Ho, S. Antonenko, A. Lauerma, et al. 2002. Human epithelial cells 4. Bouma, G., and W. Strober. 2003. The immunological and genetic basis of in- trigger dendritic cell mediated allergic inflammation by producing TSLP. Nat. flammatory bowel disease. Nat. Rev. Immunol. 3: 521–533. Immunol. 3: 673–680. 5. Manoury-Schwartz, B., G. Chiocchia, N. Bessis, O. Abehsira-Amar, F. Batteux, 33. Sekiya, T., M. Miyamasu, M. Imanishi, H. Yamada, T. Nakajima, S. Muller, S. Huang, M. C. Boissier, and C. Fournier. 1997. High susceptibility M. Yamaguchi, T. Fujisawa, R. Pawankar, Y. Sano, K. Ohta, et al. 2000. Induc- to collagen-induced arthritis in mice lacking IFN-␥ receptors. J. Immunol. 158: ible expression of a Th2-type CC chemokine thymus- and activation-regulated 5501–5506. chemokine by human bronchial epithelial cells. J. Immunol. 165: 2205–2213. 6. van den Berg, W. B. 2002. Is there a rationale for combined TNF and IL-1 34. Alferink, J., I. Lieberam, W. Reindl, A. Behrens, S. Weiss, N. Huser, K. Gerauer, blocking in arthritis? Clin. Exp. Rheumatol. 20(Suppl. 27): S21–S25. R. Ross, A. B. Reske-Kunz, P. Ahmad-Nejad, et al. 2003. Compartmentalized 7. Papadakis, K. A., and S. R. Targan. 2000. Role of cytokines in the pathogenesis production of CCL17 in vivo: strong inducibility in peripheral dendritic cells of inflammatory bowel disease. Annu. Rev. Med. 51: 289–298. contrasts selective absence from the spleen. J. Exp. Med. 197: 585–599. 6588 SPHK1 IS A NEGATIVE REGULATOR OF Th1 CELLS

35. Zhang, S., N. W. Lukacs, V. A. Lawless, S. L. Kunkel, and M. H. Kaplan. 2000. vention of thymus and activation-regulated chemokine attenuates the develop- Cutting edge: differential expression of chemokines in Th1 and Th2 cells is de- ment of allergic airway inflammation and hyperresponsiveness in mice. J. Im- pendent on Stat6 but not Stat4. J. Immunol. 165: 10–14. munol. 166: 2055–2062. 36. Xie, J. H., N. Nomura, S. L. Koprak, E. J. Quackenbush, M. J. Forrest, and 42. Berin, M. C., M. B. Dwinell, L. Eckmann, and M. F. Kagnoff. 2001. Production H. Rosen. 2003. Sphingosine-1-phosphate receptor agonism impairs the effi- of MDC/CCL22 by human intestinal epithelial cells. Am. J. Physiol. 280: ciency of the local immune response by altering trafficking of naive and antigen- G1217–G1226. activated CD4ϩ T cells. J. Immunol. 170: 3662–3670. 43. Idzko, M., E. Panther, S. Corinti, A. Morelli, D. Ferrari, Y. Herouy, S. Dichmann, 37. Cinamon, G., M. Matloubian, M. J. Lesneski, Y. Xu, C. Low, T. Lu, R. L. Proia, M. Mockenhaupt, P. Gebicke-Haerter, F. Di Virgilio, et al. 2002. Sphingosine and J. G. Cyster. 2004. Sphingosine 1-phosphate receptor 1 promotes B cell 1-phosphate induces chemotaxis of immature and modulates cytokine-release in localization in the splenic marginal zone. Nat. Immunol. 5: 713–720. mature human dendritic cells for emergence of Th2 immune responses. FASEB J. 38. Iellem, A., M. Mariani, R. Lang, H. Recalde, P. Panina-Bordignon, F. Sinigaglia, 16: 625–627. and D. D’Ambrosio. 2001. Unique chemotactic response profile and specific 44. Jin, Y., E. Knudsen, L. Wang, Y. Bryceson, B. Damaj, S. Gessani, and expression of chemokine receptors CCR4 and CCR8 by CD4ϩCD25ϩ regulatory A. A. Maghazachi. 2003. Sphingosine 1-phosphate is a novel inhibitor of T-cell T cells. J. Exp. Med. 194: 847–853. proliferation. Blood 101: 4909–4915. 39. Andrew, D. P., N. Ruffing, C. H. Kim, W. Miao, H. Heath, Y. Li, K. Murphy, 45. Delon, C., M. Manifava, E. Wood, D. Thompson, S. Krugmann, S. Pyne, and J. J. Campbell, E. C. Butcher, and L. Wu. 2001. C-C chemokine receptor 4 N. T. Ktistakis. 2004. Sphingosine kinase 1 is an intracellular effector of phos- expression defines a major subset of circulating nonintestinal memory T cells of phatidic acid. J. Biol. Chem. 279: 44763–44774. both Th1 and Th2 potential. J. Immunol. 166: 103–111. 46. Gijsbers, S., S. Asselberghs, P. Herdewijn, and P. P. Van Veldhoven. 2002. 40. Simons, F. E., Y. Shikishima, G. Van Nest, J. J. Eiden, and K. T. HayGlass. 2004. 1-O-hexadecyl-2-desoxy-2-amino-sn-glycerol, a substrate for human sphingosine Selective immune redirection in humans with ragweed allergy by injecting Amb kinase. Biochim. Biophys. Acta 1580: 1–8. a 1 linked to immunostimulatory DNA. J. Allergy Clin. Immunol. 113: 47. Allende, M. L., T. Sasaki, H. Kawai, A. Olivera, Y. Mi, G. Echten-Deckert, 1144–1151. R. Hajdu, M. Rosenbach, C. A. Keohane, S. Mandala, et al. 2004. Mice deficient 41. Kawasaki, S., H. Takizawa, H. Yoneyama, T. Nakayama, R. Fujisawa, in sphingosine kinase 1 are rendered lymphopenic by FTY720. J. Biol. Chem. M. Izumizaki, T. Imai, O. Yoshie, I. Homma, K. Yamamoto, et al. 2001. Inter- 279: 52487–52492. Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021