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* Departments of Medicine and Immunology, University of California, San Francisco, CA 94143; and
Stanford University Medical Center, Stanford, CA 94305
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and IL-4, without affecting IL-2. A Th1 line from D011.10 TCR transgenic mice without detectable S1P1 was refractory to S1P until introduction of S1P1 by retroviral transduction. S1P then evoked chemotaxis, inhibited chemotaxis to CCL-5 and CCL-21, and suppressed Ag-stimulated proliferation and IFN- production. Thus, S1P1 signals multiple immune functions of T cells as well as migration and tissue distribution. |
Introduction |
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At concentrations of 10-9–3 x 10-8 M, S1P is directly chemotactic for T cells and enhances T cell chemotactic responses to diverse chemokines (7). At plasma and lymph concentrations of 10-7–3 x 10-6 M, S1P acts directly on T cells to suppress their chemotactic responses to chemokines in vitro and in vivo (8). Stimulation of T cells sufficient to down-regulate S1P1Rs reduces both chemotactic and chemotactic inhibitory effects of S1P. Chemotactic and chemotactic inhibitory responses of S1P1 transfectants, but not S1P4 transfectants, to S1P suggested that S1P1 is the major transducer of effects of S1P on T cells (8). An immunosuppressant drug termed FTY720, which is structurally homologous to sphingosine and after phosphorylation resembles S1P, binds to most of the S1P GPCRs with respective affinities suggesting a major effect on S1P1 GPCRs (9, 10, 11). FTY720 inhibits T cell chemotaxis to S1P in vitro (8) and in vivo suppresses lymphocyte migration out of secondary lymphoid organs, recycling of lymphocytes, and thereby lymphocyte homing to sites of Ag challenge (10, 11).
Although S1P has crucial roles in mediation and regulation of lymphocyte migration, nothing is known of its effects on other T cell functions. The present data show that S1P through S1P1 GPCRs suppresses initiation of T cell proliferation and secretion of IFN- and IL-4, without altering generation of IL-2. Thus, the S1P/S1P1R system controls not only T cell migration and tissue distribution, but also initiation of early events of differentiation into effector states.
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Materials and Methods |
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Mouse CD4 T cells were isolated from splenocytes of 6- to 8-wk-old C57BL/6 female mice at a minimum purity of 97% using metallic beads bearing anti-CD4 mAbs and two cycles of magnetic retention chromatography (Miltenyi Biotec, Auburn, CA), as described (7, 8). One milliliter of some suspensions of purified CD4 T cells in RPMI 1640 with 50 µg/ml of fatty acid-free BSA (FAF-BSA) (Calbiochem, La Jolla, CA) were prestimulated by incubation for 24 h in six-well plates on 2 µg each of adherent anti-CD3 + anti-CD28 mAbs (BD PharMingen, San Diego, CA).
Generation of Th1 lines from DO11.10 TCR transgenic mouse splenic CD4 T cells and retroviral transduction with S1P1 (Edg-1) receptor
Wild-type BALB/c mouse splenocytes in RPMI 1640 containing 10% heat-treated FBS, 10 mM HEPES, 1% nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 50 µM 2-ME, 100 U/ml penicillin G, and 100 µg/ml streptomycin (RPMI-C) were irradiated with 3000 rad, preincubated for 1 h in tissue-culture flasks at 37°C and 5% CO2 to form adherent layers of APCs, washed twice and covered again with RPMI-C. Then 0.5 µg/ml OVA substituent peptide 323–339 (OVA323–339; AnaSpec, San Jose, CA) was added and incubation continued for 1 h. CD4 T cells were selected from splenocytes of DO11.10 
TCR transgenic mice (BALB/c, H-2d haplotype) (12) with anti-CD4 mAb-metallic beads and magnetic retention chromatography, as for splenic CD4 T cells of wild-type mice. The CD4 T cells then were suspended in RPMI-C containing 20 µg/ml anti-IL-4 mAb (clone 11B11; BD PharMingen) and 40 ng/ml IL-12 (R&D Systems, Minneapolis, MN) to stimulate differentiation of Th1 cells and incubated for 72 h with APCs and OVA323–339 Ag at a 20:1 ratio of APC-Th. An equal volume of RPMI-C containing 10 U/ml mouse IL-2 (Peprotech, Rocky Hill, NJ) was added to the flask and culture continued for 96 h. The T cells then were washed, added to a fresh layer of APC (1:20) in RPMI-C with 0.5 µg/ml of OVA323–339, and further incubated for 72 h. Th1 cells were recovered, washed, and suspended in RPMI-C at 0.5–1.0 x 106/ml for repeated 7-day cycles of restimulation with irradiated APCs and OVA323–339, but without cytokines or anti-cytokines. Th1 cells were taken on day 4 or 5 for functional studies.
A cDNA encoding human full-length S1P1 (Edg-1) was inserted into the pGC-IRES retroviral vector (obtained from G. L. Costa, Stanford University, Stanford, CA), which encodes yellow fluorescent protein (YFP), and introduced into some sets of mouse Th1 cells using the EcoPack2–293 system with a HEK 293-based packaging cell line for production of rodent ecotropic retrovirus (BD Biosciences, Palo Alto, CA). Transduction efficiency was over 60%, as assessed by flow cytometric analyses of expression of YFP. Real time-PCR quantification of human S1P1 mRNA revealed levels 3- to 4-fold higher in day 4 Th1 transductants than mouse spleen naive CD4 T cells. Western blots of proteins extracted from Th1 transductants and developed with rabbit anti-human S1P1 (Edg-1) IgG Ab (ExAlpha, Boston, MA) and rat anti-mouse S1P1 mAb showed levels of human S1P1 5- to 10-fold higher than endogenous mouse S1P1 in corresponding untransducted Th1 cells.
Real-time PCR quantification of mRNA encoding S1P GPCRs
Total RNA was isolated with TRIzol (Life Technologies, Grand Island, NY), treated with DNase I, and amplified quantitatively in 50-ng replicates with TaqMan primers and probes for mouse and human S1PRs and the respective constitutive standard GAPDH (Integrated DNA Technologies, Coralville, IA). The Prism 7700 Sequence Detection System with the recommended optimal reagents and conditions provided interanalysis coefficients of variation lower than 4% (Applied Biosystems, Foster City, CA). The value of each unknown was determined by a standard threshold cycle method using GAPDH as the reference sequence and a calibration curve derived from known amounts of RNA.
Western blot analyses of S1PRs
Immunoblots of proteins extracted from 5 x 106 mouse Th1 cells, before and after retroviral transduction with human S1P1, and HTC4 rat hepatoma transfectants stably expressing S1P1 or S1P4 receptors were prepared as described (8) and developed with rabbit anti-S1P1 Ab or rabbit anti-S1P4 Ab (ExAlpha).
Quantification of migration, proliferation, and cytokine secretion by CD4 T cells and Th1 cell lines
Migration of mouse purified CD4 T cells and Th1 cells, without and with recombinant S1P1 introduced by retroviral transduction, was analyzed in Transwell chambers (Costar, Cambridge, MA) with human type IV collagen (Sigma-Aldrich, St. Louis, MO)-coated 5-µm pore width polycarbonate filters and incubation for 4 h, as described (7). T cell suspensions were 1 x 107/ml in RPMI 1640 with 5% heated and charcoal-extracted FBS, from which 0.1-ml portions of each were loaded into top compartments of chemotactic chambers. CC chemokine ligand (CCL)21 (Exodus-2) or CCL5 (RANTES) chemokines (Peprotech) were the positive chemotactic stimuli. The significance of differences between altered and control migration was calculated with a two-sample t test.
For studies of T cell proliferation and cytokine generation, replicate 0.5-ml suspensions of 1.0–1.2 x 106 CD4 T cells, and S1P1-low and recombinant human S1P1-transduced DO11.10 Th1 cells in RPMI 1640 containing 5% heated and charcoal-extracted FBS and 50 µg/ml FAF-BSA were preincubated without and with a biochemical agonist or inhibitor and without or with lysophosphatidic acid (LPA) or S1P before addition to 24-well plates. Wells were precoated with 1 µg each of adherent anti-CD3 + anti-CD28 mAbs (BD PharMingen) to activate through the TCR or anti-CD3 alone for stimulation of homeostatic proliferation by 10-10–10-8 M fluid-phase mouse recombinant IL-7 (Peprotech). After 24 h of incubation, 200-µl aliquots were removed from each well for ELISA quantification of cytokines and the T cells were incubated 16 h further in fresh medium reconstituted with the original biochemical inhibitor or other agent, LPA and/or S1P, and 1.5 uCi [3H]thymidine (New England Nuclear, Boston, MA) to assess proliferation. ELISAs for mouse IFN-, IL-2, and IL-4 were from Pierce-Endogen (Cambridge, MA) and the assays were conducted as suggested by the manufacturer. For some replicate suspensions, the T cells were recovered by centrifugation, washed and lysed after 30 min of the initial incubation for measurement of the intracellular concentration of cAMP, termed [cAMP]i. Quantification of [cAMP]i was by a solid-phase low pH immunoassay after acetylation of standards and samples (R&D Systems). Statistical significance of the effects of stimuli and inhibitors was calculated as for migration assays.
Lysophospholipids and biochemical inhibitors
LPA and S1P (Sigma-Aldrich); 8-bromo-cAMP, dibutyryl-cAMP, the cell-permeant calcium chelator BAPTA-AM, the inhibitor of phosphatidylinositol-specific phospholipase C termed 1-O-octadecyl-2-O-methyl-sn-glyceryl-3-phosphorylcholine or ET-18-OCH3 (BioMol, Plymouth Meeting, PA); the adenylyl cyclase inhibitor MDL-12,330 A HCl, the protein kinase A (PKA)-selective inhibitor KT5720, and the cAMP-selective phosphodiesterase (IV) inhibitor Ro-20-1724 (Calbiochem, San Diego, CA) were obtained from the suppliers noted.
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Results |
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i with resultant decreases in basal and evoked [cAMP]i. As S1P1 couples inefficiently or not at all to Gq, G dimers or other signaling units coupled to S1P1 are presumed to activate phospholipase C (PLC) and thereby evoke increases in intracellular Ca2+ concentration ([Ca2+]i). To better delineate these signaling pathways, several selective inhibitors were examined for their capacity to alter S1P suppression of T cell proliferation. The intracellular Ca2+ chelator BAPTA-AM and the phosphatidylinositol-specific PLC inhibitor Et-18-OCH3 both completely prevented suppression of T cell proliferation by an optimal level of S1P, suggesting an obligatory role for increases in [Ca2+]i (Fig. 3). Neither inhibition of adenylyl cyclase by MDL-12,330A nor of PKA by KT5720 had any effect on S1P suppression of T cell proliferation. In contrast, the cAMP phosphodiesterase-specific inhibitor Ro-20-1724 significantly reversed the S1P suppressive effect at a concentration which substantially elevated [cAMP]i (Fig. 3, values above columns). The level of [cAMP]i in T cells stimulated for 30 min with anti-CD3 plus anti-CD28 without and with 10-6 M S1P was not detectable at the standard T cell density (Fig. 3). In the presence of 2 and 5 µM Ro-20-1724, however, T cell [cAMP]i rose to respective mean values of 0.24 and 0.72 pmol/ml (value above column, Fig. 3). The increase in [cAMP]i attained by the lower concentration of Ro-20-1724 had no significant effect on S1P suppression of T cell proliferation (not shown). However, the higher concentration of Ro-20-1724, which elevated [cAMP]i 3-fold more, significantly reversed S1P suppression of T cell proliferation (Fig. 3). To assess the effect of cAMP directly, replicate suspensions of T cells were preincubated for 15 min with 5 µM 8-bromo-cAMP and 5 µM dibutyryl-cAMP before addition of S1P and exposure to anti-CD3 plus anti-CD28 mAbs. The resultant mean [cAMP]i 30 min after mAb stimulation was significantly increased at 2.07 and 2.83 pmol/ml, respectively. S1P suppression of T cell proliferation was prevented completely by both forms of cell-permeant exogenous cAMP (Fig. 3).
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and IL-4 are only 5–20% of their peak levels on days 4 to 6 but S1PRs are not yet fully down-regulated by TCR signals. As for analyses of proliferation, replicate suspensions of mouse splenic T cells were stimulated with anti-CD3 plus anti-CD28 mAbs, which initiates a TCR-coreceptor pathway, and anti-CD3 mAb plus IL-7, which induces homeostatic proliferation of the T cells. LPA had previously been shown to suppress IL-2 secretion by stimulated naive human blood T cells (14). Inhibition of IL-2 secretion by LPA was confirmed in mouse splenic CD4 T cells stimulated by both mechanisms, where mean maximal inhibition by 10-6 M LPA exceeded 50% (Figs. 4 and 5). In contrast, 10-10–10-6 M S1P had no effect on IL-2 secretion evoked by either signal, with the exception of marginally significant 12% suppression of that elicited by IL-7 at 10-6 M S1P (Fig. 5). Inversely, 10-8–10-6 M S1P but not LPA suppressed secretion of IFN- from T cells stimulated by both pathways to the same extent as inhibition of proliferation by identical concentrations of S1P (Figs. 4 and 5). The susceptibility of T cell secretion of IL-4 to inhibition by S1P and LPA differed for the two stimulatory pathways. The secretion of IL-4 by T cells exposed to TCR-coreceptor stimulation was inhibited significantly only by 10-6 M S1P or LPA (Fig. 4). In contrast, IL-4 secretion by T cells exposed to TCR-cytokine stimulation was inhibited significantly and progressively by 10-8–10-6 M S1P, but not by 10-6 M LPA (Fig. 5).
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generation to suppression by S1P, but not LPA, irrespective of the CD4 T cell stimulus suggested that more detailed studies be conducted with Ag-stimulated Th1 lines derived from CD4 T cells of TCR transgenic mice. Profiles of mouse S1P GPCRs in the OVA peptide Ag-stimulated and expanding Th1 line by real-time PCR showed mean levels of S1P1R mRNA relative to GAPDH of 5.1 on day 4 and 2.7 on day 7 after Ag stimulation, as contrasted with 334 for unstimulated mixed splenic CD4 T cells. In contrast, S1P4 receptor mRNA mean levels relative to GAPDH were similar for T cells of all types at 27 on day 4 and 41 on day 7 after Ag stimulation, as contrasted with 69 for unstimulated mixed splenic CD4 T cells. To further define the regulatory roles of S1P1 GPCRs in functional responses of this Th1 cell line, a subline was generated by retroviral transduction with mRNA encoding human S1P1 and YFP. Flow cytometric analyses of Th1-S1P1 transductants showed that >80% expressed YFP and thus presumably high levels of S1P1. Real-time PCR quantification of human S1P1 mRNA revealed levels in Th1-S1P1 transductants at least 30-fold higher than that of the PCR reagent background and at least 2-fold higher than mouse S1P1 mRNA in normal splenic naive CD4 T cells. Western blots confirmed that human S1P1R protein was present at much higher levels in transductants than the sham-transducted parent Th1 line (Fig. 6). These differences in S1P1 expression were sufficient to predict distinctive responses of S1P1R transductants to S1P. Indeed, S1P1-high transductants of Th1 cells exhibited significantly greater chemotactic responses to S1P than the S1P1-low parent Th1 line (Fig. 7A), whereas chemotactic responses to CCL5 (RANTES) were the same for both groups. Similarly, S1P inhibited more significantly the CCL-5-induced chemotactic responses of Th1-S1P1 transductants than control Th1 sham-transductants (Fig. 7B). The results were the same with CCL-21 (Exodus-2) as the chemotactic stimulus. S1P inhibition of proliferation evoked by OVA peptide Ag and APCs also was greater for the S1P1-high transductants than the S1P1-low parent Th1 line (Fig. 7C). S1P inhibition of IFN- secretion by Th1 cells stimulated with OVA peptide and APC was much greater for the S1P1-high transductants than the S1P1-low parent line (Fig. 7D). The marginal chemotactic, chemotactic inhibitory, and proliferation inhibitory responses of Th1 sham-transductants to the higher concentration of S1P may be transduced by very low levels of S1P1Rs not detected in Western blots or by an S1P1R-independent effect of S1P on store-operated calcium entry.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most S1P GPCRs signal cells in part through Ca2+-dependent pathways, initiated by interactions with Gq or G dimers that transduce activation of a phosphatidylinositol (PI)-specific phospholipase C (6). S1P suppression of T cell proliferation also exhibits stringent Ca2+-dependence, as demonstrated by elimination of this suppressive effect when PI-specific PLC activity is reduced by the selective inhibitor Et-18-OCH3 or downstream increases in [Ca2+]i are blunted by BAPTA-AM (Fig. 3). Similar dependence on [Ca2+]i was observed in limited investigations of the inhibition of IFN- generation by S1P. In two such studies, secretion of IFN- by Th1 cells stimulated with anti-CD3 and anti-CD28 mAbs was suppressed a mean (±range) of 44 ± 13% by 10-6 M S1P. The PI-specific PLC inhibitor Et-18-OCH3 at 1 µM reduced 10-6 M S1P suppression of IFN- secretion to 6 ± 5% of control levels, and calcium buffering by 0.2 µM BAPTA-AM similarly reduced 10-6 M S1P suppression of IFN- release to 3 ± 2%. Most S1P GPCRs also interact with Gi to lower [cAMP]i, but pharmacologically induced increases in endogenous [cAMP]i or introduction of exogenous stable and cell-permeant forms of cAMP may prevent or inhibit cellular responses to S1P. The >70-fold increase in [cAMP]i induced by selective inhibition of phosphodiesterase activity and the over 200-fold increase in [cAMP]i attributable to bioconversion of stable exogenous analogues significantly and nearly totally prevented S1P suppression of T cell proliferation (Fig. 3).
S1P prevention of cytokine production by T cells stimulated with anti-CD3 mAb and either the TCR costimulator anti-CD28 mAb or the IL-7 stimulator of homeostatic proliferation did not extend substantially to IL-2 (Figs. 4 and 5). Thus both the naive-memory and activated effector T cell regulatory mechanisms of S1P spare the IL-2 growth and survival functions, to focus control selectively on differentiation-inducing cytokines capable of evoking and maintaining Th1 or Th2 subsets. The relative effectiveness of S1P in preferentially controlling Th1 and Th2 cytokines depends both on the CD4 T cell stimulus, which determines the magnitude of the cytokine response, and the concentration of S1P. Generation of IFN- and IL-4 apparently were both more sensitive to inhibition by S1P when the T cell stimulus was anti-CD3 and IL-7, but the unregulated levels of both cytokines produced were lower in this homeostatic model than in TCR-coreceptor activation of effector T cells (Fig. 4 and 5).
The results of previous analyses of contributions of individual S1PRs to T cell migration relied on overexpression of each S1PR in the same non-T migratory cell line and on partial antagonism by polyspecific drugs and low-affinity Abs. The decreased expression of S1P1 and other S1PRs in spleen-derived and cytokine-deviated Th1 cells compared with freshly isolated splenic CD4 T cells is attributed to persistent TCR stimulation, that also down-regulates S1PRs in balanced mixtures of CD4 T cells (7). This state of depressed expression of all S1PRs in otherwise normal Th cells also provided an opportunity for selectively up-regulating the S1P1R, which had been implicated as the principal transducer of T cell migration.
Retroviral transduction of the mouse Th1 subline with human S1P1R resulted in a striking increment in S1P1R protein in the transductants compared with sham-transduced controls (Fig. 6). As a result, the functional responses of the Th1 cell transductants to S1P were markedly different from those of sham transductants in this system (Fig. 7). The recombinant S1P1R in Th1 cells was coupled to significant, but moderate, suppression of Ag-induced proliferation and generation of IFN-, which attained equivalent maxima of 
30% at 10-6 M S1P (Fig. 7). In contrast, S1P1Rs mediated very prominent effects of S1P on Th1 cell chemotaxis (Fig. 7). The maximal magnitude of chemotactic responses of S1P1 transductants was nearly three times the marginal response of sham transductants to 10-7 M S1P. Even more significant was the almost 6-fold greater inhibition by S1P of CCL-5-induced chemotaxis of S1P1-bearing Th1 cells than sham-transduced Th1 cells. These data thus establish a central role for S1P1Rs in the transduction of suppressive effects of S1P on T cell proliferation and cytokine generation, as well as migration. In contrast to the stimulation of chemotaxis by levels of S1P at or below 100 nM, these levels of S1P did not directly stimulate proliferation or cytokine generation nor significantly enhance proliferation or cytokine generation evoked by either type of TCR-dependent stimulation (Figs. 1, 4, 5, 8). At levels of S1P greater than 0.1 µM, effects on all three responses were suppressive irrespective of the type of stimulus (Fig. 8). Further, it is demonstrated that the suppressive effects of S1P on migration are quantitatively more prominent than those on proliferation and cytokine production (Fig. 1, 4, 5, 8). The two distinct effects of S1P on T cell migration, observed at different concentrations, also were evident in vivo using a cutaneous air-pouch model previously applied to S1P chemotactic studies (7, 8). The introduction of 100 nM CCL21 or 100 nM S1P into air pouches separately, resulted in mean chemotaxis at 24 h of a total of 0.32 x 106 and 0.23 x 106 lymphocytes (n = 4), respectively, relative to a saline background of 0.07 x 106. With 100 nM CCL21 plus 100 nM S1P together, the mean response was 0.45 x 106 and with 100 nM CCL21 plus 3 µM S1P together, the mean response was reduced substantially to 0.09 x 106.
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Acknowledgments |
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Footnotes |
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2 G.D. and M.H.G. contributed equally to the study.
3 Address correspondence and reprint requests to Dr. Edward J. Goetzl, University of California, UB8B, UC Box 0711, 533 Parnassus Avenue at 4th, San Francisco, CA 94143-0711. E-mail address: egoetzl{at}itsa.ucsf.edu
4 Abbreviations used in this paper: S1P, sphingosine 1-phosphate; GPCR, G protein-coupled receptor; Edg, endothelial differentiation gene-encoded receptor; FAF, fatty acid-free; YFP, yellow fluorescent protein; CCL, CC chemokine ligand; [cAMP]i, intracellular concentration of cAMP; LPA, lysophosphatidic acid; PKA, protein kinase A; PLC, phospholipase C; [Ca2+]i, intracellular Ca2+ concentration; PI, phosphatidylinositol.
Received for publication March 10, 2003. Accepted for publication July 25, 2003.
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) and 10-8 M and 10-6 M LPA (
). The range of control baseline values (0%) were 196–393 dpm and control stimulated values (100%) were 80,228–231,964 dpm. The levels of statistical significance are: + = p < 0.05, * = p < 0.01, ** = p < 0.0025. The mean levels of S1P1R mRNA relative to GAPDH were 301 without stimulation, 50 at 24 h after anti-CD3 plus anti-CD28 mAbs, and 64 at 24 h after anti-CD3 plus 10-9 M mouse IL-7.
