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Type 4 sphingosine 1-phosphate G protein-coupled receptor (S1P₄) transduces S1P effects on T cell proliferation and cytokine secretion without signaling migration

Departments of Medicine and Microbiology-Immunology, University of California, San Francisco, California, USA

¹ Correspondence: University of California, UB8B, Box 0711, 533 Parnassus at 4th Ave., San Francisco, CA 94143-0711, USA. E-mail:egoetzl{at}itsa.ucsf.edu

SPECIFIC AIMS

S1P₄ is expressed principally by leukocytes of the immune system, but the nature and mechanisms of its T cell effects in immunity have not been elucidated. The aim of this study was to investigate for the first time the functions of S1P transduced by S1P₄ in T cells.

PRINCIPAL FINDINGS

1. S1P₄ does not mediate S1P effect on T cell migration
We used two model systems to investigate the cellular functions transduced by S1P₄ in T cells. In the first model S1P₄ was introduced into two mouse T cell lines termed D10G4.1 and EL-4.IL-2, which express only marginally detectable levels of endogenous S1P GPCRs. S1P₄ mRNA and protein expression after transfection indicated that S1P₄ became the major S1P GPCR and the expression level is similar to that in primary splenic CD4 T cells, without any change in the levels of S1P₁ or any other S1P GPCRs. In the second model, CD4 T cells isolated from C57BL/6 mice were treated with 100 nM FTY720 for 16 h at 37°C. It had been demonstrated that under this condition S1P₁ is down-regulated, and S1P₄ is the only residual S1P receptor then expressed at relevant levels.

The major essential function of T cells evoked and regulated by S1P GPCRs is chemotaxis. S1P₁, the quantitatively predominant of two S1P GPCRs on T cells, has been demonstrated to influence T cell migration in vitro and trafficking in vivo. We examined whether S1P₄ might mediate T cell migration to S1P, chemotaxis, and inhibition of chemotaxis to chemokines. Migration effects transduced by S1P₄ were first examined in S1P₄-transfected D10G4.1 and EL-4.IL-2 cells. No significant chemotactic migration of S1P₄-D10G4.1 cells or S1P₄-EL-4.IL-2 cells was elicited by 10^–9 M to 10^–6 M S1P. Chemotaxis to the chemokine CCL21 was neither enhanced by lower concentrations of S1P nor inhibited by higher concentrations of S1P, as seen in S1P₁-transfected Th1 cells. FTY720-treated mouse splenic naive CD4 T cells only express substantive levels of S1P₄. There were no significant changes in migration of FTY720-treated CD4 T cells toward different concentrations of S1P. Migration of FTY720-treated CD4 T cells to chemokine CCL21 was not changed in the presence of different concentrations of S1P. This confirms our findings in T cell transfectants that S1P₄ does not play any role in migration.

2. S1P₄ mediates part of the S1P effect on T cell proliferation
S1P inhibits the proliferation of several types of mouse and human CD4 T cells and peripheral blood mixed T cells by respective means of up to 50% and 80%. Effects mediated by S1P₄ on T cell proliferation were examined using S1P₄-D10G4.1, S1P₄-EL-4.IL-2, and S1P₄-predominant FTY720-treated CD4 cells. The proliferation of S1P₄-D10G4.1 and S1P₄-EL-4.IL-2 T cells evoked by anti-CD3 plus anti-CD28 MoAbs was inhibited in a dose dependence fashion by 10^–9 to 10^–6 M S1P (Fig. 1 A, B). At 10^–7 M, S1P gave maximum inhibition of 48% and 46% for S1P4-D10G4.1 and S1P₄-EL-4.IL-2 cells, respectively (Fig. 1A, B ). Similarly, 10^–9 to 10^–6 M S1P inhibited proliferation of FTY720-treated CD4 T cells in S1P concentration dependence fashion and 10^–7 M S1P gave maximum inhibition of 47% (Fig. 1C ). These data show that S1P₄ transduces an inhibitory effect of S1P on T cell proliferation.

View larger version (15K): [in this window] [in a new window]   Figure 1. S1P4-mediated inhibitory effect of S1P on T cell proliferation. Cells were cultured in 10% charcoal-absorbed (S1P free) FBS with exogenous S1P. FTY720-treated CD4 T cells, S1P4-D10G4.1 T cells, and S1P4-EL-4.IL-2 T cells cultured without S1P (controls) incorporated 14963 ± 833, 15320 ± 783, and 25110 ± 1662 cpm of 3H-thymidine, respectively (mean±SD, n=3). Percent inhibition was calculated as 1-(cpm from cells cultured in the indicated concentrations of S1P/cpm from cells cultured in the absence of S1P) x 100% at each concentration of S1P. S1P concentration dependence inhibition of the proliferation of A)S1P4 -D10G4.1 T cells, B) S1P4-EL-4.IL-2 T cells, or C) FTY720-treated CD4 T cells. A 2-tailed t test was used to determine the significance of differences between samples and controls. *P< 0.05; **P< 0.01.

3. S1P₄ mediates some of the S1P effects of S1P on regulation of cytokine secretion
S1P regulates T cell cytokine secretion in part through S1P₁ signal transduction. The effects of the S1P-S1P₄ axis on secretion of cytokines were examined using S1P₄-D10G4.1 and S1P₄-EL-4.IL-2 cells. Of the major defining cytokines secreted by Th2 cells, D10G4.1 Th2 cells generate IL-4 and IL-10. S1P₄-D10G4.1 T cell secretion of IL-4 elicited by anti-CD3 plus anti-CD28 antibodies was significantly suppressed by 10^–8 and 10^–7 M S1P (Fig. 2 A). Maximum suppression was 45% by 10^–7 M S1P. In contrast, S1P stimulated IL-10 secretion by S1P₄-D10G4.1 cells, (Fig. 2B ). IL-10 secretion was significantly enhanced by 10^–8 to 10^–6 M S1P. A maximal stimulatory effect of 155% was attained by 10^–7 M S1P. Secretion of the principal T cell growth and trophic factor, IL-2, by S1P₄-EL4.IL-2 cells was significantly inhibited by 10^–7 and 10^–6 M S1P (Fig. 2C ). A maximal inhibitory effect of 52% was attained by 10^–7 M S1P. Effects mediated by S1P₄ on IFN- secretion by primary splenic CD4 T cells were also investigated here using FTY720-treated CD4 T cells. IFN- secretion was decreased by 10^–8–10^–6 M S1P in FTY720-treated CD4 T cells (Fig. 2D ). 10^–6 M S1P significantly suppressed of IFN- secretion at a maximum level of 31%.

View larger version (17K): [in this window] [in a new window]   Figure 2. S1P4-mediated regulatory effects of S1P on T cell cytokine secretion. A) S1P concentration dependence of inhibition of IL-4 secretion by S1P4-D10G4.1 cells. Concentrations of IL-4 secreted by S1P4-D10G4.1 cells cultured without S1P (controls) were 1620 ± 33 pg/mL (mean±SD, n=3). B) S1P concentration dependence of stimulation of IL-10 secretion by S1P4 -D10G4.1 cells. The concentrations of IL-10 secreted by S1P4-D10G4.1 cells cultured without S1P (controls) were 1614±23 pg/mL, respectively (mean±SD, n=3). C) S1P concentration dependence of inhibition of IL-2 secretion by S1P4–EL4.IL-2 cells. Concentrations of IL-2 secreted by S1P4-EL4.IL-2 cells cultured without S1P (controls) were 6578±209 pg/mL (mean±SD, n=3). D) FTY720 treatment partially blocked S1P inhibition of IFN- secretion by primary CD4 T cells. The concentrations of IFN- secreted by FTY720-treated CD4 T cells cultured without S1P (controls) were 1441 ± 80 pg/mL (mean±SD, n=3). The meaning of each column, bar, and statistical symbol are the same as in Fig. 1 .

4. S1P₄-mediated regulation of cytokine secretion contributes to the S1P₄-transduced inhibitory effect of S1P on T cell proliferation
To examine the hypothesis that the S1P-S1P₄ axis suppresses proliferation of T cells in part by inhibiting secretion of stimulatory cytokines and enhancing that of suppressive cytokines, IL-4 or antibodies capable of neutralizing IL-10 or IL-10 receptor were added in the culture medium of S1P₄-D10G4.1 cells, which had been incubated with 10^–7 M S1P for the maximal regulatory effects on cytokine secretion. IL-4 was added in concentrations of 0.5 to 2 ng/mL to mimic the range established by stimulated S1P₄-D10G4.1 T cells secreting IL-4. Neutralizing anti IL-10 or IL-10 receptor antibody was added at 10 µg/well. The maximal inhibitory effect of S1P on D10G4.1 T cell proliferation was decreased significantly by addition of 1 or 2 ng/mL of IL-4 and by addition of 10 µg/well of anti IL-10 or 10 µg/well of anti IL-10 receptor antibody alone. The inhibitory effect was nearly abolished when IL-4 and anti IL-10 or IL-10 receptor antibody were added together. IL-2 was added from 0.5 to 5 ng/mL, at which range S1P₄-EL4.IL-2 T cells secrete IL-2. The inhibitory effect of 10^–7 M S1P-conditioned T cell medium on S1P4-EL-4.IL-2 T cells was decreased when 1–5 ng/mL of IL-2 was added to the culture medium. Effects on the proliferation of splenic naive CD4 cells of supernatants from cultures of S1P₄-D10G4.1 or EV-D10G4.1 T cells were quantified. Proliferation of CD4 T cells was inhibited significantly by a mean of 44% by culture supernatant from S1P₄-D10G4.1 T cells in 10^–7 M S1P, which has a lower than EV control concentration of IL-4, but a higher than EV-control concentration of IL-10. Thus, S1P₄ transduction of S1P regulatory effects on cytokine secretion contribute to S1P suppression of cell proliferation.

CONCLUSIONS AND SIGNIFICANCE

S1P is an omnific lysophospholipid in the immune system, with diverse effects on T cell proliferation, survival, migration, and other functions. The predominant S1P GPCRs of T cells are S1P₁ and S1P_4. S1P₁ and S1P₄ differ in S1P binding affinity and signal transduction mechanisms. S1P₁ has a mean K_D of 5–8 nM and couples to Gi/o, whereas the respective descriptors for S1P₄ are 60 nM, and Gi and G_12/13. Many reports describe protean T cell effects of the S1P-S1P₁ axis with major effects on migration and trafficking of naive T cells and B cells, but much less influence on proliferation and synthetic functions. Despite the restriction of expression of S1P₄ to immune cells, its mechanisms of signal transduction and functional effects in immune cells were not known. Our results reported here describe for the first time that S1P₄ does not affect any aspect of migration of T cells. In contrast, S1P₄ transduces distinctive effects of S1P on T cell proliferation and T cell cytokine generation. The S1P-S1P₄ axis significantly suppresses proliferation of both mouse naive CD4 T cells and S1P₄-transfected T cells to a far greater extent than any signal from the S1P-S1P₁ axis. The S1P-S1P₄ axis also suppresses generation of IL-2, IFN-, and IL-4 and reciprocally enhances that of IL-10 in the same S1P₄-only T cells (Fig. 2 ). The IFN- secretion by untreated CD4 T cells was decreased by a moderately greater extent than in FTY720 treated cells, which could be due to the combined effects mediated by S1P₄ and S1P₁. In contrast, the S1P-S1P₁ axis has no effect on IL-2 and suppresses generation of IFN- moderately, with only minimal suppression of IL-4 or IL-10.

Our findings imply that the integrated effect of S1P-S1P₄ signaling is suppression of T cell proliferation and presumably other aspects of immune activation (Fig. 3 ). S1P₄ transduces inhibitory and stimulatory effects on T cell secretion of several critical cytokines. IL-4, IFN-, and IL-2 generation are suppressed significantly by S1P and that of IL-10 is enhanced by S1P in S1P₄-only T cells. It has been reported that the IL-10 secreted by Th2 cells can inhibit activation of Th2 cells themselves whereas IL-2 and IL-4 support T cell activation. Therefore the integrated effects on cytokines contributes to suppression of T cell activities by the S1P-S1P₄ axis. This is apparent in the role of cytokines in inhibition of T cell proliferation by the S1P-S1P₄ axis. Upon TCR activation, S1P₄ expression is dramatically down-regulated in T cells. By adding IL-4, IL-2 or neutralizing antibodies against IL-10 or IL-10 receptor, it was demonstrated that S1P₄-mediated regulation of cytokine secretion overall contributes to the inhibition of proliferation and potentially alters the Th2/Th1 ratio.

View larger version (15K): [in this window] [in a new window]   Figure 3. Schematic diagram depicting the roles of S1P-S1P4 axis in T cells. Solid line with end-T = suppression, Solid line with end-arrow = enhancement; double slash = no effects.

Our data demonstrate that T cells need two different S1P GPCRs to transduce various cellular functions under different physiological conditions. At the normal concentrations of 0.1–0.3 µM S1P in blood and lymph, S1P mediates and modulates T cell migration via S1P₁ and affects T cell proliferation and cytokine secretion by S1P₄. Ambient tissues appear to contain nanomolar S1P, at which concentration it usually evokes T cell migration by S1P₁, and has minimal inhibitory effects on cell proliferation or cytokine secretion by S1P₁. The integrated effects of concurrent transduction of S1P signals by both S1P₁ and S1P₄ remain to be elucidated in naive T cells. However, it is clear that S1P and its GPCRs have the potential to regulate both T cell migration and homing through S1P₁ and T cell proliferation and cytokine profiles through S1P₄.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3730fje; doi: 10.1096/fj.05-3730fje

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