Transduction of multiple effects of sphingosine 1-phosphate (S1P) on T cell functions by the S1P1 G protein-coupled receptor

Sphingosine 1-phosphate (S1P) in blood, lymph, and immune tissues stimulates and regulates T cell migration through their S1P(1) (endothelial differentiation gene encoded receptor-1) G protein-coupled receptors. We show now that S1P(1)Rs also mediate suppression of T cell proliferation and cytokine production. Uptake of [(3)H]thymidine by mouse CD4 T cells stimulated with anti-CD3 mAbs plus either anti-CD28 or IL-7 was inhibited up to 50% by 10(-9)-10(-6) M S1P. Suppression by S1P required Ca(2+) signaling and was reduced by intracellular cAMP. S1P decreased CD4 T cell generation of IFN-gamma and IL-4, without affecting IL-2. A Th1 line from D011.10 TCR transgenic mice without detectable S1P(1) was refractory to S1P until introduction of S1P(1) by retroviral transduction. S1P then evoked chemotaxis, inhibited chemotaxis to CCL-5 and CCL-21, and suppressed Ag-stimulated proliferation and IFN-gamma production. Thus, S1P(1) signals multiple immune functions of T cells as well as migration and tissue distribution.     

Th17 augmentation in OTII TCR plus T cell-selective type 1 sphingosine 1-phosphate receptor double transgenic mice

Sphingosine 1-phosphate (S1P) in blood and lymph controls lymphoid traffic and tissue migration of T cells through signals from the type 1 S1PR (S1P(1)), but less is known of effects of the S1P-S1P(1) axis on nonmigration functions of T cells. CD4 T cells from a double transgenic (DTG) mouse express OTII TCRs specific for OVA peptide 323-339 (OVA) and a high level of transgenic S1P(1), resistant to suppression by T cell activation. OVA-activated DTG CD4 T cells respond as expected to S1P by chemotactic migration and reduction in secretion of IFN-gamma. In addition, DTG CD4 T cells stimulated by OVA secrete a mean of 2.5-fold more IL-17 than those from OTII single transgenic mice with concomitantly higher levels of mRNA encoding IL-17 by real-time PCR and of CD4 T cells with intracellular IL-17 detected by ELISPOT assays. OVA challenge of s.c. air pockets elicited influx of more OTII TCR-positive T cells producing a higher level of IL-17 in DTG mice than OTII control mice. Augmentation of the number and activity of Th17 cells by the S1P-S1P(1) axis may thus enhance host defense against microbes and in other settings increase host susceptibility to autoimmune diseases.     

Regulation of the roles of sphingosine 1-phosphate and its type 1 G protein-coupled receptor in T cell immunity and autoimmunity

The lipid mediator sphingosine 1-phosphate (S1P) and its type 1 G protein-coupled receptor (S1P(1)) affect mammalian immunity through alterations in thymocyte emigration, differentiation of T cell subsets, lymphocyte trafficking in lymphoid organs and other tissues, T cell-dendritic cell and T cell-B cell interactions, and cytokine generation. Recent attention to effects of the S1P-S1P(1) axis on non-migration functions of lymphocytes includes delineation of a role in terminal differentiation and survival of Th17 effector cells and adaptive Treg cells of the CD4 T cell constellation, and a greater understanding of interactions of the S1P-S1P(1) axis with immune cytokines in lymphocyte survival and activities. This breadth of involvement of the S1P-S1P(1) axis in immune responses that often are altered in immunological diseases has provided many opportunities for novel therapeutic interventions. A spectrum of pharmacological and immunochemical agents is available that alter immunity by affecting either tissue and fluid concentrations of S1P or levels of expression and signaling activities of S1P(1). Such agents have so far been beneficial in the settings of autoimmunity and rejection of transplanted organs, and are likely to become valuable constituents of combined drug programs.     

Cutting edge: Alternative signaling of Th17 cell development by sphingosine 1-phosphate

Sphingosine 1-phosphate (S1P) in blood and lymph controls T cell traffic and proliferation through type 1 S1P receptor (S1P(1)) signals, but suppression of IFN-gamma generation has been the only consistently observed effect on T cell cytokines. The fact that S1P enhances the development of Th17 cells from Ag-challenged transgenic S1P(1)-overexpressing CD4 T cells suggested that the S1P-S1P(1) axis may promote the expansion of Th17 cells in wild-type mice. In a model of Th17 cell development from CD4 T cells stimulated by anti-CD3 plus anti-CD28 Abs and a mixture of TGF-beta1, IL-1, and IL-6, S1P enhanced their number and IL-17-generating activity the same as IL-23. As for IL-23 enhancement of Th17 cell development, that by S1P was prevented by IL-4 plus IFN-gamma and by IL-27. The prevention of S1P augmentation of Th17 cell development by the S1P receptor agonist and down-regulator FTY720 implies that FTY720 immunosuppression is attributable partially to inhibition of Th17-mediated inflammation.     

Immunological effects of transgenic constitutive expression of the type 1 sphingosine 1-phosphate receptor by mouse lymphocytes

The type 1 sphingosine 1-phosphate (S1P) G protein-coupled receptor (S1P1) normally transduces S1P effects on lymph node (LN) egress and tissue migration of naive lymphocytes. We now show that persistent expression of S1P1 by lymphocytes of S1P1-transgenic (Tg) mice suppresses delayed-type hypersensitivity and results in production of significantly more IgE Ab and less IgG2 Ab than in wild-type (wt) mice. wt host LN homing of 51Cr-labeled T cells from S1P1-Tg mice was only 30-40% of that for wt T cells. Adoptive-transfer of dye-labeled activated T cells from S1P1-Tg mice into wt mice resulted in 2.2-fold more in blood and 60% less in LNs than for activated wt T cells after 1 day. Proliferative responses of stimulated T cells from S1P1-Tg mice were only 10-34% of those for wt T cells. Disordered cellular and humoral immunity of S1P1-Tg mice thus may be attributable to both altered T cell traffic and depressed T cell functions, suggesting that S1P1-specific agonists may represent a novel therapeutic approach to autoimmunity and transplant rejection.     

Migration of CD4 T cells and dendritic cells toward sphingosine 1-phosphate (S1P) is mediated by different receptor subtypes: S1P regulates the functions of murine mature dendritic cells via S1P receptor type 3

Dendritic cells (DCs) and lymphocytes are known to show a migratory response to the phospholipid mediator, sphingosine 1-phosphate (S1P). However, it is unclear whether the same S1P receptor subtype mediates the migration of lymphocytes and DCs toward S1P. In this study, we investigated the involvement of S1P receptor subtypes in S1P-induced migration of CD4 T cells and bone marrow-derived DCs in mice. A potent S1P receptor agonist, the (S)-enantiomer of FTY720-phosphate [(S)-FTY720-P], at 0.1 nM or higher and a selective S1P receptor type 1 (S1P(1)) agonist, SEW2871, at 0.1 muM or higher induced a dose-dependent down-regulation of S1P(1). The pretreatment with these compounds resulted in a significant inhibition of mouse CD4 T cell migration toward S1P. Thus, it is revealed that CD4 T cell migration toward S1P is highly dependent on S1P(1). Mature DCs, when compared with CD4 T cells or immature DCs, expressed a relatively higher level of S1P(3) mRNA. S1P at 10-1000 nM induced a marked migration and significantly enhanced the endocytosis of FITC-dextran in mature but not immature DCs. Pretreatment with (S)-FTY720-P at 0.1 microM or higher resulted in a significant inhibition of S1P-induced migration and endocytosis in mature DCs, whereas SEW2871 up to 100 microM did not show any clear effect. Moreover, we found that S1P-induced migration and endocytosis were at an extremely low level in mature DCs prepared from S1P(3)-knockout mice. These results indicate that S1P regulates migration and endocytosis of murine mature DCs via S1P(3) but not S1P(1).     

Increased nonobese diabetic Th1:Th2 (IFN-gamma:IL-4) ratio is CD4+ T cell intrinsic and independent of APC genetic background

Autoreactive CD4(+) T cells play a major role in the pathogenesis of autoimmune diabetes in nonobese diabetic (NOD) mice. We recently showed that the non-MHC genetic background controlled enhanced entry into the IFN-gamma pathway by NOD vs B6.G7 T cells. In this study, we demonstrate that increased IFN-gamma, decreased IL-4, and decreased IL-10 production in NOD T cells is CD4 T cell intrinsic. NOD CD4(+) T cells purified and stimulated with anti-CD3/anti-CD28 Abs generated greater IFN-gamma, less IL-4, and less IL-10 than B6.G7 CD4(+) T cells. The same results were obtained in purified NOD.H2(b) vs B6 CD4(+) T cells, demonstrating that the non-MHC NOD genetic background controlled the cytokine phenotype. Moreover, the increased IFN-gamma:IL-4 cytokine ratio was independent of the genetic background of APCs, since NOD CD4(+) T cells generated increased IFN-gamma and decreased IL-4 compared with B6.G7 CD4(+) T cells, regardless of whether they were stimulated with NOD or B6.G7 APCs. Cell cycle analysis showed that the cytokine differences were not due to cycle/proliferative differences between NOD and B6.G7, since stimulated CD4(+) T cells from both strains showed quantitatively identical entry into subsequent cell divisions (shown by CFSE staining), although NOD cells showed greater numbers of IFN-gamma-positive cells with each subsequent cell division. Moreover, 7-aminoactinomycin D and 5-bromo-2'-deoxyuridine analysis showed indistinguishable entry into G(0)/G(1), S, and G(2)/M phases of the cell cycle for both NOD and B6.G7 CD4(+) cells, with both strains generating IFN-gamma predominantly in the S phase. Therefore, the NOD cytokine effector phenotype is CD4(+) T cell intrinsic, genetically controlled, and independent of cell cycle machinery.     

Existence of different Fos/Jun complexes during the G0-to-G1 transition and during exponential growth in mouse fibroblasts: differential role of Fos proteins.

We have determined the different Fos/Jun complexes present in Swiss 3T3 cells either following serum stimulation of quiescent cells or during exponential growth by immunoprecipitation analyses. We have shown that while c-Fos is the major Fos protein associated with the Jun proteins (c-Jun, JunB, and JunD) soon after serum stimulation, at later times Fra-1 and Fra-2 are the predominant Fos proteins associated with the different Jun proteins. During exponential growth, the synthesis of Fra-1 and Fra-2 is maintained at a significant level, in contrast to c-Fos and FosB, which are expressed at very low or undetectable levels. Consequently, Fra-1 and Fra-2 are the main Fos proteins complexed with the Jun proteins in asynchronously growing cells. To determine whether the Fos proteins are differentially required during the G0-to-G1 transition and exponential growth for the entrance into S phase, we microinjected affinity-purified antibodies directed against c-Fos, FosB, Fra-1, and Fra-2. We have found that while the activities of c-Fos and FosB are required mostly during the G0-to-G1 transition, Fra-1 and Fra-2 are involved both in the G0-to-G1 transition and in asynchronous growth.     

Ganglioside control over IL-4 priming and cytokine production in activated T cells

Our previous studies have shown that the enzymatic activities of Neu-1, an endogenous sialidase encoded in the murine MHC, are involved in promoting IL-4 synthesis by naive CD4(+)T cells. Our present studies have characterized responsible sialoconjugate targets of Neu-1 and questioned possible biochemical mechanisms responsible for their regulatory influences on IL-4 gene expression. These studies determined that treatment of T cells with the naturally occurring ganglioside GM3 inhibited the production of IL-4 without affecting the production of IL-2. An analysis of IL-4-primed CD4(+)T cells further demonstrated that GM3 treatment specifically inhibited the restimulated production of IL-4, IL-5 and IL-13, without inhibiting the production of IL-2 and IFN-gamma. The inhibitory effects of GM3 could be overcome by treatment with thapsigargin or ionomycin, suggesting ganglioside regulation occurs upstream of activation-induced calcium mobilization. GM3 treatment attenuated the level of calcium influx following CD3epsilon crosslinking, and CD4(+)T cells from Neu-1-deficient B10.SM strain mice (neu-1(a)and IL-4-deficient) expressed reduced levels of intracellular calcium following activation. Our results indicate that activities by membrane gangliosides can influence the cytokine programs in CD4(+)T cells, possibly through the modulation of calcium responses induced by T cell activation.     

Evaluation of murine interleukin 4 (IL-4) receptor expression using anti-receptor monoclonal antibodies and S1 nuclease protection analyses

Anti-receptor antibodies have previously been used in two cytokine systems (IL-1 and TNF alpha) to identify the existence of different cytokine receptors on different cell types. In this study, we have similarly used two approaches to evaluate whether IL-4 receptors on different cell types are identical, or whether more than one species of IL-4 receptor exists. The first approach involved production of monoclonal antibodies specific for the IL-4 receptor expressed by the murine mast cell line, MC/9. Six anti-IL-4 receptor monoclonal antibodies were produced against the purified soluble extracellular domain of the recombinant IL-4 receptor derived from MC/9 cells. These antibodies were capable of binding to and specifically immunoprecipitating the soluble extracellular domain of the recombinant mast cell IL-4 receptor. Following biotinylation of the antibodies and addition of phycoerythrin-streptavidin, their binding to cell associated IL-4 receptors on MC/9 mast cells could be readily visualized by immunofluorescence. Using this approach, the anti-mast cell IL-4R antibodies were found to specifically bind IL-4 receptors expressed on a variety of other murine cell types, including T cells, B cells, macrophages, fibroblasts, and L cells. The antibodies did not bind to two human cell lines known to bind human but not murine IL-4. The intensity of staining was directly related to the number of IL-4 binding sites identified previously by receptor-ligand equilibrium binding analyses. As a second approach to evaluating potential receptor heterogeneity, we constructed S1 nuclease protection assay probes for two separate regions of the mast cell IL-4 receptor, one located in the extracellular domain and one in the intracellular domain. Subsequent S1 analyses showed that both regions are expressed by the following types of cells: T cells, B cells, macrophages, myeloid cells, L cells, and stromal cells. The two approaches used in this study therefore indicate that the same or highly similar IL-4 receptor species is expressed by a wide variety of hemopoietic and nonhemopoietic cells. Since the anti-IL-4 receptor antibodies produced in this study did not block binding of IL-4 to its receptor, we cannot exclude the possible existence of a second type of IL-4R coexpressed on the cells tested in this study, or expressed uniquely by other cell types that were not investigated.     

IL-4 suppression of in vivo T cell activation and antibody production

Injection of mice with a foreign anti-IgD Ab stimulates B and T cell activation that results in large cytokine and Ab responses. Because most anti-IgD-activated B cells die before they can be stimulated by activated T cells, and because IL-4 prolongs the survival of B cells cultured with anti-Ig, we hypothesized that treatment with IL-4 at the time of anti-IgD Ab injection would decrease B cell death and enhance anti-IgD-induced Ab responses. Instead, IL-4 treatment before or along with anti-IgD Ab suppressed IgE and IgG1 responses, whereas IL-4 injected after anti-IgD enhanced IgE responses. The suppressive effect of early IL-4 treatment on the Ab response to anti-IgD was associated with a rapid, short-lived increase in IFN-gamma gene expression but decreased CD4+ T cell activation and decreased or delayed T cell production of other cytokines. We examined the possibilities that IL-4 stimulation of IFN-gamma production, suppression of IL-1 or IL-2 production, or induction of TNF-alpha or Fas-mediated apoptosis could account for IL-4's suppressive effect. The suppressive effect of IL-4 was not reversed by IL-1, IL-2, or anti-TNF-alpha or anti-IFN-gamma mAb treatment, or mimicked by treatment with anti-IL-2Ralpha (CD25) and anti-IL-2Rbeta (CD122) mAbs. Early IL-4 treatment failed to inhibit anti-IgD-induced Ab production in Fas-defective lpr mice; however, the poor responsiveness of lpr mice to anti-IgD made this result difficult to interpret. These observations indicate that exposure to IL-4, while T cells are first being activated by Ag presentation, can inhibit T cells activation or promote deletion of responding CD4+ T cells.