Activation Of Sphingosine-1-phosphate Receptors Research Articles

Sphingosine Kinase Type 2 Is a Putative BH3-only Protein That Induces Apoptosis

There are two isoforms of sphingosine kinase (SphK) that catalyze the formation of sphingosine 1-phosphate, a potent sphingolipid mediator. Whereas SphK1 stimulates growth and survival, here we show that SphK2 enhanced apoptosis in diverse cell types and also suppressed cellular proliferation. Apoptosis was preceded by cytochrome c release and activation of caspase-3. SphK2-induced apoptosis was independent of activation of sphingosine 1-phosphate receptors. Sequence analysis revealed that SphK2 contains a 9-amino acid motif similar to that present in BH3-only proteins, a pro-apoptotic subgroup of the Bcl-2 family. As with other BH3-only proteins, co-immunoprecipitation demonstrated that SphK2 interacted with Bcl-xL. Moreover, site-directed mutation of Leu-219, the conserved leucine residue present in all BH3 domains, markedly suppressed SphK2-induced apoptosis. Hence, the apoptotic effect of SphK2 might be because of its putative BH3 domain.

Sphingosine 1-phosphate receptor activation: evidence for distinct chemokine compartments

Introduction: FTY720 is a sphingosine derived immunomodulator that causes T cell egress from peripheral blood and spleen to peripheral lymph nodes through a mechanism dependent on activation of the sphingosine 1-phosphate receptor and sensitization of the CCL19/CCR7 chemokine system. The purposes of this study were to determine if FTY720 enhances T cell migration to other chemokine systems and by what mechanism, and if a chemokine dependent compartmental model of FTY720 enhanced T cell migration exists. Methods: In vivo migration assays were performed with wild type or various knockout (KO) mice strains receiving FTY720 (0.3 mg/kg by gavage); thymus, peripheral blood, spleen, and peripheral lymph nodes were harvested after 18 hours; and T cell distribution was determined by flow cytometry. Control mice received water only. Adoptive transfer assays were performed with CSFE labelled wild type or KO splenocytes injected into wild type or various KO mice strains. All mice were transferred with 2.0 × 107 splenocytes; the experimental group received FTY720, the control group received water. Peripheral blood, spleen, and peripheral lymph nodes were harvested after 18 hours and T cell distribution was determined by flow cytometry. All experiments were performed 3 times with 3 mice per experimental and control groups. Standard deviations and p-values were calculated with Student’s t-test. Results: As shown in Figure 1, in wild-type but not CCR2−/−, CCL2−/−, or plt mice total T cell numbers were reduced in the thymus following FTY720 administration compared to controls. Peripheral blood total T cell numbers were significantly reduced in all mice strains but plt mice had statistically more total T cells then wild type, CCR2−/−, or CCL2−/−. Sequestration of T cells in LN occurred in wild type, plt, and CCL2−/− but not CCR2−/− mice compared to controls; T cell accumulation was significantly greater in wild type compared to plt or CCL2−/− mice. T cell splenic egress was seen in wild type, plt, CCR2−/−, and CCL2−/− strains compared to controls. As shown in Figure 2, CCR2−/− transferred T cells fail to accumulate in the LN in both wild type and plt recipients following FTY720 administration compared to controls and to B6 T cells transferred into the same strains. Conclusions: We conclude that FTY720 enhancement of thymic T cell egress is dependent on the CCR7 and CCR2 chemokine system. LN T cell sequestration is dependent on both CCR2 and CCR7 chemokine systems but also display a CCR7 and CCR2 independent method of T cell accumulation. FTY720 enhance T cell migration occurs in a compartmentalized fashion where the thymus, peripheral blood, peripheral lymph node, and spleen compartments appear to be independent of one another.

Sphingosine-1-phosphate receptor agonism impairs the efficiency of the local immune response by altering trafficking of naive and antigen-activated CD4+ T cells.

FTY720 (2-amino-[2-(4-octylphenyl) ethyl]-1,3-propanediol hydrochloride) is an immunosuppressive agent that inhibits allograft rejection. We recently demonstrated that FTY-phosphate, the active metabolite of FTY720, acts as a full agonist for sphingosine-1-phosphate (S1P) receptors. Furthermore, activation of S1P receptors with their natural ligand, S1P, as well as pharmacological ligands leads to lymphopenia, probably due to sequestration of lymphocytes in secondary lymphoid organs. In the present study we used a local Ag-challenged mouse model to examine the effects of FTY720 on T cell activation in the draining lymph node (DLN) and on the release of activated T cells to the peripheral blood compartment. We showed that the number of Ag-activated CD4(+) T cells in the DLN after injection of Ag and CFA into a footpad was dramatically reduced after FTY720 treatment. However, T cell proliferation, both in vitro and in vivo, was not impaired by FTY720. Our results suggest that the reduced efficiency of T cell responses in the DLN in response to a local Ag is probably due to a defective recirculation of naive T cells caused by FTY720 treatment. Furthermore, we found that the numbers of naive and Ag-activated CD4(+) T cells in the peripheral blood of Ag-challenged mice were equally reduced with FTY720 treatment, suggesting that both T cell subsets are sequestered in the DLNs. Thus, FTY720 induces immunosuppression through inhibition of both the recirculation of naive T cells and the release of Ag-activated T cells from the DLN to lymph and to the blood compartment.

The Immune Modulator FTY720 Targets Sphingosine 1-Phosphate Receptors

Immunosuppressant drugs such as cyclosporin have allowed widespread organ transplantation, but their utility remains limited by toxicities, and they are ineffective in chronic management of autoimmune diseases such as multiple sclerosis. In contrast, the immune modulating drug FTY720 is efficacious in a variety of transplant and autoimmune models without inducing a generalized immunosuppressed state and is effective in human kidney transplantation. FTY720 elicits a lymphopenia resulting from a reversible redistribution of lymphocytes from circulation to secondary lymphoid tissues by unknown mechanisms. Using FTY720 and several analogs, we show now that FTY720 is phosphorylated by sphingosine kinase; the phosphorylated compound is a potent agonist at four sphingosine 1-phosphate receptors and represents the therapeutic principle in a rodent model of multiple sclerosis. Our results suggest that FTY720, after phosphorylation, acts through sphingosine 1-phosphate signaling pathways to modulate chemotactic responses and lymphocyte trafficking.

Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists.

Blood lymphocyte numbers, essential for the development of efficient immune responses, are maintained by recirculation through secondary lymphoid organs. We show that lymphocyte trafficking is altered by the lysophospholipid sphingosine-1-phosphate (S1P) and by a phosphoryl metabolite of the immunosuppressive agent FTY720. Both species were high-affinity agonists of at least four of the five S1P receptors. These agonists produce lymphopenia in blood and thoracic duct lymph by sequestration of lymphocytes in lymph nodes, but not spleen. S1P receptor agonists induced emptying of lymphoid sinuses by retention of lymphocytes on the abluminal side of sinus-lining endothelium and inhibition of egress into lymph. Inhibition of lymphocyte recirculation by activation of S1P receptors may result in therapeutically useful immunosuppression.