Adaptive immunity depends on T-cell exit from the thymus and T and B cells travelling between secondary lymphoid organs to survey for antigens. After activation in lymphoid organs, T cells must again return to circulation to reach sites of infection; however, the mechanisms regulating lymphoid organ exit are unknown. An immunosuppressant drug, FTY720, inhibits lymphocyte emigration from lymphoid organs, and phosphorylated FTY720 binds and activates four of the five known sphingosine-1-phosphate (S1P) receptors. However, the role of S1P receptors in normal immune cell trafficking is unclear. Here we show that in mice whose haematopoietic cells lack a single S1P receptor (S1P1; also known as Edg1) there are no T cells in the periphery because mature T cells are unable to exit the thymus. Although B cells are present in peripheral lymphoid organs, they are severely deficient in blood and lymph. Adoptive cell transfer experiments establish an intrinsic requirement for S1P1 in T and B cells for lymphoid organ egress. Furthermore, S1P1-dependent chemotactic responsiveness is strongly upregulated in T-cell development before exit from the thymus, whereas S1P1 is downregulated during peripheral lymphocyte activation, and this is associated with retention in lymphoid organs. We find that FTY720 treatment downregulates S1P1, creating a temporary pharmacological S1P1-null state in lymphocytes, providing an explanation for the mechanism of FTY720-induced lymphocyte sequestration. These findings establish that S1P1 is essential for lymphocyte recirculation and that it regulates egress from both thymus and peripheral lymphoid organs.

Sphingosine-1-phosphate (S1P), a lipid signaling molecule that regulates many cellular functions, is synthesized from sphingosine and ATP by the action of sphingosine kinase. Two such kinases have been identified, SPHK1 and SPHK2. To begin to investigate the physiological functions of sphingosine kinase and S1P signaling, we generated mice deficient in SPHK1. Sphk1 null mice were viable, fertile, and without any obvious abnormalities. Total SPHK activity in most Sphk1؊/؊ tissues was substantially, but not completely, reduced indicating the presence of multiple sphingosine kinases. S1P levels in most tissues from the Sphk1؊/؊ mice were not markedly decreased. In serum, however, there was a significant decrease in the S1P level. Although S1P signaling regulates lymphocyte trafficking, lymphocyte distribution was unaffected in lymphoid organs of Sphk1؊/؊ mice. The immunosuppressant FTY720 was phosphorylated and elicited lymphopenia in the Sphk1 null mice showing that SPHK1 is not required for the functional activation of this sphingosine analogue prodrug. The results with these Sphk1 null mice reveal that some key physiologic processes that require S1P receptor signaling, such as vascular development and proper lymphocyte distribution, can occur in the absence of SPHK1.Sphingosine-1-phosphate (S1P) 1 is a signaling molecule that influences cellular functions including proliferation, survival, migration, adhesion molecule expression, and morphogenesis (1-4). S1P binds to members of the S1P receptor family (also known as EDG receptors) and, via G proteins, triggers multiple signaling pathways (5, 6). S1P has also been shown to function intracellularly mediating calcium homeostasis, cell growth, and suppression of apoptosis (7,8). In mammals, vascular development and lymphocyte trafficking are dependent on S1P receptor signaling (9 -13).Sphingosine kinase (SPHK) catalyzes the synthesis of S1P via the phosphorylation of sphingosine. SPHK activity is elevated by several stimuli, including platelet-derived growth factor, vascular endothelial growth factor, tumor necrosis factor-␣, and phorbol ester, which trigger an increase in cellular S1P levels (14). Sphk genes have been identified in mammals (15-18), insects (19), plants (20), yeast (21), worm (22), and slime mold (23, 24). Mammals carry two known SphK genes, which in mice are encoded by Sphk1 and Sphk2. The two enzymes contain five highly conserved regions (C1-C5) and an ATP binding site within a conserved lipid kinase catalytic domain (15, 16). SPHK1 has a predominantly cytoplasm localization but can be induced to localize to the inner leaflet of the plasma membrane. Interestingly in endothelial cells SPHK1 is secreted and is capable of producing S1P extracellularly (25). Sphk1 shows a tissue distribution and developmental expression pattern different from Sphk2, although both enzymes are widely expressed (16,26).The importance of S1P receptor signaling in lymphocyte trafficking was first illuminated by the activities of FTY720, a potent immunosuppressive agent. FT...

S1P 1 is a widely distributed G protein-coupled receptor whose ligand, sphingosine 1-phosphate, is present in high concentrations in the blood. The sphingosine 1-phosphate receptor-signaling pathway is believed to have potent effects on cell trafficking in the immune system. To determine the precise role of the S1P 1 receptor on T-cells, we established a T-cell-specific S1P 1 knock-out mouse. The mutant mice showed a block in the egress of mature T-cells into the periphery. The expression of the S1P 1 receptor was up-regulated in mature thymocytes, and its deletion altered the chemotactic responses of thymocytes to sphingosine 1-phosphate. The results indicated that the expression of the S1P 1 receptor on T-cells controls their exit from the thymus and entry into the blood and, thus, has a central role in regulating the numbers of peripheral T-cells.Sphingolipids are important signaling molecules in a variety of biologic contexts (1-3). Sphingosine 1-phosphate, in one paradigm, binds to members of a family of G protein-coupled receptors (S1P 1-15 ) triggering diverse effects including proliferation, survival, migration, morphogenesis, adhesion molecule expression, and cytoskeletal changes (4 -8).S1P 1 /Edg 1 , the first of these receptors described (9, 10), couples to a G i pathway. During embryonic development, the S1P 1 receptor is highly expressed in the vascular system. Gene disruption in mice has demonstrated an essential function of the S1P 1 receptor in endothelial cells for the formation of a stable vascular network (11,12).The S1P 1 receptor is widely expressed in the adult, in particular in endothelial cells, the brain, and the heart, and also in the cells of the immune system (13, 14). S1P 1 and S1P 4 receptors have been detected in T-lymphocytes (15-17). Stimulation of receptor signaling has been found to mediate and regulate cell migration and suppress proliferation and cytokine production (17, 18). Recently, S1P 1 receptors have also been implicated in lymphocyte trafficking and homing as the result of studies using FTY720, a potent immunosuppressive agent. FTY720, a sphingosine analogue, is phosphorylated by sphingosine kinase type I and more efficiently by sphingosine kinase type II (19 -21) and functions as an agonist ligand for S1P 1 , S1P 3 , S1P 4 , and S1P 5 receptors (19,22). FTY720 causes lymphopenia through sequestration of circulating lymphocytes within lymph nodes and Peyer's patches (19,(22)(23)(24) and also blocks the egress of T-cells from the thymus (25, 26). However, it is not known at which S1P receptor and cell type FTY720 exerts its effects.To begin to address the physiologic function of the S1P 1 receptor and sphingosine 1-phosphate signaling in T-cells, we have generated a T-cell-specific mouse knock-out of the S1P 1 receptor using the Cre/loxP system. Our results with these mice reveal a crucial role for the S1P 1 receptor in the egress of mature T-cells from the thymus into the circulation. EXPERIMENTAL PROCEDURESGeneration of the S1P 1 loxP/loxP Lck-Cre Mice-We previously g...

Sphingosine-1-phosphate (S1P) elicits diverse cellular responses through a family of G-protein-coupled receptors. We have shown previously that genetic disruption of the S1P 1 receptor, the most widely expressed of the family, results in embryonic lethality because of its key role within endothelial cells in regulating the coverage of blood vessels by vascular smooth muscle cells. To understand the physiologic functions of the two other widely expressed S1P receptors, we generated S1P 2 and S1P 3 null mice. Neither the S1P 2 null mice nor the S1P 3 null mice exhibited significant embryonic lethality or obvious phenotypic abnormalities. To unmask possible overlapping or collaborative functions between the S1P 1 , S1P 2 , and S1P 3 receptors, we examined embryos with multiple S1P receptor mutations. We found that S1P 1 S1P 2 double null and S1P 1 S1P 2 S1P 3 triple null embryos displayed a substantially more severe vascular phenotype than did embryos with only S1P 1 deleted. We also found partial embryonic lethality and vascular abnormalities in S1P 2 S1P 3 double null embryos. Our results indicate that the S1P 1 , S1P 2 , and S1P 3 receptors have redundant or cooperative functions for the development of a stable and mature vascular system during embryonic development. Sphingosine-1 phosphate (S1P)1 is a sphingolipid metabolite that is present at high levels in the blood (1-3). Through the interaction with a family of five G-protein-coupled receptors (S1P 1-5 ), originally known as EDG receptors, sphingosine-1-phosphate triggers diverse cellular responses, including cytoskeletal changes, proliferation, and migration (1, 4 -8). The S1P 1 , S1P 2 , and S1P 3 receptors are widely expressed, including on embryonic endothelial cells (Table I) (9 -14). S1P 4 and S1P 5 receptor expression is more restricted and found on the cells of the immune and nervous systems (15, 16). The S1P 1 receptor couples selectively to the G i signaling pathway, whereas the S1P 2 and S1P 3 receptors both couple to the G i , G q , and G 12/13 pathways (2,(17)(18)(19)(20). In addition to these five S1P receptors, GPR3, GPR6, GPR12, and GPR63 have been characterized as G-protein-coupled receptors that interact with sphingosine-1-phosphate (21-23).The major physiological effects of S1P receptor signaling defined thus far have been localized to the immune and vascular systems. A global deletion of the S1P 1 receptor in mice results in lethality beginning at E12.5 due to severe hemorrhage as the result of deficient coverage of vessels by vascular smooth muscle cells, a process that occurs during the last stages of angiogenesis and is necessary for stabilizing the vascular system (14). Through analysis of endothelial cell-specific S1P 1 receptor knock-out mice, we have shown that the S1P 1 receptor functions within endothelial cells to regulate vascular smooth muscle cell coverage (24). The function of the S1P 1 receptor in the developing vasculature is also essential for proper limb development (25). Deletion of the S1P 1 receptor in T-cells has re...