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...

Sphingosine-1-phosphate (S1P) stimulates signaling pathways via G-proteincoupled receptors and triggers diverse cellular processes, including growth, survival, and migration. In S1P 1 receptordeficient embryos, blood vessels were incompletely covered by vascular smooth muscle cells (VSMCs), indicating the S1P 1 receptor regulates vascular maturation.Because S1P 1 receptor expression is not restricted to a particular cell type, it was not known whether the S1P 1 receptor controlled VSMC coverage of vessels in a cell-autonomous fashion by functioning directly in VSMCs or indirectly through its activity in endothelial cells (ECs). By using the Cre/loxP system, we disrupted the S1P 1 gene solely in ECs. The phenotype of the conditional mutant embryos mimicked the one obtained in the embryos globally deficient in S1P 1 . Thus, vessel coverage by VSMCs is directed by the activity of the S1P 1 receptor in ECs. IntroductionEmbryonic blood vessel development occurs via vasculogenesis, angiogenesis, and maturation. [1][2][3] In vasculogenesis, endothelial cells (ECs) differentiate de novo and form the primary vascular plexus. In angiogenesis, the pre-existing network is remodeled by splitting and sprouting, producing a complex vascular tree of variably sized vessels. Finally, during maturation vascular smooth muscle cells (VSMCs) differentiate and are recruited to the channel wall to stabilize the new vessel, to protect it against rupture, and to provide hemostatic control. This final maturation process requires cell-to-cell communication and interactions between ECs and VSMCs. 4,5 Although many of the signaling pathways that become engaged during blood vessel formation have been defined, 1-3,6-14 the pathways regulating the interactions between ECs and VSMCs during vascular maturation, especially in larger vessels, remain unclear. The sphingosine-1-phosphate receptor (S1P 1 ) (formally known as Edg1) was shown to be essential for vascular maturation during embryonic development. 15 S1P 1 is a widely distributed G-proteincoupled receptor for sphingosine-1-phosphate, a blood-borne bioactive lipid. Stimulation of the S1P 1 receptor triggers a Gi-linked pathway, leading to growth, survival, migration, and morphogenesis. [16][17][18] Disruption of the S1P 1 gene in mice caused embryonic lethality because of massive hemorrhage at embryonic day (E) 12.5 to E14.5. 15 Although in the S1P 1 Ϫ/Ϫ embryos, VSMCs were in the vicinity of aortae and other vessels, they failed to completely and productively surround the nascent endothelial tubes. Because the S1P 1 receptor is expressed in both ECs and VSMCs, the cell type in which the receptor regulated vascular maturation was uncertain. It was not known whether the S1P 1 receptor controlled VSMC coverage of vessels in a cell-autonomous fashion by functioning directly in VSMCs or indirectly through its activity in ECs.To address this issue we have established mice with the S1P 1 gene deleted specifically in ECs. Our results demonstrate that vessel coverage by VSMCs is directed by the ac...

Although much is known about the migration of T cells from blood to lymph nodes, less is known about the mechanisms regulating the migration of T cells from tissues into lymph nodes through afferent lymphatics. Here we investigated T cell egress from nonlymphoid tissues into afferent lymph in vivo and developed an experimental model to recapitulate this process in vitro. Agonism of sphingosine 1-phosphate receptor 1 inhibited the entry of tissue T cells into afferent lymphatics in homeostatic and inflammatory conditions and caused the arrest, mediated at least partially by interactions of the integrin LFA-1 with its ligand ICAM-1 and of the integrin VLA-4 with its ligand VCAM-1, of polarized T cells at the basal surface of lymphatic but not blood vessel endothelium. Thus, the increased sphingosine 1-phosphate present in inflamed peripheral tissues may induce T cell retention and suppress T cell egress.

Gangliosides are a family of glycosphingolipids that contain sialic acid. Although they are abundant on neuronal cell membranes, their precise functions and importance in the central nervous system (CNS) remain largely undefined. We have disrupted the gene encoding GD3 synthase (GD3S), a sialyltransferase expressed in the CNS that is responsible for the synthesis of b-series gangliosides. GD3S؊/؊ mice, even with an absence of b-series gangliosides, appear to undergo normal development and have a normal life span. To further restrict the expression of gangliosides, the GD3S mutant mice were crossbred with mice carrying a disrupted GalNAcT gene encoding ␤1,4-N-acetylgalactosaminyltransferase. These double mutant mice expressed GM3 as their major ganglioside. In contrast to the single mutant mice, the double mutants displayed a sudden death phenotype and were extremely susceptible to induction of lethal seizures by sound stimulus. These results demonstrate unequivocally that gangliosides play an essential role in the proper functioning of the CNS.Gangliosides are glycosphingolipids that contain sialic acid (reviewed in Ref. 1). They are found on the external leaflet of the plasma membrane on eucaryotic cells and are most abundant in the central nervous system (CNS) 1 where they represent the major sialoglycoconjugate. Because of their dramatic changes in expression during neuronal development and differentiation (3-6), as well as their prominence in the mature CNS, gangliosides have long been assumed to have fundamental roles in CNS development and function.In the ganglioside biosynthetic pathway (1) (see Fig. 2A), lactosylceramide serves as the core structure. The first ganglioside synthesized, GM3, 2 is produced by the transfer of an ␣2,3-linked sialic acid residue to lactosylceramide. Subsequently, GM3 can be modified by the action of ␤1,4-N-acetylgalactosaminyltransferase (GalNAcT, EC 2.4.1.92) to produce GM2 and other complex gangliosides. Alternatively, GM3 can be modified by the action of GD3 synthase (CMP-sialic acid: GM3 ␣-2,8-sialyltransferase, EC 2.4.99.8) to produce the disialoganglioside GD3, which diverts the pathway to the synthesis of b-and c-series gangliosides. Gene targeting in mice has been a particularly fertile approach for uncovering the functions of gangliosides in the CNS. Disruption of the GalNAcT gene (7) blocks the synthesis of complex gangliosides and results in the expression of only the simple gangliosides GM3 and GD3. Surprisingly, these mutant mice are viable, with a normal life span and a CNS that is largely intact both morphologically and functionally (8, 9). These mice do, however, exhibit an agerelated dysmyelination process that is associated with axonal degeneration (10). The mechanism for dysmyelination may be the absence of neuronal ganglioside ligands for myelin-associated glycoprotein (MAG) resulting in myelin instability. Ultimately, motor defects are observed in aged, 12-month-old GalNAcTϪ/Ϫ mice, suggesting a role for complex gangliosides in long-term CNS maintenance ...

The cleavage of sphingoid base phosphates by sphingosine-1-phosphate (S1P) lyase to produce phosphoethanolamine and a fatty aldehyde is the final degradative step in the sphingolipid metabolic pathway. We have studied mice with an inactive S1P lyase gene and have found that, in addition to the expected increase of sphingoid base phosphates, other sphingolipids (including sphingosine, ceramide, and sphingomyelin) were substantially elevated in the serum and/or liver of these mice. This latter increase is consistent with a reutilization of the sphingosine backbone for sphingolipid synthesis due to its inability to exit the sphingolipid metabolic pathway. Furthermore, the S1P lyase deficiency resulted in changes in the levels of serum and liver lipids not directly within the sphingolipid pathway, including phospholipids, triacyglycerol, diacylglycerol, and cholesterol. Even though lipids in serum and lipid storage were elevated in liver, adiposity was reduced in the S1P lyasedeficient mice. Microarray analysis of lipid metabolism genes in liver showed that the S1P lyase deficiency caused widespread changes in their expression pattern, with a significant increase in the expression of PPAR␥, a master transcriptional regulator of lipid metabolism. However, the mRNA expression of the genes encoding the sphingosine kinases and S1P phosphatases, which directly control the levels of S1P, were not significantly changed in liver of the S1P lyase-deficient mice. These results demonstrate that S1P lyase is a key regulator of the levels of multiple sphingolipid substrates and reveal functional links between the sphingolipid metabolic pathway and other lipid metabolic pathways that may be mediated by shared lipid substrates and changes in gene expression programs. The disturbance of lipid homeostasis by altered sphingolipid levels may be relevant to metabolic diseases.Sphingolipid metabolism generates diverse lipid molecules that are utilized by cells in multiple ways (Fig. 1A) (1, 2). Complex sphingolipids, such as sphingomyelin and glycosphingolipids, are structural components of cell membranes and drive the formation of plasma membrane lipid domains by virtue of their interactions with sterols. Metabolic intermediates, notably sphingosine, ceramide, and sphingosine-1-phosphate (S1P), 3 serve as bioactive molecules by regulating cellular signaling pathways. An additional function of sphingolipid metabolism is the synthesis of substrates that are utilized by other lipid metabolic hubs. However, the functional, regulatory, and physiological significance of the intersection of sphingolipid metabolism with other lipid pathways is not well understood.A decisive step in the sphingolipid metabolic pathway is carried out by S1P lyase, encoded by the Sgpl1 gene (3, 4). S1P lyase, which resides in the endoplasmic reticulum and is widely distributed in tissues, catalyzes the final degradative step in the sphingolipid metabolic pathway with the cleavage of phosphorylated sphingoid bases to generate phosphoethanolamine and a fatty aldehyd...