The sphingosine 1-phosphate receptor agonist FTY720 differentially affects the sequestration of CD4+/CD25+ T-regulatory cells and enhances their functional activity

The sphingosine 1-phosphate (S1P) receptor agonist FTY720 is well known for its immunomodulatory activity, sequestering lymphocytes from blood and spleen into secondary lymphoid organs and thereby preventing their migration to sites of inflammation. Because inflammation is critically dependent on a balance between Ag-specific Th/effector cells and T-regulatory cells, we investigated the effect of FTY720 on T-regulatory cell trafficking and functional activity. An increased number of CD4+/CD25+ T cells was found in blood and spleens of FTY720-treated mice, and transfer of these cells resulted in a significantly more pronounced accumulation in spleens but not lymph nodes after treatment, suggesting that this compound differentially affects the homing properties of T-regulatory cells compared with other T cell subsets. Indeed, CD4+/CD25+ T cells express lower levels of S1P1 and S1P4 receptors and demonstrate a reduced chemotactic response to S1P. Moreover, analysis of the functional response of FTY720-treated CD4+/CD25+ T cells revealed an increased suppressive activity in an in vitro Ag-specific proliferation assay. This correlated with enhanced function in vivo, with T-regulatory cells obtained from FTY720-treated mice being able to suppress OVA-induced airway inflammation. Thus, FTY720 differentially affects the sequestration of T-regulatory cells and importantly, increases the functional activity of T-regulatory cells, suggesting that it may have disease-modifying potential in inflammatory disorders.     

Roles for lysophospholipid S1P receptors in multiple sclerosis

Sphingosine 1-phosphate (S1P) signaling in the treatment of multiple sclerosis (MS) has been highlighted by the efficacy of FTY720 (fingolimod), which upon phosphorylation can modulate S1P receptor activities. FTY720 has become the first oral treatment for relapsing MS that was approved by the FDA in September 2010. Phosphorylated FTY720 modulates four of the five known S1P receptors (S1P(1), S1P(3), S1P(4), and S1P(5)) at high affinity. Studies in human MS and its animal model, experimental autoimmune encephalomyelitis (EAE), have revealed that FTY720 exposure alters lymphocyte trafficking via sequestration of auto-aggressive lymphocytes within lymphoid organs, representing the current understanding of its mechanism of action. These effects primarily involve S1P(1), which is thought to attenuate inflammatory insults in the central nervous system (CNS). In addition, FTY720's actions may involve direct effects on S1P receptor-mediated signaling in CNS cells, based upon the known expression of S1P receptors in CNS cell types relevant to MS, access to the CNS through the blood-brain barrier (BBB), and in vitro studies. These data implicate lysophospholipid signaling--via S1P(1) and perhaps other lysophospholipid receptors--in therapeutic approaches to MS and potentially other diseases with immunological and/or neurological components.     

Sphingosine kinase 2 is required for modulation of lymphocyte traffic by FTY720

Immunotherapeutic drugs that mimic sphingosine 1-phosphate (S1P) disrupt lymphocyte trafficking and cause T helper and T effector cells to be retained in secondary lymphoid tissue and away from sites of inflammation. The prototypical therapeutic agent, 2-alkyl-2-amino-1,3-propanediol (FTY720), stimulates S1P signaling pathways only after it is phosphorylated by one or more unknown kinases. We generated sphingosine kinase 2 (SPHK2) null mice to demonstrate that this kinase is responsible for FTY720 phosphorylation and thereby its subsequent actions on the immune system. Both systemic and lymphocyte-localized sources of SPHK2 contributed to FTY720 induced lymphopenia. Although FTY720 was selectively activated in vivo by SPHK2, other S1P pro-drugs can be phosphorylated to cause lymphopenia through the action of additional sphingosine kinases. Our results emphasize the importance of SPHK2 expression in both lymphocytes and other tissues for immune modulation and drug metabolism.     

Immunosuppressive and anti-angiogenic sphingosine 1-phosphate receptor-1 agonists induce ubiquitinylation and proteasomal degradation of the receptor

Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator, regulates lymphocyte trafficking, vascular permeability, and angiogenesis by activation of the S1P1 receptor. This receptor is activated by FTY720-P, a phosphorylated derivative of the immunosuppressant and vasoactive compound FTY720. However, in contrast to the natural ligand S1P, FTY720-P appears to act as a functional antagonist, even though the mechanisms involved are poorly understood. In this study, we investigated the fate of endogenously expressed S1P1 receptor in agonist-activated human umbilical vein endothelial cells and human embryonic kidney 293 cells expressing green fluorescent protein-tagged S1P1. We show that FTY720-P is more potent than S1P at inducing receptor degradation. Pretreatment with an antagonist of S1P1, VPC 44116, prevented receptor internalization and degradation. FTY720-P did not induce degradation of internalization-deficient S1P1 receptor mutants. Further, small interfering RNA-mediated down-regulation of G protein-coupled receptor kinase-2 and beta-arrestins abolished FTY720-P-induced S1P1 receptor degradation. These data suggest that agonist-induced phosphorylation of S1P1 and subsequent endocytosis are required for FTY720-P-induced degradation of the receptor. S1P1 degradation is blocked by MG132, a proteasomal inhibitor. Indeed, FTY720-P strongly induced polyubiquitinylation of S1P1 receptor, whereas S1P at concentrations that induced complete internalization was not as efficient, suggesting that receptor internalization is required but not sufficient for ubiquitinylation and degradation. We propose that the ability of FTY720-P to target the S1P1 receptor to the ubiquitinylation and proteasomal degradation pathway may at least in part underlie its immunosuppressive and anti-angiogenic properties.     

Production and release of sphingosine 1-phosphate and the phosphorylated form of the immunomodulator FTY720

The bioactive lipid molecule sphingosine 1-phosphate (S1P) binds to specific cell surface receptors and regulates several cellular processes. S1P is abundant in plasma, and physiologically its most important target cells are lymphocytes and vascular endothelial cells. S1P plays a pivotal role in the immune system by regulating lymphocyte egress from the thymus and secondary lymphoid organs. The immunomodulator FTY720 impairs this egress, causing lymphopenia. Platelets had long been considered to be the major source of plasma S1P, however recent studies revealed the importance of erythrocytes as a major supply. The sphingosine analog FTY720 is a prodrug, and FTY720 phosphate (FTY720-P) its functional form. Although both erythrocytes and platelets can produce S1P, only platelets synthesize and release FTY720-P. This review will focus on the recent advances in our understanding of the metabolism and release of S1P and FTY720-P, especially in platelets and erythrocytes.     

Down-regulation of S1P1 Receptor Surface Expression by Protein Kinase C Inhibition

The sphingosine 1-phosphate receptor type 1 (S1P₁) is important for the maintenance of lymphocyte circulation. S1P₁ receptor surface expression on lymphocytes is critical for their egress from thymus and lymph nodes. Premature activation-induced internalization of the S1P₁ receptor in lymphoid organs, mediated either by pharmacological agonists or by inhibition of the S1P degrading enzyme S1P-lyase, blocks lymphocyte egress and induces lymphopenia in blood and lymph. Regulation of S1P₁ receptor surface expression is therefore a promising way to control adaptive immunity. Hence, we analyzed potential cellular targets for their ability to alter S1P₁ receptor surface expression without stimulation. The initial observation that preincubation of mouse splenocytes with its natural analog sphingosine was sufficient to block Transwell^TM chemotaxis to S1P directed subsequent investigations to the underlying mechanism. Sphingosine is known to inhibit protein kinase C (PKC), and PKC inhibition with nanomolar concentrations of staurosporine, calphostin C, and GF109203X down-regulated surface expression of S1P₁ but not S1P₄ in transfected rat hepatoma HTC₄ cells. The PKC activator phorbol 12-myristate 13-acetate partially rescued FTY720-induced down-regulation of the S1P₁ receptor, linking PKC activation with S1P₁ receptor surface expression. FTY720, but not FTY720 phosphate, efficiently inhibited PKC. Cell-based efficacy was obvious with 10 nm FTY720, and in vivo treatment of mice with 0.3–3 mg/kg/day FTY720 showed increasing concentration-dependent effectiveness. PKC inhibition therefore may contribute to lymphopenia by down-regulating S1P₁ receptor cell surface expression independently from its activation.     

Enhanced FTY720-mediated lymphocyte homing requires G alpha i signaling and depends on beta 2 and beta 7 integrin

The immunomodulatory drug FTY720 interferes with sphingosine-1-phosphate (S1P) receptor signaling leading to lymphocyte retention in secondary lymphoid organs and consequently to profound lymphopenia in the peripheral blood. The molecular mechanisms transduced by S1P receptors upon being triggered by its native ligand, S1P, or by FTY720, are largely unknown. In this study we analyze the role of beta2 and beta7 integrin and their ligands ICAM-1, VCAM-1, and MadCAM-1 on lymphocyte homing in the presence of FTY720. We demonstrate that this drug facilitates homing of lymphocytes single-deficient of either beta2 or beta7 integrin but not of beta2-deficient lymphocytes, which in addition were blocked by anti-beta7 integrin Abs. Enhanced lymphocyte homing is preceded by increased adherence of integrin-deficient as well as wild-type lymphocytes to high endothelial venules (HEV) in FTY720-treated animals. Elevated adherence to HEV requires intact lymphocyte Galphai signaling that cannot be stably imprinted on lymphocytes even after prolonged exposure to FTY720. Thus, FTY720 influences lymphocyte homeostasis not only by suppressing lymphocyte egress from lymph nodes but also by facilitating lymphocyte homing across HEV in an integrin-dependent fashion.     

Sphingosine 1-phosphate receptors regulate chemokine-driven transendothelial migration of lymph node but not splenic T cells

Chemokines and chemokine receptors are required for T cell trafficking and migration. Recent evidence shows that sphingosine 1-phosphate (S1P) and S1PRs are also important for some aspects of T cell migration, but how these two important receptor-ligand systems are integrated and coregulated is not known. In this study, we have investigated CCL19-CCR7 and CXCL12-CXCR4-driven migration of both splenic and peripheral lymph node (PLN) nonactivated and naive T cells, and used both S1P and the S1PR ligand, FTY720, to probe these interactions. The results demonstrate that splenic T cell migration to CCL19 or CXCL12 is enhanced by, but does not require, S1PR stimulation. In contrast, PLN T cell migration to CXCL12, but not CCL19, requires both chemokine and S1PR stimulation, and the requirement for dual receptor stimulation is particularly important for steps involving transendothelial migration. The results also demonstrate that: 1) splenic and PLN nonactivated and naive T cells use different molecular migration mechanisms; 2) CCR7 and CXCR4 stimulation engage different migration mechanisms; and 3) S1P and FTY720 have distinct S1PR agonist and antagonist properties. The results have important implications for understanding naive T cell entry into and egress from peripheral lymphoid organs, and we present a model for how S1P and chemokine receptor signaling may be integrated within a T cell.     

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.     

B cell-specific S1PR1 deficiency blocks prion dissemination between secondary lymphoid organs

Many prion diseases are peripherally acquired (e.g., orally or via lesions to skin or mucous membranes). After peripheral exposure, prions replicate first upon follicular dendritic cells (FDC) in the draining lymphoid tissue before infecting the brain. However, after replication upon FDC within the draining lymphoid tissue, prions are subsequently propagated to most nondraining secondary lymphoid organs (SLO), including the spleen, by a previously underdetermined mechanism. The germinal centers in which FDC are situated produce a population of B cells that can recirculate between SLO. Therefore, we reasoned that B cells were ideal candidates by which prion dissemination between SLO may occur. Sphingosine 1-phosphate receptor (S1PR)1 stimulation controls the egress of T and B cells from SLO. S1PR1 signaling blockade sequesters lymphocytes within SLO, resulting in lymphopenia in the blood and lymph. We show that, in mice treated with the S1PR modulator FTY720 or with S1PR1 deficiency restricted to B cells, the dissemination of prions from the draining lymph node to nondraining SLO is blocked. These data suggest that B cells interacting with and acquiring surface proteins from FDC and recirculating between SLO via the blood and lymph mediate the initial propagation of prions from the draining lymphoid tissue to peripheral tissues.     

AKP-11 - A Novel S1P1 Agonist with Favorable Safety Profile Attenuates Experimental Autoimmune Encephalomyelitis in Rat Model of Multiple Sclerosis

Sphingosine-1-phosphate receptor 1 (S1P1) mediated regulation of lymphocyte egress from lymphoid organs is recognized as the mechanism of FTY720 (Fingolimod, Gilenya) efficacy in relapsing-remitting forms of multiple sclerosis (RRMS). In this study we describe a novel S1P1 agonist AKP-11, next generation of S1P1 agonist, with immunomodulatory activities in cell culture model and for therapeutic efficacy against an animal model of MS, i.e. experimental autoimmune encephalomyelitis (EAE) but without the adverse effects observed with FTY720. Like FTY720, AKP-11 bound to S1P1 is internalized and activates intracellular AKT and ERKs cellular signaling pathways. In contrast to FTY720, AKP-11 mediated S1P1 downregulation is independent of sphingosine kinase activity indicating it to be a direct agonist of S1P1. The S1P1 loss and inhibition of lymphocyte egress by FTY720 leads to lymphopenia. In comparison with FTY720, oral administration of AKP-11 caused milder and reversible lymphopenia while providing a similar degree of therapeutic efficacy in the EAE animal model. Consistent with the observed reversible lymphopenia with AKP-11, the S1P1 recycled back to cell membrane in AKP-11 treated cells following its withdrawal, but not with withdrawal of FTY720. Accordingly, a smaller degree of ubiquitination and proteolysis of S1P1 was observed in AKP-11 treated cells as compared to FTY720. Consistent with previous observations, FTY720 treatment is associated with adverse effects of bradycardia and lung vascular leaks in rodents, whereas AKP-11 treatment had undetectable effects on bradycardia and reduced lung vascular leaks as compared to FTY720. Taken together, the data documents that AKP-11 treatment cause milder and reversible lymphopenia with milder adverse effects while maintaining therapeutic efficacy similar to that observed with FTY720, thus indicating therapeutic potential of AKP-11 for treatment of MS and related autoimmune disorders.     

A splicing isoform of LPP1, LPP1a, exhibits high phosphatase activity toward FTY720 phosphate

The sphingolipid metabolite sphingosine 1-phosphate (S1P) plays an essential function in the egress of T cells from the thymus and secondary lymphoid organs. The novel immunomodulating agent FTY720 is phosphorylated in vivo to the functional form FTY720 phosphate (FTY720-P), which is structurally similar to S1P. FTY720-P inhibits the S1P-mediated T cell egress as an agonist of S1P receptors. FTY720-P is not stable in plasma and is dephosphorylated to FTY720. In the present study, we investigated activities toward FTY720-P of LPP family members (LPP1, LPP1a, LPP2, and LPP3), which exhibit broad substrate specificity. Of the four, LPP1a, the splicing isoform of LPP1, had the highest activity toward FTY720-P, and the highest affinity. Among blood-facing cells tested, only endothelial cells displayed high phosphatase activity for FTY720-P. Significant levels of LPP1a expression were found in endothelial cells, suggesting that LPP1a is important for the dephosphorylation of FTY720-P in plasma.     

FTY720 exerts a survival advantage through the prevention of end-stage glomerular inflammation in lupus-prone BXSB mice

FTY720 is a novel investigational agent targeting the sphingosine 1-phosphate (S1P) receptors with an ability to cause immunosuppression by inducing lymphocyte sequestration in lymphoid organs. Systemic lupus erythematosus (SLE) is refractory autoimmune disease characterized by the production of a wide variety of autoantibodies and immune complex (IC)-mediated lupus nephritis. Among several SLE-prone strains of mice, BXSB is unique in terms of the disease-associated monocytosis in periphery and the reduced frequency of marginal zone B (MZ B) cells in spleen. In the present study, we examined the effect of FTY720 on lupus nephritis of BXSB mice. FTY720 treatment resulted in a marked decrease in lymphocytes, but not monocytes, in peripheral blood, and caused relocalization of marginal zone B (MZ B) cells into the follicle in the spleen. These changes did not affect the production of autoantibodies, thus IgG and C3 were deposited in glomeruli in FTY720-treated mice. Despite these IC depositions, FTY720-treated mice showed survival advantage with the improved proteinuria. Histological analysis revealed that FTY720 suppressed mesangial cell proliferation and inflammatory cell infiltration. These results suggest that FTY720 ameliorates lupus nephritis by inhibiting the end-stage inflammatory process following IC deposition in glomeruli.     

FTY720 suppresses CD4+CD44highCD62L- effector memory T cell-mediated colitis

FTY720, a sphingosine-derived immunomodulator, causes immunosuppression via enhancement of lymphocyte sequestration into secondary lymphoid organs, thereby preventing their antigen-activated T cell egress to sites of inflammation. FTY720 is highly effective in inhibiting autoimmunity in various animal models. However, there is little known about how FTY720 controls the migration property of memory T cells. Here, we demonstrated that FTY720 prevents the development of colitis induced by the adoptive transfer of lamina propria (LP) colitogenic effector memory CD4+ T cells (TEM cells; CD45RB(low)CD44(high)CD62L-) into severe combined immunodeficiency (SCID) mice and suppresses interferon-gamma, interleukin-2, and tumor necrosis factor-alpha production by LP CD4+ T cells. The numbers of spleen, peripheral blood, mesenteric lymph node, and LP CD4+ T cells in FTY720-treated mice were significantly reduced compared with those in control mice. Notably, LP CD4+ TEM cells as well as splenic CD4+CD45RBhigh T cells expressed several spingosine-1-phosphate receptors that are targets for FTY720. Furthermore, FTY720 also prevented the development of colitis induced by the adoptive transfer of splenic CD4+CD45RBhigh T cells into SCID mice. Collectively, the present data indicate that FTY720 treatment may offer the potential not only to prevent the onset of disease but also to treat memory T cell-mediated autoimmune diseases including inflammatory bowel diseases.     

Novel insights into the mechanism of action of FTY720 in a transgenic model of allograft rejection: implications for therapy of chronic rejection

FTY720 is a high-affinity agonist at the sphingosine 1-phosphate receptor 1 that prevents lymphocyte egress from lymphoid tissue and prolongs allograft survival in several animal models of solid organ transplantation. In this study we used a recently developed adoptive transfer model of TCR transgenic T cells to track allospecific CD4+ T cell expansion and trafficking characteristics, cytokine secretion profiles, and surface phenotype in vivo in the setting of FTY720 administration. We report that FTY720 administration had no effect on alloantigen-driven T cell activation, proliferation, acquisition of effector-memory function, or T cell apoptosis. However, FTY720 caused a reversible sequestration of alloantigen-specific effector-memory T cells in regional lymphoid tissue associated with a decrease in T cell infiltration within the allograft and a subsequent prolongation in allograft survival. Furthermore, delayed administration of FTY720 in a cardiac model of chronic allograft rejection attenuated the progression of vasculopathy and tissue fibrosis consistent with the hypothesis that FTY720 interrupts the trafficking of activated effector-memory T cells. These data have important implications for targeting the sphingosine 1-phosphate receptor 1 in solid organ transplantation.     

Sphingosine 1-phosphate-mediated trafficking of pathogenic Th2 and mast cells for the control of food allergy

Sphingosine 1-phosphate (S1P) has been proposed as a regulator of lymphocyte trafficking, but its role in mucosa-associated diseases, such as in food allergies, remains to be elucidated. To examine the role of S1P in allergic diseases in the intestine, we used a Th2 cell-mediated Ag-specific allergic diarrhea model and demonstrated that type 1 S1P receptor (S1P(1)) expression was preferentially associated with pathogenic CD4(+) T cells for the development of allergic reactions. Consistent with this demonstration, treatment with FTY720, a modulator of the S1P(1), prevented allergic diarrhea by inhibiting the migration of systemically primed pathogenic CD4(+) T cells induced by oral challenge with allergen into the large intestine. In addition, FTY720 hampered mast cell infiltration into the large intestine, whereas eosinophil infiltration into the large intestine and total and allergen-specific serum IgE production were comparable between mock- and FTY720-treated groups. These results suggest that modulation of the S1P-mediated pathway to inhibit the migration of pathogenic CD4(+) T cells and mast cells into the large intestine could be a novel strategy for preventing allergic diarrhea.     

The Sphingosine 1-Phosphate Transporter,SPNS2, Functions as a Transporter of the Phosphorylated Form of the Immunomodulating Agent FTY720

FTY720 is a novel immunomodulating drug that can be phosphorylated inside cells; its phosphorylated form, FTY720-P, binds to a sphingosine 1-phosphate (S1P) receptor, S1P₁, and inhibits lymphocyte egress into the circulating blood. Although the importance of its pharmacological action has been well recognized, little is known about how FTY720-P is released from cells after its phosphorylation inside cells. Previously, we showed that zebrafish Spns2 can act as an S1P exporter from cells and is essential for zebrafish heart formation. Here, we demonstrate that human SPNS2 can transport several S1P analogues, including FTY720-P. Moreover, FTY720-P is transported by SPNS2 through the same pathway as S1P. This is the first identification of an FTY720-P transporter in cells; this finding is important for understanding FTY720 metabolism.     

The immunosuppressant FTY720 inhibits tumor angiogenesis via the sphingosine 1-phosphate receptor 1

FTY720, a sphingosine 1-phosphate (S1P) analog, acts as an immunosuppressant through trapping of T cells in secondary lymphoid tissues. FTY720 was also shown to prevent tumor growth and to inhibit vascular permeability. The MTT proliferation assay illustrated that endothelial cells are more susceptible to the anti-proliferative effect of FTY720 than Lewis lung carcinoma (LLC1) cells. In a spheroid angiogenesis model, FTY720 potently inhibited the sprouting activity of VEGF-A-stimulated endothelial cells even at concentrations that apparently had no anti-proliferative effect. Mechanistically, the anti-angiogenic effect of the general S1P receptor agonist FTY720 was mimicked by the specific S1P1 receptor agonist SEW2871. Moreover, the anti-angiogenic effect of FTY720 was abrogated in the presence of CXCR4-neutralizing antibodies. This indicates that the effect was at least in part mediated by the S1P1 receptor and involved transactivation of the CXCR4 chemokine receptor. Additionally, we could illustrate in a coculture spheroid model, employing endothelial and smooth muscle cells (SMCs), that the latter confer a strong protective effect regarding the action of FTY720 upon the endothelial cells. In a subcutaneous LLC1 tumor model, the anti-angiogenic capacity translated into a reduced tumor size in syngeneic C57BL/6 mice. Consistently, in the Matrigel plug in vivo assay, 10 mg/kg/d FTY720 resulted in a strong inhibition of angiogenesis as demonstrated by a reduced capillary density. Thus, in organ transplant patients, FTY720 may prove efficacious in preventing graft rejection as well as tumor development.     

Mice deficient in sphingosine kinase 1 are rendered lymphopenic by FTY720

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.