The immune modulator FTY720 inhibits sphingosine-1-phosphate lyase activity

FTY720 is a novel immunomodulatory agent that inhibits lymphocyte trafficking and prevents allograft rejection. FTY720 is phosphorylated in vivo, and the phosphorylated drug acts as agonist for a family of G protein-coupled receptors that recognize sphingosine 1-phosphate. Evidence suggests that FTY720-phosphate-induced activation of S1P1 is responsible for its mechanism of action. FTY720 was rationally designed by modification of myriocin, a naturally occurring sphingoid base analog that causes immunosuppression by interrupting sphingolipid metabolism. In this study, we examined interactions between FTY720, FTY720-phosphate, and sphingosine-1-phosphate lyase, the enzyme responsible for irreversible sphingosine 1-phosphate degradation. FTY720-phosphate was stable in the presence of active sphingosine-1-phosphate lyase, demonstrating that the lyase does not contribute to FTY720 catabolism. Conversely, FTY720 inhibited sphingosine-1-phosphate lyase activity in vitro. Treatment of mice with FTY720 inhibited tissue sphingosine-1-phosphate lyase activity within 12 h, whereas lyase gene and protein expression were not significantly affected. Tissue sphingosine 1-phosphate levels remained stable or increased throughout treatment. These studies raise the possibility that disruption of sphingosine 1-phosphate metabolism may account for some effects of FTY720 on immune function and that sphingosine-1-phosphate lyase may be a potential target for immunomodulatory therapy.     

High Density Lipoprotein Stimulated Migration of Macrophages Depends on the Scavenger Receptor Class B,Type I,PDZK1 and Akt1 and Is Blocked by Sphingosine 1 Phosphate Receptor Antagonists

HDL carries biologically active lipids such as sphingosine-1-phosphate (S1P) and stimulates a variety of cell signaling pathways in diverse cell types, which may contribute to its ability to protect against atherosclerosis. HDL and sphingosine-1-phosphate receptor agonists, FTY720 and SEW2871 triggered macrophage migration. HDL-, but not FTY720-stimulated migration was inhibited by an antibody against the HDL receptor, SR-BI, and an inhibitor of SR-BI mediated lipid transfer. HDL and FTY720-stimulated migration was also inhibited in macrophages lacking either SR-BI or PDZK1, an adaptor protein that binds to SR-BI''s C-terminal cytoplasmic tail. Migration in response to HDL and S1P receptor agonists was inhibited by treatment of macrophages with sphingosine-1-phosphate receptor type 1 (S1PR1) antagonists and by pertussis toxin. S1PR1 activates signaling pathways including PI3K-Akt, PKC, p38 MAPK, ERK1/2 and Rho kinases. Using selective inhibitors or macrophages from gene targeted mice, we demonstrated the involvement of each of these pathways in HDL-dependent macrophage migration. These data suggest that HDL stimulates the migration of macrophages in a manner that requires the activities of the HDL receptor SR-BI as well as S1PR1 activity.     

Acid sphingomyelinase is required for efficient phago-lysosomal fusion

The acid sphingomyelinase (ASMase) localizes to the lumen of endosomes, phagosomes and lysosomes as well as to the outer leaflet of the plasma membrane and hydrolyses sphingomyelin to ceramide and phosphorylcholine. Using the facultative intracellular bacterium Listeria monocytogenes , we show that maturation of phagosomes into phagolysosomes is severely impaired in macrophages genetically deficient for ASMase. Unlike in wild-type macrophages, phagosomes containing L. monocytogenes in ASMase^−/− macrophages remained positive for the late phagosomal markers mannose-6-phosphate receptor (M6PR) and Rab7 for at least 2 h and, correspondingly, showed delayed acquisition of lysosomal markers like lysosome associated membrane protein 1 (Lamp1). The transfer of lysosomal fluid phase markers into phagosomes containing L. monocytogenes was severely impaired in ASMase^−/− macrophages and decreased with increasing size of the cargo. Moreover, phagosomes containing L. monocytogenes from ASMase^−/− cells acquired significantly less listeriocidal proteases cathepsin D, B and L. The results of this study suggest that ASMase is required for the proper fusion of late phagosomes with lysosomes, which is crucial for efficient transfer of lysosomal antibacterial hydrolases into phagosomes.     

Relevance and potential of sphingosine-1-phosphate in vascular inflammatory disease

The typical pathological feature of atherosclerosis is inflammation. In the last years, it has become evident that inhibition of inflammation is one important therapeutic option in atherosclerosis. Recently, sphingolipid sphingosine-1-phosphate (S1P) was identified as a crucial molecule with potent anti-inflammatory properties. Indeed, S1P activates various G protein-coupled receptors, namely S1P1-S1P5. In the vasculature, mainly S1P1-3 receptors are present. FTY720, after phosphorylation to FTY720-P, is an orally active S1P mimetic. FTY720 has been developed for therapy in the field of autoimmune diseases and organ transplantation. In analogy to S1P, FTY720 shows potent anti-inflammatory effects and several groups have tested the in vivo effects of FTY720 on the progression of inflammatory vascular diseases. They could show that S1P receptor activation might lead to a partial inhibition of the progression of atherosclerotic lesions. S1P receptor activation therefore might be a concept for anti-inflammatory drug treatment. However, it is not clear how S1P and FTY720 exactly act on vascular inflammation. This review article gives a brief overview over the known actions of S1P in vascular inflammatory disease.     

The immunosuppressant FTY720 is phosphorylated by sphingosine kinase type 2

The potent immunosuppressive drug FTY720, a sphingosine analog, induces redistribution of lymphocytes from circulation to secondary lymphoid tissues. FTY720 is phosphorylated in vivo and functions as an agonist for four G-protein-coupled sphingosine-1-phosphate receptors. The identity of the kinase that phosphorylates FTY720 is still not known. Here we report that although both sphingosine kinase type 1 (SphK1) and type 2 (SphK2) can phosphorylate FTY720 with low efficiency, SphK2 is much more effective than SphK1. FTY720 inhibited phosphorylation of sphingosine catalyzed by SphK2 to a greater extent than it inhibits SphK1. Thus, SphK2 may be the relevant enzyme that is responsible for in vivo phosphorylation of FTY720.     

Cross-talk between PDGF and S1P signalling elucidates the inhibitory effect and potential antifibrotic action of the immunomodulator FTY720 in activated HSC-cultures

Platelet-derived growth factor (PDGF) has been shown to be essential in the activation of hepatic stellate cells (HSCs), contributing to the onset and development of hepatic fibrosis. Recently, sphingosine-1-phosphate (S1P) has been shown to be a mitogen and stimulator of chemotaxis also for HSCs. Since it has been demonstrated in several cell types that cross-talk between PDGF and S1P signalling pathways occurs, our aim was to investigate the potential antifibrotic effect of FTY720, whose phosphorylated form acts as a potent S1P receptor (S1PR) modulator, on HSCs. FTY720 inhibits cell proliferation and migration after PDGF stimulation on HSCs in a concentration range between 0.1 and 1 muM. By using compounds that block S1P signalling (PTX and VPC23019), we assessed that FTY720 also acts in an S1P receptor-independent way by decreasing the level of tyrosine phosphorylation of PDGF receptor, with subsequent inhibition of the PDGF signalling pathway. In addition, inhibition of sphingosine kinase2 (SphK2), which is responsible for FTY720 phosphorylation, by DMS/siRNA unveils a mechanism of action irrespective of its phosphorylation, in particular decreasing the level of S1P(1) on the plasma membrane. These findings led us to hypothesize a potential use of FTY720 as a potential antifibrotic drug for further clinical application.     

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.     

Acid sphingomyelinase and inhibition by phosphate ion: role of inhibition by phosphatidyl-myo-inositol 3,4,5-triphosphate in oligodendrocyte cell signaling

There is ample evidence that both acid (ASMase) and neutral (NSMase) sphingomyelinases play a role in cell death so inhibitors of either enzyme could have significant value as protectors against neurodegeneration. We used a fluorogenic sphingomyelinase substrate, 6-hexadecanoylamino-4-methylumbelliferyl-phosphorylcholine, and a [(14)C]choline-labeled sphingomyelin substrate to screen large numbers of phosphocompounds for inhibition of ASMase in extracts of human oligodendroglioma cells (HOG) and neonatal rat oligodendrocytes. Non-competitive inhibition was observed with inorganic phosphate and AMP, which was a more potent inhibitor of ASMase than cyclic AMP, ADP or ATP. However, other nucleotide phosphates, sugar phosphates, nucleotide sugars and glycerol phosphate did not inhibit ASMase. Our key finding was that phosphatidyl-myo-inositol 3,4,5-triphosphate [PtdIns (3,4,5)P(3)] was a much more potent inhibitor of ASMase than lysophosphatidic acid or phosphatidyl-myo-inositol 4,5-diphosphate [PtdIns(4,5)P(2)]. When PtdIns(3,4,5)P(3) was added to cultured cells we observed 50% inhibition of ASMase but no inhibition of other lysosomal hydrolases. After transfection of HOG cells with the tumor supressor phosphatase and tensin homolog protein (PTEN), which hydrolyses PtdIns(3,4,5)P(3) to PtdIns(4,5)P(2), we observed a two-fold increase in ASMase activity. Furthermore, the phosphatidylinositol-3-kinase inhibitor wortmannin (which reduces PtdIns(3,4,5)P(3) levels) also resulted in activation of ASMase. We propose that the small amount of ASMase activity associated with detergent-resistant cell membranes (Rafts) is regulated by PtdIns(3,4,5)P(3) and is most likely involved in receptor clustering and capping.     

Fingolimod Phosphate Attenuates Oligomeric Amyloid β–Induced Neurotoxicity via Increased Brain-Derived Neurotrophic Factor Expression in Neurons

The neurodegenerative processes that underlie Alzheimer''s disease are mediated, in part, by soluble oligomeric amyloid β, a neurotoxic protein that inhibits hippocampal long-term potentiation, disrupts synaptic plasticity, and induces the production of reactive oxygen species. Here we show that the sphingosine-1-phosphate (S1P) receptor (S1PR) agonist fingolimod phosphate (FTY720-P)-a new oral drug for multiple sclerosis-protects neurons against oligomeric amyloid β-induced neurotoxicity. We confirmed that primary mouse cortical neurons express all of the S1P receptor subtypes and FTY720-P directly affects the neurons. Treatment with FTY720-P enhanced the expression of brain-derived neurotrophic factor (BDNF) in neurons. Moreover, blocking BDNF-TrkB signaling with a BDNF scavenger, TrkB inhibitor, or ERK1/2 inhibitor almost completely ablated these neuroprotective effects. These results suggested that the neuroprotective effects of FTY720-P are mediated by upregulated neuronal BDNF levels. Therefore, FTY720-P may be a promising therapeutic agent for neurodegenerative diseases, such as Alzheimer''s disease.     

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.     

Phosphorylation of the immunomodulatory drug FTY720 by sphingosine kinases

The immunomodulatory drug FTY720 is phosphorylated in vivo, and the resulting FTY720 phosphate as a ligand for sphingosine-1-phosphate receptors is responsible for the unique biological effects of the compound. So far, phosphorylation of FTY720 by murine sphingosine kinase (SPHK) 1a had been documented. We found that, while FTY720 is also phosphorylated by human SPHK1, the human type 2 isoform phosphorylates the drug 30-fold more efficiently, because of a lower Km of FTY720 for SPHK2. Similarly, murine SPHK2 was more efficient than SPHK1a. Among splice variants of the human SPHKs, an N-terminally extended SPHK2 isoform was even more active than SPHK2 itself. Further SPHK superfamily members, namely ceramide kinase and a "SPHK-like" protein, failed to phosphorylate sphingosine and FTY720. Thus, only SPHK1 and 2 appear to be capable of phosphorylating FTY720. Using selective assay conditions, SPHK1 and 2 activities in murine tissues were measured. While activity of SPHK2 toward sphingosine was generally lower than of SPHK1, FTY720 phosphorylation was higher under conditions favoring SPHK2. In human endothelial cells, while activity of SPHK1 toward sphingosine was 2-fold higher than of SPHK2, FTY720 phosphorylation was 7-fold faster under SPHK2 assay conditions. Finally, FTY720 was poorly phosphorylated in human blood as compared with rodent blood, in line with the low activity of SPHK1 and in particular of SPHK2 in human blood. To conclude, both SPHK1 and 2 are capable of phosphorylating FTY720, but SPHK2 is quantitatively more important than SPHK1.     

Enhancement of neoangiogenesis and follicle survival by sphingosine-1-phosphate in human ovarian tissue xenotransplants

Ovarian transplantation is one of the key approaches to restoring fertility in women who became menopausal as a result of cancer treatments. A major limitation of human ovarian transplants is massive follicular loss during revascularization. Here we investigated whether sphingosine-1-phosphate or its receptor agonists could enhance neoangiogenesis and follicle survival in ovarian transplants in a xenograft model. Human ovarian tissue xenografts in severe-combined-immunodeficient mice were treated with sphingosine-1-phosphate, its analogs, or vehicle for 1-10 days. We found that sphingosine-1-phosphate treatment increased vascular density in ovarian transplants significantly whereas FTY720 and SEW2871 had the opposite effect. In addition, sphingosine-1-phosphate accelerated the angiogenic process compared to vehicle-treated controls. Furthermore, sphingosine-1-phosphate treatment was associated with a significant proliferation of ovarian stromal cell as well as reduced necrosis and tissue hypoxia compared to the vehicle-treated controls. This resulted in a significantly lower percentage of apoptotic follicles in sphingosine-1-phosphate-treated transplants. We conclude that while sphingosine-1-phosphate promotes neoangiogenesis in ovarian transplants and reduces ischemic reperfusion injury, sphingosine-1-phosphate receptor agonists appear to functionally antagonize this process. Sphingosine-1-phosphate holds great promise to clinically enhance the survival and longevity of human autologous ovarian transplants.     

There is More to a Lipid than just Being a Fat: Sphingolipid-Guided Differentiation of Oligodendroglial Lineage from Embryonic Stem Cells

Dr. Robert K. Yu’s research showed for the first time that the composition of glycosphingolipids is tightly regulated during embryo development. Studies in our group showed that the glycosphingolipid precursor ceramide is also critical for stem cell differentiation and apoptosis. Our new studies suggest that ceramide and its derivative, sphingosine-1-phosphate (S1P), act synergistically on embryonic stem (ES) cell differentiation. When using neural precursor cells (NPCs) derived from ES cells for transplantation, residual pluripotent stem (rPS) cells pose a significant risk of tumor formation after stem cell transplantation. We show here that rPS cells did not express the S1P receptor S1P1, which left them vulnerable to ceramide or ceramide analog (N-oleoyl serinol or S18)-induced apoptosis. In contrast, ES cell-derived NPCs expressed S1P1 and were protected in the presence of S1P or its pro-drug analog FTY720. Consistent with previous studies, FTY720-treated NPCs differentiated predominantly toward oligodendroglial lineage as tested by the expression of the oligodendrocyte precursor cell (OPC) markers Olig2 and O4. As the consequence, a combined administration of S18 and FTY720 to differentiating ES cells eliminated rPS cells and promoted oligodendroglial differentiation. In addition, we show that this combination promoted differentiation of ES cell-derived NPCs toward oligodendroglial lineage in vivo after transplantation into mouse brain.     

S1P lyase regulates DNA damage responses through a novel sphingolipid feedback mechanism

The injurious consequences of ionizing radiation (IR) to normal human cells and the acquired radioresistance of cancer cells represent limitations to cancer radiotherapy. IR induces DNA damage response pathways that orchestrate cell cycle arrest, DNA repair or apoptosis such that irradiated cells are either repaired or eliminated. Concomitantly and independent of DNA damage, IR activates acid sphingomyelinase (ASMase), which generates ceramide, thereby promoting radiation-induced apoptosis. However, ceramide can also be metabolized to sphingosine-1-phosphate (S1P), which acts paradoxically as a radioprotectant. Thus, sphingolipid metabolism represents a radiosensitivity pivot point, a notion supported by genetic evidence in IR-resistant cancer cells. S1P lyase (SPL) catalyzes the irreversible degradation of S1P in the final step of sphingolipid metabolism. We show that SPL modulates the kinetics of DNA repair, speed of recovery from G2 cell cycle arrest and the extent of apoptosis after IR. SPL acts through a novel feedback mechanism that amplifies stress-induced ceramide accumulation, and downregulation/inhibition of either SPL or ASMase prevents premature cell cycle progression and mitotic death. Further, oral administration of an SPL inhibitor to mice prolonged their survival after exposure to a lethal dose of total body IR. Our findings reveal SPL to be a regulator of ASMase, the G2 checkpoint and DNA repair and a novel target for radioprotection.     

Protective Effect of FTY720 Against Sevoflurane-Induced Developmental Neurotoxicity in Rats

Sevoflurane, a common used inhaled anaesthetic, induces neuronal apoptosis in preclinical studies and correlates with functional neurological impairment. We investigated whether FTY720, a known sphingosine-1 phosphate (S1P) receptor agonist, could exert neuroprotective effect against sevoflurane-induced neurotoxicity. Neuroprotective effect of FTY720 was evaluated in vitro in hippocampal neuronal cells from neonatal rats and in vivo in rat pups. In vitro cell apoptosis was determined by flow cytometry after exposure to 3 % sevoflurane for different period of time, or after 6-h exposure to sevoflurane with the presence of FTY720, SEW2871 (selective S1P1 receptor agonist) or combination of FTY720 and VPC23019 (S1P antagonist). Western blot analysis was performed with hippocampal tissue from rat pups exposed to 3 % sevoflurane for 6 h with or without pre-treatment with FTY720 injection. Neurological function tests were also performed with rat pups exposed to 3 % sevoflurane for 6 h with or without pre-treatment with FTY720 injection. FTY720, at nanomolar concentration, significantly prevents sevoflurane-induced neuronal apoptosis. SEW2871 showed similar neuroprotective effect to FTY720, whereas VPC23019 abrogated the neuroprotective effect of FTY720 when given together. Western blots results demonstrated that FTY710 significantly preserved the level of phosphorylated ERK1/2, Bcl-2 and Bax. Although anaesthetic treatment did not affect general health and emotional status, sevoflurane-induced cognitive impairment in rat models. Administration of FTY720 at 1 mg/kg significantly attenuated sevoflurane-induced neurocognitive impairment. Although further studies are needed to evaluate the feasibility of clinical usage of FTY720 as neuroprotective agent, the study provides preclinical experimental evidence for the efficacy of FTY720 against sevoflurane-induced developmental neurotoxicity.     

Regulation of cell survival by sphingosine-1-phosphate receptor S1P1 via reciprocal ERK-dependent suppression of Bim and PI-3-kinase/protein kinase C-mediated upregulation of Mcl-1

Although the ability of bioactive lipid sphingosine-1-phosphate (S1P) to positively regulate anti-apoptotic/pro-survival responses by binding to S1P₁ is well known, the molecular mechanisms remain unclear. Here we demonstrate that expression of S1P₁ renders CCL39 lung fibroblasts resistant to apoptosis following growth factor withdrawal. Resistance to apoptosis was associated with attenuated accumulation of pro-apoptotic BH3-only protein Bim. However, although blockade of extracellular signal-regulated kinase (ERK) activation could reverse S1P₁-mediated suppression of Bim accumulation, inhibition of caspase-3 cleavage was unaffected. Instead S1P₁-mediated inhibition of caspase-3 cleavage was reversed by inhibition of phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC), which had no effect on S1P₁ regulation of Bim. However, S1P₁ suppression of caspase-3 was associated with increased expression of anti-apoptotic protein Mcl-1, the expression of which was also reduced by inhibition of PI3K and PKC. A role for the induction of Mcl-1 in regulating endogenous S1P receptor-dependent pro-survival responses in human umbilical vein endothelial cells was confirmed using S1P receptor agonist FTY720-phosphate (FTY720P). FTY720P induced a transient accumulation of Mcl-1 that was associated with a delayed onset of caspase-3 cleavage following growth factor withdrawal, whereas Mcl-1 knockdown was sufficient to enhance caspase-3 cleavage even in the presence of FTY720P. Consistent with a pro-survival role of S1P₁ in disease, analysis of tissue microarrays from ER⁺ breast cancer patients revealed a significant correlation between S1P₁ expression and tumour cell survival. In these tumours, S1P₁ expression and cancer cell survival were correlated with increased activation of ERK, but not the PI3K/PKB pathway. In summary, pro-survival/anti-apoptotic signalling from S1P₁ is intimately linked to its ability to promote the accumulation of pro-survival protein Mcl-1 and downregulation of pro-apoptotic BH3-only protein Bim via distinct signalling pathways. However, the functional importance of each pathway is dependent on the specific cellular context.     

Potent S1P receptor agonists replicate the pharmacologic actions of the novel immune modulator FTY720

Alteration in lymphocyte trafficking and prevention of graft rejection in rodents observed on exposure to FTY720 (1) or its corresponding phosphate ester 2 can be induced by the systemic administration of potent sphingosine-1-phosphate receptor agonists exemplified by 19. The similar S1P receptor profiles of 2 and 19 coupled with their comparable potency in vivo supports a connection between S1P receptor agonism and immunosuppressive efficacy.     

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.