sew2871 and sphingosine-1-phosphate

The recent identification of a cellular balance between ceramide and sphingosine 1-phosphate (S1P) as a critical regulator of cell growth and death has stimulated increasing research effort to clarify the role of ceramide and S1P in various diseases associated with dysregulated cell proliferation and apoptosis. S1P acts mainly, but not exclusively, by binding to and activating specific cell surface receptors, the so-called S1P receptors. These receptors belong to the class of G protein-coupled receptors that constitute five subtypes, denoted as S1P(1)-S1P(5), and represent attractive pharmacological targets to interfere with S1P action. Whereas classical receptor antagonists will directly block S1P action, S1P receptor agonists have also proven useful, as recently shown for the sphingolipid-like immunomodulatory substance FTY720. When phosphorylated by sphingosine kinase to yield FTY720 phosphate, it acutely acts as an agonist at S1P receptors, but upon prolonged presence, it displays antagonistic activity by specifically desensitizing the S1P(1) receptor subtype. This commentary will cover the most recent developments in the field of S1P receptor pharmacology and highlights the potential therapeutic benefit that can be expected from these novel drug targets in the future.

Topics: Animals; Fingolimod Hydrochloride; Humans; Lysophospholipids; Oxadiazoles; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Sphingosine; Sulfhydryl Compounds; Thiazolidines; Thiophenes

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that regulates fundamental cellular processes such as proliferation, migration, apoptosis, and differentiation through 5 cognate G protein-coupled receptors (S1P1-S1P5). We previously demonstrated that blockade of S1P2 signaling in S1P2-deficient mice attenuates high-fat diet-induced adipocyte hypertrophy and glucose intolerance and an S1P2-specific antagonist JTE-013 inhibits, whereas an S1P1/S1P3 dual antagonist (VPC23019) activates, adipogenic differentiation of preadipocytes. Based on those observations, this study examined whether an S1P1-specific agonist, SEW-2871, VPC23019, or their combination acts on obesity and glucose intolerance in leptin-deficient ob/ob mice. The oral administration of SEW-2871 or JTE-013 induced significant reductions in body/epididymal fat weight gains and epididymal/inguinal fat adipocyte sizes and improved glucose intolerance and adipocyte inflammation in ob/ob mice but not in their control C57BL/6J mice. Both SEW-2871 and JTE-013 decreased messenger RNA levels of tumor necrosis factor-α and CD11c, whereas they increased those of CD206 and adiponectin in the epididymal fats isolated from ob/ob mice with no changes in the levels of peroxisome proliferator activated receptor γ and its regulated genes. By contrast, VPC23019 did not cause any such alterations but counteracted with all those SEW-2871 actions in these mice. In conclusion, the S1P1 agonist SEW-2871 acted like the S1P2 antagonist JTE-013 to reduce body/epididymal fats and improve glucose tolerance in obese mice. Therefore, this study raises the possibility that endogenous S1P could promote obesity/type 2 diabetes through the S1P2, whereas exogenous S1P could act against them through the S1P1.

Topics: Animals; Diabetes Mellitus, Type 2; Glucose; Glucose Intolerance; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Obese; Obesity; Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors

Cardiac fibrosis and myocyte hypertrophy are hallmarks of the cardiac remodelling process in cardiomyopathies such as heart failure (HF). Dyslipidemia or dysregulation of lipids contribute to HF. The dysregulation of high density lipoproteins (HDL) could lead to altered levels of other lipid metabolites that are bound to it such as sphingosine-1- phosphate (S1P). Recently, it has been shown that S1P and its analogue dihydrosphingosine-1-phosphate (dhS1P) are bound to HDL in plasma. The effects of dhS1P on cardiac cells have been obscure. In this study, we show that extracellular dhS1P is able to increase collagen synthesis in neonatal rat cardiac fibroblasts (NCFs) and cause hypertrophy of neonatal cardiac myocytes (NCMs). The janus kinase/signal transducer and activator (JAK/STAT) signalling pathway was involved in the increased collagen synthesis by dhS1P, through sustained increase of tissue inhibitor of matrix metalloproteinase 1 (TIMP1). Extracellular dhS1P increased phosphorylation levels of STAT1 and STAT3 proteins, also caused an early increase in gene expression of transforming growth factor-β (TGFβ), and sustained increase in TIMP1. Inhibition of JAKs led to inhibition of TIMP1 and TGFβ gene and protein expression. We also show that dhS1P is able to cause NCM hypertrophy through S1P-receptor-1 (S1PR1) signalling which is opposite to that of its analogue, S1P. Taken together, our results show that dhS1P increases collagen synthesis in cardiac fibroblasts causing fibrosis through dhS1P-JAK/STAT-TIMP1 signalling.

Topics: Animals; Animals, Newborn; Biomarkers; Cell Differentiation; Collagen; Fibroblasts; Gene Expression Regulation; Hypertrophy; Janus Kinases; Lysophospholipids; Matrix Metalloproteinase 2; Models, Biological; Myocardium; Myocytes, Cardiac; Oxadiazoles; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Rats, Sprague-Dawley; RNA, Messenger; Signal Transduction; Smad2 Protein; Sphingosine; STAT Transcription Factors; Thiophenes; Time Factors; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta

Endothelial barrier dysfunction is a hallmark in the pathogenesis of sepsis. Sphingosine-1-phosphate (S1P) has been proposed to be critically involved in the maintenance of endothelial barrier function predominately by activating S1P receptor-1 (S1P1). Previous studies have shown that the specific S1P1 agonist SEW2871 improves endothelial barrier function under inflammatory conditions. However, the effectiveness of SEW2871 and potential side effects remained largely unexplored in a clinically relevant model of sepsis. Therefore, this study aimed to evaluate the effects of SEW2871 in the Colon ascendens stent peritonitis (CASP) model.. Polymicrobial sepsis was induced in Sprague-Dawley rats using CASP model that enabled the monitoring of macro-hemodynamic parameters. Twelve hours after surgery, animals received either SEW2871 or sodium chloride. Mesenteric endothelial barrier function was evaluated 24 h after sepsis induction by intravital microscopy. Organ pathology was assessed in lungs. S1P levels, blood gas analyses, and blood values were measured at different time points. In parallel the effect of SEW2871 was evaluated in human dermal microvascular endothelial cells.. In vitro SEW2871 partially stabilized TNF-α-induced endothelial barrier breakdown. However, in vivo SEW2871 caused severe cardiac side effects in septic animals leading to an increased lethality. Sepsis-induced endothelial barrier dysfunction was not attenuated by SEW2871 as revealed by increased FITC-albumin extra-vasation, requirement of intravasal fluid replacement, and pulmonary edema. Interestingly, Sham-operated animals did not present any side effects after SEW2871 treatment.. Our study demonstrates that the application of SEW2871 causes severe cardiac side effects and cannot attenuate the inflammation-induced endothelial barrier breakdown in a clinically relevant sepsis model, suggesting that the time point of administration and the pro-inflammatory milieu play a pivotal role in the therapeutic benefit of SEW2871.

Topics: Animals; Disease Models, Animal; Humans; Lysophospholipids; Male; Oxadiazoles; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Sepsis; Sphingosine; Sphingosine-1-Phosphate Receptors; Thiophenes; Tumor Necrosis Factor-alpha

Alzheimer's disease (AD) is characterized by alterations of amyloid precursor protein (APP) metabolism, accumulation of amyloid  peptides (A), hyperphosphorylation of Tau proteins and also by sphingolipids disturbances. These changes lead to oxidative stress, mitochondria dysfunction, synaptic loss and neuro-inflammation. It is known that A may promote ceramides formation and reversely, ceramides could stimulate A peptides release. However, the effect of ceramide and sphingosine-1-phosphate (S1P) on APP metabolism has not been fully elucidated. In this study we investigated the role of ceramide and S1P on APP metabolism. Moreover, the effect of ceramide and SEW 2871 (agonist for S1P receptor-1) on Sirt1 (NAD+-dependent nuclear enzyme responsible for stress response) gene expression under A toxicity was analyzed. Experiments were carried out using pheochromocytoma cells (PC-12) transfected with: an empty vector (used as a control), human wild-type APP gene (APPwt) and Swedish mutated (K670M/N671L) APP gene (APPsw). Our results indicated that C2-ceramide significantly decreased the viability of the APPwt, APPsw as well as empty vector-transfected PC12 cells. It was observed that C2-ceramide had no significant effect on the mRNA level of - and -secretase in APPwt and APPsw cells. However, it significantly decreased transcription of -secretase in control cells. Results also showed a significant increase in Psen1 (crucial subunit of -secretase) gene expression in APPsw cells after incubation with C2-ceramide. We observed that SEW 2871 significantly upregulated the mRNA level of -secretase in control-empty vector-transfected cells subjected to C2-ceramide toxicity. The same tendency, though insignificant, was observed in APPwt and APPsw cells. Moreover, SEW 2871 enhanced the mRNA level of -secretase and Psen1 in APPsw cells after C2-ceramide treatment. Additionally, SEW 2871 significantly upregulated a gene expression of Sirt1 in APPwt and also APPsw cells subjected to C2-ceramide toxicity. Furthermore, it was observed that SEW 2871 significantly enhanced the viability of all investigated cells' lines probably through its positive influence on Sirt1.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Ceramides; Humans; Lysophospholipids; Models, Theoretical; Neurons; Oxadiazoles; PC12 Cells; Rats; Receptors, Lysosphingolipid; Sphingosine; Thiophenes; Transcription, Genetic

In this study, the wound closure of mouse skin defects was examined in terms of recruitment of mesenchymal stem cells (MSC) and macrophages. For the cells recruitment, stromal derived factor-1 (SDF-1) of a MSC recruitment agent and sphingosine-1 phosphate agonist (SEW2871) of a macrophages recruitment agent were incorporated into gelatin hydrogels, and then released in a controlled fashion. When applied to a skin wound defect of mice, gelatin hydrogels incorporating mixed 500 ng SDF-1 and 0.4, 0.8, or 1.6 mg SEW2871-micelles recruited a higher number of both MSC and macrophages than those incorporating SDF-1 or phosphate buffered saline. However, the number of M1 phenotype macrophages for the hydrogel incorporating mixed SDF-1 and SEW2871-micelles recruited was remarkably low to a significant extent compared with that for those hydrogel incorporating 0.4, 0.8, or 1.6 mg SEW2871-micelles. On the other hand, the number of M2 macrophages 3 days after the implantation of the hydrogels incorporating SDF-1 and 0.4 mg SEW2871-micelles significantly increased compared with that for other hydrogels. In vivo experiments revealed the hydrogels incorporating SDF-1 and 0.4 mg SEW2871-micelles promoted the wound closure of skin defect to a significant stronger extent than those incorporating SEW2871-micelles, SDF-1, and a mixture of SDF-1 and higher doses of SEW2871-micelles. It is concluded that the in vivo recruitment of MSC and macrophages to the defects may contribute to the tissue regeneration of skin wound.

Topics: Animals; Cattle; Cell Movement; Cells, Cultured; Chemokine CXCL12; Delayed-Action Preparations; Gelatin; Hydrogels; Lysophospholipids; Macrophages; Male; Mesenchymal Stem Cells; Mice, Inbred C57BL; Oxadiazoles; Skin; Sphingosine; Swine; Thiophenes; Wound Healing

Recent evidence suggests that an extreme shift may occur in sphingosine metabolism in neuroinflammatory contexts. Sphingosine 1-phosphate (S1P)-metabolizing enzymes (SMEs) regulate the level of S1P. We recently found that FTY720, a S1P analogue, and SEW2871, a selective S1P receptor 1 (S1P1) agonist, provide protection against neural damage and memory deficit in amyloid beta (Aβ)-injected animals. This study aimed to evaluate the effects of these two analogues on the expression of SMEs as well as their anti-inflammatory roles.. Rats were treated with intracerebral lipopolysaccharide (LPS) or Aβ. Memory impairment was assessed by Morris water maze and the effects of drugs on SMEs as well as inflammatory markers, TNF- α and COX-II, were determined by immunoblotting.. Aβ and LPS differentially altered the expression profile of SMEs. In Aβ-injected animals, FTY720 and SEW2871 treatments exerted anti-inflammatory effects and restored the expression profile of SMEs, in parallel to our previous findings. In LPS animals however, in spite of anti-inflammatory effects of the two analogues, only FTY720 restored the levels of SMEs and prevented memory deficit.. The observed ameliorating effects of FTY720 and SEW7821 can be partly attributed to the interruption of the vicious cycle of abnormal S1P metabolism and neuro-inflammation. The close imitation of the FTY720 effects by SW2871 in Aβ-induced neuro-inflammation may highlight the attractive role of S1P1 as a potential target to restore S1P metabolism and inhibit inflammatory processes.

Topics: Amyloid beta-Peptides; Animals; Anti-Inflammatory Agents; Cyclooxygenase 2; Fingolimod Hydrochloride; Inflammation; Lipopolysaccharides; Lysophospholipids; Male; Maze Learning; Memory Disorders; Oxadiazoles; Rats; Rats, Wistar; Receptors, Lysosphingolipid; Sphingosine; Thiophenes; Tumor Necrosis Factor-alpha

Both immunosuppressive and cytoreductive effects of γ-irradiation contribute to engraftment of allogeneic haematopoietic stem and progenitor cells. We hypothesized that a release of host stem and progenitor cells from the niche prior to conditioning would permit engraftment after less intensive conditioning. Administration of AMD3100 and SEW2871 on days -4 to -2 followed by irradiation on day -1 in a non-myeloablative zebrafish transplant model resulted in a reduced radiation minimum dose of 10 Gy from 15 Gy being sufficient for engraftment. Targeting the SDF-1 (CXCL12)/CXCR4- and S1P/S1P1 -axis increased the efficacy of allografting in an experimental transplant model.

Topics: Animal Experimentation; Animals; Benzylamines; Cyclams; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Heterocyclic Compounds; Lysophospholipids; Oxadiazoles; Receptors, CXCR4; Signal Transduction; Sphingosine; Thiophenes; Transplantation Chimera; Transplantation, Homologous; Zebrafish

Sphingosine-1-phosphate (S1P) modulates many cell functions such as lymphocyte trafficking and signaling as well as keratinocyte proliferation. However, less is known about the specific effects of S1P on cytokine production, particularly on the interaction between dendritic cells (DCs) and keratinocytes, cell types which are crucial for the initiation and maintenance of chronic inflammatory skin diseases like atopic dermatitis or psoriasis. Especially the cytokines of the IL-12 family play a dominant role in many inflammatory diseases as they have a significant impact on T-helper cell function. In the present study we show that S1P decreased the production of the pro-inflammatory cytokines IL-12 and IL-23 in LPS-stimulated DCs via the common subunit p40 as well as in the crosstalk with activated keratinocytes. By using specific S1P receptor agonists (SEW2871, FTY720-P) and antagonist (JTE013) we identified an important role for S1P receptor 1 in the modulation of the cytokine profile. While diminishing IL-12 and IL-23 secretion, S1P enhanced IL-27 production in DCs. To elucidate the mechanism of the different impact on the IL-12 family cytokine production, we investigated the mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinase (PI3K) pathways in DCs. By using specific MAPK-Inhibitors (U0126, SB202190, SP600125) we demonstrated that ERK, p38 and JNK differently regulate each pathway of each cytokine. While p38 and JNK did not seem to play a role in the modulation properties of S1P on cytokine production, ERK is at least partially involved in the S1P mediated modulation of IL-12 and IL-27. The PI3K-Inhibitor abrogated the S1P-induced decrease of IL-12 and IL-23 secretion, while it had no influence on the S1P-induced increase of IL-27 production. These data implicate, that S1P has an anti-inflammatory impact on the production of IL-12 family cytokines, indicating therapeutic potential for S1P treatment of several inflammatory diseases like psoriasis.

Topics: Androstadienes; Animals; Bone Marrow Cells; Cells, Cultured; Dendritic Cells; Enzyme Inhibitors; Gene Expression; Immunoblotting; Interleukin-12; Interleukin-23; Interleukin-27; Lipopolysaccharides; Lysophospholipids; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; Oxadiazoles; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sphingosine; Thiophenes; Wortmannin

The ceramide-sphingosine 1-phosphate (S1P) rheostat is important in regulating cell fate. Several chemotherapeutic agents, including paclitaxel (Taxol), involve pro-apoptotic ceramide in their anticancer effects. The ceramide-to-S1P pathway is also implicated in the development of pain, raising the intriguing possibility that these sphingolipids may contribute to chemotherapy- induced painful peripheral neuropathy, which can be a critical dose-limiting side effect of many widely used chemotherapeutic agents.We demonstrate that the development of paclitaxel-induced neuropathic pain was associated with ceramide and S1P formation in the spinal dorsal horn that corresponded with the engagement of S1P receptor subtype 1 (S1PR(1))- dependent neuroinflammatory processes as follows: activation of redox-sensitive transcription factors (NFκB) and MAPKs (ERK and p38) as well as enhanced formation of pro-inflammatory and neuroexcitatory cytokines (TNF-α and IL-1β). Intrathecal delivery of the S1PR1 antagonist W146 reduced these neuroinflammatory processes but increased IL-10 and IL-4, potent anti-inflammatory/ neuroprotective cytokines. Additionally, spinal W146 reversed established neuropathic pain. Noteworthy, systemic administration of the S1PR1 modulator FTY720 (Food and Drug Administration- approved for multiple sclerosis) attenuated the activation of these neuroinflammatory processes and abrogated neuropathic pain without altering anticancer properties of paclitaxel and with beneficial effects extended to oxaliplatin. Similar effects were observed with other structurally and chemically unrelated S1PR1 modulators (ponesimod and CYM-5442) and S1PR1 antagonists (NIBR-14/15) but not S1PR1 agonists (SEW2871). Our findings identify for the first time the S1P/S1PR1 axis as a promising molecular and therapeutic target in chemotherapy-induced painful peripheral neuropathy, establish a mechanistic insight into the biomolecular signaling pathways, and provide the rationale for the clinical evaluation of FTY720 in chronic pain patients.

Topics: Anilides; Animals; Antineoplastic Agents, Phytogenic; Cytokines; Enzyme Activation; Fingolimod Hydrochloride; Humans; Immunosuppressive Agents; Indans; Lysophospholipids; Male; Neuralgia; Organophosphonates; Oxadiazoles; Paclitaxel; Propylene Glycols; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Thiazoles; Thiophenes

The objective of this study is to design a drug delivery system (DDS) for the in vivo promotion of macrophage recruitment. As the drug, a water-insoluble agonist of sphingosine-1-phosphate type 1 receptor (SEW2871) was selected. SEW2871 (SEW) was water-solubilized by micelle formation with gelatin grafted by L-lactic acid oligomer. SEW micelles were mixed with gelatin, followed by dehydrothermal crosslinking of gelatin to obtain gelatin hydrogels incorporating SEW micelles. SEW was released from the hydrogels incorporating SEW micelles in vitro and in vivo. The water-solubilized SEW showed in vitro macrophage migration activity. When implanted into the back subcutis or the skin wound defect of mice, the hydrogel incorporating SEW micelles promoted macrophage migration toward the tissue around the implanted site to a significantly great extent compared with SEW-free hydrogel and that mixed with SEW micelles. The hydrogel is a promising DDS to enhance macrophage recruitment in vivo.

Topics: Animals; Cattle; Cell Count; Cell Movement; Delayed-Action Preparations; Gelatin; Humans; Hydrogels; Lactic Acid; Lysophospholipids; Macrophages; Male; Mice, Inbred C57BL; Micelles; Oxadiazoles; Solubility; Sphingosine; Sus scrofa; Thiophenes; Time Factors; Water

Pathologically swollen lymph nodes (LNs), or buboes, characterize Yersinia pestis infection, yet how they form and function is unknown. We report that colonization of the draining LN (dLN) occurred due to trafficking of infected dendritic cells and monocytes in temporally distinct waves in response to redundant chemotactic signals, including through CCR7, CCR2, and sphingosine-1-phospate (S1P) receptors. Retention of multiple subsets of phagocytes within peripheral LNs using the S1P receptor agonist FTY720 or S1P1-specific agonist SEW2871 increased survival, reduced colonization of downstream LNs, and limited progression to transmission-associated septicemic or pneumonic disease states. Conditional deletion of S1P1 in mononuclear phagocytes abolished node-to-node trafficking of infected cells. Thus, Y. pestis-orchestrated LN remodeling promoted its dissemination via host cells through the lymphatic system but can be blocked by prevention of leukocyte egress from DLNs. These findings define a novel trafficking route of mononuclear phagocytes and identify S1P as a therapeutic target during infection.

Topics: Animals; CD11 Antigens; CD11b Antigen; Cell Movement; Chemokine CCL21; Dendritic Cells; Female; Fingolimod Hydrochloride; Integrin alpha Chains; Lymph Nodes; Lysophospholipids; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Monocytes; Oxadiazoles; Phagocytes; Plague; Propylene Glycols; Receptors, CCR2; Receptors, CCR7; Receptors, Lysosphingolipid; Sphingosine; Thiophenes; Yersinia pestis

Synthetic sphingosine 1-phosphate receptor 1 modulators constitute a new class of drugs for the treatment of autoimmune diseases. Sphingosine 1-phosphate (S1P) signaling, however, is also involved in the development of fibrosis. Using normal human lung fibroblasts, we investigated the induction of fibrotic responses by the S1P receptor (S1PR) agonists S1P, FTY720-P, ponesimod, and SEW2871 and compared them with the responses induced by the known fibrotic mediator TGF-β1. In contrast to TGF-β1, S1PR agonists did not induce expression of the myofibroblast marker α-smooth muscle actin. However, TGF-β1, S1P, and FTY720-P caused robust stimulation of extracellular matrix (ECM) synthesis and increased pro-fibrotic marker gene expression including connective tissue growth factor. Ponesimod showed limited and SEW2871 showed no pro-fibrotic potential in these readouts. Analysis of pro-fibrotic signaling pathways showed that in contrast to TGF-β1, S1PR agonists did not activate Smad2/3 signaling but rather activated PI3K/Akt and ERK1/2 signaling to induce ECM synthesis. The strong induction of ECM synthesis by the nonselective agonists S1P and FTY720-P was due to the stimulation of S1P2 and S1P3 receptors, whereas the weaker induction of ECM synthesis at high concentrations of ponesimod was due to a low potency activation of S1P3 receptors. Finally, in normal human lung fibroblast-derived myofibroblasts that were generated by TGF-β1 pretreatment, S1P and FTY720-P were effective stimulators of ECM synthesis, whereas ponesimod was inactive, because of the down-regulation of S1P3R expression in myofibroblasts. These data demonstrate that S1PR agonists are pro-fibrotic via S1P2R and S1P3R stimulation using Smad-independent pathways.

Topics: Actins; Animals; CHO Cells; Cricetinae; Cricetulus; Down-Regulation; Extracellular Matrix; Humans; Lung; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myofibroblasts; Organophosphates; Oxadiazoles; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Pulmonary Fibrosis; Receptors, Lysosphingolipid; Signal Transduction; Smad2 Protein; Smad3 Protein; Sphingosine; Thiazoles; Thiophenes; Transforming Growth Factor beta1

Activated protein C (APC) exerts anticoagulant effects via inactivation of factors Va and VIIIa and cytoprotective effects via protease activated receptor (PAR)1. Inhibition of endogenous APC in endotoxemia and sepsis results in exacerbation of coagulation and inflammation, with consequent enhanced lethality.. We here sought to dissect the distinct roles of the anticoagulant and cytoprotective functions of endogenous APC in severe Gram-negative pneumonia-derived sepsis (melioidosis).. We infected wild-type (WT) mice with Burkholderia pseudomallei, a common sepsis pathogen in southeast Asia, and treated them with antibodies inhibiting both the anticoagulant and cytoprotective functions of APC (MPC1609) or the anticoagulant functions of APC (MAPC1591) only. Additionally, we administered SEW2871 (stimulating the S1P1-pathway downstream from PAR1) to control and MPC1609-treated mice.. MPC1609, but not MAPC1591, significantly worsened survival, increased coagulation activation, facilitated bacterial growth and dissemination and enhanced the inflammatory response. The effects of MPC1609 could not be reversed by SEW2871, suggesting that S1P1 does not play a major role in this model.. These results suggest that the mere inhibition of the anticoagulant function of APC does not interfere with its protective role during Gram-negative pneumosepsis, suggesting a more prominent role for cytoprotective effects of APC .

Topics: Animals; Antibodies, Monoclonal; Bacterial Load; Blood Coagulation; Burkholderia pseudomallei; Cytokines; Cytoprotection; Disease Models, Animal; Female; Inflammation; Inflammation Mediators; Liver; Lung; Lysophospholipids; Melioidosis; Mice; Mice, Inbred C57BL; Oxadiazoles; Protein C; Receptor, PAR-1; Sepsis; Signal Transduction; Sphingosine; Thiophenes; Time Factors

T cells produce a number of cytokines and chemokines upon stimulation with TLR agonists in the presence or absence of TCR signals. Here, we show that secretion of neutrophil chemoattractant CXCL8 from human T cell line Jurkat in response to stimulation with TLR agonists is reduced when cell stimulation is carried out in presence of serum. Serum does not, however, inhibit TCR-activated secretion of CXCL8 nor does it down-regulate TLR-costimulated IL-2 secretion from activated T cells. The molecule that can mimic the ability to bring about suppression in CXCL8 from TLR-activated T cells is serum-borne bioactive lipid, S1P. Serum and S1P-mediated inhibition require intracellular calcium. S1P also suppresses CXCL8 secretion from peripheral blood-derived human T cells activated ex vivo with various TLR ligands. Our findings reveal a previously unrecognized role for S1P in regulating TLR-induced CXCL8 secretion from human T cells.

Topics: Calcium; Fingolimod Hydrochloride; Flagellin; Gene Expression Regulation; Humans; Interleukin-2; Interleukin-8; Jurkat Cells; Lipopeptides; Lymphocyte Activation; Lysophospholipids; Molecular Mimicry; Oxadiazoles; Propylene Glycols; Receptors, Antigen, T-Cell; Serum; Signal Transduction; Sphingosine; T-Lymphocytes; Thiophenes; Toll-Like Receptors; U937 Cells

Exogenous sphingosine-1-phosphate (S1P), a lipid mediator in blood, attenuates acute microvascular permeability increases via receptor S1P1 to stabilize the endothelium. To evaluate the contribution of erythrocytes as an endogenous source of S1P to the regulation of basal permeability, we measured permeability coefficients in intact individually perfused venular microvessels of rat mesentery. This strategy also enabled the contributions of other endogenous S1P sources to be evaluated. Apparent permeability coefficients (P(S)) to albumin and α-lactalbumin and the hydraulic conductivity of mesenteric microvessels were measured in the presence or absence of rat erythrocytes or rat erythrocyte-conditioned perfusate. Rat erythrocytes added to the perfusate were the principal source of S1P in these microvessels. Basal P(S) to albumin was stable and typical of blood-perfused microvessels (mean 0.5 × 10(-6) cm/s) when erythrocytes or erythrocyte-conditioned perfusates were present. When they were absent, P(S) to albumin or α-lactalbumin increased up to 40-fold (over 10 min). When exogenous S1P was added to perfusates, permeability returned to levels comparable with those seen in the presence of erythrocytes. Addition of SEW 2871, an agonist specific for S1P1, in the absence of red blood cells reduced P(S)(BSA) (40-fold reduction) toward basal. The specific S1P1 receptor antagonist (W-146) reversed the stabilizing action of erythrocytes and increased permeability (27-fold increase) in a manner similar to that seen in the absence of erythrocytes. Erythrocytes are a primary source of S1P that maintains normal venular microvessel permeability. Absence of erythrocytes or conditioned perfusate in in vivo and in vitro models of endothelial barriers elevates basal permeability.

Topics: Albumins; Animals; Capillary Permeability; Endothelium, Vascular; Erythrocytes; Lactalbumin; Lysophospholipids; Male; Mesentery; Oxadiazoles; Paracrine Communication; Pressure; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Sphingosine; Thiophenes; Time Factors; Venules

Directed migration of hepatic myofibroblasts (hMFs) contributes to the development of liver fibrosis. However, the signals regulating the motility of these cells are incompletely understood. We have recently shown that sphingosine 1-phosphate (S1P) and S1P receptors (S1PRs) are involved in mouse liver fibrogenesis. Here, we investigated the role of S1P/S1PRs signals in human liver fibrosis involving motility of human hMFs.. S1P level in the liver was examined by high-performance liquid chromatography. Expression of S1PRs was characterized, in biopsy specimens of human liver and cultured hMFs, by immunofluorescence and real-time RT-PCR or Western blot analysis. Cell migration was determined in Boyden chambers, by using the selective S1P receptor agonist or antagonist and silencing of S1PRs expression with small interfering RNA.. S1P level in the human fibrotic liver was increased through up-regulation of sphingosine kinase (SphK), irrespective of the etiology of fibrosis. S1P receptors type 1, 2, and 3 (S1P(1,2,3)) were expressed in human hMFs in vivo and in vitro. Interestingly, S1P(1,3) were strongly induced in human fibrotic samples, whereas expression of S1P(2) was massively decreased. S1P exerted a powerful migratory action on human hMFs. Furthermore, the effect of S1P was mimicked by SEW2871 (an S1P(1) agonist), and blocked by suramin (an S1P(3) antagonist) and by silencing S1P(1,3) expression. In contrast, JTE-013 (an S1P(2) antagonist) and silencing of S1P(2) expression enhanced S1P-induced migration.. SphK/S1P/S1PRs signaling axis plays an important role in human liver fibrosis and is involved in the directed migration of human hMFs into the damaged areas.

Topics: Base Sequence; Cell Movement; Cells, Cultured; Humans; Liver Cirrhosis; Lysophospholipids; Myofibroblasts; Oxadiazoles; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Suramin; Thiophenes

Sphingosine-1-phosphate (S1P) is an important mediator of inflammation recently shown in in vitro studies to increase the excitability of small-diameter sensory neurons, at least in part, via activation of the S1P(1) receptor subtype. Activation of S1PR(1) has been reported to increase the formation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived superoxide (O(2)(·-)) and nitric oxide synthase (NOS)-derived nitric oxide (NO). This process favors the formation of peroxynitrite (ONOO(-) [PN]), a potent mediator of hyperalgesia associated with peripheral and central sensitization. The aims of our study were to determine whether S1P causes peripheral sensitization and thermal hyperalgesia via S1PR(1) activation and PN formation. Intraplantar injection of S1P in rats led to a time-dependent development of thermal hyperalgesia that was blocked by the S1PR(1) antagonist W146, but not its inactive enantiomer W140. The hyperalgesic effects of S1P were mimicked by intraplantar injection of the well-characterized S1PR(1) agonist SEW2871. The development of S1P-induced hyperalgesia was blocked by apocynin, a NADPH oxidase inhibitor; N(G)-nitro-l-arginine methyl ester, a nonselective NOS inhibitor; and by the potent PN decomposition catalysts (FeTM-4-PyP(5+) and MnTE-2-PyP(5+)). Our findings provide mechanistic insight into the signaling pathways engaged by S1P in the development of hyperalgesia and highlight the contribution of the S1P(1) receptor-to-PN signaling in this process. Sphingosine-1-phosphate (S1P)-induced hyperalgesia is mediated by S1P1 receptor activation and mitigated by inhibition or decomposition of peroxynitrite, providing a target pathway for novel pain management strategies.

Topics: Acetophenones; Anilides; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Activation; Enzyme Inhibitors; Hyperalgesia; Lysophospholipids; Male; Metalloporphyrins; NG-Nitroarginine Methyl Ester; Organophosphonates; Oxadiazoles; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Reaction Time; Receptors, Lysosphingolipid; Sphingosine; Thiophenes; Time Factors

Elevated intraocular pressure is the main risk factor in primary open-angle glaucoma, involving an increased resistance to aqueous humor outflow in the juxtacanalicular region of the conventional outflow pathway which includes the trabecular meshwork (TM) and the inner wall of Schlemm's canal (SC). Previously, sphingosine-1-phosphate (S1P) was shown to decrease outflow facility in porcine and human eyes, thus increasing outflow resistance and intraocular pressure. Owing to S1P's known effect of increasing barrier function in endothelial cells and the robust expression of the S1P₁ receptor on the inner wall of SC, we hypothesized that S1P₁ receptor activation promotes junction formation and decreases outflow facility. The effects of subtype-specific S1P receptor compounds were tested in human and porcine whole-eye perfusions and human primary cultures of SC and TM cells to determine the receptor responsible for S1P effects on outflow resistance. The S1P₁-specific agonist SEW2871 failed to both mimic S1P effects in paired human eye perfusions, as well as increase myosin light chain (MLC) phosphorylation in cell culture, a prominent outcome in S1P-treated SC and TM cells. In contrast, the S1P₂ antagonist JTE-013, but not the S1P₁ or S1P₁,₃ antagonists, blocked the S1P-promoted increase in MLC phosphorylation. Moreover, JTE-013 prevented S1P-induced decrease in outflow facility in perfused human eyes (P < 0.05, n = 6 pairs). Similarly, porcine eyes perfused with JTE-013 + S1P did not differ from eyes with JTE-013 alone (P = 0.53, n = 3). These results demonstrate that S1P₂ , and not S1P₁ or S1P₃, receptor activation increases conventional outflow resistance and is a potential target to regulate intraocular pressure.

Topics: Animals; Aqueous Humor; Cells, Cultured; Glaucoma, Open-Angle; Humans; Intraocular Pressure; Lysophospholipids; Myosin Light Chains; Oxadiazoles; Phosphorylation; Pyrazoles; Pyridines; Receptors, Lysosphingolipid; Sphingosine; Swine; Thiophenes; Trabecular Meshwork

Sphingosine-1-phosphate (S1P) has been implicated in angiogenesis, inflammation, cancerogenesis, neurological excitability and immune regulation and is synthesized by two different sphingosine kinases (SphK). It was suggested that mice lacking the gene for SphK1 exhibit no obvious phenotype, because SphK2 compensates for its absence. However, recent investigations revealed that under challenge SphK1 contributed to pro-inflammatory processes favoring Th2 and Th17 rather than Th1-type reactions. To investigate the immune modulatory role of SphK1 as opposed to SphK2 specifically for the Th1 propagating IL-12p70 we compared WT and SphK1(-/-) splenocytes and Flt3-ligand differentiated BMCs of WT and SphK1(-/-), representing dendritic cells as major producers of IL-12p70, incubated with LPS. We determined the impact on IL-12p70 in comparison to other inflammatory cytokines, and on DC and macrophage surface marker expression, SphK mRNA, protein expression and enzymatic activity in splenocytes. Our data demonstrated that SphK1 deficiency enhanced LPS-induced IL-12p70 production although SphK2 was present. To further characterize SphK1-dependent IL-12p70 regulation we exogenously applied S1P, SEW2871 and the new potent S1P1 agonist CYM5442. Both S1P and S1P1-specific analogs fully compensated the increase of IL-12p70 production in SphK1-deficient splenocytes. The use of pertussis toxin, to block G(i)-coupled signaling downstream of S1P1, again increased IL-12p70 and neglected the compensation achieved by addition of S1P and S1P1 agonists pointing on the importance of this specific S1P-receptor. Given that, in parallel to a prominent IL-12p35 increase following LPS stimulation, LPS also enhanced SphK expression and total SphK activity, we concluded that SphK1-derived S1P acting via S1P1 is a major mechanism of this negative IL-12p70 feedback loop, which did not affect other cytokines. Moreover, our data showed that SphK2 activity failed to compensate for SphK1 deficiency. These findings clearly point to a divergent and cytokine-specific impact of immune cell SphK1 and SphK2 in chronic inflammation and cancer.

Topics: Animals; Bone Marrow Cells; CD8 Antigens; Cell Differentiation; Dendritic Cells; Enzyme Assays; Flow Cytometry; Gene Deletion; Gene Expression Regulation; Indans; Interleukin-12; Interleukin-12 Receptor beta 2 Subunit; Interleukin-12 Subunit p40; Lipopolysaccharides; Lymphocyte Activation; Lysophospholipids; Membrane Proteins; Mice; Oxadiazoles; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; Sphingosine; Spleen; Thiophenes; Toll-Like Receptors

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.

Topics: Animals; Apoptosis; Cryopreservation; Female; Fingolimod Hydrochloride; Graft Survival; Humans; Immunohistochemistry; Immunosuppressive Agents; Lysophospholipids; Mice; Mice, SCID; Neovascularization, Physiologic; Ovarian Follicle; Ovary; Oxadiazoles; Propylene Glycols; Sphingosine; Thiophenes; Transplantation, Heterologous

Endothelial cell (EC) barrier dysfunction induced by inflammatory agonists is a frequent pathophysiologic event in multiple diseases. The platelet-derived phospholipid sphingosine-1 phosphate (S1P) reverses this dysfunction by potently enhancing the EC barrier through a process involving Rac GTPase-dependent cortical actin rearrangement as an integral step. In this study we explored the role of the ezrin, radixin, and moesin (ERM) family of actin-binding linker protein in modulating S1P-induced human pulmonary EC barrier enhancement. S1P induces ERM translocation to the EC periphery and promotes ERM phosphorylation on a critical threonine residue (Ezrin-567, Radixin-564, Moesin-558). This phosphorylation is dependent on activation of PKC isoforms and Rac1. The majority of ERM phosphorylation on these critical threonine residues after S1P occurs in moesin and ezrin. Baseline radixin phosphorylation is higher than in the other two ERM proteins but does not increase after S1P. S1P-induced moesin and ezrin threonine phosphorylation is not mediated by the barrier enhancing receptor S1PR1 because siRNA downregulation of S1PR1 fails to inhibit these phosphorylation events, while stimulation of EC with the S1PR1-specific agonist SEW2871 fails to induce these phosphorylation events. Silencing of either all ERM proteins or radixin alone (but not moesin alone) reduced S1P-induced Rac1 activation and phosphorylation of the downstream Rac1 effector PAK1. Radixin siRNA alone, or combined siRNA for all three ERM proteins, dramatically attenuates S1P-induced EC barrier enhancement (measured by transendothelial electrical resistance (TER), peripheral accumulation of di-phospho-MLC, and cortical cytoskeletal rearrangement. In contrast, moesin depletion has the opposite effects on these parameters. Ezrin silencing partially attenuates S1P-induced EC barrier enhancement and cytoskeletal changes. Thus, despite structural similarities and reported functional redundancy, the ERM proteins differentially modulate S1P-induced alterations in lung EC cytoskeleton and permeability. These results suggest that ERM activation is an important regulatory event in EC barrier responses to S1P.

Topics: Actins; Amides; Antigens, CD; Bacterial Proteins; Bacterial Toxins; Cadherins; Cell Membrane; Cells, Cultured; Chelating Agents; Cytoskeletal Proteins; Cytoskeleton; Egtazic Acid; Electric Impedance; Endothelial Cells; Gene Knockdown Techniques; Humans; Imidazoles; Lysophospholipids; Membrane Proteins; Microfilament Proteins; Oxadiazoles; p38 Mitogen-Activated Protein Kinases; Permeability; Phosphorylation; Protein Kinase C; Pulmonary Artery; Pyridines; Receptors, Lysosphingolipid; rho GTP-Binding Proteins; rho-Associated Kinases; RNA Interference; Sphingosine; Thiophenes

The aims of this study are to examine the effect of sphingosine 1-phosphate (S1P) on IL-2-activated natural killer (NK) cell lysis of K562 tumor cells and immature dendritic cells (iDCs), and to investigate the mechanisms involved in S1P activity. Our results show that S1P protected K562 cells or iDCs from NK cell lysis, which was reversed by FTY720 and SEW2871, the antagonists of S1P(1). S1P did not modulate the expression of NKG2D, NKp30, NKp44 or CD158 on the surface of NK cells, and neither affected the expression of CD80, CD83, or CD86 on the surface of DCs. In contrast, it increased the expression of HLA-I and HLA-E on DCs, an activity that was inhibited by FTY720 or SEW2871. Similarly, the inhibitory effect of S1P for NK cell lysis of K562 cells was directed toward S1P(1) expressed on the tumor cells but not on NK cells. Further analysis indicates that NK cells secreted various cytokines and chemokines with various intensities: (1) low (IL-4, IL-6, IL-12, TNF-alpha and MCP-1); (2) intermediate (IL-1beta, IL-10, TGF-beta1, and IL-17A); (3) high (IFN-gamma, and MIP-1alpha); and (4) very high (MIP-1beta). S1P significantly reduced the release of IL-17A and IFN-gamma from NK cells, but this inhibition was S1P(1)-independent. These results indicate that S1P is an anti-inflammatory molecule, and that S1P(1) is important for the interaction among NK cells and tumor cells or DCs leading to up-regulation of HLA-I and HLA-E on the surface of DCs, but not in S1P inhibition of the release of inflammatory cytokines from NK cells. Further, the results suggest that FTY720 and SEW2871 may potentially be used as prophylactic and/or therapeutic drugs to treat cancer patients.

Topics: Chemokines; Cytokines; Cytotoxicity, Immunologic; Dendritic Cells; Enzyme-Linked Immunosorbent Assay; Fingolimod Hydrochloride; Flow Cytometry; Humans; Immunoblotting; Immunosuppressive Agents; K562 Cells; Killer Cells, Natural; Lysophospholipids; Oxadiazoles; Propylene Glycols; Sphingosine; Thiophenes; Up-Regulation

Sphingosine 1-phosphate (S1P) is a key endogenous regulator of the response to lung injury, maintaining endothelial barrier integrity through interaction with one of its receptors, S1P(1). The short-term administration of S1P or S1P(1) receptor agonists enhances endothelial monolayer barrier function in vitro, and attenuates injury-induced vascular leak in the lung and other organ systems in vivo. Although S1P(1) agonists bind to and activate S1P(1), several of these agents also induce receptor internalization and degradation, and may therefore act as functional antagonists of S1P(1) after extended exposure. Here we report on the effects of prolonged exposure to these agents in bleomycin-induced lung injury. We demonstrate that repeated administration of S1P(1) agonists dramatically worsened lung injury after bleomycin challenge, as manifested by increased vascular leak and mortality. Consistent with these results, prolonged exposure to S1P(1) agonists in vitro eliminated the ability of endothelial cell monolayers to respond appropriately to the barrier-protective effects of S1P, indicating a loss of normal S1P-S1P(1) signaling. As bleomycin-induced lung injury progressed, continued exposure to S1P(1) agonists also resulted in increased pulmonary fibrosis. These data indicate that S1P(1) agonists can act as functional antagonists of S1P(1) on endothelial cells in vivo, which should be considered in developing these agents as therapies for vascular leak syndromes. Our findings also support the hypothesis that vascular leak is an important component of the fibrogenic response to lung injury, and suggest that targeting the S1P-S1P(1) pathway may also be an effective therapeutic strategy for fibrotic lung diseases.

Topics: Animals; beta-Alanine; Bleomycin; Blood Coagulation; Endothelial Cells; Fibrosis; Fingolimod Hydrochloride; Humans; Lung Injury; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Oxadiazoles; Pneumonia; Propylene Glycols; Pulmonary Alveoli; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Survival Analysis; Thiophenes; Vascular Diseases

Liver failure due to ischemia and reperfusion (IR) and subsequent acute kidney injury are significant clinical problems. We showed previously that liver IR selectively reduced plasma sphinganine-1-phosphate levels without affecting sphingosine-1-phosphate (S1P) levels. Furthermore, exogenous sphinganine-1-phosphate protected against both liver and kidney injury induced by liver IR. In this study, we elucidated the signaling mechanisms of sphinganine-1-phosphate-mediated renal and hepatic protection. A selective S1P(1) receptor antagonist blocked the hepatic and renal protective effects of sphinganine-1-phosphate, whereas a selective S1P(2) or S1P(3) receptor antagonist was without effect. Moreover, a selective S1P(1) receptor agonist, SEW-2871, provided similar degree of liver and kidney protection compared with sphinganine-1-phosphate. Furthermore, in vivo gene knockdown of S1P(1) receptors with small interfering RNA abolished the hepatic and renal protective effects of sphinganine-1-phosphate. In contrast to sphinganine-1-phosphate, S1P's hepatic protection was enhanced with an S1P(3) receptor antagonist. Inhibition of extracellular signal-regulated kinase, Akt or pertussis toxin-sensitive G-proteins blocked sphinganine-1-phosphate-mediated liver and kidney protection in vivo. Taken together, our results show that sphinganine-1-phosphate provided renal and hepatic protection after liver IR injury in mice through selective activation of S1P(1) receptors and pertussis toxin-sensitive G-proteins with subsequent activation of ERK and Akt.

Topics: Acute Kidney Injury; Animals; Extracellular Signal-Regulated MAP Kinases; Ischemia; Kidney; Liver; Liver Diseases; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Oxadiazoles; Proto-Oncogene Proteins c-akt; Receptors, Lysosphingolipid; Reperfusion Injury; Signal Transduction; Sphingosine; Thiophenes

Sphingosine 1-phosphate (S1P) and its receptor, S1P receptor type 1 (S1P(1)), are essential for lymphocyte egress from secondary lymphoid organs (SLO). Fingolimod (FTY720), the S1P receptor modulator, inhibits lymphocyte egress from SLO and decreases circulating lymphocytes; however, it also induces a significant decrease in the number of peripheral blood lymphocytes in alymphoplasia (aly/aly) mice lacking SLO. In this study, we demonstrated that the administration of FTY720 induced sequestration of mature lymphocytes, particularly T cells, into the bone marrow (BM) in aly/aly mice, implying that the reduction of circulating lymphocytes in these mice by FTY720 was due to inhibition of lymphocyte egress from the BM. Since sequestration of mature T cells into the BM was also induced in normal mice by selective S1P(1) agonist or S1P lyase inhibitor, it is suggested that S1P(1) expression and the S1P gradient play an important role in egress of mature T cells from the BM. Prophylactic administration of FTY720 to ovalbumin (OVA)-immunized mice significantly inhibited footpad swelling induced by OVA challenging with a marked reduction of OVA-specific T(h) cells in the BM, indicating that immunomodulation by FTY720 is likely due to reduced circulation of antigen-specific T(h) cells. On the other hand, OVA-specific T(h) cells, like naive T cells, were also sequestered into the BM and SLO of OVA-immunized mice by a short exposure of FTY720 after OVA challenging. These results suggest that the S1P-S1P(1) axis plays a regulatory role in egress of mature T cells including antigen-specific T(h) cells from the BM.

Topics: Adoptive Transfer; Animals; Bone Marrow; Cell Count; Cell Movement; Fingolimod Hydrochloride; Hypersensitivity, Delayed; Immunization; Lymphoid Tissue; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Ovalbumin; Oxadiazoles; Propylene Glycols; Receptors, Lysosphingolipid; Sphingosine; T-Lymphocytes; Thiophenes

Sphingosine 1-phosphate (S1P) through its interaction with a family of G protein-coupled receptors (S1PR) is proving to have a significant impact on the activation of a variety of cell types, most notably those cells mediating the inflammatory response. Previously, we showed that S1P enhanced the excitability of small diameter sensory neurons, and mRNA for S1PR(1-4) was expressed in sensory neurons. These initial findings did not determine which S1PR subtype(s) mediated the increased excitability. Here, we report that exposure to the selective S1PR(1) agonist, SEW2871, produced a significant increase in excitability of some, but not all, sensory neurons. To further examine the role of S1PR(1), neurons were treated with siRNA targeted to S1PR(1). siRNA reduced S1PR(1) protein expression by 75% and blocked the sensitization produced by SEW2871, although some neurons remained responsive to subsequent exposure to S1P. Treatment with scramble siRNA did not alter S1PR(1) expression. Recordings from siRNA- and scramble-treated neurons suggested three distinct populations based on their sensitivities to SEW2871 and S1P. Approximately 50% of the neurons exhibited a significant increase in excitability after exposure to SEW2871 and subsequent S1P produced no additional increase; ∼25% were not affected by SEW2871 but S1P significantly increased excitability; and ∼25% of the neurons were not sensitized by either SEW2871 or S1P. RT-PCR measurements obtained from single neurons showed that 50% of the small diameter neurons expressed the mRNA for S1PR(1). These results indicate that S1PR(1) plays a prominent, although not exclusive, role in mediating the enhancement of excitability produced by S1P.

Topics: Analysis of Variance; Animals; Blotting, Western; Cells, Cultured; Electrophysiology; Lysophospholipids; Male; Membrane Potentials; Oxadiazoles; Posterior Horn Cells; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Signal Transduction; Sphingosine; Thiophenes

Migration of Langerhans cells (LCs) from the skin to the lymph node is an essential step in the pathogenesis of allergic contact dermatitis (ACD). Therefore, inhibition of LC-migration could be a promising strategy to improve this skin disease. Effects of sphingosine-1-phosphate (S1P) and the immunomodulator FTY720 on LC trafficking is not well defined, yet. Thus, we investigated the action of topically administered S1P and FTY720 in a murine model of ACD. Most interestingly, FTY720 as well as S1P inhibited the inflammatory reaction in the elicitation phase of ACD. In the sensitization phase, FTY720, and S1P reduced the weight and cell count of the draining auricular lymph node, as well as immigrated dendritic cells provoked by repetitive topical administration of the hapten. Correspondingly, the density of LCs in the epidermis was higher in FTY720- and S1P-treated mice compared to vehicle treatment. A skin dendritic cell migration assay confirmed the significant inhibition of dendritic cell migration by FTY720 and S1P. These data supply conclusive evidence that the strategy of targeting the migratory response of LCs with locally acting S1P or FTY720 represents an emerging option in the treatment of allergic skin diseases like contact hypersensitivity and atopic dermatitis.

Topics: Administration, Topical; Animals; Cell Movement; Dendritic Cells; Dermatitis, Allergic Contact; Female; Fingolimod Hydrochloride; Histocompatibility Antigens Class II; Immunosuppressive Agents; Lysophospholipids; Mice; Mice, Inbred BALB C; Oxadiazoles; Propylene Glycols; Receptors, Lysosphingolipid; Skin; Sphingosine; Thiophenes; Toluene 2,4-Diisocyanate

Several experimental studies have demonstrated protection against cardiac ischaemia-reperfusion injury achieved by pre-treatment with exogenous sphingosine-1-phosphate (S1P). We tested the hypothesis that pharmacological S1P receptor agonists improve recovery of function when applied with reperfusion.. Isolated rat cardiomyocytes were stimulated with exogenous S1P, the selective S1P1 receptor agonist SEW2871, or the S1P1/3 receptor agonist FTY720. Western blot analysis was performed to analyse downstream signalling pathways. Ischaemia-reperfusion studies were conducted in rat cardiomyocytes, isolated Langendorff-perfused rat hearts, and in human myocardial muscle strip preparations to evaluate the effect of S1P receptor agonists on cell death and recovery of mechanical function. All S1P receptor agonists were able to activate Akt. This was associated with transactivation of the epidermal growth factor receptor. In isolated cardiomyocytes, selective stimulation of the S1P1 receptor by SEW2871 induced protection against cell death when administered either before or after ischaemia-reperfusion. In isolated rat hearts, treatment with FTY720 during reperfusion attenuated the rise in left ventricular end-diastolic pressure (LVEDP) and improved the recovery of left ventricular developed pressure without limiting infarct size. However, selective S1P1 receptor stimulation did not improve functional recovery but rather increased LVEDP. Additional experiments employing a human myocardial ischaemia-reperfusion model also demonstrated improved functional recovery induced by FTY720 treatment during reperfusion.. Pharmacological S1P receptor agonists have distinct effects on ischaemia-reperfusion injury. Their efficacy when applied during reperfusion makes them potential candidates for pharmaceutical postconditioning therapy after cardiac ischaemia.

Topics: Animals; Animals, Newborn; Cardiotonic Agents; Cell Death; Cells, Cultured; ErbB Receptors; Fingolimod Hydrochloride; Humans; Lysophospholipids; Myocardial Reperfusion Injury; Myocytes, Cardiac; Oxadiazoles; Perfusion; Propylene Glycols; Proto-Oncogene Proteins c-akt; Rats; Receptors, Lysosphingolipid; Recovery of Function; Signal Transduction; Sphingosine; Thiophenes; Time Factors; Ventricular Function, Left; Ventricular Pressure

Sphingosine-1-phosphate (S1P) is a ubiquitous, lipophilic cellular mediator that acts in part by activation of G-protein-coupled receptor. Modulation of S1P signaling is an emerging pharmacotherapeutic target for immunomodulatory drugs. Although multiple S1P receptor types exist in the CNS, little is known about their function. Here, we report that S1P stimulated G-protein activity in the CNS, and results from [(35)S]GTPgammaS autoradiography using the S1P(1)-selective agonist SEW2871 and the S1P(1/3)-selective antagonist VPC44116 show that in several regions a majority of this activity is mediated by S1P(1) receptors. S1P receptor activation inhibited glutamatergic neurotransmission as determined by electrophysiological recordings in cortical neurons in vitro, and this effect was mimicked by SEW2871 and inhibited by VPC44116. Moreover, central administration of S1P produced in vivo effects resembling the actions of cannabinoids, including thermal antinociception, hypothermia, catalepsy and hypolocomotion, but these actions were independent of CB(1) receptors. At least one of the central effects of S1P, thermal antinociception, is also at least partly S1P(1) receptor mediated because it was produced by SEW2871 and attenuated by VPC44116. These results indicate that CNS S1P receptors are part of a physiologically relevant and widespread neuromodulatory system, and that the S1P(1) receptor contributes to S1P-mediated antinociception.

Topics: Animals; Animals, Newborn; Binding, Competitive; Cannabinoids; Cells, Cultured; Cerebral Cortex; Coculture Techniques; Guanosine 5'-O-(3-Thiotriphosphate); Hyperalgesia; Lysophospholipids; Neurons; Oxadiazoles; Patch-Clamp Techniques; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; Receptors, Lysosphingolipid; Receptors, Neurotransmitter; Signal Transduction; Sphingosine; Sulfur Radioisotopes; Synaptic Transmission; Thiophenes

Evidence is emerging indicating that sphingosine-1-phosphate (S1P) participates in signaling in the retina. To determine whether S1P might be involved in signaling in the inner retina specifically, we examine the effects of this sphingolipid on cultured retinal amacrine cells. Whole cell voltage-clamp recordings reveal that S1P activates a cation current that is dependent on signaling through G(i) and phospholipase C. These observations are consistent with the involvement of members of the S1P receptor family of G-protein-coupled receptors in the production of the current. Immunocytochemistry and PCR amplification provide evidence for the expression of S1P1R and S1P3R in amacrine cells. The receptor-mediated channel activity is shown to be highly sensitive to blockade by lanthanides consistent with the behavior of transient receptor potential canonical (TRPC) channels. PCR products amplified from amacrine cells reveal that TRPCs 1 and 3-7 channel subunits have the potential to be expressed. Because TRPC channels provide a Ca(2+) entry pathway, we asked whether S1P caused cytosolic Ca(2+) elevations in amacrine cells. We show that S1P-dependent Ca(2+) elevations do occur in these cells and that they might be mediated by S1P1R and S1P3R. The Ca(2+) elevations are partially due to release from internal stores, but the largest contribution is from influx across the plasma membrane. The effect of inhibition of sphingosine kinase suggests that the production of cytosolic S1P underlies the sustained nature of the Ca(2+) elevations. Elucidation of the downstream effects of these signals will provide clues to the role of S1P in regulating inner retinal function.

Topics: Amacrine Cells; Animals; Calcium; Calcium Signaling; Cells, Cultured; Chick Embryo; Estrenes; Heparin; Inositol 1,4,5-Trisphosphate Receptors; Ion Channel Gating; Lanthanoid Series Elements; Lysophospholipids; Membrane Potentials; Models, Biological; Oxadiazoles; Patch-Clamp Techniques; Pertussis Toxin; Phosphodiesterase Inhibitors; Potassium Channel Blockers; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Retina; Sphingosine; Tetraethylammonium; Thiophenes; Type C Phospholipases

Sphingosine 1-phosphate (S1P) selectively and potently constricts isolated cerebral arteries, but this response has not been pharmacologically characterized.. The receptor subtype(s) involved in S1P-induced cerebrovascular constriction were characterized using genetic (S1P(2) and S1P(3) receptor null mice) and pharmacological tools (phospho-FTY720, a S1P(1/3/4/5) receptor agonist; SEW2871, a S1P(1) receptor agonist, JTE-013, a S1P(2) receptor antagonist, VPC23019, a S1P(1/3) receptor antagonist). Isolated basilar or peripheral (femoral, mesenteric resistance) arteries, from either rat or mouse, were studied in a wire myograph.. S1P concentration-dependently constricted basilar artery in rat, wild-type (WT) and S1P(2) null mice, but barely affected vascular tone in S1P(3) null mice. Vasoconstriction to U46619 (a thromboxane analogue) or to endothelin-1 did not differ between WT, S1P(2) and S1P(3) null mice. JTE-013 inhibited not only S1P-induced vasoconstriction, but also KCl-, U46619- and endothelin-1-induced constriction. This effect was observed in WT as well as in S1P(2) null mice. VPC23019 increased the concentration-dependent vasoconstriction to S1P in both rat and mouse basilar arteries with intact endothelium, but not in rat basilar artery without endothelium. Phospho-FTY720 concentration-dependently constricted rat basilar arteries, but not femoral or mesenteric resistance arteries, while SEW2871 did not induce any response in the same arteries.. S1P constricts cerebral arteries through S1P(3) receptors. The purported S1P(2) receptor antagonist JTE-013 does not appear to be selective, at least in rodents. Enhancement of S1P-induced contraction by VPC23019 might be related to blockade of S1P(1) receptors and NO generation.

Topics: Animals; Cerebral Arteries; Dose-Response Relationship, Drug; Fingolimod Hydrochloride; In Vitro Techniques; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Oxadiazoles; Propylene Glycols; Pyrazoles; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Sphingosine; Thiophenes; Vasoconstriction

Adult mouse ventricular myocytes express S1P(1), S1P(2), and S1P(3) receptors. S1P activates Akt and ERK in adult mouse ventricular myocytes through a pertussis toxin-sensitive (G(i/o)-mediated) pathway. Akt and ERK activation by S1P are reduced approximately 30% in S1P(3) and 60% in S1P(2) receptor knock-out myocytes. With combined S1P(2,3) receptor deletion, activation of Akt is abolished and ERK activation is reduced by nearly 90%. Thus the S1P(1) receptor, while present in S1P(2,3) receptor knock-out myocytes, is unable to mediate Akt or ERK activation. In contrast, S1P induces pertussis toxin-sensitive inhibition of isoproterenol-stimulated cAMP accumulation in both WT and S1P(2,3) receptor knock-out myocytes demonstrating that the S1P(1) receptor can functionally couple to G(i). An S1P(1) receptor selective agonist, SEW2871, also decreased cAMP accumulation but failed to activate ERK or Akt. To determine whether localization of the S1P(1) receptor mediates this signaling specificity, methyl-beta-cyclodextrin (MbetaCD) treatment was used to disrupt caveolae. The S1P(1) receptor was concentrated in caveolar fractions, and associated with caveolin-3 and this localization was disrupted by MbetaCD. S1P-mediated activation of ERK or Akt was not diminished but inhibition of cAMP accumulation by S1P and SEW2871 was abolished by MbetaCD treatment. S1P inhibits the positive inotropic response to isoproterenol and this response is also mediated through the S1P(1) receptor and lost following caveolar disruption. Thus localization of S1P(1) receptors to caveolae is required for the ability of this receptor to inhibit adenylyl cyclase and contractility but compromises receptor coupling to Akt and ERK.

Topics: Animals; beta-Cyclodextrins; Cyclic AMP; Enzyme Activation; Gene Expression Regulation; GTP-Binding Protein alpha Subunits, Gi-Go; Isoproterenol; Lysophospholipids; Male; Mice; Mice, Knockout; Mitogen-Activated Protein Kinases; Myocardial Contraction; Myocytes, Cardiac; Oxadiazoles; Pertussis Toxin; Protein Transport; Proto-Oncogene Proteins c-akt; Receptors, Lysosphingolipid; RNA, Messenger; Sphingosine; Thiophenes

The phosphorylated derivative of the immunosuppressant FTY720 interacts with and modulates the function of sphingosine-1-phosphate (S1P)-receptors. We observed a significant reduction of endothelial surface binding of a S1P(1)-specific antibody after FTY720 treatment of 6h and longer, which was associated with a reduced healing after mechanic injury, impaired angiogenesis and enhanced adhesion molecule expression. FTY720 (5h) had no impact on the expression of S1P(1)- or S1P(3)-encoding transcripts. Notably, pre-treatment of cells with FTY720 for only 30min, which did not reduce S1P(1) surface expression, inhibited the rapid S1P- and SEW2871- (a S1P(1) agonist) induced cortical actin formation and cell migration. FTY720 was effective at concentrations as low as 5nM. FTY720 at therapeutic concentrations may be harmful by impairing important endothelial functions. Interestingly, FTY720 inhibited endothelial actin remodelling and cell migration without decreasing S1P(1) surface expression.

Topics: Actins; Cell Adhesion Molecules; Cell Membrane; Cell Movement; Cells, Cultured; Endothelium, Vascular; Fingolimod Hydrochloride; Humans; Immunosuppressive Agents; Lysophospholipids; Neovascularization, Physiologic; Oxadiazoles; Propylene Glycols; Receptors, Lysosphingolipid; Sphingosine; Thiophenes; Wound Healing

Sphingosine 1-phosphate subtype 1 (S1P(1)) receptor agonists alter lymphocyte trafficking and endothelial barrier integrity in vivo. Among these is the potent, non-selective agonist, FTY720-P, whose mechanism of action has been suggested to correlate with S1P(1) down-regulation. Discovery of the in vivo active S1P(1)-selective agonist, SEW2871, has broadened our understanding of minimal requirements for S1P(1) function while highlighting differences regarding agonist effect on S1P(1) fate, because SEW2871 does not degrade S1P(1). To further understand the mechanism of agonist-induced S1P(1) down-regulation, we compared signaling and fate of human S1P(1)-green fluorescent protein (GFP) in stable 293 cells, using AFD-R, a chiral analog of FTY720-P, SEW2871, and S1P. Although all agonists acutely internalized S1P(1) to late endosomal vesicles and activated GTPgammaS(35) binding and pERK to similar maxima, only AFD-R led to significant S1P(1) down-regulation, as shown by GFP immunoprecipitation studies. Down-regulation was time- and concentration-dependent, was partially blocked by proteasomal inhibition and reversed by chloroquine and an antagonist to S1P(1). All agonists induced a receptor-associated increase in ubiquitination, with AFD-R inducing 3-fold more accumulation than S1P and being 3-4 logs more potent than SEW2871. The formation of AFD-R-receptor ubiquitin complex was inhibited by antagonist and chloroquine and was enhanced by proteasomal inhibition. Identification of proteins by PAGE liquid chromatography-tandem mass spectrometry in cells treated with AFD-R confirmed the co-migration of ubiquitin peptides with those of S1P(1) and GFP, relative to vehicle alone. These data suggest that the hierarchy of ubiquitin recruitment to S1P(1) (AFD-R > S1P > SEW2871) correlates with the efficiency of lysosomal receptor degradation and reflects intrinsic differences between agonists.

Topics: Cells, Cultured; Chloroquine; Fingolimod Hydrochloride; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Ligands; Lysophospholipids; Oxadiazoles; Propylene Glycols; Protein Transport; Proteomics; Receptors, G-Protein-Coupled; Receptors, Lysosphingolipid; Sphingosine; Thiophenes; Ubiquitin

Dendritic cells (DCs) and lymphocytes are known to show a migratory response to the phospholipid mediator, sphingosine 1-phosphate (S1P). However, it is unclear whether the same S1P receptor subtype mediates the migration of lymphocytes and DCs toward S1P. In this study, we investigated the involvement of S1P receptor subtypes in S1P-induced migration of CD4 T cells and bone marrow-derived DCs in mice. A potent S1P receptor agonist, the (S)-enantiomer of FTY720-phosphate [(S)-FTY720-P], at 0.1 nM or higher and a selective S1P receptor type 1 (S1P(1)) agonist, SEW2871, at 0.1 muM or higher induced a dose-dependent down-regulation of S1P(1). The pretreatment with these compounds resulted in a significant inhibition of mouse CD4 T cell migration toward S1P. Thus, it is revealed that CD4 T cell migration toward S1P is highly dependent on S1P(1). Mature DCs, when compared with CD4 T cells or immature DCs, expressed a relatively higher level of S1P(3) mRNA. S1P at 10-1000 nM induced a marked migration and significantly enhanced the endocytosis of FITC-dextran in mature but not immature DCs. Pretreatment with (S)-FTY720-P at 0.1 microM or higher resulted in a significant inhibition of S1P-induced migration and endocytosis in mature DCs, whereas SEW2871 up to 100 microM did not show any clear effect. Moreover, we found that S1P-induced migration and endocytosis were at an extremely low level in mature DCs prepared from S1P(3)-knockout mice. These results indicate that S1P regulates migration and endocytosis of murine mature DCs via S1P(3) but not S1P(1).

Topics: Animals; CD4-Positive T-Lymphocytes; Cell Movement; Cells, Cultured; Dendritic Cells; Dose-Response Relationship, Drug; Down-Regulation; Endocytosis; Fingolimod Hydrochloride; Immunosuppressive Agents; Lysophospholipids; Mice; Mice, Knockout; Oxadiazoles; Propylene Glycols; Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors; Thiophenes

Endothelial activation and monocyte adhesion to endothelium are key events in inflammation. Sphingosine-1-phosphate (S1P) is a sphingolipid that binds to G protein-coupled receptors on endothelial cells (ECs). We examined the role of S1P in modulating endothelial activation and monocyte-EC interactions in vivo.. We injected C57BL/6J mice intravenously with tumor necrosis factor (TNF)-alpha in the presence and absence of the S1P1 receptor agonist SEW2871 and examined monocyte adhesion. Aortas from TNF-alpha-injected mice had a 4-fold increase in the number of monocytes bound, whereas aortas from TNF-alpha plus SEW2871-treated mice had few monocytes bound (P<0.0001). Using siRNA, we found that inhibiting the S1P1 receptor in vascular ECs blocked the ability of S1P to prevent monocyte-EC interactions in response to TNF-alpha. We examined signaling pathways downstream of S1P1 and found that 100 nM S1P increased phosphorylation of Akt and decreased activation of c-jun.. Thus, we provide the first evidence that S1P signaling through the endothelial S1P1 receptor protects the vasculature against TNF-alpha-mediated monocyte-EC interactions in vivo.

Topics: Animals; Aorta; Cell Adhesion; Cells, Cultured; Chemokines; E-Selectin; Endothelium, Vascular; Intercellular Adhesion Molecule-1; Lysophospholipids; Mice; Mice, Inbred C57BL; Monocytes; Oxadiazoles; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Thiophenes; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1; Vasculitis

Using high-throughput screening, Jo et al. in this issue of Chemistry & Biology [1] have identified SEW2871 as a structurally unique sphingosine 1-phosphate(1) (S1P(1)) receptor agonist. SEW2871 binds to and activates the S1P(1) receptor and initiates a survival signaling pathway similar to that of S1P.

Topics: Drug Evaluation, Preclinical; Lysophospholipids; Oxadiazoles; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Thiophenes

The essential role of the sphingosine 1-phosphate (S1P) receptor S1P(1) in regulating lymphocyte trafficking was demonstrated with the S1P(1)-selective nanomolar agonist, SEW2871. Despite its lack of charged headgroup, the tetraaromatic compound SEW2871 binds and activates S1P(1) through a combination of hydrophobic and ion-dipole interactions. Both S1P and SEW2871 activated ERK, Akt, and Rac signaling pathways and induced S1P(1) internalization and recycling, unlike FTY720-phosphate, which induces receptor degradation. Agonism with receptor recycling is sufficient for alteration of lymphocyte trafficking by S1P and SEW2871. S1P(1) modeling and mutagenesis studies revealed that residues binding the S1P headgroup are required for kinase activation by both S1P and SEW2871. Therefore, SEW2871 recapitulates the action of S1P in all the signaling pathways examined and overlaps in interactions with key headgroup binding receptor residues, presumably replacing salt-bridge interactions with ion-dipole interactions.

Topics: Animals; Binding Sites; Cell Line; Cell Membrane; Cricetinae; Drug Evaluation, Preclinical; Enzyme Activation; Humans; Ligands; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Models, Molecular; Molecular Probes; Mutation; Oxadiazoles; Phosphorylation; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; rac GTP-Binding Proteins; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Thiophenes

Sphingosine 1-phosphate type 1 (S1P(1)) receptor agonists cause sequestration of lymphocytes in secondary lymphoid organs by a mechanism that is not well understood. One hypothesis proposes that agonists act as 'functional antagonists' by binding and internalizing S1P(1) receptors on lymphocytes; a second hypothesis proposes instead that S1P(1) agonists act on endothelial cells to prevent lymphocyte egress from lymph nodes. Here, two-photon imaging of living T cells in explanted lymph nodes after treatment with S1P(1) agonists or antagonists has provided insight into the mechanism by which S1P(1) agonists function. The selective S1P(1) agonist SEW2871 caused reversible slowing and 'log-jamming' of T cells between filled medullary cords and empty sinuses, whereas motility was unaltered in diffuse cortex. Removal or antagonist competition of SEW2871 permitted recovery of T cell motility in the parenchyma of the medulla and resumption of migration across the stromal endothelial barrier, leading to refilling of sinuses. Our results provide visualization of transendothelial migration of T cells into lymphatic sinuses and suggest that S1P(1) agonists act mainly on endothelial cell S1P(1) receptors to inhibit lymphocyte migration.

Topics: Animals; Cell Migration Inhibition; Cell Movement; Cells, Cultured; Lymph Nodes; Lymphatic Vessels; Lysophospholipids; Mice; Mice, Inbred BALB C; Oxadiazoles; Receptors, Lysosphingolipid; Sphingosine; T-Lymphocyte Subsets; Thiophenes