sphingosine-1-phosphate and Lymphopenia

FTY720 is a recently approved first line therapy for relapsing forms of multiple sclerosis. In this context, FTY720 is a pro-drug, with its anti-multiple sclerosis, immunosuppressive effects largely elicited following its phosphorylation by sphingosine kinase 2 and subsequent modulation of G protein-coupled sphingosine 1-phosphate (S1P) receptor 1 that induces lymphopenia by altering lymphocyte trafficking. A number of other biological effects of FTY720 have, however, been described, including considerable evidence that this drug also has anti-cancer properties. These other effects of FTY720 are independent of S1P receptors, and appear facilitated by modulation of a range of other recently described protein targets by nonphosphorylated FTY720. Here, we review the direct targets of FTY720 that contribute to its anti-cancer properties. We also discuss other recently described protein effectors that, in combination with S1P receptors, appear to contribute to its immunosuppressive effects.

Topics: Antineoplastic Agents; Apoptosis; Fingolimod Hydrochloride; Humans; Immunosuppressive Agents; Lymphopenia; Lysophospholipids; Multiple Sclerosis; Neoplasms; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Receptors, Lysosphingolipid; Sphingosine

Sphingosine 1-phosphate (S1P) is an important mediator of cancer cell growth and proliferation. Production of S1P is catalyzed by sphingosine kinase 1 (SphK). Safingol, (l-threo-dihydrosphingosine) is a putative inhibitor of SphK. We conducted a phase I trial of safingol (S) alone and in combination with cisplatin (C).. A 3 + 3 dose escalation was used. For safety, S was given alone 1 week before the combination. S + C were then administered every 3 weeks. S was given over 60 to 120 minutes, depending on dose. Sixty minutes later, C was given over 60 minutes. The C dose of 75 mg/m(2) was reduced in cohort 4 to 60 mg/m(2) due to excessive fatigue.. Forty-three patients were treated, 41 were evaluable for toxicity, and 37 for response. The maximum tolerated dose (MTD) was S 840 mg/m(2) over 120 minutes C 60 mg/m(2), every 3 weeks. Dose-limiting toxicity (DLT) attributed to cisplatin included fatigue and hyponatremia. DLT from S was hepatic enzyme elevation. S pharmacokinetic parameters were linear throughout the dose range with no significant interaction with C. Patients treated at or near the MTD achieved S levels of more than 20 μmol/L and maintained levels greater than and equal to 5 μmol/L for 4 hours. The best response was stable disease in 6 patients for on average 3.3 months (range 1.8-7.2 m). One patient with adrenal cortical cancer had significant regression of liver and lung metastases and another had prolonged stable disease. S was associated with a dose-dependent reduction in S1P in plasma.. Safingol, the first putative SphK inhibitor to enter clinical trials, can be safely administered in combination with cisplatin. Reversible dose-dependent hepatic toxicity was seen, as expected from preclinical data. Target inhibition was achieved with downregulation of S1P. The recommended phase II dose is S 840 mg/m(2) and C 60 mg/m(2), every 3 weeks.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Area Under Curve; Cisplatin; Dose-Response Relationship, Drug; Drug Administration Schedule; Fatigue; Female; Humans; Lymphopenia; Lysophospholipids; Male; Metabolic Clearance Rate; Middle Aged; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Treatment Outcome

Thymocyte egress is a critical determinant of T cell homeostasis and adaptive immunity. Despite the roles of G protein-coupled receptors in thymocyte emigration, the downstream signaling mechanism remains poorly defined. Here, we report the discrete roles for the two branches of mevalonate metabolism-fueled protein prenylation pathway in thymocyte egress and immune homeostasis. The protein geranylgeranyltransferase Pggt1b is up-regulated in single-positive thymocytes, and loss of Pggt1b leads to marked defects in thymocyte egress and T cell lymphopenia in peripheral lymphoid organs in vivo. Mechanistically, Pggt1b bridges sphingosine-1-phosphate and chemokine-induced migratory signals with the activation of Cdc42 and Pak signaling and mevalonate-dependent thymocyte trafficking. In contrast, the farnesyltransferase Fntb, which mediates a biochemically similar process of protein farnesylation, is dispensable for thymocyte egress but contributes to peripheral T cell homeostasis. Collectively, our studies establish context-dependent effects of protein prenylation and unique roles of geranylgeranylation in thymic egress and highlight that the interplay between cellular metabolism and posttranslational modification underlies immune homeostasis.

Topics: Alkyl and Aryl Transferases; Animals; cdc42 GTP-Binding Protein; Cell Movement; Cells, Cultured; Farnesyltranstransferase; Homeostasis; Lymphopenia; Lysophospholipids; Mevalonic Acid; Mice; Mice, Inbred C57BL; Mice, Knockout; p21-Activated Kinases; Protein Prenylation; Signal Transduction; Sphingosine; T-Lymphocytes; Thymocytes; Thymus Gland

Multiple sclerosis (MS) is a chronic, inflammatory, autoimmune disease of the central nervous system (CNS) which is associated with lower life expectancy and disability. The experimental antigen-induced encephalomyelitis (EAE) in mice is a useful animal model of MS, which allows exploring the etiopathogenetic mechanisms and testing novel potential therapeutic drugs. A new therapeutic paradigm for the treatment of MS was introduced in 2010 through the sphingosine 1-phosphate (S1P) analogue fingolimod (FTY720, Gilenya

Topics: Animals; Central Nervous System; CHO Cells; Cricetulus; Disease Models, Animal; Encephalomyelitis; Encephalomyelitis, Autoimmune, Experimental; Fingolimod Hydrochloride; Immunosuppressive Agents; Ligands; Lymphopenia; Lysophospholipids; Mice; Morpholinos; Multiple Sclerosis; Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors; Spinal Cord; T-Lymphocytes

T cell dysfunction contributes to tumor immune escape in patients with cancer and is particularly severe amidst glioblastoma (GBM). Among other defects, T cell lymphopenia is characteristic, yet often attributed to treatment. We reveal that even treatment-naïve subjects and mice with GBM can harbor AIDS-level CD4 counts, as well as contracted, T cell-deficient lymphoid organs. Missing naïve T cells are instead found sequestered in large numbers in the bone marrow. This phenomenon characterizes not only GBM but a variety of other cancers, although only when tumors are introduced into the intracranial compartment. T cell sequestration is accompanied by tumor-imposed loss of S1P1 from the T cell surface and is reversible upon precluding S1P1 internalization. In murine models of GBM, hindering S1P1 internalization and reversing sequestration licenses T cell-activating therapies that were previously ineffective. Sequestration of T cells in bone marrow is therefore a tumor-adaptive mode of T cell dysfunction, whose reversal may constitute a promising immunotherapeutic adjunct.

Topics: Animals; Bone Marrow; Brain Neoplasms; Endocytosis; Glioblastoma; Humans; Lymphoid Tissue; Lymphopenia; Lysophospholipids; Mice, Inbred C57BL; Sphingosine; Spleen; T-Lymphocytes

Sepsis mouse models revealed thymus atrophy, characterised by decreased thymus weight and loss of thymocytes due to apoptosis. Mice suffered from lymphopenia, a lack of T cells in the periphery, which attenuates their ability to fight against recurring and secondary infections during sepsis progression. Key players in thymus atrophy are IL-6, which is directly involved in thymus involution, and the sphingosine-1-phosphate - sphingosine-1-phosphate receptor 1 signaling, influencing thymocytes emigration. In healthy individuals a sphingosine-1-phosphate (S1P) gradient from lymphoid organs to the circulatory system serves as signal for mature T cell egress. In the present study we investigated, whether inhibition of S1P generation improves thymus involution. In sepsis, induced by cecal ligation and puncture (CLP), S1P in the thymus increased, while it decreased in serum, thus disrupting the naturally occurring S1P gradient. As a potential source of S1P we identified increased numbers of apoptotic cells in the thymic cortex of septic mice. Pharmacological inhibition of the S1P generating sphingosine kinases, by 4- [[4-(4-Chlorophenyl)-2-thiazolyl]amino]phenol (SK I-II), administered directly following CLP, prevented thymus atrophy. This was reflected by lymphocytosis, diminished apoptosis, decreased IL-6 expression, and an unaltered thymus weight. In addition SK I-II-treatment preserved the S1P balance and prevented S1P-dependent internalization of the sphingosine-1-phosphate receptor 1. Our data suggest that inhibition of sphingosine kinase and thus, S1P generation during sepsis restores thymic T cell egress, which might improve septic outcome.

Topics: Aminophenols; Animals; Apoptosis; Atrophy; Cecum; Disease Models, Animal; Interleukin-6; Lymphocytosis; Lymphopenia; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Phosphotransferases (Alcohol Group Acceptor); Receptors, Lysosphingolipid; Sepsis; Sphingosine; Thiazoles; Thymocytes; Thymus Gland

Metastasis is the leading cause of death for cancer patients. This multi-stage process requires tumour cells to survive in the circulation, extravasate at distant sites, then proliferate; it involves contributions from both the tumour cell and tumour microenvironment ('host', which includes stromal cells and the immune system). Studies suggest the early steps of the metastatic process are relatively efficient, with the post-extravasation regulation of tumour growth ('colonization') being critical in determining metastatic outcome. Here we show the results of screening 810 mutant mouse lines using an in vivo assay to identify microenvironmental regulators of metastatic colonization. We identify 23 genes that, when disrupted in mouse, modify the ability of tumour cells to establish metastatic foci, with 19 of these genes not previously demonstrated to play a role in host control of metastasis. The largest reduction in pulmonary metastasis was observed in sphingosine-1-phosphate (S1P) transporter spinster homologue 2 (Spns2)-deficient mice. We demonstrate a novel outcome of S1P-mediated regulation of lymphocyte trafficking, whereby deletion of Spns2, either globally or in a lymphatic endothelial-specific manner, creates a circulating lymphopenia and a higher percentage of effector T cells and natural killer (NK) cells present in the lung. This allows for potent tumour cell killing, and an overall decreased metastatic burden.

Topics: Animals; Anion Transport Proteins; Cell Line, Tumor; Cell Movement; Disease Models, Animal; Female; Genome; Genomics; Killer Cells, Natural; Lung Neoplasms; Lymphopenia; Lysophospholipids; Male; Mice; Neoplasm Metastasis; Sphingosine; T-Lymphocytes; Tumor Microenvironment

Caramel color is widely used in the food industry, and its many variations are generally considered to be safe. It has been known for a long time that THI (2-acetyl-4-(tetrahydroxybutyl)imidazole), a component of caramel color III, causes lymphopenia in animals through sphingosine 1-phosphate (S1P) lyase (S1PL) inhibition. However, this mechanism of action has not been fully validated because THI does not inhibit S1PL in vitro. To reconcile this situation, we examined molecular details of THI mechanism of action using "smaller" THI derivatives. We identified a bioactive derivative, A6770, which has the same lymphopenic effect as THI via S1PL inhibition. In the case of A6770 we observe this effect both in vitro and in vivo, and demonstrate that A6770 is phosphorylated and inhibits S1PL in the same way as 4-deoxypyridoxine. In addition, A6770 was detected in rat plasma following oral administration of THI, suggesting that A6770 is a key metabolic intermediate of THI.

Topics: Animals; Cell Line; Dose-Response Relationship, Drug; Food Coloring Agents; Imidazoles; Lymphopenia; Lysophospholipids; Mice; Mice, Inbred C57BL; Rats; Rats, Inbred Lew; Sphingosine; Structure-Activity Relationship

Growing evidence supports that the immunomodulatory drug fingolimod is protective in stroke. Fingolimod binds to 4 of 5 sphingosine-1-phosphate (S1P) receptors and, among other actions, it induces lymphopenia. In this study, we investigated whether a selective S1P1 agonist is protective in experimental stroke.. Drug selectivity was studied in vitro in cells overexpressing the human S1P receptors. Mice (n=54) received different doses of LASW1238 (3 or 10 mg/kg), fingolimod (1 mg/kg), or the vehicle intraperitoneal, and lymphopenia was studied at different time points. After intraluminal middle cerebral artery occlusion for 45 minutes and immediately after reperfusion, mice (n=56) received the drug treatment. At 24 hours, a neurological test was performed and infarct volume was measured. Treatment and all the analyses were performed in a blind fashion.. In vitro functional assays showed that LASW1238 is a selective agonist of the S1P1 receptor. At 10 mg/kg, this compound induced sustained lymphopenia in mice comparable with fingolimod, whereas at 3 mg/kg it induced short-lasting lymphopenia. After ischemia, both LASW1238 (10 mg/kg) and fingolimod reduced infarct volume, but only LASW1238 (10 mg/kg) showed statistically significant differences versus the vehicle. The neurological function and plasma cytokine levels were not different between groups.. The selective S1P1 agonist LASW1238 reduces infarct volume after ischemia/reperfusion in mice, but only when lymphopenia is sustained for at least 24 hours. S1P1 and lymphocytes are potential targets for drug treatment in stroke. Defining the best drug dosing regimens to control the extent and duration of lymphopenia is critical to achieve the desired effects.

Topics: Animals; Cerebral Infarction; Fingolimod Hydrochloride; Immunosuppressive Agents; Lymphopenia; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Receptors, Lysosphingolipid; Reperfusion Injury; Sphingosine

Sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) is critical for lymphocyte egress from lymphoid organs. Lymphocytes encounter low S1P concentrations near exit sites before transmigration, yet S1PR1 signaling is rapidly terminated after exposure to S1P. How lymphocytes maintain S1PR1 signaling in a low S1P environment near egress sites is unknown. Here we identify dynamin 2, an essential component of endocytosis, as a novel regulator of T cell egress. Mice with T cell-specific dynamin 2 deficiency had profound lymphopenia and impaired egress from lymphoid organs. Dynamin 2 deficiency caused impaired egress through regulation of S1PR1 signaling, and transgenic S1PR1 overexpression rescued egress in dynamin 2 knockout mice. In low S1P concentrations, dynamin 2 was essential for S1PR1 internalization, which enabled continuous S1PR1 signaling and promoted egress from both thymus and lymph nodes. In contrast, dynamin 2-deficient cells were only capable of a pulse of S1PR1 signaling, which was insufficient for egress. Our results suggest a possible mechanism by which T lymphocytes positioned at exit portals sense low S1P concentrations, promoting their egress into circulatory fluids.

Topics: Animals; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Chemotaxis; Dynamin II; Endocytosis; GTP-Binding Protein alpha Subunits, Gi-Go; Lectins, C-Type; Ligands; Lymph Nodes; Lymphopenia; Lysophospholipids; Mice; Mice, Knockout; Receptors, Chemokine; Receptors, G-Protein-Coupled; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Thymocytes

In the quest to discover a potent and selective class of direct agonists to the sphingosine-1-phosphate receptor, we explored the carboxylate functional group as a replacement to previously reported lead phosphates. This has led to the discovery of potent and selective direct agonists with moderate to substantial in vivo lymphopenia. The previously reported selectivity enhancing moiety (SEM) and selectivity enhancing orientation (SEO) in the phenylamide and phenylimidazole scaffolds were crucial to obtaining selectivity for S1P receptor subtype 1 over 3.

Topics: Administration, Oral; Amino Acids; Animals; Inhibitory Concentration 50; Lymphopenia; Mice; Molecular Structure; Protein Binding; Receptors, Lysosphingolipid

Altered sphingosine 1-phosphate (S1P) homeostasis and signaling is implicated in various inflammatory diseases including atherosclerosis. As S1P levels are tightly controlled by S1P lyase, we investigated the impact of hematopoietic S1P lyase (Sgpl1(-/-)) deficiency on leukocyte subsets relevant to atherosclerosis.. LDL receptor deficient mice that were transplanted with Sgpl1(-/-) bone marrow showed disrupted S1P gradients translating into lymphopenia and abrogated lymphocyte mitogenic and cytokine response as compared to controls. Remarkably however, Sgpl1(-/-) chimeras displayed mild monocytosis, due to impeded stromal retention and myelopoiesis, and plasma cytokine and macrophage expression patterns, that were largely compatible with classical macrophage activation. Collectively these two phenotypic features of Sgpl1 deficiency culminated in diminished atherogenic response.. Here we not only firmly establish the critical role of hematopoietic S1P lyase in controlling S1P levels and T cell trafficking in blood and lymphoid tissue, but also identify leukocyte Sgpl1 as critical factor in monocyte macrophage differentiation and function. Its, partly counterbalancing, pro- and anti-inflammatory activity spectrum imply that intervention in S1P lyase function in inflammatory disorders such as atherosclerosis should be considered with caution.

Topics: Aldehyde-Lyases; Animals; Atherosclerosis; Bone Marrow Cells; Cell Differentiation; Female; Hematopoiesis; Lymphocyte Count; Lymphopenia; Lysophospholipids; Macrophages; Mice; Mice, Knockout; Neutrophils; Phenotype; Plaque, Atherosclerotic; Receptors, LDL; Sphingosine; Spleen

Sphingosine 1-phosphate (S1P) regulates lymphocyte trafficking through the type 1 sphingosine 1-phosphate receptor (S1P(1)) and participates in many pathological conditions, including autoimmune diseases. We developed a novel S1P(1)-selective antagonist, TASP0277308, which is structurally unrelated to S1P. This antagonist competitively inhibited S1P-induced cellular responses, such as chemotaxis and receptor internalization. Furthermore, differing from previously reported S1P(1) antagonists, TASP0277308 demonstrated in vivo activities to induce lymphopenia, a block in T cell egress from the thymus, displacement of marginal zone B cells, and upregulation of CD69 expression on both T and B cells, all of which recapitulate phenotypes of S1P(1)-deficient lymphocytes. In a mouse collagen-induced arthritis model, TASP0277308 significantly suppressed the development of arthritis, even after the onset of disease. These findings provide the first chemical evidence to our knowledge that S1P(1) antagonism is responsible for immunosuppression in the treatment of autoimmune diseases and also resolve the discrepancies between genetic and chemical studies on the functions of S1P(1) in lymphocytes.

Topics: Animals; Arthritis, Experimental; B-Lymphocytes; Cricetinae; Cricetulus; HEK293 Cells; Humans; Immune Tolerance; Immunosuppressive Agents; Lymphopenia; Lysophospholipids; Male; Mice; Sphingosine; Sulfones; T-Lymphocytes; Thymus Gland; Triazoles

Sphingosine 1-phosphate (S1P) regulates lymphocyte trafficking via type-1 S1P receptor (S1P(1)) and participates in many pathological conditions. We developed a novel type S1P(1)-selective antagonist, TASP0251078, which is structurally unrelated to S1P. This competitive antagonist inhibited binding of S1P to S1P(1) resulting in reduced signaling downstream of S1P(1), including GTPγS-binding and cAMP formation. TASP0251078 also inhibited S1P-induced cellular responses such as chemotaxis and receptor-internalization. Furthermore, when administered in vivo, TASP0251078 induced lymphopenia in blood, which is different from previously reported effects of other S1P(1)-antagonists. In a mouse contact hypersensitivity model, TASP0251078 effectively suppressed ear swelling, leukocyte infiltration, and hyperplasia. These findings provide the chemical evidence that S1P(1) antagonism is responsible for lymphocyte sequestration from the blood, and suggest that the effect of S1P(1) agonists on lymphocyte sequestration results from their functional antagonism.

Topics: Animals; Chemotaxis; CHO Cells; Cricetinae; Cricetulus; Cyclic AMP; Dermatitis, Contact; Ear; Edema; Female; Guanosine 5'-O-(3-Thiotriphosphate); HEK293 Cells; Humans; Hyperplasia; Leukocytes; Lymphopenia; Lysophospholipids; Male; Mice; Mice, Inbred BALB C; Molecular Structure; Protein Binding; Rats; Rats, Inbred Lew; Receptors, Lysosphingolipid; Sphingosine; Sulfonamides; Triazoles

CS-0777 is a selective sphingosine 1-phosphate receptor-1 modulator under investigation for treatment of multiple sclerosis (MS). We conducted an open-label, pilot study in 25 MS patients to assess the safety, pharmacokinetics, pharmacodynamics and exploratory efficacy of oral CS-0777 (0.1, 0.3 and 0.6 mg), administered once weekly or every other week for 12 weeks. CS-0777 resulted in a pronounced, dose-dependent decrease in lymphocytes and CD4 T cell subsets, which returned to baseline within 4 weeks after the last dose. Overall, CS-0777 was safe and well-tolerated. These results require confirmation in a double-blind, placebo-controlled and adequately powered phase 2 study in MS.

Topics: Administration, Oral; Amino Alcohols; CD4-Positive T-Lymphocytes; Dose-Response Relationship, Immunologic; Down-Regulation; Humans; Lymphocyte Subsets; Lymphopenia; Lysophospholipids; Multiple Sclerosis; Pilot Projects; Pyrroles; Receptors, Lysosphingolipid; Sphingosine

Sphingosine-1-phosphate (S1P) is lipid messenger involved in the regulation of embryonic development, immune system functions, and many other physiological processes. However, the mechanisms of S1P transport across cellular membranes remain poorly understood, with several ATP-binding cassette family members and the spinster 2 (Spns2) member of the major facilitator superfamily known to mediate S1P transport in cell culture. Spns2 was also shown to control S1P activities in zebrafish in vivo and to play a critical role in zebrafish cardiovascular development. However, the in vivo roles of Spns2 in mammals and its involvement in the different S1P-dependent physiological processes have not been investigated. In this study, we characterized Spns2-null mouse line carrying the Spns2(tm1a(KOMP)Wtsi) allele (Spns2(tm1a)). The Spns2(tm1a/tm1a) animals were viable, indicating a divergence in Spns2 function from its zebrafish ortholog. However, the immunological phenotype of the Spns2(tm1a/tm1a) mice closely mimicked the phenotypes of partial S1P deficiency and impaired S1P-dependent lymphocyte trafficking, with a depletion of lymphocytes in circulation, an increase in mature single-positive T cells in the thymus, and a selective reduction in mature B cells in the spleen and bone marrow. Spns2 activity in the nonhematopoietic cells was critical for normal lymphocyte development and localization. Overall, Spns2(tm1a/tm1a) resulted in impaired humoral immune responses to immunization. This study thus demonstrated a physiological role for Spns2 in mammalian immune system functions but not in cardiovascular development. Other components of the S1P signaling network are investigated as drug targets for immunosuppressive therapy, but the selective action of Spns2 may present an advantage in this regard.

Topics: Animals; Anion Transport Proteins; Cell Differentiation; Crosses, Genetic; Gene Targeting; Immunophenotyping; Lymphocyte Subsets; Lymphopenia; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutagenesis, Insertional; Protein Transport; Sphingosine

The synthetic sphingosine-1-phosphate (S1P) analogue, FTY720, attenuates ischaemia/reperfusion (I/R) injury by inducing peripheral lymphopaenia. Recent studies suggest that FTY720 may also exert protective effects by modulating dendritic cell (DC) function or directly affecting regulatory T cells (Tregs). The purpose of the present study was to examine whether the beneficial effect of FTY720 in I/R-induced acute kidney injury (AKI) involves modulation of DCs or Tregs.. Mice underwent bilateral ischaemia, and FTY720 or vehicle was then administered. Biochemical values, histological kidney damage and tissue inflammation were assessed. Phenotype and function of DCs in blood/spleen or kidney were also examined by flow cytometry or mixed lymphocyte reaction (MLR) assay. Percent Tregs or FoxP3 mRNA expression was examined in kidney and spleen, and depletion and adoptive transfer of Tregs were also performed.. Treatment with FTY720 attenuated I/R kidney injury and reduced inflammation. The beneficial effect of FTY720 was associated with expansion of peripheral CD11b( +) CD11c( +) DC and with maturation of spleen CD11c( +) DC, which showed impaired allostimulatory capacity. FTY720-treated animals also showed a higher frequency of CD4( +) CD25( +) Tregs and an upregulation of FoxP3 mRNA expression in spleen and kidney. In vitro experiments showed that FTY720 induced expansion of Tregs, possibly via conversion from non-Tregs to Tregs. Depletion and adoptive transfer of Tregs were associated with loss and recovery of the beneficial effects of FTY720.. These results suggest that the beneficial effects of FTY720 in I/R injury may be partially mediated by DC modulation or by increasing Treg activity. Further studies that identify tolerance induction mechanisms will be useful for developing strategies for the prevention or treatment of AKI.

Topics: Acute Kidney Injury; Animals; CD4-Positive T-Lymphocytes; Dendritic Cells; Fingolimod Hydrochloride; Flow Cytometry; Forkhead Transcription Factors; Immunosuppressive Agents; Interleukin-2 Receptor alpha Subunit; Kidney; Lymphocyte Culture Test, Mixed; Lymphopenia; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Propylene Glycols; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sphingosine; Spleen; T-Lymphocytes, Regulatory

Sphingosine 1-phosphate (S1P) initiates T and B cell exit from lymphoid tissues by activating the S1P(1) receptor on lymphocytes. To define the mechanistic details of this ligand-receptor interaction, the biological activity of the S1P-blocking Ab Sphingomab was investigated. Treatment of mice with Sphingomab resulted in blood B and T cell lymphopenia. Although Sphingomab blocked S1P(1)-mediated calcium flux and receptor downregulation by S1P in vitro, plasma from Sphingomab-treated mice demonstrated a 4-fold increase in S1P concentration and largely retained its stimulating activity on S1P receptors. Plasma-borne S1P was obviously not sufficiently inactivated by Sphingomab to account for the observed lymphopenia. Therefore, we addressed the local S1P-blocking activity of Sphingomab in spleen and peripheral lymph nodes (pLNs) as a potential cause of PBL depletion. Transwell chemotaxis assays revealed the migration of freshly isolated splenocytes, but not pLN cells to S1P. However, chemotaxis of pLN cells was regained after culture in S1P-low medium, and pLN cells isolated from Sphingomab-treated mice also revealed enhanced chemotaxis to S1P, indicating substantial local inactivation of S1P in pLN after Sphingomab treatment. We conclude that treatment with the S1P-blocking Ab Sphingomab induces lymphopenia by inactivating S1P locally in pLN and not systemically in plasma. Consequently, the presence of local S1P amounts in secondary lymphoid organs contributes to B and T cell egress.

Topics: Animals; Antibodies, Monoclonal; B-Lymphocyte Subsets; Cell Line, Tumor; Cell Movement; Cells, Cultured; Down-Regulation; Humans; Lymph Nodes; Lymphopenia; Lysophospholipids; Mice; Mice, Inbred C57BL; Rats; Receptors, Lysosphingolipid; Sphingolipids; Sphingosine; T-Lymphocyte Subsets

GPCR inhibitors are highly prevalent in modern therapeutics. However, interference with complex GPCR regulatory mechanisms leads to both therapeutic efficacy and adverse effects. Recently, the sphingosine-1-phosphate (S1P) receptor inhibitor FTY720 (also known as Fingolimod), which induces lymphopenia and prevents neuroinflammation, was adopted as a disease-modifying therapeutic in multiple sclerosis. Although highly efficacious, dose-dependent increases in adverse events have tempered its utility. We show here that FTY720P induces phosphorylation of the C-terminal domain of S1P receptor 1 (S1P₁) at multiple sites, resulting in GPCR internalization, polyubiquitinylation, and degradation. We also identified the ubiquitin E3 ligase WWP2 in the GPCR complex and demonstrated its requirement in FTY720-induced receptor degradation. GPCR degradation was not essential for the induction of lymphopenia, but was critical for pulmonary vascular leak in vivo. Prevention of receptor phosphorylation, internalization, and degradation inhibited vascular leak, which suggests that discrete mechanisms of S1P receptor regulation are responsible for the efficacy and adverse events associated with this class of therapeutics.

Topics: Animals; Capillary Leak Syndrome; Dose-Response Relationship, Drug; Endocytosis; Fingolimod Hydrochloride; Gene Knock-In Techniques; Lymphopenia; Lysophospholipids; Mice; Organophosphates; Peptide Hydrolases; Phosphorylation; Propylene Glycols; Protein Processing, Post-Translational; Protein Structure, Tertiary; Pulmonary Edema; Receptors, G-Protein-Coupled; Receptors, Lysosphingolipid; Recombinant Fusion Proteins; Sphingosine; Sphingosine-1-Phosphate Receptors; Ubiquitin-Protein Ligases; Ubiquitination

T cell chemotaxis to sphingosine 1-phosphate (S1P) and the chemokines CCL21 and CCL5 was studied in ten adults with T lymphocytopenia, other immunological abnormalities (nine of ten), and frequent bacterial infections (seven of ten). Mean chemotactic responses to S1P of CD4 T cells from CD4 T lymphocytopenic patients and of CD8 T cells from CD8 T lymphocytopenic patients were significantly lower than those of healthy matched controls. Chemotaxis to CCL21 was lower than that of controls for CD4 T cells of three CD4 T lymphocytopenic patients and for CD8 T cells of three CD8 T lymphocytopenic patients, but none of the T cells of patients had diminished chemotaxis to CCL5. Defective T cell chemotactic responses to S1P and some chemokines may lead to subset-selective abnormal T cell trafficking and chronic T cell lymphocytopenia.

Topics: Adult; Aged; Autoantibodies; Bacterial Infections; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cells, Cultured; Chemokine CCL21; Chemokine CCL5; Chemotaxis; Humans; Immunoglobulin M; Lymphopenia; Lysophospholipids; Male; Middle Aged; Receptors, CCR7; Receptors, Lysosphingolipid; Recurrence; Sphingosine; T-Lymphocyte Subsets

Modifying FTY720, an immunosuppressant modulator, led to a new series of well phosphorylated tetralin analogs as potent S1P1 receptor agonists. The stereochemistry effect of tetralin ring was probed, and (-)-(R)-2-amino-2-((S)-6-octyl-1,2,3,4-tetrahydronaphthalen-2-yl)propan-1-ol was identified as a good SphK2 substrate and potent S1P1 agonist with good oral bioavailability.

Topics: Administration, Oral; Animals; Crystallography, X-Ray; Immunosuppressive Agents; Lymphopenia; Mice; Models, Molecular; Multiple Sclerosis; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Prodrugs; Receptors, Lysosphingolipid; Structure-Activity Relationship; Tetrahydronaphthalenes

Sphingosine kinases (SKs) 1 and 2 produce high concentrations of sphingosine 1-phosphate (S1P) in blood and lymph. In contrast, S1P concentrations in lymphoid tissues are kept low by the S1P-degrading activity of the S1P-lyase. These differences in S1P concentrations drive lymphocyte circulation. Inhibition of the S1P-lyase prevents lymphocyte egress and causes lymphopenia because of increased S1P levels in lymphoid tissues. In this study, we investigated the source of this accumulating S1P in lymphoid tissues by using SK2-deficient (SK2(-/-)) mice. In contrast to wild-type mice, SK2(-/-) mice exhibited attenuated lymphopenia after S1P-lyase inhibition by 4-deoxypyridoxine (DOP). Consistently, S1P concentrations were only modestly increased in lymphoid tissues of SK2(-/-) mice compared with a significantly higher increase in wild-type mice after DOP treatment. Low S1P concentrations in lymphoid tissues of DOP-treated SK2(-/-) mice were accompanied by higher S1P concentrations in blood, suggesting that SK2(-/-) mice display defective S1P transport from blood into lymphoid tissues. To investigate this potential new role of SK2, RBCs loaded with traceable C17-S1P were transfused into wild-type and SK2(-/-) mice, resulting in much higher C17-S1P concentrations in blood of SK2(-/-) mice compared with wild-type mice 2 h after transfusion. Moreover, cocultures of RBCs with mouse splenocytes and endothelial cells demonstrated that SK2 regulated cellular uptake of S1P from RBCs. Collectively, our data suggest that S1P in lymphoid tissues derives from blood and point to an essential role of SK2 in S1P transport.

Topics: Animals; B-Lymphocyte Subsets; Catalysis; Cells, Cultured; Coculture Techniques; Endothelium, Vascular; Humans; Lymphoid Tissue; Lymphopenia; Lysophospholipids; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Transport; Sphingosine; T-Lymphocyte Subsets

The sphingosine 1-phosphate receptor 1 (S1P(1)) promotes lymphocyte egress from lymphoid organs. Previous work showed that agonist-induced internalization of this G protein-coupled receptor correlates with inhibition of lymphocyte egress and results in lymphopenia. However, it is unclear if S1P(1) internalization is necessary for this effect. We characterize a knockin mouse (S1p1r(S5A/S5A)) in which the C-terminal serine-rich S1P(1) motif, which is important for S1P(1) internalization but dispensable for S1P(1) signaling, is mutated. T cells expressing the mutant S1P(1) showed delayed S1P(1) internalization and defective desensitization after agonist stimulation. Mutant mice exhibited significantly delayed lymphopenia after S1P(1) agonist administration or disruption of the vascular S1P gradient. Adoptive transfer experiments demonstrated that mutant S1P(1) expression in lymphocytes, rather than endothelial cells, facilitated this delay in lymphopenia. Thus, cell-surface residency of S1P(1) on T cells is a primary determinant of lymphocyte egress kinetics in vivo.

Topics: Amino Acid Substitution; Animals; Base Sequence; Cell Membrane; Chemotaxis, Leukocyte; Endocytosis; Fingolimod Hydrochloride; Kinetics; Lymphopenia; Lysophospholipids; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Mutant Proteins; Propylene Glycols; Receptors, Lysosphingolipid; Sphingosine; T-Lymphocytes

S1PL is an aldehyde-lyase that irreversibly cleaves sphingosine 1-phosphate (S1P) in the terminal step of sphingolipid catabolism. Because S1P modulates a wide range of physiological processes, its concentration must be tightly regulated within both intracellular and extracellular environments.. In order to better understand the function of S1PL in this regulatory pathway, we assessed the in vivo effects of different levels of S1PL activity using knockout (KO) and humanized mouse models.. Our analysis showed that all S1PL-deficient genetic models in this study displayed lymphopenia, with sequestration of mature T cells in the thymus and lymph nodes. In addition to the lymphoid phenotypes, S1PL KO mice (S1PL(-/-)) also developed myeloid cell hyperplasia and significant lesions in the lung, heart, urinary tract, and bone, and had a markedly reduced life span. The humanized knock-in mice harboring one allele (S1PL(H/-)) or two alleles (S1PL(H/H)) of human S1PL expressed less than 10 and 20% of normal S1PL activity, respectively. This partial restoration of S1PL activity was sufficient to fully protect both humanized mouse lines from the lethal non-lymphoid lesions that developed in S1PL(-/-) mice, but failed to restore normal T-cell development and trafficking. Detailed analysis of T-cell compartments indicated that complete absence of S1PL affected both maturation/development and egress of mature T cells from the thymus, whereas low level S1PL activity affected T-cell egress more than differentiation.. These findings demonstrate that lymphocyte trafficking is particularly sensitive to variations in S1PL activity and suggest that there is a window in which partial inhibition of S1PL could produce therapeutic levels of immunosuppression without causing clinically significant S1P-related lesions in non-lymphoid target organs.

Topics: Aldehyde-Lyases; Animals; Biomarkers; Bone Marrow Cells; Hematopoiesis; Humans; Immune System; Lymphopenia; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutation; Sphingosine; Spleen; T-Lymphocytes; Thymus Gland

Resident immune cells are a hallmark of atherosclerotic lesions. The sphingolipid analogue drug FTY720 mediates retrafficking of immune cells and inhibits their homing to inflammatory sites. We have evaluated the effect of FTY720 on atherogenesis and lipid metabolism.. ApoE-/- mice on a normal laboratory diet received oral FTY720 for 12 weeks, which led to a 2.4-fold increase in serum cholesterol (largely VLDL fraction) and a 1.8-fold increase in hepatic HMGCoA reductase mRNA. FTY720 increased plasma sphingosine-1-phosphate and induced marked peripheral blood lymphopenia. A discoordinate modulation of B, T and monocyte cell numbers was found in peripheral lymphoid organs. Overall depletion of T cells was accompanied by a relative (2-fold) increase in regulatory T cell content paralleled by a similar increase in effector memory T cells (CD4+ CD44hi CD62lo) as absolute numbers of both subpopulations remained essentially unchanged. Lymphocyte function was unaltered as indicated by anti-OxLDL antibodies and T cell proliferation. There were no changes in atherosclerotic lesions in early and established atherosclerosis.. FTY720 mediated peripheral lymphocyte depletion and retrafficking without altering function and overall balance of pro- and antiatherogenic lymphocyte populations. A net decrease in lymphocyte numbers occurred concomitantly with a more proatherogenic hypercholesterolemia resulting in unaltered atherogenesis.

Topics: Animals; Apolipoproteins E; Atherosclerosis; Fingolimod Hydrochloride; Hypercholesterolemia; Immunosuppressive Agents; Lipid Metabolism; Lymphocyte Subsets; Lymphocytes; Lymphopenia; Lysophospholipids; Male; Mice; Mice, Knockout; Propylene Glycols; Sphingosine; Spleen

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

Topics: Animals; Fingolimod Hydrochloride; Flow Cytometry; Homozygote; Immunosuppressive Agents; Lymphocytes; Lymphopenia; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Prodrugs; Propylene Glycols; Reverse Transcriptase Polymerase Chain Reaction; Sphingosine

Lymphocyte egress from the thymus and from peripheral lymphoid organs depends on sphingosine 1-phosphate (S1P) receptor-1 and is thought to occur in response to circulatory S1P. However, the existence of an S1P gradient between lymphoid organs and blood or lymph has not been established. To further define egress requirements, we addressed why treatment with the food colorant 2-acetyl-4-tetrahydroxybutylimidazole (THI) induces lymphopenia. We found that S1P abundance in lymphoid tissues of mice is normally low but increases more than 100-fold after THI treatment and that this treatment inhibits the S1P-degrading enzyme S1P lyase. We conclude that lymphocyte egress is mediated by S1P gradients that are established by S1P lyase activity and that the lyase may represent a novel immunosuppressant drug target.

Topics: Aldehyde-Lyases; Animals; B-Lymphocytes; Chemotaxis, Leukocyte; Enzyme Inhibitors; Food Coloring Agents; Hematopoietic Stem Cells; Imidazoles; Immunosuppressive Agents; Lymph; Lymph Nodes; Lymphoid Tissue; Lymphopenia; Lysophospholipids; Mice; Mice, Inbred C57BL; Pyridoxine; Receptors, Lysosphingolipid; RNA Interference; Sphingosine; T-Lymphocytes; Thymus Gland; Vitamin B 6

Sphingosine 1-phosphate (S1P) influences heart rate, coronary artery caliber, endothelial integrity, and lymphocyte recirculation through five related high affinity G-protein-coupled receptors. Inhibition of lymphocyte recirculation by non-selective S1P receptor agonists produces clinical immunosuppression preventing transplant rejection but is associated with transient bradycardia. Understanding the contribution of individual receptors has been limited by the embryonic lethality of the S1P(1) knock-out and the unavailability of selective agonists or antagonists. A potent, S1P(1)-receptor selective agonist structurally unrelated to S1P was found to activate multiple signals triggered by S1P, including guanosine 5'-3-O-(thio)triphosphate binding, calcium flux, Akt and ERK1/2 phosphorylation, and stimulation of migration of S1P(1)- but not S1P(3)-expressing cells in vitro. The agonist also alters lymphocyte trafficking in vivo. Use of selective agonism together with deletant mice lacking S1P(3) receptor reveals that agonism of S1P(1) receptor alone is sufficient to control lymphocyte recirculation. Moreover, S1P(1) receptor agonist plasma levels are causally associated with induction and maintenance of lymphopenia. S1P(3), and not S1P(1), is directly implicated in sinus bradycardia. The sustained bradycardia induced by S1P receptor non-selective immunosuppressive agonists in wild-type mice is abolished in S1P(3)-/- mice, whereas S1P(1)-selective agonist does not produce bradycardia. Separation of receptor subtype usage for control of lymphocyte recirculation and heart rate may allow the identification of selective immunosuppressive S1P(1) receptor agonists with an enhanced therapeutic window. S1P(1)-selective agonists will be of broad utility in understanding cell functions in vitro, and vascular physiology in vivo, and the success of the chemical approach for S1P(1) suggests that selective tools for the resolution of function across this broad lipid receptor family are now possible.

Topics: Animals; Calcium; Cell Movement; CHO Cells; Cricetinae; Fingolimod Hydrochloride; Guanosine 5'-O-(3-Thiotriphosphate); Heart Rate; Immunosuppressive Agents; Lymphatic System; Lymphocytes; Lymphopenia; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Phosphorylation; Propylene Glycols; Protein Kinases; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Signal Transduction; Sphingosine

FTY720 is an immunosuppressant that reduces circulating levels of naïve lymphocytes by increasing their localization and sequestration in secondary lymphoid organs. It is considered to be an agonist for sphingosine 1-phosphate (S1P) G protein-coupled receptors (GPCRs) after phosphorylation at micromolar concentrations. We now describe its nonagonist and noncompetitive inhibitory activity at low nanomolar concentrations for types 1 and 5 S1P-GPCRs and of moderate potency for type 2 S1P-GPCRs. FTY720 blocks S1P signaling through S1P1,2,5 by inducing their internalization and intracellular partial degradation without affecting S1P3 or S1P4. S1P-R internalization is maximal several hours after only seconds of incubation with FTY720 at 37 degrees C and washing, and continues for days before recovery of surface expression and functions. The timing and extent of S1P-R internalization are highly dependent on FTY720 concentration. FTY720 is therefore an S1P-GPCR-selective and noncompetitive inhibitor with a unique mechanism of action.

Topics: Animals; Chemotaxis; Dose-Response Relationship, Drug; Down-Regulation; Drug Design; Endocytosis; Fingolimod Hydrochloride; Gene Targeting; Humans; Immunosuppressive Agents; Jurkat Cells; Lymphocyte Subsets; Lymphopenia; Lysophospholipids; Mice; Propylene Glycols; Protein Isoforms; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Recombinant Fusion Proteins; Signal Transduction; Sphingosine; Transduction, Genetic; Transfection

Sphingosine-1-phosphate (S1P), a lipid signaling molecule that regulates many cellular functions, is synthesized from sphingosine and ATP by the action of sphingosine kinase. Two such kinases have been identified, SPHK1 and SPHK2. To begin to investigate the physiological functions of sphingosine kinase and S1P signaling, we generated mice deficient in SPHK1. Sphk1 null mice were viable, fertile, and without any obvious abnormalities. Total SPHK activity in most Sphk1-/-tissues was substantially, but not completely, reduced indicating the presence of multiple sphingosine kinases. S1P levels in most tissues from the Sphk1-/- mice were not markedly decreased. In serum, however, there was a significant decrease in the S1P level. Although S1P signaling regulates lymphocyte trafficking, lymphocyte distribution was unaffected in lymphoid organs of Sphk1-/- mice. The immunosuppressant FTY720 was phosphorylated and elicited lymphopenia in the Sphk1 null mice showing that SPHK1 is not required for the functional activation of this sphingosine analogue prodrug. The results with these Sphk1 null mice reveal that some key physiologic processes that require S1P receptor signaling, such as vascular development and proper lymphocyte distribution, can occur in the absence of SPHK1.

Topics: Animals; Base Sequence; DNA; Fingolimod Hydrochloride; Immunosuppressive Agents; Lymphocytes; Lymphopenia; Lysophospholipids; Mice; Mice, Knockout; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Prodrugs; Propylene Glycols; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine

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.

Topics: Animals; Apoptosis; Cell Line; Cell Membrane; Chemotaxis; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Fingolimod Hydrochloride; Guanosine 5'-O-(3-Thiotriphosphate); Insecta; Kidney; Lipid Metabolism; Lymphocytes; Lymphopenia; Mice; Models, Chemical; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Rats; Rats, Inbred Lew; Rats, Wistar; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Recombinant Proteins; Signal Transduction; Sphingosine; Time Factors

Blood lymphocyte numbers, essential for the development of efficient immune responses, are maintained by recirculation through secondary lymphoid organs. We show that lymphocyte trafficking is altered by the lysophospholipid sphingosine-1-phosphate (S1P) and by a phosphoryl metabolite of the immunosuppressive agent FTY720. Both species were high-affinity agonists of at least four of the five S1P receptors. These agonists produce lymphopenia in blood and thoracic duct lymph by sequestration of lymphocytes in lymph nodes, but not spleen. S1P receptor agonists induced emptying of lymphoid sinuses by retention of lymphocytes on the abluminal side of sinus-lining endothelium and inhibition of egress into lymph. Inhibition of lymphocyte recirculation by activation of S1P receptors may result in therapeutically useful immunosuppression.

Topics: Animals; B-Lymphocytes; Binding, Competitive; Calcium; CHO Cells; Cricetinae; Cyclic AMP; Fingolimod Hydrochloride; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Immunosuppressive Agents; Ligands; Lymph Nodes; Lymphocyte Count; Lymphopenia; Lysophospholipids; Mice; Organophosphates; Organophosphonates; Phosphorylation; Propylene Glycols; Rats; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Sphingosine; Spleen; Stereoisomerism; T-Lymphocytes