FTY720 ameliorates Th1-mediated colitis in mice by directly affecting the functional activity of CD4+CD25+ regulatory T cells

Following the present concepts, the synthetic sphingosine analog of myriocin FTY720 alters migration and homing of lymphocytes via sphingosine 1-phosphate receptors. However, several studies indicate that the immunosuppressive properties of FTY720 may alternatively be due to tolerogenic activities via modulation of dendritic cell differentiation or based on direct effects on CD4(+)CD25(+) regulatory T cells (Treg). As Treg play an important role for the cure of inflammatory colitis, we used the Th1-mediated 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis model to address the therapeutic potential of FTY720 in vivo. A rectal enema of TNBS was given to BALB/c mice. FTY720 was administered i.p. from days 0 to 3 or 3 to 5. FTY720 substantially reduced all clinical, histopathologic, macroscopic, and microscopic parameters of colitis analyzed. The therapeutic effects of FTY720 were associated with a down-regulation of IL-12p70 and subsequent Th1 cytokines. Importantly, FTY720 treatment resulted in a prominent up-regulation of FoxP3, IL-10, TGFbeta, and CTLA4. Supporting the hypothesis that FTY720 directly affects functional activity of CD4(+)CD25(+) Treg, we measured a significant increase of CD25 and FoxP3 expression in isolated lamina propria CD4(+) T cells of FTY720-treated mice. The impact of FTY720 on Treg induction was further confirmed by concomitant in vivo blockade of CTLA4 or IL-10R which significantly abrogated its therapeutic activity. In conclusion, our data provide clear evidence that in addition to its well-established effects on migration FTY720 leads to a specific down-regulation of proinflammatory signals while simultaneously inducing functional activity of CD4(+)CD25(+) Treg. Thus, FTY720 may offer a promising new therapeutic strategy for the treatment of IBD.     

Sphingosine-1-phosphate receptor agonism impairs the efficiency of the local immune response by altering trafficking of naive and antigen-activated CD4+ T cells

FTY720 (2-amino-[2-(4-octylphenyl) ethyl]-1,3-propanediol hydrochloride) is an immunosuppressive agent that inhibits allograft rejection. We recently demonstrated that FTY-phosphate, the active metabolite of FTY720, acts as a full agonist for sphingosine-1-phosphate (S1P) receptors. Furthermore, activation of S1P receptors with their natural ligand, S1P, as well as pharmacological ligands leads to lymphopenia, probably due to sequestration of lymphocytes in secondary lymphoid organs. In the present study we used a local Ag-challenged mouse model to examine the effects of FTY720 on T cell activation in the draining lymph node (DLN) and on the release of activated T cells to the peripheral blood compartment. We showed that the number of Ag-activated CD4(+) T cells in the DLN after injection of Ag and CFA into a footpad was dramatically reduced after FTY720 treatment. However, T cell proliferation, both in vitro and in vivo, was not impaired by FTY720. Our results suggest that the reduced efficiency of T cell responses in the DLN in response to a local Ag is probably due to a defective recirculation of naive T cells caused by FTY720 treatment. Furthermore, we found that the numbers of naive and Ag-activated CD4(+) T cells in the peripheral blood of Ag-challenged mice were equally reduced with FTY720 treatment, suggesting that both T cell subsets are sequestered in the DLNs. Thus, FTY720 induces immunosuppression through inhibition of both the recirculation of naive T cells and the release of Ag-activated T cells from the DLN to lymph and to the blood compartment.     

The immunomodulatory sphingosine 1-phosphate analog FTY720 reduces lesion size and improves neurological outcome in a mouse model of cerebral ischemia

Cerebral ischemia is accompanied by fulminant cellular and humoral inflammatory changes in the brain which contribute to lesion development after stroke. A tight interplay between the brain and the peripheral immune system leads to a biphasic immune response to stroke consisting of an early activation of peripheral immune cells with massive production of proinflammatory cytokines followed by a systemic immunosuppression within days of cerebral ischemia that is characterized by massive immune cell loss in spleen and thymus. Recent work has documented the importance of T lymphocytes in the early exacerbation of ischemic injury. The lipid signaling mediator sphingosine 1-phosphate-derived stable analog FTY720 (fingolimod) acts as an immunosuppressant and induces lymphopenia by preventing the egress of lymphocytes, especially T cells, from lymph nodes. We found that treatment with FTY720 (1 mg/kg) reduced lesion size and improved neurological function after experimental stroke in mice, decreased the numbers of infiltrating neutrophils, activated microglia/macrophages in the ischemic lesion and reduced immunohistochemical features of apoptotic cell death in the lesion.     

Anti-inflammatory effects of FTY720 against viral-induced immunopathology: role of drug-induced conversion of T cells to become Foxp3+ regulators

FTY720 has been used to control inflammatory lesions, but the mechanisms by which the drug acts in vivo are poorly understood. Such mechanisms may result primarily from effects on lymphocyte and dendritic cell homing to lymphoid and inflammatory sites. We demonstrate that FTY720 may also act by causing the conversion of TCR-stimulated nonregulatory CD4(+) T cells to Foxp3(+)CD4(+) regulatory T cells and by enhancing their suppressive activity. In a model in which mice were ocularly infected with HSV, daily treatment with FTY720 resulted in significantly diminished ocular lesions. The treated animals showed increased frequencies of Foxp3(+) T cells in lymphoid organs and at two inflammatory sites, namely cornea and trigeminal ganglia. In a second series of experiments, immunized DO11.10RAG2(-/-) animals, normally lacking endogenous Foxp3(+) T cells, that were given FTY720 treatment developed high frequencies of Foxp3(+) regulatory T cells in lymph nodes. Some converted cells persisted in treated animals for several weeks after drug administration was discontinued. Finally, FTY720 could effectively induce Foxp3-expressing cells from Foxp3(-) cells in vitro, an effect inhibited by anti-TGF-beta or the proinflammatory cytokine IL-6. Accordingly, the anti-inflammatory effects of FTY720 could be mediated at least in part by its ability to cause the conversion of Ag-stimulated conventional T cells to become Foxp3(+) regulators. The use of FTY720 along with Ag administration could represent a useful therapeutic means to selectively expand Ag-specific regulators, which could be valuable in many clinical situations such as allotransplants, some autoimmunities, as well as with some chronic infections.     

Lymphocyte modulation with FTY720 improves hemorrhagic shock survival in swine

The inflammatory response to severe traumatic injury results in significant morbidity and mortality. Lymphocytes have recently been identified as critical mediators of the early innate immune response to ischemia-reperfusion injury. Experimental manipulation of lymphocytes following hemorrhagic shock may prevent secondary immunologic injury in surgical and trauma patients. The objective of this study is to evaluate the lymphocyte sequestration agent FTY720 as an immunomodulator following experimental hemorrhagic shock in a swine liver injury model. Yorkshire swine were anesthetized and underwent a grade III liver injury with uncontrolled hemorrhage to induce hemorrhagic shock. Experimental groups were treated with a lymphocyte sequestration agent, FTY720, (n = 9) and compared to a vehicle control group (n = 9). Animals were observed over a 3 day survival period after hemorrhage. Circulating total leukocyte and neutrophil counts were measured. Central lymphocytes were evaluated with mesenteric lymph node and spleen immunohistochemistry (IHC) staining for CD3. Lung tissue infiltrating neutrophils were analyzed with myeloperoxidase (MPO) IHC staining. Relevant immune-related gene expression from liver tissue was quantified using RT-PCR. The overall survival was 22.2% in the vehicle control and 66.7% in the FTY720 groups (p = 0.081), and reperfusion survival (period after hemorrhage) was 25% in the vehicle control and 75% in the FTY720 groups (p = 0.047). CD3(+) lymphocytes were significantly increased in mesenteric lymph nodes and spleen in the FTY720 group compared to vehicle control, indicating central lymphocyte sequestration. Lymphocyte disruption significantly decreased circulating and lung tissue infiltrating neutrophils, and decreased expression of liver immune-related gene expression in the FTY720 treated group. There were no observed infectious or wound healing complications. Lymphocyte sequestration with FTY720 improves survival in experimental hemorrhagic shock using a porcine liver injury model. These results support a novel and clinically relevant lymphocyte immunomodulation strategy to ameliorate secondary immune injury in hemorrhagic shock.     

Disparate in vivo efficacy of FTY720 in xenograft models of Philadelphia positive and negative B-lineage acute lymphoblastic leukemia

Most patients with acute lymphoblastic leukemia (ALL) respond well to standard chemotherapy-based treatments. However a significant proportion of patients, particularly adult patients, relapse with the majority dying of leukemia. FTY720 is an immunosuppressive drug that was recently approved for the treatment of multiple sclerosis and is currently under pre-clinical investigation as a therapy for a number of hematological malignancies. Using human ALL xenografts in NOD/SCIDγc(-/-) mice, we show for the first time that three Ph(+) human ALL xenografts responded to FTY720 with an 80 ± 12% (p = 0.048) reduction in overall disease when treatment was commenced early. In contrast, treatment of mice with FTY720 did not result in reduced leukemia compared to controls using four separate human Ph(-) ALL xenografts. Although FTY720 reactivated PP2A in vitro, this reactivation was not required for death of Ph(-) ALL cells. The plasma levels of FTY720 achieved in the mice were in the high nanomolar range. However, the response seen in the Ph(+) ALL xenografts when treatment was initiated early implies that in vivo efficacy may be obtained with substantially lower drug concentrations than those required in vitro. Our data suggest that while FTY720 may have potential as a treatment for Ph(+) ALL it will not be a useful agent for the treatment of Ph(-) B-ALL.     

Inhibition of P2X7 receptors improves outcomes after traumatic brain injury in rats

Traumatic brain injury (TBI) is the leading cause of death and disability for people under the age of 45 years worldwide. Neuropathology after TBI is the result of both the immediate impact injury and secondary injury mechanisms. Secondary injury is the result of cascade events, including glutamate excitotoxicity, calcium overloading, free radical generation, and neuroinflammation, ultimately leading to brain cell death. In this study, the P2X7 receptor (P2X7R) was detected predominately in microglia of the cerebral cortex and was up-regulated on microglial cells after TBI. The microglia transformed into amoeba-like and discharged many microvesicle (MV)-like particles in the injured and adjacent regions. A P2X7R antagonist (A804598) and an immune inhibitor (FTY720) reduced significantly the number of MV-like particles in the injured/adjacent regions and in cerebrospinal fluid, reduced the number of neurons undergoing apoptotic cell death, and increased the survival of neurons in the cerebral cortex injured and adjacent regions. Blockade of the P2X7R and FTY720 reduced interleukin-1βexpression, P38 phosphorylation, and glial activation in the cerebral cortex and improved neurobehavioral outcomes after TBI. These data indicate that MV-like particles discharged by microglia after TBI may be involved in the development of local inflammation and secondary nerve cell injury.     

FTY720 produces caspase-independent cell death of acute lymphoblastic leukemia cells

Acute lymphoblastic leukemia (ALL), the most common form of childhood cancer, usually responds to chemotherapy but patients who develop disease relapse have a poor prognosis. New agents to treat ALL are urgently required. FTY720 is an immunosuppressive drug that has promising in vitro activity in a number of malignancies, with the proposed mechanism being the reactivation of the protein serine/threonine phosphatase, PP2A. FTY720 reduced the proliferation and viability of Ph(+) and Ph(-) ALL cell lines and patient samples with IC 50 values for viability between 5.3 to 7.9 μM. Cell death was caspase-independent with negligible activation of caspase-3 and no inhibition of FTY720-induced cell death by the caspase inhibitor Z-VAD-FMK. The cytotoxic effects of FTY720 were independent of PP2A reactivation as determined by the lack of effect of the PP2A inhibitor okadaic acid. Features of autophagy, including autophagosomes, LC3II expression and increased autophagic flux, were induced by FTY720. However the phosphorylated form of FTY720 (FTY720-P) similarly induced autophagy but not cell death. This suggests that autophagy is prosurvival in this setting, a finding supported by protection from cell death induced by the cytotoxic agent vincristine. FTY720 also induced reactive oxygen species (ROS) and the antioxidant N-acetyl-cysteine (NAC) partially reversed the cytotoxic effects, demonstrating a role for ROS generation in the cell death mechanism. FTY720 is an active drug in vitro in ALL cell lines and patient samples. Evidence supports a caspase-independent mechanism of cell death with the occurrence of autophagy and necrosis.     

Reduction of the peripheral blood CD56(bright) NK lymphocyte subset in FTY720-treated multiple sclerosis patients

FTY720 (fingolimod) treatment of multiple sclerosis (MS) results in lymphopenia due to increased recruitment into and decreased egress from secondary lymphoid organs of CCR7(+) lymphocytes. Although absolute numbers of NK lymphocytes were reported as being unaltered in FTY720-treated MS patients (MS-FTY), such analyses did not detect a change in a minor subset. Because expression of CCR7 has been described on CD56(bright) NK cells, a minority population of NK cells, we investigated the effect of FTY720 treatment on the phenotype and function of human NK cells in the peripheral circulation of MS patients. MS-FTY patients displayed a decreased proportion of peripheral CD56(bright)CD62L(+)CCR7(+) NK cells compared with untreated MS and healthy donors. In vitro treatment with FTY720-P increased migration of untreated donor NK cells to CXCL12 while reducing the response to CX3CL1 with similar migration responses seen in NK cells from MS-FTY patients. FTY720-P inhibited sphingosine 1-phosphate-directed migration of CD56(bright) and CD56(dim) NK cells subsets from untreated healthy donors. IL-12- and IL-15-stimulated NK cells from MS-FTY patients displayed similar capacity to produce IFN-γ, TNF, IL-10, and MIP-1α cytokines/chemokines compared with NK cells from untreated healthy donors and displayed comparable levels of degranulation in response to K562 tumor cells compared with untreated donors. Subset alterations and function of NK cell populations will need to be considered as part of assessing overall immunosurveillance capacity of patients with MS who will receive sustained FTY720 therapy.     

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

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

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

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

Preclinical evaluation of the immunomodulatory lymphocyte trafficking drug FTY720 for HIV prevention in the female genital mucosa of macaques

FTY720 has been shown to reduce inflammatory cytokines and immune cells in the genital mucosa of macaques. This pilot study examined the ability of FTY720 to inhibit HIV acquisition. Systemic treatment with FTY720 failed to prevent or delay vaginal SHIV transmission.     

Antinociceptive activity of the S1P-receptor agonist FTY720

FTY720 is a novel immunosuppressive drug that inhibits the egress of lymphocytes from secondary lymphoid tissues and thymus. In its phosphorylated form FTY720 is a potent S1P receptor agonist. Recently it was also shown that FTY720 can reduce prostaglandin synthesis through the direct inhibition of the cytosolic phospholipase A2 (cPLA2). Since prostaglandins are important mediators of nociception, we studied the effects of FTY720 in different models of nociception. We found that intraperitoneal administration of FTY720 reduced dose-dependently the nociceptive behaviour of rats in the formalin assay. Although the antinociceptive doses of FTY720 were too low to alter the lymphocyte count, prostanoid concentrations in the plasma were dramatically reduced. Surprisingly, intrathecally administered FTY720 reduced the nociceptive behaviour in the formalin assay without altering spinal prostaglandin synthesis, indicating that additional antinociceptive mechanisms beside the inhibition of prostaglandin synthesis are involved. Accordingly, FTY720 reduced also the nociceptive behaviour in the spared nerve injury model for neuropathic pain which does not depend on prostaglandin synthesis. In this model the antinociceptive effect of FTY720 was similar to gabapentin, a commonly used drug to treat neuropathic pain. Taken together we show for the first time that FTY720 possesses antinociceptive properties and that FTY720 reduces nociceptive behaviour during neuropathic pain.     

Pulmonary endothelial cell barrier enhancement by FTY720 does not require the S1P1 receptor

Novel therapeutic strategies are needed to reverse the loss of endothelial cell (EC) barrier integrity that occurs during inflammatory disease states such as acute lung injury. We previously demonstrated potent EC barrier augmentation in vivo and in vitro by the platelet-derived phospholipid, sphingosine 1-phosphate (S1P) via ligation of the S1P1 receptor. The S1P analogue, FTY720, similarly exerts barrier-protective vascular effects via presumed S1P1 receptor ligation. We examined the role of the S1P1 receptor in sphingolipid-mediated human lung EC barrier enhancement. Both S1P and FTY-induced sustained, dose-dependent barrier enhancement, reflected by increases in transendothelial electrical resistance (TER), which was abolished by pertussis toxin indicating Gi-coupled receptor activation. FTY-mediated increases in TER exhibited significantly delayed onset and intensity relative to the S1P response. Reduction of S1P1R expression (via siRNA) attenuated S1P-induced TER elevations whereas the TER response to FTY was unaffected. Both S1P and FTY rapidly (within 5 min) induced S1P1R accumulation in membrane lipid rafts, but only S1P stimulated S1P1R phosphorylation on threonine residues. Inhibition of PI3 kinase activity attenuated S1P-mediated TER increases but failed to alter FTY-induced TER elevation. Finally, S1P, but not FTY, induced significant myosin light chain phosphorylation and dramatic actin cytoskeletal rearrangement whereas reduced expression of the cytoskeletal effectors, Rac1 and cortactin (via siRNA), attenuated S1P-, but not FTY-induced TER elevations. These results mechanistically characterize pulmonary vascular barrier regulation by FTY720, suggesting a novel barrier-enhancing pathway for modulating vascular permeability.     

Macrophage-T Cell Interactions Mediate Neuropathic Pain through the Glucocorticoid-induced Tumor Necrosis Factor Ligand System

Peripheral neuroinflammation caused by activated immune cells can provoke neuropathic pain. Herein, we investigate the actions of macrophages and T cells through glucocorticoid-induced tumor neurosis factor receptor ligand (GITRL) and its receptor (GITR) in neuropathic pain. After partial sciatic nerve ligation (PSL) in enhanced green fluorescent protein (eGFP) chimeric mice generated by the transplantation of eGFP⁺ bone marrow cells, eGFP⁺ macrophages, and T cells markedly migrated to the injured site after PSL. Administration of agents to deplete macrophages (liposome-clodronate and Clophosome-A^TM) or T cells (anti-CD4 antibody and FTY720) could suppress PSL-induced thermal hyperalgesia and tactile allodynia. The expression levels of co-stimulatory molecules GITRL and GITR were increased on infiltrating macrophages and T cells, respectively. The perineural injection of a GITRL neutralizing antibody that could inhibit the function of the GITRL-GITR pathway attenuated PSL-induced neuropathic pain. Additionally, the induction of inflammatory cytokines and the accumulation of GITR⁺ T cells in the injured SCN were abrogated after macrophage depletion by Clophosome-A^TM. In conclusion, GITRL expressed on macrophages drives cytokine release and T cell activation, resulting in neuropathic pain via GITR-dependent actions. The GITRL-GITR pathway might represent a novel target for the treatment of neuropathic pain.     

FTY720 induces cell cycle arrest and apoptosis of rat glomerular mesangial cells

The present study aimed to examine the effect of FTY720, a new immunosuppressive agent, on the proliferation and apoptosis of glomerular mesangial cells (GMC), and investigate the underlying mechanisms. Cultured rat GMC were treated by FTY720, and the cell viability, apoptosis and cell cycle progression were examined. Furthermore, cell cycle related gene expression profile was analyzed by cDNA microarray, and the protein expression of cell cycle related genes as well as Bax and Bcl-2 were examined by Western blot. The results showed that FTY720 inhibited GMC proliferation and induced apoptosis of GMC in a dose- and time-dependent manner, and induced G(1) phase cell cycle arrest in GMC in a dose-dependent manner as well. cDNA microarray analysis revealed that FTY720 regulated the expression of cell cycle-related gene. Western blot analysis showed that FTY720 induced the downregulation of cyclin D1, cyclin E, CDK2, CDK4, Bcl-2 and E2F1 and the upregulation of Kip1/p27, Cip1/p21, Bax and Rb in GMC in a dose-dependent manner. These results demonstrated that FTY720 could inhibit the proliferation of GMC through inducing cell cycle arrest and apoptosis, probably via the regulation of the expression of cell cycle-related genes and Bax/Bcl-2.     

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

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

Differential activation of c-Jun NH2-terminal kinase and p38 pathways during FTY720-induced apoptosis of T lymphocytes that is suppressed by the extracellular signal-regulated kinase pathway

FTY720 is a novel immunosuppressive drug derived from a metabolite from Isaria sinclairii that is known to induce apoptosis of rat splenic T cells. In this study, we examined the intracellular signaling pathway triggered by FTY720. Treatment of human Jurkat T lymphocytes with FTY720-induced apoptosis characterized by DNA fragmentation. The same treatment induced activation of protein kinases such as c-Jun NH2-terminal kinase (JNK), p38/CSBP (CSAID-binding protein), and a novel 36-kDa myelin basic protein (MBP) kinase, but not extracellular signal-regulated kinase (ERK). Pretreatment of Jurkat cells with DEVD-CHO blocked FTY720-induced DNA fragmentation as well as the activation of p38/CSBP. However, DEVD-CHO treatment failed to inhibit FTY720-induced activation of JNK and the 36-kDa MBP kinase. We have also demonstrated that activation of the ERK signaling pathway completely suppressed the FTY720-induced apoptotic process including activation of caspase 3 and activation of JNK and the 36-kDa MBP kinase. Furthermore, transient expression of constitutively active mitogen-activated protein kinase/ERK kinase (MEK) protected the cells from FTY720-induced cell death. The effect of MEK was canceled by coexpression of a mitogen-activated protein kinase phosphatase, CL100. These results indicate that JNK and p38 pathways are differentially regulated during FTY720-induced apoptosis and that activation of ERK pathway alone is sufficient to cancel the FTY720-induced death signal.     

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

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

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

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