FTY720 stimulates multidrug transporter- and cysteinyl leukotriene-dependent T cell chemotaxis to lymph nodes

FTY720 is a sphingosine-derived immunosuppressant. Phosphorylated FTY720 promotes T cell homing from spleen and peripheral blood to LNs by acting as an agonist for sphingosine-1-phosphate (S1P) receptors. Here we demonstrate that FTY720 enhances the activity of the sphingosine transporter Abcb1 (Mdr1) and the leukotriene C(4) transporter Abcc1 (Mrp1). Both transporters must be active for FTY720-mediated T cell migration and LN homing. Migration and homing driven by FTY720, phosphorylated FTY720, or S1P also require 5-lipoxygenase-mediated synthesis of cysteinyl leukotrienes and their efflux from the cell. FTY720-mediated LN homing events further downstream are dependent on CCL19, CCL21, VLA-4alpha, and CD44. Use of T cells deficient in 5-lipoxygenase, Abcb1, and Abcc1, and comparison of the effects of FTY720 with those of S1P, suggest a model of sequential engagement of Abcb1, SP1 receptors, 5-lipoxygenase, and Abcc1 to enhance T cell migration and homing.     

ABCA1, ABCG1, and SR-BI: Transit of HDL-associated sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) is a zwitterionic lysophospholipid generated by the sphingosine kinase-catalyzed phosphorylation of sphingosine. A number of the biological effects of S1P are mediated by its binding to five specific G protein-coupled receptors located on the cell surface or intracellular targets. However, the synthesis and secretion of S1P require release out of cells for binding with receptors by certain transporters and carriers. High-density lipoprotein (HDL) is an important carrier of S1P in the blood, but the mechanism by which it does so is unclear. This review discusses the mechanism how S1P is transported, and focuses particularly on how the formation of HDL-associated S1P (HDL-S1P) is mediated by certain transporters and carriers. A hypothesis that the ATP-binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor class B memberI (SR-BI) play pivotal roles in HDL-S1P formation is also described.     

The balance between sphingosine and sphingosine-1-phosphate is decisive for mast cell activation after Fc epsilon receptor I triggering.

Over the last few years, sphingolipids have been identified as potent second messenger molecules modulating cell growth and activation. A newly emerging facet to this class of lipids suggests a picture where the balance between two counterregulatory lipids (as shown in the particular example of ceramide and sphingosine-1-phosphate in T lymphocyte apoptosis) determines the cell fate by setting the stage for various protein signaling cascades. Here, we provide a further example of such a decisive balance composed of the two lipids sphingosine and sphingosine-1-phosphate that determines the allergic responsiveness of mast cells. High intracellular concentrations of sphingosine act as a potent inhibitor of the immunoglobulin (Ig)E plus antigen-mediated leukotriene synthesis and cytokine production by preventing activation of the mitogen-activated protein kinase pathway. In contrast, high intracellular levels of sphingosine-1-phosphate, also secreted by allergically stimulated mast cells, activate the mitogen-activated protein kinase pathway, resulting in hexosaminidase and leukotriene release, or in combination with ionomycin, give cytokine production. Equivalent high concentrations of sphingosine-1-phosphate are dominant over sphingosine as they counteract its inhibitory potential. Therefore, it might be inferred that sphingosine-kinase is pivotal to the activation of signaling cascades initiated at the Fc epsilon receptor I by modulating the balance of the counterregulatory lipids.     

1-磷酸鞘氨醇对T细胞功能的影响

1-磷酸鞘氨醇（S1P）是鞘磷脂代谢过程中产生的一种重要信号分子，可与多条信号通路交联而产生广泛的生物学效应。T细胞表达5种S1P受体，即S1P1—S1P5。S1P信号可以调节T细胞的多种免疫效应，包括T细胞增殖和凋亡、T细胞向Th2细胞分化、细胞因子分泌及T细胞迁移等。抑制S1P信号通路的药物FTY720可将T细胞阻滞于次级淋巴器官，造成外周血T细胞显著减少而发挥强烈的免疫抑制作用。本文对S1P的合成和降解、S1P受体及其介导的信号途径、T细胞S1P受体的表达、S1P对T细胞功能的调节及S1P信号通路的免疫抑制药物作了概述．     

1磷酸鞘氨醇及其G蛋白偶联受体的免疫调节功能研究进展

1磷酸鞘氨醇（sphingosine—l—phosphate，S1P）是一种具有重要生物学活性的溶血磷脂，它作为第一信使与各种免疫细胞膜上相应的G蛋白偶联受体相互作用发挥不同的免疫调节功能。S1P可抑制T细胞的增殖，并抑制活化的CD4^＋ T细胞分泌IFN-γ和IL-4。SIP对T细胞、B细胞、树突状细胞和NK细胞都具有趋化性，其主要效应是通过其受体SIP1介导调节淋巴细胞再循环和组织分布。S1P对调节性T细胞趋化性弱，是调节性T细胞发挥最佳活性所必需的。新型免疫抑制剂FFY720进入人体后快速磷酸化，形成具有生物学活性的FFY720-P，与SIP相似，是其受体S1P1、S1P3、S1P。和S1P，的真正激动剂，可影响成熟T细胞、B细胞、树突状细胞及调节性T细胞的组织分布与功能活性，具有低毒高效可逆的免疫抑制效应，能够预防异体移植物排斥及治疗自身免疫病。本文就S1P的合成、代谢及其G蛋白偶联受体在免疫细胞的表达、对各种免疫细胞的影响、以S1PGPCRs为靶点的药物及其临床意义进行综述。     

Regulation of blood and vascular cell function by bioactive lysophospholipids

Summary.  Lysophosphatidic acid (LPA), its sphingolipid homolog sphingosine 1-phosphate (S1P) and several other related molecules constitute a family of bioactive lipid phosphoric acids that function as receptor-active mediators with roles in cell growth, differentiation, inflammation, immunomodulation, apoptosis and development. LPA and S1P are present in physiologically relevant concentrations in the circulation. In isolated cell culture systems or animal models, these lipids exert a range of effects that suggest that S1P and LPA could play important roles in maintaining normal vascular homeostasis and in vascular injury responses. LPA and S1P act on a series of G protein-coupled receptors, and LPA may also be an endogenous regulator of PPARγ activity. In this review, we discuss potential roles for lysolipid signaling in the vasculature and mechanisms by which these bioactive lipids could contribute to cardiovascular disease.     

First evidence of sphingosine 1-phosphate lyase protein expression and activity downregulation in human neoplasm: implication for resistance to therapeutics in prostate cancer

This is the first report of sphingosine 1-phosphate lyase (SPL) protein expression and enzymatic activity in human neoplasm. This enzyme drives irreversible degradation of sphingosine 1-phosphate (S1P), a bioactive lipid associated with resistance to therapeutics in various cancers, including prostate adenocarcinoma. In fresh human prostatectomy specimens, a remarkable decrease in SPL enzymatic activity was found in tumor samples, as compared with normal adjacent tissues. A significant relationship between loss of SPL expression and higher Gleason score was confirmed in tissue microarray (TMA) analysis. Moreover, SPL protein expression and activity were inversely correlated with those of sphingosine kinase-1 (SphK1), the enzyme producing S1P. SPL and SphK1 expressions were independently predictive of aggressive cancer on TMA, supporting the relevance of S1P in prostate cancer. In human C4-2B and PC-3 cell lines, silencing SPL enhanced survival after irradiation or chemotherapy by decreasing expression of proteins involved in sensing and repairing DNA damage or apoptosis, respectively. In contrast, enforced expression of SPL sensitized cancer cells to irradiation or docetaxel by tilting the ceramide/S1P balance toward cell death. Interestingly, the S1P degradation products failed to sensitize to chemo- and radiotherapy, supporting the crucial role of ceramide/S1P balance in cancer. Of note, the combination of SPL enforced expression with a SphK1 silencing strategy by further decreasing S1P content made prostate cancer cells even more sensitive to anticancer therapies, suggesting that a dual strategy aimed at stimulating SPL, and inhibiting SphK1 could represent a future approach to sensitize cancer cells to cancer treatments. Mol Cancer Ther; 11(9); 1841-51. ?2012 AACR.     

Engagement of major histocompatibility complex class II molecules induces sustained, lymphocyte function-associated molecule 1-dependent cell adhesion

Antigenic stimulation is associated with enhanced adhesion between T cells and antigen-presenting cells (APC). Binding of ligands to the T cell antigen receptor activates the adhesion function of lymphocyte function-associated molecule 1 (LFA-1; CD11a/CD18). We demonstrate here that ligand binding to major histocompatibility complex class II (Ia) molecules also activates LFA-1 function, providing a reciprocal mechanism for the induction of adhesion between T cells and Ia+ APC. Adhesion was affected by a qualitative change in LFA-1 molecules and was reversed by the protein kinase C inhibitor sphingosine. These results define a novel role for Ia molecules as signal transducing receptors that regulate LFA-1-dependent adhesion via a putative, Ia-coupled protein kinase(s).     

A central role for DOCK2 during interstitial lymphocyte motility and sphingosine-1-phosphate-mediated egress

Recent observations using multiphoton intravital microscopy (MP-IVM) have uncovered an unexpectedly high lymphocyte motility within peripheral lymph nodes (PLNs). Lymphocyte-expressed intracellular signaling molecules governing interstitial movement remain largely unknown. Here, we used MP-IVM of murine PLNs to examine interstitial motility of lymphocytes lacking the Rac guanine exchange factor DOCK2 and phosphoinositide-3-kinase (PI3K)gamma, signaling molecules that act downstream of G protein-coupled receptors, including chemokine receptors (CKRs). T and B cells lacking DOCK2 alone or DOCK2 and PI3Kgamma displayed markedly reduced motility inside T cell area and B cell follicle, respectively. Lack of PI3Kgamma alone had no effect on migration velocity but resulted in increased turning angles of T cells. As lymphocyte egress from PLNs requires the sphingosine-1-phosphate (S1P) receptor 1, a G(alphai) protein-coupled receptor similar to CKR, we further analyzed whether DOCK2 and PI3Kgamma contributed to S1P-triggered signaling events. S1P-induced cell migration was significantly reduced in T and B cells lacking DOCK2, whereas T cell-expressed PI3Kgamma contributed to F-actin polymerization and protein kinase B phosphorylation but not migration. These findings correlated with delayed lymphocyte egress from PLNs in the absence of DOCK2 but not PI3Kgamma, and a markedly reduced cell motility of DOCK2-deficient T cells in close proximity to efferent lymphatic vessels. In summary, our data support a central role for DOCK2, and to a lesser extent T cell-expressed PI3Kgamma, for signal transduction during interstitial lymphocyte migration and S1P-mediated egress.     

FTY720, a new class of immunomodulator, inhibits lymphocyte egress from secondary lymphoid tissues and thymus by agonistic activity at sphingosine 1-phosphate receptors

FTY720 is the first of a new immunomodulator class: sphingosine 1-phosphate (S1P) receptor agonist. In 1994, an immunosuppressive natural product, ISP-I (myriocin), was isolated from the culture broth of Isaria sinclairii, a type of vegetative wasp. The chemical modification of ISP-I yielded a new compound, FTY720, which has more potent immunosuppressive activity and less toxicity than ISP-I does. FTY720 has been shown to be highly effective in experimental allotransplantation models and autoimmune disease models. A striking feature of FTY720 is the induction of a marked decrease in peripheral blood T- and B-cells at doses that show immunosuppressive activity in these models. Reportedly, FTY720 is rapidly converted to FTY720-phosphate (FTY720-P) by sphingosine kinase 2 in vivo, and FTY720-P acts as a potent agonist at S1P receptors. Recently, it has been suggested that FTY720-P internalizes S1P1 on lymphocytes and thereby inhibits the migration of lymphocytes toward S1P. Thus, it is likely that the reduction of circulating lymphocytes by FTY720 is due to the inhibition of S1P/S1P1-dependent lymphocyte egress from secondary lymphoid tissues and thymus. Because FTY720 displays a novel mechanism of action that has not been observed with other immunosuppressive agents and shows a synergism with cyclosporin A (CsA) and tacrolimus, it is presumed that FTY720 provides a useful tool for the prevention of transplant rejection and a new therapeutic approach for autoimmune diseases including multiple sclerosis and rheumatoid arthritis.     

Elevated sphingosine-1-phosphate promotes sickling and sickle cell disease progression

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates multicellular functions through interactions with its receptors on cell surfaces. S1P is enriched and stored in erythrocytes; however, it is not clear whether alterations in S1P are involved in the prevalent and debilitating hemolytic disorder sickle cell disease (SCD). Here, using metabolomic screening, we found that S1P is highly elevated in the blood of mice and humans with SCD. In murine models of SCD, we demonstrated that elevated erythrocyte sphingosine kinase 1 (SPHK1) underlies sickling and disease progression by increasing S1P levels in the blood. Additionally, we observed elevated SPHK1 activity in erythrocytes and increased S1P in blood collected from patients with SCD and demonstrated a direct impact of elevated SPHK1-mediated production of S1P on sickling that was independent of S1P receptor activation in isolated erythrocytes. Together, our findings provide insights into erythrocyte pathophysiology, revealing that a SPHK1-mediated elevation of S1P contributes to sickling and promotes disease progression, and highlight potential therapeutic opportunities for SCD.     

Transactivation of sphingosine-1-phosphate receptors by FcepsilonRI triggering is required for normal mast cell degranulation and chemotaxis

Mast cells secrete various substances that initiate and perpetuate allergic responses. Cross-linking of the high-affinity receptor for IgE (FcepsilonRI) in RBL-2H3 and bone marrow-derived mast cells activates sphingosine kinase (SphK), which leads to generation and secretion of the potent sphingolipid mediator, sphingosine-1-phosphate (S1P). In turn, S1P activates its receptors S1P1 and S1P2 that are present in mast cells. Moreover, inhibition of SphK blocks FcepsilonRI-mediated internalization of these receptors and markedly reduces degranulation and chemotaxis. Although transactivation of S1P1 and Gi signaling are important for cytoskeletal rearrangements and migration of mast cells toward antigen, they are dispensable for FcepsilonRI-triggered degranulation. However, S1P2, whose expression is up-regulated by FcepsilonRI cross-linking, was required for degranulation and inhibited migration toward antigen. Together, our results suggest that activation of SphKs and consequently S1PRs by FcepsilonRI triggering plays a crucial role in mast cell functions and might be involved in the movement of mast cells to sites of inflammation.     

线粒体神经酰胺酶上调K562细胞Bcl-2蛋白表达水平

最近，克隆了一种新的选择性定位于线粒体的神经酰胺酶，提示线粒体存在着神经酰胺代谢途径，可能对线粒体的功能，特别对凋亡产生影响。本研究将线粒体神经酰胺酶基因转染到K562细胞，以此了解线粒体神经酰胺酶的细胞生物学效应。以脂质体介导将含有线粒体神经酰胺酶cDNA的pcDNA3．1／His—CDase质粒转染到K562细胞，G418筛选阳性克隆，建立稳定表达线粒体神经酰胺酶的K562TC细胞株。用MTT法、annexin V／PI法、FCM及Westernblot分别检测K562与K562TC细胞在细胞毒药物抗性、血清剥夺耐受性及Bcl-2蛋白表达水平方面的差异。结果表明：虽然K562TC与K562细胞对阿霉素、足叶乙甙及亚砷酸的敏感性没有差异，但是K562TC细胞Bcl-2蛋白水平明显升高，抗血清剥夺能力明显增强。以硫代反义寡核苷酸特异性封闭K562TC细胞线粒体神经酰胺酶，下调Bcl-2蛋白表达水平；二甲基鞘氨醇(鞘氨醇激酶抑制剂，降低细胞内1-磷酸鞘氨醇水平)同样下调K562TC细胞Bcl-2蛋白表达水平，而1-磷酸鞘氨醇明显上调K562细胞Bcl-2表达水平。结论：线粒体神经酰胺酶将线粒体神经酰胺代谢为鞘氨醇，进而在鞘氨醇激酶作用下生成1-磷酸鞘氨醇，此代谢途径上调K562细胞Bcl-2蛋白表达水平。     

Development of small-molecule inhibitors of sphingosine-1-phosphate signaling

The pleiotropic sphingolipid mediator, sphingosine-1-phosphate, produced in cells by two sphingosine kinase isoenzymes, SphK1 and SphK2, regulates many cellular and physiological processes important for homeostasis and development and pathophysiology. Many of the actions of S1P are mediated by a family of five specific cell surface receptors that are ubiquitously and specifically expressed, although important direct intracellular targets of S1P have also recently been identified. S1P, SphK1, and or S1P receptors have been linked to onset and progression of numerous diseases, including many types of cancer, and especially inflammatory disorders, such as multiple sclerosis, asthma, rheumatoid arthritis, inflammatory bowel disease, and sepsis. S1P formation and signaling are attractive targets for development of new therapeutics. The effects of a number of inhibitors of SphKs and S1PRs have been examined in animal models of human diseases. The effectiveness of the immunosuppressant FTY720 (known as Fingolimod or Gilenya), recently approved for the treatment of multiple sclerosis, whose actions are mediated by downregulation of S1PR1, has become the gold standard for S1P-centric drugs. Here, we review S1P biology and signaling with an emphasis on potential therapeutic benefits of specific interventions and discuss recent development of small molecule antagonists and agonists that target specific subtypes of S1P receptors as well as inhibitors of SphKs.     

Direct interactions between B and T lymphocytes bearing complementary receptors

A murine cloned Th cell line specific for the antigen conalbumin in the context of self I-A molecules can be activated by low concentrations of soluble antireceptor mAb. By using an antireceptor mAb to shared antigenic determinants on T cell receptors, we have shown that the ability to be activated by soluble antireceptor mAb is an unusual, although not unique, feature of this cloned T cell line. This activation does not involve occult APC, FcR, or interaction between individual cloned T cells, as limiting-dilution analysis shows that individual cells of this clone will grow in the presence of the antireceptor antibody and IL-1 as stimulus. This cloned T cell line is highly immunogenic in vivo, giving rise to antireceptor antibodies that stimulate its growth in both mice and rats. This response is not dependent upon exogenous T cells. Rather, the clone directly interacts with complementary B cells, as shown by the production of mAb in nude mice, and by production of stimulating antireceptor antibodies by purified B cells cultured with cloned Th cells in vitro. Several features of this cloned Th cell line, most especially its ability to be activated, rather than inhibited, by antireceptor antibodies, may account for its striking ability to directly activate B cells bearing complementary receptors. The direct interaction of the cloned Th cell with B cells bearing complementary receptors may serve as a model for receptor-receptor interactions in the generation of both T and B cell repertoires.     

Discovery and evaluation of inhibitors of human ceramidase

The ceramide/sphingosine-1-phosphate (S1P) rheostat has been hypothesized to play a critical role in regulating tumor cell fate, with elevated levels of ceramide inducing death and elevated levels of S1P leading to survival and proliferation. Ceramidases are key enzymes that control this rheostat by hydrolyzing ceramide to produce sphingosine and may also confer resistance to drugs and radiation. Therefore, ceramidase inhibitors have excellent potential for development as new anticancer drugs. In this study, we identify a novel ceramidase inhibitor (Ceranib-1) by screening a small molecule library and describe the synthesis of a more potent analogue (Ceranib-2). In a cell-based assay, both compounds were found to inhibit cellular ceramidase activity toward an exogenous ceramide analogue, induce the accumulation of multiple ceramide species, decrease levels of sphingosine and S1P, inhibit the proliferation of cells alone and in combination with paclitaxel, and induce cell-cycle arrest and cell death. In vivo, Ceranib-2 was found to delay tumor growth in a syngeneic tumor model without hematologic suppression or overt signs of toxicity. These data support the selection of ceramidases as suitable targets for anticancer drug development and provide the first nonlipid inhibitors of human ceramidase activity.     

Sphingosine 1-phosphate signalling via the endothelial differentiation gene family of G-protein-coupled receptors

Sphingosine 1-phosphate (S1P) is stored in and released from platelets in response to cell activation. However, recent studies show that it is also released from a number of cell types, where it can function as a paracrine/autocrine signal to regulate cell proliferation, differentiation, survival, and motility. This review discusses the role of S1P in cellular regulation, both at the molecular level and in terms of health and disease. The main biochemical routes for S1P synthesis (sphingosine kinase) and degradation (S1P lyase and S1P phosphatase) are described. The major focus is on the ability of S1P to bind to a novel family of G-protein-coupled receptors (endothelial differentiation gene [EDG]-1, -3, -5, -6, and -8) to elicit signal transduction (via G(q)-, G(i)-, G(12)-, G(13)-, and Rho-dependent routes). Effector pathways regulated by S1P are divergent, such as extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, phospholipases C and D, adenylyl cyclase, and focal adhesion kinase, and occur in multiple cell types, such as immune cells, neurones, smooth muscle, etc. This provides a molecular basis for the ability of S1P to act as a pleiotropic bioactive lipid with an important role in cellular regulation. We also give an account of the expanding role for S1P in health and disease; in particular, with regard to its role in atherosclerosis, angiogenesis, cancer, and inflammation. Finally, we describe future directions for S1P research and novel approaches whereby S1P signalling can be manipulated for therapeutic intervention in disease.     

Idiotype-recognizing T helper cells that are not idiotype specific

In this study T helper cells that recognize idiotypes as carriers for a hapten-specific B cell response were analyzed under limiting dilution conditions. T helper cells, induced by phosphorylcholine-hemocyanin (PC-Hy) priming, recognize trinitrophenylated TEPC-15 and MOPC-167 (TNP-T15, TNP-167) equally well. Limiting dilution analysis indicates identical frequencies of helper cells for TNP-T15 and TNP-167. Double immunization protocols using TNP-T15 and TNP-167 fail to demonstrate additive effects. Inhibition of carrier recognition in vitro using free hapten, PC, and unconjugated T15 or M167 indicates identical specificities of helper cells for T15 and M167. Collectively, these results provide strong evidence that PC-Hy priming induces only one population of idiotype-recognizing helper cells that are unable to distinguish between the T15 and the M167 idiotopes. The helper cell induction circuit was further analyzed. PC-Hy priming induces T15/167-specific helper T cells in X-linked immune defect-expressing F1 mice. This indicates that a B cell response to PC is not required to induce idiotype-recognizing T cells. Adoptive cotransfer of B cells from PC-Hy-primed mice together with normal T cells fails to induce idiotype-recognizing T cells. These results indicate the existence of a T helper1-T helper2 induction loop. In this scheme, the T helper1 cell carries T15-like receptors and the T helper2 cells, anti-T15-like receptors. Monoclonal antiidiotypic antibodies specific for T15 also induce a T15/167-recognizing T helper cell population. This finding demonstrates that idiotope-specific priming induces non-idiotype-specific T cells. Evidently, the idiotypic T cell network is based on a different selection of idiotope determinants than the selection of the B cell idiotype network.     

B7-DC, a new dendritic cell molecule with potent costimulatory properties for T cells

Dendritic cells (DCs), unique antigen-presenting cells (APCs) with potent T cell stimulatory capacity, direct the activation and differentiation of T cells by providing costimulatory signals. As such, they are critical regulators of both natural and vaccine-induced immune responses. A new B7 family member, B7-DC, whose expression is highly restricted to DCs, was identified among a library of genes differentially expressed between DCs and activated macrophages. B7-DC fails to bind the B7.1/2 receptors CD28 and cytotoxic T lymphocyte-associated antigen (CTLA)-4, but does bind PD-1, a receptor for B7-H1/PD-L1. B7-DC costimulates T cell proliferation more efficiently than B7.1 and induces a distinct pattern of lymphokine secretion. In particular, B7-DC strongly costimulates interferon gamma but not interleukin (IL)-4 or IL-10 production from isolated naive T cells. These properties of B7-DC may account for some of the unique activity of DCs, such as their ability to initiate potent T helper cell type 1 responses.