<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is known to play a pivotal role in the regulation of lymphocyte emigration from organized lymphoid tissues such as the peripheral lymph nodes and thymus, but its immunologic role in unorganized and diffused tissues remains to be elucidated. Here we show that the trafficking of peritoneal B cells is principally regulated by S<em>1</em>P. All peritoneal B cells including B<em>1</em>a, B<em>1</em>b, and B2 B cells express comparable levels of the type <em>1</em> S<em>1</em>P receptor. Thus, treatment with FTY720, an S<em>1</em>P receptor modulator, caused the rapid disappearance of peritoneal B cells by inhibiting both their emigration from parathymic lymph nodes and their recirculation from the blood into the peritoneal cavity without affecting their progenitor populations. These changes did not affect natural plasma antibody production or phosphorylcholine (PC)-specific antibody production in serum after peritoneal immunization with heat-killed Streptococcal pneumoniae (R36A). However, FTY720 dramatically reduced peritoneal B cell-derived natural intestinal secretory IgA production without affecting the expression of J-chain and polyimmunoglobulin receptors. Additionally, FTY720 impaired the generation of PC-specific fecal IgA responses after oral immunization with R36A. These findings point to a pivotal role for S<em>1</em>P in connecting peritoneal B cells with intestinal B-cell immunity.

BACKGROUND

The lysosphingolipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is carried in the blood in association with lipoproteins, predominantly high density lipoproteins (HDL). Emerging evidence suggests that many of the effects of HDL on cardiovascular function may be attributable to its S<em>1</em>P cargo.

METHODS

Here we have evaluated how levels of S<em>1</em>P and related sphingolipids in an HDL-containing fraction of human serum correlate with occurrence of ischemic heart disease (IHD). To accomplish this we used liquid chromatography-mass spectrometry to measure S<em>1</em>P levels in the HDL-containing fraction of serum (depleted of LDL and VLDL) from 204 subjects in the Copenhagen City Heart Study (CCHS). The study group consisted of individuals having high serum HDL cholesterol (HDL-C) (females:≥ 73.5 mg/dL; males:≥ 6<em>1</em>.9 mg/dL) and verified IHD; subjects with high HDL-C and no IHD; individuals with low HDL-C (females:≤ 38.7 mg/dL; males:≤ 34.<em>1</em> mg/dL) and IHD, and subjects with low HDL-C and no IHD.

RESULTS

The results show a highly significant inverse relationship between the level of S<em>1</em>P in the HDL-containing fraction of serum and the occurrence of IHD. Furthermore, an inverse relationship with IHD was also observed for two other sphingolipids, dihydro-S<em>1</em>P and C24:<em>1</em>-ceramide, in the HDL-containing fraction of serum. Additionally, we demonstrated that the amount of S<em>1</em>P on HDL correlates with the magnitude of HDL-induced endothelial cell barrier signaling.

CONCLUSIONS

These findings indicate that compositional differences of sphingolipids in the HDL-containing fraction of human serum are related to the occurrence of IHD, and may contribute to the putative protective role of HDL in IHD.

Activation of the GPCR <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor <em>1</em> (S<em>1</em>P<em>1</em>) by <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) regulates key physiological processes. S<em>1</em>P<em>1</em> activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S<em>1</em>P<em>1</em> activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S<em>1</em>P<em>1</em> activation. These mice, known as S<em>1</em>P<em>1</em> GFP signaling mice, produce a S<em>1</em>P<em>1</em> fusion protein containing a transcription factor linked by a protease cleavage site at the C terminus as well as a β-arrestin/protease fusion protein. Activated S<em>1</em>P<em>1</em> recruits the β-arrestin/protease, resulting in the release of the transcription factor, which stimulates the expression of a GFP reporter gene. Under normal conditions, S<em>1</em>P<em>1</em> was activated in endothelial cells of lymphoid tissues and in cells in the marginal zone of the spleen, while administration of an S<em>1</em>P<em>1</em> agonist promoted S<em>1</em>P<em>1</em> activation in endothelial cells and hepatocytes. In S<em>1</em>P<em>1</em> GFP signaling mice, LPS-mediated systemic inflammation activated S<em>1</em>P<em>1</em> in endothelial cells and hepatocytes via hematopoietically derived S<em>1</em>P. These data demonstrate that S<em>1</em>P<em>1</em> GFP signaling mice can be used to evaluate S<em>1</em>P<em>1</em> activation and S<em>1</em>P<em>1</em>-active compounds in vivo. Furthermore, this strategy could be potentially applied to any GPCR to identify sites of receptor activation during normal physiology and disease.

Novel isosteric analogs of the ceramidase inhibitors (<em>1</em>S,2R)-N-myristoylamino-phenylpropanol-<em>1</em> (d-e-MAPP) and (<em>1</em>R,2R)-N-myristoylamino-4'-nitro-phenylpropandiol-<em>1</em>,3 (B<em>1</em>3) with modified targeting and physicochemical properties were developed and evaluated for their effects on endogenous bioactive sphingolipids: ceramide, <em>sphingosine</em>, and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (Cer, Sph, and S<em>1</em>P) in MCF7 cells as determined by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS). Time- and dose-response studies on the effects of these compounds on Cer species and Sph levels, combined with structure-activity relationship (SAR) data, revealed 4 distinct classes of analogs which were predominantly defined by modifications of the N-acyl-hydrophobic interfaces: N-acyl-analogs (class A), urea-analogs (class B), N-alkyl-analogs (class C), and omega-cationic-N-acyl analogs (class D). Signature patterns recognized for two of the classes correspond to the cellular compartment of action of the new analogs, with class D acting as mitochondriotropic agents and class C compounds acting as lysosomotropic agents. The neutral agents, classes A and B, do not have this compartmental preference. Moreover, we observed a close correlation between the selective increase of C(<em>1</em>6)-, C(<em>1</em>4)-, and C(<em>1</em>8)-Cers and inhibitory effects on MCF7 cell growth. The results are discussed in the context of compartmentally targeted regulators of Sph, Cer species, and S<em>1</em>P in cancer cell death, emphasizing the role of C(<em>1</em>6)-Cer. These novel analogs should be useful in cell-based studies as specific regulators of Cer-Sph-S<em>1</em>P inter-metabolism, in vitro enzymatic studies, and for therapeutic development.

Lysophosphatidic acid (LPA) serves as the prototypic lysophospholipid mediator that acts through G-protein-coupled receptors to evoke a host of responses in numerous target cells. The hormone- and growth-factor-like activities of LPA, mediated by distinct G proteins, were discovered about <em>1</em>0 years ago. Since then, considerable progress has been made in our understanding of LPA receptor signaling, culminating in the recent identification of a growing family of heptahelical receptors specific for LPA and the structurally related lysolipid, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P). In addition to stimulating Gi-Ras-mediated cell proliferation, LPA and S<em>1</em>P induce rapid G alpha <em>1</em>2/<em>1</em>3-RhoA-mediated cytoskeletal changes underlying such diverse responses as neurite retraction, cell rounding, and enhanced tumor cell invasiveness. LPA also triggers inhibition of gap-junctional communication. This overview focuses on how our understanding of LPA as an intercellular lipid mediator has developed during the last decade.

OBJECTIVE

We have previously described that bioactive lysophospholipids-lysophosphatidic acid (LPA), <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), and sphingosylphosphorylcholine (SPC)-are present in ascitic fluids from patients with ovarian cancer. To understand the role of these lipids in ovarian cancer, we investigated the effects of these lipids on interleukin-8 (IL-8) production in ovarian cancer cells. IL-8 is a proinflammatory and proangiogenic factor, which is potentially involved in ovarian cancer development.

METHODS

The Clontech PCR-Select cDNA subtraction method (Clontech Laboratories, Inc., Palo Alto, CA) was used to identify genes potentially regulated by LPA in HEY and OCC<em>1</em> ovarian cancer cell lines. Northern blot analysis was used to confirm and examine IL-8 mRNA regulation by lysolipids. Enzyme-linked immunosorbent assay (ELISA) was used for detecting secreted IL-8.

RESULTS

We describe here that LPA, S<em>1</em>P, and SPC increased mRNA levels (2- to 7-fold) and protein secretion (2- to <em>1</em>2-fold) of IL-8 from ovarian cancer cells (HEY, OCC<em>1</em>, and SKOV3) in vitro. These regulations were both dose- and time-dependent. All three lipids increased the stability IL-8 mRNA in HEY cells. In contrast to malignant ovarian cancer cells, immortalized human ovarian epithelial cells did not respond to any of these lipids to increase the secretion of IL-8, although these cells secreted similar basal levels of IL-8 (3<em>1</em>0 pg/ml/<em>1</em>0,000 cells). Two breast cancer cell lines (MCF7 and T47D) secreted lower basal levels of IL-8 (48-80 pg/ml/<em>1</em>0,000 cells), compared with ovarian cancer cells (200-500 pg/ml/<em>1</em>0,000 cells). MCF7 cells responded to LPA, but not S<em>1</em>P and SPC, by increasing the secretion of IL-8. T47D and MCF<em>1</em>0A, an immortalized breast cell line, did not respond to LPA, S<em>1</em>P, or SPC to increase IL-8 secretion.

CONCLUSIONS

LPA, S<em>1</em>P, and SPC regulate the mRNA and protein levels of the proinflammatory and proangiogenic factor IL-8 in ovarian cancer cells. The pathological significance of these regulations in ovarian cancer remains to be further investigated.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP), the product of <em>sphingosine</em> kinase, is an important signaling molecule with intra- and extracellular functions. The cDNA for the mouse <em>sphingosine</em> kinase has recently been reported. In this paper we describe the cloning, expression and characterization of the human <em>sphingosine</em> kinase (huSPHK<em>1</em>). Sequence analysis comparison revealed that this kinase is evolutionarily very conserved, having a high degree of homology with the murine enzyme, and presenting several conserved regions with bacteria, yeast, plant, and mammalian proteins. Expressed huSPHK<em>1</em> cDNA specifically phosphorylates D-erythro-<em>sphingosine</em> and, to a lesser extent, D, L-erythro-dihydro<em>sphingosine</em>, and not at all the 'threo' isoforms of dihydro<em>sphingosine</em>; hydroxy-ceramide or non-hydroxy-ceramide; diacylglycerol (DAG); phosphatidylinositol (PI); phosphatidylinositol-4-<em>phosphate</em> (PIP); or phosphatidylinositol-4, 5-bis<em>phosphate</em> (PIP(2)). huSPHK<em>1</em> shows typical Michaelis-Menten kinetics (V(max)=56microM and K(m)=5microM). The kinase is inhibited by D,L-threo-dihydro<em>sphingosine</em> (K(i)=3microM), and by N, N-dimethyl-<em>sphingosine</em> (K(i)=5microM). Northern blots indicate highest expression in adult lung and spleen, followed by peripheral blood leukocyte, thymus and kidney, respectively. It is also expressed in brain and heart. In addition, database searches with the stSG2854 sequence indicate that huSPHK<em>1</em> is also expressed in endothelial cells, retinal pigment epithelium, and senescent fibroblasts.

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

<strong class="sub-title"> Objective: </strong> To report outcomes on patients with multiple sclerosis (MS) and related disorders with coronavirus disease 20<em>1</em>9 (COVID-<em>1</em>9) illness.

<strong class="sub-title"> Methods: </strong> From March <em>1</em>6 to April 30, 2020, patients with MS or related disorders at NYU Langone MS Comprehensive Care Center were identified with laboratory-confirmed or suspected COVID-<em>1</em>9. The diagnosis was established using a standardized questionnaire or by review of in-patient hospital records.

<strong class="sub-title"> Results: </strong> We identified 76 patients (55 with relapsing MS, of which 9 had pediatric onset; <em>1</em>7 with progressive MS; and 4 with related disorders). Thirty-seven underwent PCR testing and were confirmed positive. Of the entire group, 64 (84%) patients were on disease-modifying therapy (DMT) including anti-CD20 therapies (n = 34, 44.7%) and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor modulators (n = <em>1</em>0, <em>1</em>3.5%). The most common COVID-<em>1</em>9 symptoms were fever and cough, but 2<em>1</em>.<em>1</em>% of patients had neurologic symptom recrudescence preceding or coinciding with the infection. A total of <em>1</em>8 (23.7%) were hospitalized; 8 (<em>1</em>0.5%) had COVID-<em>1</em>9 critical illness or related death. Features more common among those hospitalized or with critical illness or death were older age, presence of comorbidities, progressive disease, and a nonambulatory status. No DMT class was associated with an increased risk of hospitalization or fatal outcome.

<strong class="sub-title"> Conclusions: </strong> Most patients with MS with COVID-<em>1</em>9 do not require hospitalization despite being on DMTs. Factors associated with critical illness were similar to the general at-risk patient population. DMT use did not emerge as a predictor of poor COVID-<em>1</em>9 outcome in this preliminary sample.

The endothelial differentiation gene-6 (Edg-6) was recently identified as an orphan G-protein-coupled receptor. Its predicted amino acid sequence is very close to Edg family of receptor proteins whose ligand is supposed to be lysophosphatidic acid (LPA) or lysosphingolipid such as <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) and sphingosylphosphorylcholine (SPC). Transfection of the Edg-6 into Chinese hamster ovary (CHO) cells and K562 cells resulted in the appearance of high-affinity [(3)H]S<em>1</em>P binding activity. Among lipids employed, S<em>1</em>P and, even though less potent, SPC, displaced the [(3)H]S<em>1</em>P binding, but LPA was inactive. In Edg-6-transfected CHO cells, an increase in cytosolic Ca(2+) concentration in response to S<em>1</em>P or SPC was clearly enhanced without change in the LPA-induced action as compared with the vector-transfected cells. The enhancement of the Ca(2+) response was associated with a significant accumulation of inositol <em>phosphate</em>, reflecting activation of phospholipase C. Similar enhancement of Ca(2+) response to S<em>1</em>P or SPC was also observed in Edg-6-expressing K562 cells. These lipid-induced actions in CHO cells and K562 cells expressing Edg-6 were markedly suppressed by pertussis toxin treatment. We conclude that Edg-6 is one of S<em>1</em>P or lysosphingolipid receptors that couple to phospholipase C-Ca(2+) system through pertussis toxin-sensitive G-proteins.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a lipid that functions as a metabolic intermediate and a cellular signaling molecule. These roles are integrated when compartments with differing extracellular S<em>1</em>P concentrations are formed that serve to regulate functions within the immune and vascular systems, as well as during pathologic conditions. Gradients of S<em>1</em>P concentration are achieved by the organization of cells with specialized expression of S<em>1</em>P metabolic pathways within tissues. S<em>1</em>P concentration gradients underpin the ability of S<em>1</em>P signaling to regulate in vivo physiology. This review will discuss the mechanisms that are necessary for the formation and maintenance of S<em>1</em>P gradients, with the aim of understanding how a simple lipid controls complex physiology. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

ERM proteins are regulated by phosphorylation of the most C-terminal threonine residue, switching them from an activated to an inactivated form. However, little is known about the control of this regulation. Previous work in our group demonstrated that secretion of acid sphingomyelinase acts upstream of ERM dephosphorylation, suggesting the involvement of sphingomyelin (SM) hydrolysis in ERM regulation. To define the role of specific lipids, we employed recombinant bacterial sphingomyelinase (bSMase) as a direct probe of SM metabolism at the plasma membrane. bSMase induced a rapid dose- and time-dependent decrease in ERM dephosphorylation. ERM dephosphorylation was driven by ceramide generation and not by sphingomyelin depletion, as shown using recombinant sphingomyelinase D. The generation of ceramide at the plasma membrane was sufficient for ERM regulation, and no intracellular SM hydrolysis was required, as was visualized using Venus-tagged lysenin probe, which specifically binds SM. Interestingly, hydrolysis of plasma membrane bSMase-induced ceramide using bacterial ceramidase caused ERM hyperphosphorylation and formation of cell surface protrusions. The effects of plasma membrane ceramide hydrolysis were due to <em>sphingosine</em> <em>1</em>-<em>phosphate</em> formation, as ERM phosphorylation was blocked by an inhibitor of <em>sphingosine</em> kinase and induced by <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. Taken together, these results demonstrate a new regulatory mechanism of ERM phosphorylation by sphingolipids with opposing actions of ceramide and <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. The approach also defines a tool kit to probe sphingolipid signaling at the plasma membrane.

Osteoclasts are multinucleated myeloid lineage cells formed in response to macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) by fusion of bone marrow-derived precursors that circulate in the blood and are attracted to sites of bone resorption in response to factors, such as <em>sphingosine</em>-<em>1</em> <em>phosphate</em> signaling. Major advances in understanding of the molecular mechanisms regulating osteoclast functions have been made in the past 20 years, mainly from mouse and human genetic studies. These have revealed that osteoclasts express and respond to proinflammatory and anti-inflammatory cytokines. Some of these cytokines activate NF-κB and nuclear factor of activated T cells, cytoplasmic <em>1</em> (NFATc<em>1</em>) signaling to induce osteoclast formation and activity and also regulate communication with neighboring cells through signaling proteins, including ephrins and semaphorins. Osteoclasts also positively and negatively regulate immune responses and osteoblastic bone formation. These advances have led to development of new inhibitors of bone resorption that are in clinical use or in clinical trials; and more should follow, based on these advances. This article reviews current understanding of how bone resorption is regulated both positively and negatively in normal and pathologic states.

Lysophosphatidic acid (<em>1</em>-acyl-2-lyso-sn-glycero-3-<em>phosphate</em>; LPA) and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) are bioactive phospholipids which respectively act as agonists for the G-protein-coupled lpA receptors (LPA<em>1</em>, LPA2, and LPA3) and s<em>1</em>p receptors (S<em>1</em>P<em>1</em>, S<em>1</em>P2, S<em>1</em>P3, S<em>1</em>P4, and S<em>1</em>P5), collectively referred to as lysophospholipid receptors (lpR). Since astrocytes are responsive to LPA and S<em>1</em>P, we examined mechanisms of lpR signaling in rat cortical secondary astrocytes. Rat cortical astrocyte mRNA expression by quantitative TaqMan polymerase chain reaction (PCR) analysis revealed the following order of relative expression of lpR mRNAs: s<em>1</em>p3>s<em>1</em>p<em>1</em>)lpa<em>1</em>)s<em>1</em>p2=lpa3>)s<em>1</em>p5. Activation of lpRs by LPA or S<em>1</em>P led to multiple pharmacological effects, including the influx of calcium, phosphoinositide (PI) hydrolysis, phosphorylation of extracellular receptor regulated kinase (ERK) and release of [3H]-arachidonic acid (AA). These signalling events downstream of lpR activation were inhibited to varying degrees by pertussis toxin (PTX) pretreatment or by the inhibition of <em>sphingosine</em> kinase (SK), a rate-limiting enzyme in the biosynthesis of S<em>1</em>P from <em>sphingosine</em>. These results suggest that astrocyte lpR signalling mechanisms likely involve both Gi- and Gq-coupled GPCRs and that receptor-mediated activation of SK leads to intracellular generation of S<em>1</em>P, which in turn amplifies the lpR signalling in a paracrine/autocrine manner.

Gaucher disease causes pathologic skeletal changes that are not fully explained. Considering the important role of mesenchymal stromal cells (MSCs) in bone structural development and maintenance, we analyzed the cellular biochemistry of MSCs from an adult patient with Gaucher disease type <em>1</em> (N370S/L444P mutations). Gaucher MSCs possessed a low glucocerebrosidase activity and consequently had a 3-fold increase in cellular glucosylceramide. Gaucher MSCs have a typical MSC marker phenotype, normal osteocytic and adipocytic differentiation, growth, exogenous lactosylceramide trafficking, cholesterol content, lysosomal morphology, and total lysosomal content, and a marked increase in COX-2, prostaglandin E2, interleukin-8, and CCL2 production compared with normal controls. Transcriptome analysis on normal MSCs treated with the glucocerebrosidase inhibitor conduritol B epoxide showed an up-regulation of an array of inflammatory mediators, including CCL2, and other differentially regulated pathways. These cells also showed a decrease in <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. In conclusion, Gaucher disease MSCs display an altered secretome that could contribute to skeletal disease and immune disease manifestations in a manner distinct and additive to Gaucher macrophages themselves.

Liver failure due to ischemia and reperfusion (IR) and subsequent acute kidney injury are significant clinical problems. We showed previously that liver IR selectively reduced plasma sphinganine-<em>1</em>-<em>phosphate</em> levels without affecting <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) levels. Furthermore, exogenous sphinganine-<em>1</em>-<em>phosphate</em> protected against both liver and kidney injury induced by liver IR. In this study, we elucidated the signaling mechanisms of sphinganine-<em>1</em>-<em>phosphate</em>-mediated renal and hepatic protection. A selective S<em>1</em>P(<em>1</em>) receptor antagonist blocked the hepatic and renal protective effects of sphinganine-<em>1</em>-<em>phosphate</em>, whereas a selective S<em>1</em>P(2) or S<em>1</em>P(3) receptor antagonist was without effect. Moreover, a selective S<em>1</em>P(<em>1</em>) receptor agonist, SEW-287<em>1</em>, provided similar degree of liver and kidney protection compared with sphinganine-<em>1</em>-<em>phosphate</em>. Furthermore, in vivo gene knockdown of S<em>1</em>P(<em>1</em>) receptors with small interfering RNA abolished the hepatic and renal protective effects of sphinganine-<em>1</em>-<em>phosphate</em>. In contrast to sphinganine-<em>1</em>-<em>phosphate</em>, S<em>1</em>P's hepatic protection was enhanced with an S<em>1</em>P(3) receptor antagonist. Inhibition of extracellular signal-regulated kinase, Akt or pertussis toxin-sensitive G-proteins blocked sphinganine-<em>1</em>-<em>phosphate</em>-mediated liver and kidney protection in vivo. Taken together, our results show that sphinganine-<em>1</em>-<em>phosphate</em> provided renal and hepatic protection after liver IR injury in mice through selective activation of S<em>1</em>P(<em>1</em>) receptors and pertussis toxin-sensitive G-proteins with subsequent activation of ERK and Akt.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) was first identified as a lysophospholipid metabolite whose formation is required for the irreversible degradation of sphingolipids. Years later, it was discovered that S<em>1</em>P is a bioactive lipid that provokes varied cell responses by acting through cell-surface receptors to drive cell signaling. More recent findings in model organisms have now established that S<em>1</em>P metabolism and signaling are integrated into many physiological systems. We describe here the surprising breadth of function of S<em>1</em>P in mammalian development and the underlying biologic processes that S<em>1</em>P regulates.

Sphingolipids were once regarded as inert structural components of cell membranes. Now these metabolites are generally believed to be important bioactive molecules that control a wide repertoire of cellular processes such as proliferation and survival of cells. Along with these ubiquitous cell functions observed in many peripheral tissues sphingolipid metabolites, especially <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, exert important neuron-specific functions such as regulation of neurotransmitter release. This review summarizes physiological and pathological roles of sphingolipid metabolites emphasizing the role of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> in the central nervous system.

OBJECTIVE

The <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor agonist fingolimod reduces infarct size in rodent models of stroke and enhances blood-brain barrier integrity. Based on these observations, we hypothesized that combination of fingolimod with tissue plasminogen activator (tPA) would reduce the risk of hemorrhagic transformation associated with delayed administration of tPA.

METHODS

We evaluated the effects of fingolimod in a mouse model of thromboembolic stroke, in which both the beneficial effect of reperfusion associated with early tPA treatment and hemorrhagic transformation associated with delayed administration mimic clinical observations in humans.

RESULTS

Our results demonstrate that fingolimod treatment attenuates the neurological deficit and reduces infarct volume after in situ thromboembolic occlusion of the middle cerebral artery. Combination of fingolimod and tPA improves the neurological outcome of the thrombolytic therapy and reduces the risk of hemorrhagic transformation associated with delayed administration of tPA.

CONCLUSIONS

This study confirms the protective efficacy of fingolimod as a treatment against ischemic stroke in another rodent model of stroke (thromboembolic occlusion), and suggests that fingolimod could potentially be used in combination with tPA to reduce the risk of brain hemorrhage.

The ceramide-<em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) rheostat is important in regulating cell fate. Several chemotherapeutic agents, including paclitaxel (Taxol), involve pro-apoptotic ceramide in their anticancer effects. The ceramide-to-S<em>1</em>P pathway is also implicated in the development of pain, raising the intriguing possibility that these sphingolipids may contribute to chemotherapy- induced painful peripheral neuropathy, which can be a critical dose-limiting side effect of many widely used chemotherapeutic agents.We demonstrate that the development of paclitaxel-induced neuropathic pain was associated with ceramide and S<em>1</em>P formation in the spinal dorsal horn that corresponded with the engagement of S<em>1</em>P receptor subtype <em>1</em> (S<em>1</em>PR(<em>1</em>))- dependent neuroinflammatory processes as follows: activation of redox-sensitive transcription factors (NFκB) and MAPKs (ERK and p38) as well as enhanced formation of pro-inflammatory and neuroexcitatory cytokines (TNF-α and IL-<em>1</em>β). Intrathecal delivery of the S<em>1</em>PR<em>1</em> antagonist W<em>1</em>46 reduced these neuroinflammatory processes but increased IL-<em>1</em>0 and IL-4, potent anti-inflammatory/ neuroprotective cytokines. Additionally, spinal W<em>1</em>46 reversed established neuropathic pain. Noteworthy, systemic administration of the S<em>1</em>PR<em>1</em> modulator FTY720 (Food and Drug Administration- approved for multiple sclerosis) attenuated the activation of these neuroinflammatory processes and abrogated neuropathic pain without altering anticancer properties of paclitaxel and with beneficial effects extended to oxaliplatin. Similar effects were observed with other structurally and chemically unrelated S<em>1</em>PR<em>1</em> modulators (ponesimod and CYM-5442) and S<em>1</em>PR<em>1</em> antagonists (NIBR-<em>1</em>4/<em>1</em>5) but not S<em>1</em>PR<em>1</em> agonists (SEW287<em>1</em>). Our findings identify for the first time the S<em>1</em>P/S<em>1</em>PR<em>1</em> axis as a promising molecular and therapeutic target in chemotherapy-induced painful peripheral neuropathy, establish a mechanistic insight into the biomolecular signaling pathways, and provide the rationale for the clinical evaluation of FTY720 in chronic pain patients.

Although much is described about the molecules involved in neutrophil migration from circulation into tissues, less is known about the molecular mechanisms that regulate neutrophil entry into lymph nodes (LNs) draining a local inflammatory site. In this study, we investigated neutrophil migration toward LNs in a context of inflammation induced by immunization of BALB/c mice with OVA emulsified in CFA. We demonstrated that neutrophils can enter LNs of OVA/CFA-immunized mice not only via lymphatic vessels but also from blood, across high endothelial venules. By adoptive transfer experiments, we showed that this influx was dependent on an inflammatory-state condition and previous neutrophil stimulation with OVA/anti-OVA immune complexes. Importantly, we have demonstrated that, in the migratory pattern to LNs, neutrophils used L-selectin and P-selectin glycoprotein ligand-<em>1</em>, macrophage-<em>1</em> Ag and LFA-<em>1</em> integrins, and CXCR4 to get access across high endothelial venules, whereas macrophage-<em>1</em> Ag, LFA-<em>1</em>, and CXCR4 were involved in their trafficking through afferent lymphatics. Strikingly, we found that stimulation with immune complexes significantly upregulated the expression of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor 4 on neutrophils, and that treatment with the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> agonist FTY720 altered neutrophil LN-homing ability. These findings summarized in this article disclose the molecular pattern that controls neutrophil recruitment to LNs.

The neural membranes contain phospholipids, sphingolipids, cholesterol, and proteins. Glycerophospholipids and sphingolipids are precursors for lipid mediators involved in signal transduction processes. Degradation of glycerophospholipids by phospholipase A(2) (PLA(2)) generates arachidonic acid (AA) and docosahexaenoic acids (DHA). Arachidonic acid is metabolized to eicosanoids and DHA is metabolized to docosanoids. The catabolism of glycosphingolipids generates ceramide, ceramide <em>1</em>-<em>phosphate</em>, <em>sphingosine</em>, and <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. These metabolites modulate PLA(2) activity. Arachidonic acid, a product derived from glycerophospholipid catabolism by PLA(2), modulates sphingomyelinase (SMase), the enzyme that generates ceramide and phosphocholine. Furthermore, <em>sphingosine</em> <em>1</em>-<em>phosphate</em> modulates cyclooxygenase, an enzyme responsible for eicosanoid production in brain. This suggests that an interplay and cross talk occurs between lipid mediators of glycerophospholipid and glycosphingolipid metabolism in brain tissue. This interplay between metabolites of glycerophospholipid and sphingolipid metabolism may play an important role in initiation and maintenance of oxidative stress associated with neurologic disorders as well as in neural cell proliferation, differentiation, and apoptosis. Recent studies indicate that PLA(2) and SMase inhibitors can be used as neuroprotective and anti-apoptotic agents. Development of novel inhibitors of PLA(2) and SMase may be useful for the treatment of oxidative stress, and apoptosis associated with neurologic disorders such as stroke, Alzheimer disease, Parkinson disease, and head and spinal cord injuries.

Lysophospholipids (LPs) such as lysophosphatidic acid (LPA) and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) are known to mediate various biological responses, including cell proliferation, migration, and differentiation. To better understand the role of these lipids in mammalian early development, we applied whole-mount in situ hybridization techniques to E8.5 to E<em>1</em>2.5 mouse embryos. We determined the expression patterns of the following LP receptor genes, which belong to the G protein-coupled receptor (GPCR) family: EDG<em>1</em> to EDG8 (S<em>1</em>P<em>1</em> to S<em>1</em>P5 and LPA<em>1</em> to LPA3), LPA4 (GPR23/P2Y9), and LPA5 (GPR92). We found that the S<em>1</em>P/LPA receptor genes exhibit overlapping expression patterns in a variety of organ primordia, including the developing brain and cardiovascular system, presomitic mesoderm and somites, branchial arches, and limb buds. These results suggest that multiple receptor systems for LPA/S<em>1</em>P lysophospholipids may be functioning during organogenesis.

Given that TLRs and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) are key players in inflammation, we explored the potential interplay between TLRs and S<em>1</em>P in the adhesion/inflammatory pathways in primary human endothelial cells. As determined by Western blot and flow cytometry, cells treated with LPS (a TLR4 ligand) and S<em>1</em>P showed significantly enhanced expression of adhesion molecules such as ICAM-<em>1</em> and E-selectin compared with the effect of either ligand alone. Cell-type differences on E-selectin upregulation were observed. In contrast, no cooperation effect on ICAM-<em>1</em> or E-selectin was observed with a TLR2/TLR<em>1</em> ligand. Consistent with an increase in adhesion molecule expression, endothelial cell treatment with LPS plus S<em>1</em>P significantly enhanced adhesion of PBMCs under shear stress conditions compared with the effect of either ligand alone and exhibited comparable levels of cell adhesion strength as those after TNF-α treatment. Moreover, LPS and S<em>1</em>P cooperated to increase the expression of proinflammatory molecules such as IL-6, cyclooxygenase-2, and prostacyclin, as determined by ELISA and Western blot. The analysis of signaling pathways revealed the synergistic phosphorylation of ERK upon LPS plus S<em>1</em>P treatment of HUVEC and human aortic endothelial cells and cell-type differences on p38 and NF-κB activation. Moreover, pharmacological and small interfering RNA experiments disclosed the involvement of S<em>1</em>P(<em>1</em>/3) and NF-κB in the cooperation effect and that cell origin determines the S<em>1</em>P receptors and signaling routes involved. <em>Sphingosine</em> kinase activity induction upon LPS plus S<em>1</em>P treatment suggests S<em>1</em>P- <em>Sphingosine</em> kinase axis involvement. In summary, LPS and S<em>1</em>P cooperate to increase proinflammatory molecules in endothelial cells and, in turn, to augment leukocyte adhesion, thus exacerbating S<em>1</em>P-mediated proadhesive/proinflammatory properties.