Sphingolipids and the regulation of the immune response

seminars in IMMUNOLOGY, Vol. 14, 2002: pp. 57–63 doi:10.1006/smim.2001.0342, available online at http://www.idealibrary.com on

Sphingolipids and the regulation of the immune response Thomas Baumruker∗ and Eva. E. Prieschl

long chain base (sphingoid) backbone, commonly with 18–20 carbon atoms and several hydroxyl groups. 1,2 Acylation of the amine with a fatty acid or 2-hydroxy fatty acids results in more complex compounds such as ceramide. Addition of polar head groups (sugars) in position 1 of ceramide further enhances this complexity which is exemplified by the group of cerebrosides and gangliosides. 3 Sphingolipids are components of all eukaryotic membranes, but their diversity suggested early on that further functions besides serving solely as membrane building blocks were apparent. This hypothesis has been verified by research over the last decade that has resulted in several surprising findings and insights for this compound class. Nowadays it is known that sphingolipids employ at least three different modes of action to modify cellular responses and a rheostat concept describing counter-regulatory lipids has been introduced. 2 In immunology sphingolipids regulate diverse processes such as apoptosis, differentiation and cell responsiveness after receptor triggering and therefore are regarded as highly specific signalling molecules.

Over the last decade evidence has accumulated that sphingolipids are important and specific signalling molecules for cell-to-cell communication (mediator function) as well as for intracellular signalling processes (second messenger function). In addition, glycosylated sphingolipids are essential building blocks of rafts thereby participating in the initiation of receptor mediated signalling events. In immunology, processes such as T cell apoptosis, Th1 versus Th2 T cell differentiation, phagocytosis, and allergic excitability are either influenced or directly regulated by this class of lipids. Models such as the ‘dual function concept’ (differentiation of structural components versus signalling molecules) and the ‘rheostat concept’ (the balance of two or more sphingolipids is essential for the biological function) describe the multiple properties of these signalling molecules. Key words: Edg receptors / rheostat / signalling / sphingolipids c 2002 Academic Press

Introduction A wide array of lipids and their metabolites including diacylglycerol, platelet activating factor, phosphatidic acid, arachidonic acid, prostaglandines, leukotrienes, eicosanoids, thromboxanes, lipoxins, inositol phosphates, and inositol glycans, are of essential importance in complex cellular signalling processes. Among them, sphingolipids comprise a class of about 400 naturally occurring compounds, which were only recently added to the group of bioactive lipid molecules. Structurally, they are characterized by a

From structural components to signalling molecules In 1884 Tudichum 4 first isolated and characterized sphingolipids from brain extracts. For nearly 100 years these lipids have had a shadowy existence and were only regarded as uniformly distributed structural components of eukaryotic cell membranes. No additional assigned biological effect was known apart from one exception: in 1942 Klenk 5 found that sphingolipids are the pathological basis for certain lipid storage diseases. As a consequence the general toxicity of sphingolipids was considered the cause of these disorders resulting in the psychosine hypothesis for Krabbe disease. 6 Soon this explanation was extended to other lipid storage

From the Novartis Research Institute, Brunner Str. 59, A-1235 Vienna, Austria. *Corresponding author. E-mail: [email protected] c

2002 Academic Press 1044–5323 / 02 / 010057+ 07 / $35.00 / 0

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the internal catabolism and anabolism of lipids, a close link between the extracellular and intracellular signalling paths/routes exists (dual mode of action). This is exemplified by recent findings that S1P receptors lead to an increase in intracellular S1P, which replaces Ins(3,4,5)P3 for the mobilization of internal Ca2+ .

abnormalities, although the different phenotypes of the diseases already suggested more specific actions of particular sphingolipids instead of pure toxicity. This thinking was supported by findings of Hannun and colleagues 7 in 1987. They described how high doses of some sphingolipids such as D-erythro-sphingosine (S) inhibit PKC and that PMA activation of neutrophils provoked intracellular changes of sphingolipid levels. Finally, in 1996 the ground breaking findings by Spiegel 8 led to the definition of a rheostat being composed of ceramide and sphingosine-1-phosphate (S1P), which regulates T cell apoptosis.

Sphingolipids as ligands for receptors The structural basis for the extracellular route—the receptors In 1996 van Koppen et al. 16,17 and Moolenaar’s group established that GPCRs with an unknown structure are required for some actions of S1P. Their conclusion was based on EC50 concentrations in the very low nanomolar range (a hint for a high affinity receptor), a sensitivity of the biological effects to pertussis toxin and the fact that microinjection of S1P failed to provoke identical responses (see also later for Ca2+ ). Later, the cloning of the Edg (endothelial differentiation gene) family of receptors provided the structural basis for this extracellular action of S1P. 18 Nowadays eight different structures are known with Edg 1, 3, 5, 6 and 8 being S1P specific, while Edg 2, 4 and 7 preferably interact with lysophosphatidic acid (LPA). 11 The ubiquitously expressed S1P-specific Edg receptors are closely related with an identity of about 50%. Edg 6, on the other hand, is structurally less conserved (35–42% identical) and restricted to the haematopoietic system. The only other sphingolipid interacting with the Edg receptors is sphingosylphosphorylcholin (SPC), however, with an at least 10–50 fold lesser affinity in most cases; no agonistic or antagonistic binding is observed for ceramide, sphingomyelin, or dimethyl-S. 11 This exclusivity can be explained by a recent computational model of Edg 1 and a mutational analysis. It suggests that Arg120 and Arg292 ions pair with the phosphate group and Glu121 with the ammonium moiety of S1P to allow a high affinity interaction. 19

The various faces of sphingolipids in signalling Today it is known that sphingolipids contribute to cellular signalling processes by at least three different modes of action. First, certain cell types such as platelets, mononuclear phagocytes and mast cells secrete certain sphingolipids upon activation, which then bind as ligands to a subfamily of the seven-spanning G-protein coupled receptors. 9–11 In this respect they act as ‘classical’ mediators. Second, analogous to the phosphoinositides, sphingolipids function as intracellular second messengers. 12,13 And third, in their glycosylated status they constitute the specific structural components of ‘rafts’, which are cell membrane compartments implicated in the initiation of signalling cascades. 14 There, the activated/ligated receptors are brought into close proximity/vicinity to doubly acetylated protein tyrosine kinases, this being regarded as the first step of cellular activation. The importance of compartmentalized glycosphingolipids has been demonstrated by increasing the concentration of galactosyl-S (psychosine) in mast cells, in a process we call ‘enforced raft formation’. 15 This resulted in a receptor-independent triggering of the cells suggesting that the concentration of glycosylated sphingolipids in the cell membrane strongly affects the activation status of certain immune cells. Consequently these findings extend the psychosine hypothesis and for the first time provide an alternative explanation besides toxicity. As raft biology will be the topic covered in a whole issue of this series we will concentrate exclusively on the first two mechanisms in the following. For the general background we keep the categorization of mediator versus second messenger. However, as sphingolipid-mediators originate primarily from

The basis for the diversity of biological effects Different Edg receptors, although high in structural homology, convey diverse biological effects. This can be explained structurally by the coupling of the receptors to different G-proteins. Edg 1 and Edg 8 exclusively employ Gi , while Edg 3 and Edg 5 are 58

Sphingolipids and immune response regulation

NH3 + HO

CO2 -

AN

L-Serine

+ CH (CH ) COS Co A 3

2 14

1

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AB

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OL

IC

RO

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O

UT

2

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NH2

Serine Palmitoyl transferase 3-Ketosphinganine reductase Sphinganine -N-Acyltransferase Dihydroceramide -Desaturase Gucosyltransferase Galactosyltransferase I

CH3

OH

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D-erythro-sphinganine O HN

CH3

HO

H3 C

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D-erythro-Dihydroceramide

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Sphingomyelin

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-O

-O

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Hexadec -2-enal

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P O

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10

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H

P O

E UT O R

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NH3 +

Sphingomyelinase Ceramidase Sphingosine kinase Sphingosine -1-phosphate -Lyase

Ethanolamin phosphate

Figure 1. The sphingolipid metabolism: selected parts of the anabolic and catabolic route of sphingolipids are shown, with the emphasis on the sphingomyelin pathway and the first steps in the build up. Arrows/numbers give the corresponding enzymes catalysing these reactions. Note the characteristic 4/5 trans double bond in the catabolic route versus the anabolic one.

In contrast to other Ca2+ fluxes, S1P-mediated Ca2+ increase is not dependent on Ca2+ influx and unlike Ins(3,4,5)P3 , its action is insensitive to heparin. 13 Intracellularly sphingolipids are generated along a complex anabolic pathway, which starts with the condensation of palmitoyl-CoA with serine. Alternatively, a catabolic route, which degrades more complex structures such as sphingomyelin and glycosphingolipids, also gives rise to S and S1P. Both pathways, however, are separated to facilitate the dual function of these lipids: serving as structural components and as signalling molecules. Structurally, the 4/5 double bond allows the discrimination between newly synthesized sphingolipids versus catabolic products as the dehydrogenation occurs relatively late in the anabolic route (see Figure 1). Therefore the pathways only converge at the level

more promiscuous (Edg 3 is linked to Gq, G12, G13; Edg 5 is linked to Gi, G12, G13, Gq). As a consequence Gi -dependent receptors (Edg 1 and Edg 8) most likely activate Rac. In contrast, Edg 3 mediates transient Ca2+ fluxes, Ins(3,4,5)P3 increase and Rho activation while Edg 5 leads to the stimulation of PLC, the MAPK pathways and an increase of cAMP 11 .

Sphingolipids as second messenger molecules To elucidate the intracellular role of S1P, microinjections into cells were performed. In the presence of pertussis toxin, which eliminates most G-protein mediated signalling events, Ca2+ mobilization was observed as a response. This is a strong argument for S1P playing the role of an intracellular messenger. 59

T. Baumruker and E. E. Prieschl

S1PP

CK

Choline

aSMase

SK1 Ceramidase

Sphingomyelin

nSMase

Sphingosine

Ceramide

SDK

CAPK

SK2

P

Sphingosine-1phosphate

Edg R

Figure 2. The sphingolipid/protein interface: shown are the (three) layers of proteins grouped around sphingolipids as signalling molecules. The central sphingomyelin pathway, further anabolic/catabolic enzymes and intracellular and extracellular effector molecules. a, acidic; CAPK ceramide-activated protein kinase; CK, ceramide kinase; Edg, endothelial differentiation gene; n, neutral; S1P, sphingosine-1-phosphate; S1PP, S1P phosphatase; SDK, S-dependent kinase; SK1,2, S-kinase 1 and 2; SMase, sphingomyelinase.

S and ceramide induce apoptosis in T cells. 8,22 In contrast, S1P emerged as a counter-regulatory principle, which prevents the programmed cell death after a variety of stimuli, thereby defining the first rheostat composed of sphingolipids. Currently it is a matter of debate, if the capacity of S1P reflects its action as an extracellular or intracellular signalling molecule. 23 Recently, Goetzl et al. 24 investigated the anti-apoptotic contribution of the S1P receptors Edg 3 and Edg 5 in Tsup-1 human T lymphoblastoma cells after Fas, CD2, as well as CD3 plus CD28 induced programmed cell death. Blocking Edg expression concomitantly abolished the protective dose-dependent effect of S1P (10−10 to 10−7 M), indicating that it exerts its effect via Edg receptors as an extracellular mediator. Interestingly, downregulation of Edg 2 and Edg 4, two LPA receptors, showed that this lipid has a similar potential as S1P in this particular cellular system with the exception that it does not encounter a C6 ceramide induced apoptosis. C6 ceramide itself, however, suppresses the expression of Edg 2/Edg 4 but not of Edg 3 thereby explaining this difference. While this data clearly points towards S1P acting as a mediator, studies with SK demonstrate that enhanced kinase activity shifts the intracellular balance to S1P and thereby promotes cell survival in Jurkat T cells after ceramide or Fas induced apoptosis. 25

of dihydroceramide, which is dehydrogenated to ceramide. In contrast, S is never produced by dehydrogenation of the corresponding anabolic dihydro-S and vice versa (see Figure 1). 13 Although many sphingolipids may have the potential to modify signalling processes, the major focus of signalling studies is put on the sphingomyelin pathway. It is composed of three enzymes: sphingomyelinase, ceramidase, and S-kinase (SK), which convert sphingomyelin to ceramide, ceramide to S, and S to S1P, respectively. All three enzymes respond to a variety of stimuli, such as TNFα or IL-1β, and contribute to the initial signalling steps, which lead to activation, differentiation and apoptosis. 20 The products of this pathway are further modified by enzymes such as ceramide kinase generating derivatives with a yet unknown function. The link to the protein signalling cascade finally depends either on a number of intracellular effector proteins exemplified by ceramide-activated protein kinase or by surface bound molecules such as the Edg receptors 21 (see Figure 2).

Sphingolipids in T cell apoptosis The interest in sphingolipids as signalling molecules in immune cells arose as it became evident that 60

Sphingolipids and immune response regulation

the Mg2+ -dependent neutral sphingomyelinase is proportionally activated in those cells. The maximum level of intracellular ceramide is found at the end of the phagocytic process, which reflects the negative feedback of this lipid mediator on antigen uptake. At the molecular level this corresponds to an inhibition of PLD, an enzyme required for FcR-mediated phagocytosis. fMLP, however, additionally activates a Ca2+ -dependent ceramide kinase, resulting in a maximum conversion of ceramide to ceramide-1phosphate 15 min after stimulation. This molecule itself is fusogenic for vesicles implying that it contributes to the generation of phagolysosomes in the process of phagocytosis. In contrast to other proteins (annexins) or free fatty acids implicated in this process, however, ceramide-1-phosphate influences the rate of membrane fusion directly, independent of Ca2+ . As 0.5% ceramide-1-phosphate in the membranes is sufficient to exert its effect, a mediator role for this phospholipid is suggested besides its general lipid property. 29 Taken together this data proposes that a rheostat composed of ceramide-1-phosphate and ceramide regulates phagocytosis; the first acting pro-phagocytotic, the second anti-phagocytotic. In addition, evidence exists that this lipid pair also regulates the oxidative burst of neutrophils. In monocytes/macrophages the Fcγ RI comprises an essential receptor for the clearance of immune complexes. Besides internalization of the IgG antigen complex, however, a signalling cascade is initiated which leads to target cell killing via antibody directed cellular cytotoxicity. In cytokine-primed U937 cells, PLD and SK are implicated to participate in the activation process, particularly to elicit a Ca2+ signal. 30 Interestingly, Fcγ RII triggering, which is an alternative, low affinity IgG receptor depends on PLC with the subsequent production of DAG and Ins(3,4,5)P3 to provoke a rise in Ca2+ in the same cells.

To exclude an indirect effect due to secretion of S1P and subsequent binding to Edg receptors, S1P was determined in the supernatant and additionally pertussis toxin, as a G protein inhibitor, was applied. No detectable amounts of S1P were released from the cells and the process is insensitive to pertussis toxin. Altogether this suggests that S1P may act anti-apoptotically via both routes, as an extracellular mediator and as a second messenger. Besides direct regulation of apoptosis, sphingolipids are generated along the sphingomyelin pathway in response to CD95 cross-linking. One potential mechanism is the activation of acidic sphingomyelinase (ASM) resulting in ceramide release and subsequently the block of CRAC channels and Ca2+ influx. This finding was substantiated with thymocytes from ASM knockout mice, which do not show this blockage. 26

Sphingolipids in T helper balance S, but not ceramide, specifically inhibits the proliferation of Th2 T cells, while Th1 T cells are not affected. In a model of murine contact photosensitivity, S at a concentration of 10 and 3 µM suppresses the DNA synthesis of antigen specific CD4+ suppressor T cells (Th2), but leaves the corresponding Th1 cells untouched 27 . This block is supposed to be a result of a delayed G1 to S-phase progression in Th2 T cells. Surprisingly, the same cell population responds to S with an elevation of intracellular free Ca2+ and enhanced mRNA expression of IL-4 and IL-4R. Therefore the S-mediated effects in Th2 T cells are different from normal apoptosis induction and rather reflect a modulatory potential of this lipid class. These findings imply that individual immunological responses depend on the concentrations of particular sphingolipids and on the balance between counterregulatory lipids in the serum.

A rheostat of sphingolipids regulating allergic excitability

Sphingolipids in phagocytosis and receptors for IgG—another rheostat?

Based on the findings by Kinet 10,31 that in RBL cells SK is induced after FcεRI triggering, we investigated the role of this enzyme as well as its substrate and product in the course of a mast cell activation in depth. Initially the product of this enzymatic reaction, S1P, was regarded as an alternative second messenger to Ins(3,4,5)P3 in the Ca2+ mobilization, analogous to the situation described

Phagocytosis of antibody coated erythrocytes, which serve as a model antigen for IgG-dependent uptake, is accompanied by an increase of the internal ceramide concentration within the first 2 h in fMLP stimulated PMNs. 28 Concomitantly, 61

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maturation is incomplete due to a deficiency of vascular smooth muscle cells/pericytes. Furthermore, in zebrafish both the importance and evolutionary conservation of the S1P system is demonstrated by knock outs of miles (mil), an S1P receptor. 38 These transgenics exhibit an impaired heart development, due to a failure of heart precursor cells to migrate to the midline.

in Fcγ RI activated monocytes. This explained the so-called Ins(3,4,5)P3 gap in mast cells, meaning that Ins(3,4,5)P3 levels and Ca2+ levels do not correlate early in activation. 32,33 Besides this second messenger role, the overall influence of SK turned out to be far more reaching. High intracellular concentrations of S abolish signalling initiated at the FcεRI by blocking the MAP kinase pathway already at the level of Raf kinase. Consequently this prevents the induction of AP1 transcription factors, which are required as co-factors for NF-AT to promote cytokine induction. In contrast, S1P activates the MAP kinase pathway and reverts the inhibitory effects of S if applied in equimolar concentrations. These findings imply that SK is pivotal to the generation of an intracellular lipid milieu, which permits protein based signalling cascades to trigger activation. Therefore a rheostat, composed of S and S1P, that works to regulate the allergic excitability of mast cells is proposed. 2,10

Acknowledgement We thank Ruth Ponting for critical reading of the manuscript.

References 1. Hannun YA, Bell RM (1989) Functions of sphingolipids and sphingolipid breakdown products in cellular regulation. Science 243:500–507 2. Prieschl EE, Baumruker T (2000) Sphingolipids: second messengers, mediators and raft constituents in signaling. Immunol Today 21:555–560 3. Sakai T, Koezuka Y (1998) Ceramide derivatives as therapeutic agents. Exp Opin Ther Patents 8:1673–1682 4. Merrill AH, Schmelz EM, Dillehay DL, Spiegel S, Shayman JA, Schroeder JJ, Riley RT, Voss KA, Wang E (1997) Sphingolipids—the enigmatic lipid class: biochemistry, physiology, and pathophysiology. Toxicol Appl Pharmacol 142:208–225 5. Kolter T, Sandhoff K (1999) Sphingolipide—ihre Stoffwechselwege und die Pathobiochemie neurodegenerativer Erkrankungen. Angew Chem 111:1632–1670 6. Miyatake T, Suzuki K (1972) Globoid cell leukodystrophy: additional deficiency of psychosine galactosidase. Biochem Biophys Res Commun 48:539–543 7. Hannun YA, Bell RM (1987) Lysosphingolipids inhibit protein kinase C: implications for the sphingolipidoses. Science 235:670–674 8. Cuvillier O, Pirianov G, Kleuser B, Vanek PG, Coso OA, Gutkind S, Spiegel S (1996) Suppression of ceramidemediated programmed cell death by sphingosine-1phosphate. Nature 381:800–803 9. Yatomi Y, Ruan F, Hakomori S, Igarashi Y (1995) Sphingosine1-phosphate: a platelet-activating sphingolipid released from agonist-stimulated human platelets. Blood 86:193–202 10. Prieschl EE, Csonga R, Novotny V, Kikuchi GE, Baumruker T (1999) The balance between sphingosine and sphingosine-1-phosphate is decisive for mast cell activation after FcεRI triggering. J Exp Med 190:1–9 11. Hla T (2001) Sphingosine 1-phosphate receptors. Prostaglandins Other Lipid Mediat 64:135–142 12. Shayman JA (1996) Sphingolipids: the role in intracellular signaling and renal growth. J Am Soc Neph 2:171–182 13. Olivera A, Spiegel S (2001) Sphingosine kinase: a mediator of vital cellular functions. Prostaglandins Other Lipid Mediat 64:123–134 14. Ilangumaran S, He HT, Hoessli DC (2000) Microdomains in lymphocyte signalling: beyond GPI-anchored proteins. Immunol Today 21:2–7

Sphingolipids in signalling outside of the immune system but provoked by immune cells Macrophages and mast cells are the major producers of the pleiotropic cytokine TNFα. Its prime target, the vascular endothelium, responds to it with the secretion of various further cytokines and the expression of adhesion molecules such as VCAM-1. In HUVEC cells, which serve as a model system for endothelial cells, SK is induced upon TNFα triggering and S1P promotes the enhanced expression of adhesion molecules. 20 By analogy, in human umbilical vein endothelial cells IL-1β induces sphingomyelinase leading to elevated intracellular ceramide levels, which amplify the IL-1β signalling. 34 Ceramide alone, however, had no signalling potential. In contrast, S1P, which is released by some immune cells after activation, has profound effects on the endothelium by acting pro-angiogenically without the requirement of another co-factor. 35 Again, the high affinity receptors Edg 1 and Edg 3 are implicated in transmitting elevated extracellular S1P levels into adhesion, spreading and migration. At the molecular level the GTPase Rho is employed to induce αV β3 or β1 integrins. 36 Interestingly, a concerted action of both receptors is required for this effect. The importance of S1P and its receptors is further underlined by the finding that Edg 1 knock out mice exhibit embryonic haemorrhage. 37 Whilst vasculogenesis and angiogenesis are normal in those mice, vascular 62

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15. Prieschl EE, Csonga R, Novotny V, Kikuchi GE, Baumruker T (2000) Glycosphingolipid induced relocation of Lyn and Syk into detergent resistant membranes results in mast cell activation. J Immunol 164:5389–5397 16. Postma FR, Jalink K, Hengeveld T, Moolenaar WH (1996) Sphingosine-1-phosphate rapidly induces Rhodependent neurite retraction: action through a specific cell surface receptor. EMBO J 15:2388–2392 17. van Koppen C, Meyer zu HM, Laser KT, Zhang C, Jakobs KH, Bunemann M, Pott L (1996) Activation of a high affinity Gi protein-coupled plasma membrane receptor by sphingosine1-phosphate. J Biol Chem 271:2082–2087 18. Lee MJ, Van Brocklyn JR, Thangada S, Liu CH, Hand AR, Menzeleev R, Spiegel S, Hla T (1998) Sphingosine-1phosphate as a ligand for the G protein-coupled receptor EDG-1. Science 279:1552–1555 19. Parrill AL, Wang D, Bautista DL, Van Brocklyn JR, Lorincz Z, Fischer DJ, Baker DL, Liliom K, Spiegel S, Tigyi G (2000) Identification of Edg1 receptor residues that recognize sphingosine 1-phosphate. J Biol Chem 275:39379–39384 20. Xia P, Wang L, Gamble JR, Vadas MA (1999) Activation of sphingosine kinase by tumor necrosis factor-alpha inhibits apoptosis in human endothelial cells. J Biol Chem 274:34499–34505 21. Zhang Y, Yao B, Delikat S, Bayoumy S, Lin XH, Basu S, McGinnley M, Chang-Hui PY, Lichtenstein H, Kolesnick RN (1997) Kinase suppressor of ras is ceramideactivated protein kinase. Cell 89:63–72 22. Cuvillier O, Edsall L, Spiegel S (2000) Involvement of sphingosine in mitochondria-dependent Fas-induced apoptosis of type II Jurkat T cells. J Biol Chem 275:15691–15700 23. Van Brocklyn JR et al. (1998) Dual actions of sphingosine1-phosphate: extracellular through the Gi-coupled receptor edg-1 and intracellular to regulate proliferation and survival [In Process Citation]. J Cell Biol 13:229–240 24. Goetzl EJ, Kong Y, Mei B (1999) Lysophosphatidic acid and sphingosine 1-phosphate protection of T cells from apoptosis in association with suppression of Bax. J Immunol 162:2049–2056 25. Olivera A, Kohama T, Edsall L, Nava V, Cuvillier O, Poulton S, Spiegel S (1999) Sphingosine kinase expression increases intracellular sphingosine-1-phosphate and promotes cell growth and survival. J Cell Biol 147:545–558 26. Kirschnek S, Paris F, Weller M, Grassme H, Ferlinz K, Riehle A, Fuks Z, Kolesnick R, Gulbins E (2000) CD95mediated apoptosis in vivo involves acid sphingomyelinase. J Biol Chem 275:27316–27323 27. Tokura Y, Wakita H, Yagi H, Nishimura K, Furukawa F, Taki-

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29.

30.

31.

32.

33.

34.

35.

36.

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38.

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gawa M (1996) Th2 suppressor cells are more susceptible to sphingosine than Th1 cells in murine contact photosensitivity. J Invest Dermatol 107:34–40 Suchard SJ, Hinkovska-Galcheva V, Mansfield PJ, Boxer LA, Shayman JA (1997) Ceramide inhibits IgG-dependent phagocytosis in human polymorphonuclear leukocytes. Blood 89:2139–2147 Hinkovska-Galcheva VT, Boxer LA, Mansfield PJ, Harsh D, Blackwood A, Shayman JA (1998) The formation of ceramide1-phosphate during neutrophil phagocytosis and its role in liposome fusion. J Biol Chem 273:33203–33209 Melendez A, Floto RA, Gillooly DJ, Harnett MM, Allen JM (1998) Fc gamma RI coupling to phospholipase D initiates sphingosine kinase-mediated calcium mobilization and vesicular trafficking. J Biol Chem 273:9393–9402 Choi OH, Kim JH, Kinet JP (1996) Calcium mobilization via sphingosine kinase in signalling by the Fc epsilon RI antigen receptor. Nature 380:634–636 Jones SV, Choi OH, Beaven MA (1991) Carbachol induces secretion in a mast cell line (RBL-2H3) transfected with the ml muscarinic receptor gene. FEBS Lett 289: 47–50 Choi OH, Lee JH, Kassessinoff T, Cunha-Melo JR, Jones SVP, Beaven MA (1993) Antigen and carbachol mobilize calcium by similar mechanisms in a transfected mast cell line (RBL2H3 cells) that expresses mI muscarinic receptors. J Immunol 151:5586–5595 Masamune A, Igarashi Y, Hakomori S (1996) Regulatory role of ceramide in interleukin (IL)-1 beta-induced Eselectin expression in human umbilical vein endothelial cells. Ceramide enhances IL-1 beta action, but is not sufficient for E-selectin expression. J Biol Chem 271:9368–9375 Lee MJ, Thangada S, Claffey KP, Ancellin N, Liu CH, Kluk M, Volpi M, Sha’afi RI, Hla T (1999) Vascular endothelial cell adherens junction assembly and morphogenesis induced by sphingosine-1-phosphate. Cell 99:301–312 Paik JH, Chae S, Lee MJ, Thangada S, Hla T (2001) Sphingosine 1-phosphate-induced endothelial cell migration requires the expression of EDG-1 and EDG-3 receptors and Rhodependent activation of alpha vbeta3- and beta1-containing integrins. J Biol Chem 276:11830–11837 Liu Y et al. (2000) Edg-1, the G protein-coupled receptor for sphingosine-1-phosphate, is essential for vascular maturation. J Clin Invest 106:951–961 Kupperman E, An S, Osborne N, Waldron S, Stainier DY (2000) A sphingosine-1-phosphate receptor regulates cell migration during vertebrate heart development. Nature 406:192–195