THE JOURNAL OF BIOLOGICAL CHEMISTRY
`© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
`
`Vol. 279, No. 14, Issue of April 2, pp. 13839–13848, 2004
`Printed in U.S.A.
`
`Sphingosine 1-Phosphate (S1P) Receptor Subtypes S1P1 and S1P3,
`Respectively, Regulate Lymphocyte Recirculation and Heart Rate*
`Received for publication, October 27, 2003, and in revised form, January 13, 2004
`Published, JBC Papers in Press, January 19, 2004, DOI 10.1074/jbc.M311743200
`
`M. Germana Sanna‡, Jiayu Liao§, Euijung Jo‡, Christopher Alfonso‡, Min-Young Ahn‡,
`Melissa S. Peterson‡, Bill Webb¶, Sophie Lefebvre储, Jerold Chun**‡‡, Nathanael Gray储,
`and Hugh Rosenत
`From the Departments of ‡Immunology, §Chemistry, and **Molecular Biology, ¶The Center for Mass Spectrometry,
`The Scripps Research Institute, La Jolla, California 92037 and 储The Genomics Institute of the Novartis Research
`Foundation, San Diego, California 92121
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`widespread effects upon multiple physiological systems (1). It
`regulates heart rate (2), coronary artery blood flow (3), blood
`pressure (4), endothelial integrity in lung (5, 6) and most re-
`cently has been shown to regulate the recirculation of lympho-
`cytes (7–11). Many of the physiologically relevant functions
`occur in the low nanomolar range, including activation of en-
`dothelial nitric oxide synthase (12, 13), vasorelaxation (14), and
`inhibition of thymic egress and lymphocyte recirculation (11).
`Free plasma levels of S1P are tightly regulated by protein
`binding to albumin and high density lipoprotein to avoid the
`deleterious effects of systemic S1P receptor subtype activation
`at high concentrations of ligand, such as bradycardia and cor-
`onary artery vasospasm (3, 15). The choice of S1P, through its
`receptors, as an acute regulator of the number of blood lym-
`phocytes may represent an interesting evolutionary choice by
`the immune system, which evolved after the circulatory sys-
`tem. The pleiotropic responses to S1P, mediated by distinct
`receptor subtypes in a tissue-specific manner, require the def-
`inition of the minimal receptor activation and minimal signals
`necessary and sufficient to regulate these key physiological
`functions. Advances in understanding the contribution of indi-
`vidual receptors has been limited by the current unavailability
`of selective agonists or antagonists. Although S1P2 and S1P3
`were successfully deleted in mice (16, 17), deletion of the S1P1
`gene is embryonic lethal at day 13.5 (18), due to the failure of
`vascular maturation. New tools that elucidate biological func-
`tions of receptor subtypes are needed.
`S1P receptor function in the immune system has experimen-
`tal advantages in approaching selective S1P receptor function
`at vascular interfaces. The maintenance of efficient immune
`responses depends upon maximizing the probabilities of T lym-
`phocytes encountering their cognate antigens (7). The dis-
`persed anatomy of the immune system requires recirculation of
`naive lymphocytes, which emerge from their organs of primary
`lymphopoiesis (thymus and bone marrow), traversing the
`blood, entering secondary lymphoid organs across high endo-
`thelium, and finally crossing sinus-lining endothelium to re-
`turn to the blood stream via lymph and thoracic duct (19–22).
`S1P receptors play a critical role as the molecular gatekeeper
`regulating lymphocyte recirculation. Relatively non-selective
`S1P receptor agonists active on S1P1, S1P3, S1P4, and S1P5 but
`not S1P2, such as the phosphate-ester of the immunomodula-
`tory prodrug FTY720, induce rapid lymphopenia by inhibiting
`the egress of lymphocytes from lymph nodes into lymph (9), as
`
`Sphingosine 1-phosphate (S1P) influences heart rate,
`coronary artery caliber, endothelial integrity, and lym-
`phocyte recirculation through five related high affinity
`G-protein-coupled receptors. Inhibition of lymphocyte re-
`circulation by non-selective S1P receptor agonists pro-
`duces clinical immunosuppression preventing transplant
`rejection but is associated with transient bradycardia.
`Understanding the contribution of individual receptors
`has been limited by the embryonic lethality of the S1P1
`knock-out and the unavailability of selective agonists or
`antagonists. A potent, S1P1-receptor selective agonist
`structurally unrelated to S1P was found to activate mul-
`tiple signals triggered by S1P, including guanosine 5ⴕ-3-
`O-(thio)triphosphate binding, calcium flux, Akt and
`ERK1/2 phosphorylation, and stimulation of migration of
`S1P1- but not S1P3-expressing cells in vitro. The agonist
`also alters lymphocyte trafficking in vivo. Use of selective
`agonism together with deletant mice lacking S1P3 recep-
`tor reveals that agonism of S1P1 receptor alone is suffi-
`cient to control lymphocyte recirculation. Moreover, S1P1
`receptor agonist plasma levels are causally associated
`with induction and maintenance of lymphopenia. S1P3,
`and not S1P1, is directly implicated in sinus bradycardia.
`The sustained bradycardia induced by S1P receptor non-
`selective immunosuppressive agonists in wild-type mice
`is abolished in S1P3ⴚ/ⴚ mice, whereas S1P1-selective ag-
`onist does not produce bradycardia. Separation of recep-
`tor subtype usage for control of lymphocyte recirculation
`and heart rate may allow the identification of selective
`immunosuppressive S1P1 receptor agonists with an en-
`hanced therapeutic window. S1P1-selective agonists will
`be of broad utility in understanding cell functions in vitro,
`and vascular physiology in vivo, and the success of the
`chemical approach for S1P1 suggests that selective tools
`for the resolution of function across this broad lipid re-
`ceptor family are now possible.
`
`Sphingosine 1-phosphate (S1P),1 through its high affinity
`G-protein-coupled receptors, is a physiological mediator with
`
`* The costs of publication of this article were defrayed in part by the
`payment of page charges. This article must therefore be hereby marked
`“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
`indicate this fact.
`‡‡ Supported by the NIMH.
`§§ To whom correspondence should be addressed: Dept. of Immunol-
`ogy, The Center for Mass Spectrometry, The Scripps Research Institute,
`10550 North Torrey Pines Rd., ICND 118, La Jolla, CA 92037. Tel.:
`858-784-2396; Fax: 858-784-2988; E-mail: hrosen@scripps.edu.
`fluorescence-activated cell sorting; CHAPS, 3-[(3-cholamidopropyl)di-
`1 The abbreviations used are: S1P, sphingosine 1-phosphate; hS1P,
`human S1P; GTP␥S, guanosine 5⬘-3-O-(thio)triphosphate; SEW2871,
`methylammonio]-1-propanesulfonic acid; ERK, extracellular signal-
`regulated kinase; PBS, phosphate-buffered saline; GC-MS, gas chroma-
`5-(4-phenyl-5-trifluoromethylthiophen-2-yl)-3-(3-trifluoromethylphe-
`tography-mass spectrometry; AFD-(R), phosphate ester of AAL; AAL-
`nyl)-(1,2,4)-oxadiazole; CHO, Chinese hamster ovary; BSA, bovine se-
`(R), 2-amino-4-(4-heptyloxyphenyl)-2-methyl butanol.
`rum albumin; FLIPR, Fluorescence Imaging Plate Reader; FACS,
`13839
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`This paper is available on line at http://www.jbc.org
`
`Apotex v. Novartis
`IPR2017-00854
`NOVARTIS 2012
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`13840
`
`Discrete Functions for S1P1 and S1P3 Receptors
`min. The ratio fluorescence at 420 nm to that at 510 nm was used to
`well as from thymus into blood (11, 23). Inhibition of lympho-
`measure calcium flux of propidium iodide-negative cells.
`cyte egress is associated with clinically useful immunosuppres-
`sion in both transplantation and autoimmune disease models
`(24–26). Pleiotropic responses at low nanomolar plasma con-
`centrations is seen in this system, because FTY720 mediates
`both lymphopenia and a transient dose-dependent bradycardia
`on initial dosing in humans (27). The therapeutic window for
`S1P receptor agonists may therefore depend on the association
`of single receptors with critical functions.
`We have combined a chemical approach with the use of S1P
`receptor null mice to help define receptor selectivity. We chose
`a chemical approach for S1P1, because of the absence of the
`knock-out. Published data on FTY720 phosphonate (respective
`IC50 values for human S1P1 (8.2 nM), S1P2 (⬎10,000 nM), S1P3
`(151 nM), S1P4 (33 nM), and S1P5 (178 nM)) suggested that S1P1
`is responsible for inhibition of lymphocyte egress (9), a fact that
`was subsequently strengthened by structure-activity correla-
`tions among a collection of semi-selective S1PR agonists (43,
`44).2 We now show that the discovery of selective S1P receptor
`agonists is useful in demonstrating that selective biochemical
`signals can regulate complex in vivo biology.
`
`EXPERIMENTAL PROCEDURES
`S1P Receptor Agonists
`AFD-(R) was the kind gift of Novartis Pharma (Basel, Switzerland,
`Volker Brinkmann). 5-(4-Phenyl-5-trifluoromethylthiophen-2-yl)-3-(3-
`trifluoromethylphenyl)-(1,2,4)-oxadiazole was purchased from May-
`bridge (Tintagel, Cornwall).
`
`Cells and Plasmids
`CHO cells stably expressing human S1P receptors (hS1P) hS1P1,
`hS1P2, hS1P3, hS1P4, and hS1P5, were kindly provided by Danilo
`Guerini (Novartis Pharma).
`Membrane Preparations
`Membranes were prepared from CHO cells expressing human or
`murine S1P1, S1P2, S1P3, S1P4, and S1P5, for use in ligand and
`[35S]GTP␥S binding studies as described previously (9) and suspended
`in Buffer B with 15% glycerol and stored at ⫺80 °C.
`
`Agonist Assays
`Measurements of [35S]GTP␥S Binding—Serial dilutions of S1P (di-
`luted in 4% BSA) or SEW2871 (diluted in Me2SO) were added to
`membranes (1–10 ␮g of protein/well) and assayed as described (9).
`Measurements of Ca⫹ Flux—Calcium flux assays in the Fluorescence
`Imaging Plate Reader (FLIPR; Molecular Devices) format were per-
`formed as described (9). The assay was initiated by transferring an
`equal volume of ligand to the cell plate, and calcium flux was recorded
`over a 3-min interval. Cellular response was quantitated as maximal
`peak height by averaging triplicate wells and expressing as percent
`response relative to S1P activation without pretreatment. Spleen cells,
`after lysis of erythrocytes with 0.17 M NH4Cl, were separated by adher-
`ence to tissue-culture plastic and adherent (stromal cells, macrophages,
`and neutrophils), and non-adherent (lymphocytes) were assayed for
`calcium flux in response to SEW2871 or S1P or ionomycin separately.
`Fluorescence intensity is an absolute measure of fluorescence emission
`upon laser excitation. Flow cytometric measurement of calcium flux
`was performed on cells isolated by teasing apart lymph nodes to single
`cell suspensions, followed by loading with Fluo-3 (Molecular Probes,
`Cell-permeant fluorescence dye 3, calcium binding, 1 ␮M at 4 ⫻ 108
`viable cells/ml). Cells were then labeled with non-activating antibodies
`to CD4 (CD4-PE, BD Pharmingen) and CD8 (CD8-PE), respectively, in
`the presence of propidium iodide. Absolute fluorescence intensity over a
`four-log scale is the standard method for comparisons of fluorescence
`intensity by FACS. Agonist challenge with 1 ␮M SEW2871, S1P, or
`ionomycin (Sigma) at 37 °C was performed in a temperature-controlled
`FACSCalibur flow cytometer (BD Bioscience, Mountain View, CA).
`FACS events were collected using CELLQUEST software (BD Bio-
`science), and then analyzed with FLOWJO (Treestar, San Carlos, CA).
`FACS events were collected for 30 s, and then ionomycin, S1P, or
`SEW2871 were added and events were collected for an additional 10
`
`2 S. Mandala, J. Hale, R. Hajdu, and H. Rosen, unpublished results.
`
`Western Blotting of S1P-activated Kinases
`Control CHO cells and the CHO cells stably transfected with human
`S1P1 or S1P3 were cultured to 50% confluence on 6-well plate in com-
`plete RPMI 1640 supplemented with 10% fetal bovine serum. Cells
`were serum-starved for 16 h and stimulated with SEW2871 diluted to
`various concentrations in the serum-free medium with 0.1% fatty acid-
`free BSA. At 5 min, cells were lysed in 50 mM Tris, pH 8.0, 125 mM
`NaCl, 20 mM CHAPS, 2 mM dithiothreitol, 1 mM EDTA, 2 mM Na3VO4,
`10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor
`mixture. Cell lysates were analyzed by Western blotting after separa-
`tion on 10% SDS-PAGE using mouse monoclonal anti-phospho-ERK1/2
`antibody (sc-7383; Santa Cruz Biotechnology) and rabbit polyclonal
`anti-phospho-Akt antibody (BD Biosciences). Total ERK1 and ERK2
`were detected using a rabbit affinity-purified polyclonal anti-ERK an-
`tibody (sc-94; Santa Cruz Biotechnology), and total Akt was detected
`using a rabbit affinity-purified polyclonal anti-Akt1 antibody (BD Bio-
`sciences). Band intensities corresponding to pERK1, pERK2, and pAkt
`were quantitated by imaging (Kodak 1D Scientific Imaging Systems).
`Amounts of pERK1/2 and pAkt were normalized for the total amounts
`of ERK1/2 and Akt. Primary lymph node lymphocytes were teased from
`the peripheral and mesenteric nodes of C57 BL6 mice kept rigorously at
`4 °C. Cells were warmed to 37 °C, stimulated for 5 min with S1P or
`SEW2871 (50 nM and 500 nM), or 50 ng/ml phorbol myristate acetate or
`vehicle, and Akt and ERK phosphorylation was determined as above.
`
`Assay of S1P Receptors-dependent Cell Migration
`Cell adhesion and migration assays were performed as follows. Cells
`expressing CHO, CHO-S1P1, or CHO-S1P3 were starved overnight in
`regular medium without fetal bovine serum prior to migration assay.
`Cell migration assays were performed using modified Boyden chambers
`(tissue culture-treated, 6.5-mm diameter, 10-␮m thickness, 8-␮m pores,
`Transwell®; Costar Corp., Cambridge, MA) containing polycarbonate
`membranes coated on the underside of the membrane with 5 ␮g/ml
`fibronectin in PBS for 2 h at 37 °C, rinsed once with PBS, and then
`placed into the lower chamber containing 500 ␮l of migration buffer
`(RPMI with 0.5% BSA; Invitrogen, San Diego, CA). Serum-starved cells
`were removed from culture dishes with Hanks’ balanced salt solution
`containing 5 mM EDTA and 25 mM Hepes, pH 7.2, and 0.01% trypsin,
`washed twice with migration buffer, and then resuspended in Migration
`buffer (106 cells/ml). 75,000 cells were then added to the top of each
`migration chamber and allowed to migrate to the underside of the top
`chamber for 3 h in the presence or absence of either S1P or SEW2871
`(100 nm or 1 ␮M), which had been added to the lower chamber. The
`non-migratory cells on the upper membrane surface were removed with
`a cotton swab, and the migratory cells attached to the bottom surface of
`the membrane were fixed with 4% paraformaldehyde and stained with
`propidium iodide (1 ␮g/ml) in PBS for 20 min at room temperature. The
`number of migratory cells per membrane was evaluated by looking at
`five different fields with an inverted microscope using a 40⫻ objective.
`Each determination represents the average of three individual wells. In
`control experiments, cell migration on vehicle control was less than
`0.01% of the total cell population.
`
`Pharmacokinetic Analysis
`All samples were analyzed after CHCl3 extraction, evaporation to
`dryness, and redissolution in 0.1 ml of CHCl3, followed by splitless
`injection on an Agilent 6890N gas chromatograph. Sample detection
`was carried out by using a 5973 mass-selective detector with single ion
`monitoring at 440 m/z for SEW2871 and 372 m/z for a spiked and
`structurally related internal standard, SEW2898. Limit of quantitation
`was 0.4 ng/␮l in plasma, based on spikes into human serum. Sample
`amounts were determined by comparison to a standard curve, R2 ⫽
`0.99. Non-compartmental pharmacokinetic analysis of plasma levels
`was performed by using PK Solutions 2.0 software (Summit Research
`Services, Montrose, CO).
`
`Induction of Lymphopenia in Mice
`C57BL6 or S1P3⫺/⫺ mice (16) or their S1P3⫹/⫹ litter mate controls
`were gavaged with increasing doses of SEW2871 or vehicle (10%
`Me2SO/25% Tween 20 v/v), and blood collected into EDTA tubes (BD
`Biosciences). Full blood counts were determined by veterinary autoana-
`lyzer calibrated for mouse blood (H2000, CARESIDE, Culver City, CA)
`at times stated as described previously (9). All animal studies were
`approved by the Institutional Animal Care and Use Committee.
`
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`Discrete Functions for S1P1 and S1P3 Receptors
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`FIG. 1. SEW2871 is a selective agonist for hS1P1 receptor. A, structures of S1P and SEW2871. B, S1P and SEW2871 activation of hS1P1
`receptor. CHO cell membranes expressing stably transfected hS1P1 receptor were tested for agonism in a GTP␥S binding assay. SEW2871 (upright
`triangles, Œ) was compared with the physiological ligand S1P (closed squares, f) and results normalized to percentage of GTP␥S induced at
`maximal S1P concentrations. EC50 values (mean ⫾ S.D.; n) were 13.8 ⫾ 8.3 nM (n ⫽ 3) for SEW2871 and 0.4 ⫾ 0.24 nM (n ⫽ 6) for S1P. C, SEW2871
`does not agonize hS1P2–5. CHO cell membranes stably expressing one of hS1P2–5 were assayed for ligand-induced GTP␥S binding. All receptor EC50
`values for S1P are shown in the text. SEW2871 had no effect on hS1P3 (closed squares, f); hS1P2 (inverted triangles, ); hS1P4 (upright triangles,
`Œ); hS1P5 (diamonds, ⽧) at concentrations up to 10 ␮M. D, SEW2871 induced a concentration-dependent ligand-dependent calcium flux on hS1P1
`(clear bars, 䡺) but not hS1P2–5 in a FLIPR format assay.
`
`Histology
`C57Bl/6 mice were gavaged with 0.1 ml of vehicle or SEW 2871 (10
`mg/kg). Sixteen hours later, mesenteric and inguinal lymph nodes were
`fixed in 10% formalin in PBS and paraffin-embedded, and 5-␮m sec-
`tions were stained with hematoxylin and eosin. Images were acquired
`by Metamorph software on an Olympus AX70 microscope.
`
`Measurement of Heart Rate in Conscious Mice
`Effects on heart rate in S1P3⫺/⫺ or wild type littermates or C57BL6
`controls were measured by ECG analysis in conscious mice using the
`ANONYmouse ECG screening system (MouseSpecifics, Boston, MA),
`before and after injection of the non-selective S1P receptor agonist
`AFD-(R) or vehicle control. No difference between WT littermates and
`C57BL6 mice were seen.
`
`RESULTS
`High Throughput Screening Identifies S1P1-selective Ago-
`nists—Published binding studies on hS1P1 with FTY720 and
`FTY-P (9), as well as mutagenesis and modeling with natural
`ligand S1P (28, 29), suggested a two-site binding model. The
`hydrophobic-aromatic residues bind within receptor trans-
`membrane domains and the ligand headgroups form salt
`bridges with glutamate and arginine side chains. Specifically,
`FTY720 has a measurable IC50 (300 nM) for S1P1 that is en-
`hanced 1000-fold by the enantioselective addition of the phos-
`phate ester (9). FTY720 binding implies that G-protein-coupled
`
`receptor privileged structures, structurally unrelated to S1P,
`could likely access the transmembrane site as agonists, with
`sequence differences between receptor subtypes making the
`discovery of selective agonists probable (30). Indeed, such
`agents, including the featured compound in this report, have
`previously been identified and characterized (43).2
`SEW2871 Activates Signals and Responses through S1P1
`Alone Comparable to S1P in GTP␥S Activation, Calcium Flux,
`Kinase Phosphorylation, and Cell Migration—5-(4-Phenyl-5-
`trifluoromethylthiophen-2-yl)-3-(3-trifluoromethylphenyl)-
`(1,2,4)-oxadiazole (SEW2871) (Fig. 1A) is a novel selective ag-
`onist for hS1P1 structurally unrelated to S1P. Unlike S1P, it
`has no solubilizing or charged headgroups. S1P showed 50%
`maximal receptor activation in the GTP␥S binding assays
`(EC50) of 0.4 ⫾ 0.24 nM (mean ⫾ S.D.; n ⫽ 6) on human S1P1
`(hS1P1), whereas EC50 values for SEW2871 on hS1P1 were
`13 ⫾ 8.58 nM (mean ⫾ S.D.; n ⫽ 3) (Fig. 1B). Like S1P,
`SEW2871 was a full agonist with levels of receptor activation
`comparable to S1P (Fig. 1B). Although S1P is a non-selective
`agonist with EC50 values (mean ⫾ S.D.) of 3.8 ⫾ 3.5 nM (hS1P2;
`n ⫽ 4), 0.6 ⫾ 0.35 nM (hS1P3; n ⫽ 6), 67 ⫾ 13 nM (hS1P4; n ⫽
`4), 0.5 ⫾ 0.39 nM (hS1P5; n ⫽ 3) on the respective human
`receptors, SEW2871 was inactive at 10,000 nM on hS1P2,
`hS1P3, hS1P4, and hS1P5 (Fig. 1C).
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`13842
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`Discrete Functions for S1P1 and S1P3 Receptors
`
`FIG. 2. SEW2871 is a selective ago-
`nist for mS1P1 receptor. A, S1P and
`SEW2871 activation of mS1P1 receptor.
`CHO cell membranes expressing tran-
`siently transfected mS1P1 receptor were
`tested for agonism in a GTP␥S binding
`assay. SEW2871 (open squares) was com-
`pared with the physiological ligand S1P
`(closed squares) and results normalized to
`percentage of GTP␥S induced at maximal
`S1P concentrations. EC50 values were
`20.7 nM for SEW2871 and 1.4 nM for S1P.
`B, SEW2871 is not an agonist on
`mS1P2–5. CHO cell membranes tran-
`siently expressing one of mS1P2–5 were
`assayed for ligand-induced GTP␥S bind-
`ing. Responses to S1P (see figure for sym-
`bol key) are shown at 10 and 100 nM for:
`mS1P2, mS1P3, mS1P4, and mS1P5, re-
`spectively. SEW2871 had no effect on
`mS1P2, mS1P3, mS1P4, and mS1P5 at
`concentrations up to 10 ␮M. C, compari-
`son of calcium flux stimulation on the mu-
`rine S1P receptors by 100 nM S1P (black
`columns) or SEW2871 (gray column) by
`FLIPR. (Only mS1P1 showed a significant
`calcium flux to SEW2871.) Fluorescence
`intensity is intended as the absolute
`measure of fluorescence emission upon la-
`ser excitation.
`
`We confirmed full selective agonism for hS1P1 alone in the
`ligand-dependent calcium flux assay (Fig. 1D) for SEW2871 in
`stably transfected CHO cell lines, with no significant activation
`of hS1P2–5 (Fig. 1D) up to ⱖ 10 ␮M. We found evidence for
`selective but full agonism of murine S1P1 (mS1P1), EC50 ⫽ 20.7
`nM (Fig. 2A), with no activity at 10 ␮M on mS1P2–5 (Fig. 2B).
`EC50 values for S1P on the transiently transfected murine
`receptors S1P1–5 were 1.4 nM (mS1P1), 2.0 nM (mS1P2), 2.3 nM
`(mS1P3), 75 nM (mS1P4), and 16 nM (mS1P5), respectively.
`Identical selectivity was seen both in membranes of CHO cells
`transiently transfected with the respective murine receptors
`and assayed for agonism in GTP␥S binding assays, as well as in
`intact cells in calcium flux assays (Fig. 2C). Pretreatment with
`30 ng/ml pertussis toxin in the GTP␥S assay fully inhibited
`GTP␥S binding induced by either S1P or SEW2871, confirming
`that SEW2871 is also acting through the Gi-coupled receptor.
`We also compared kinase phosphorylation in response to S1P
`and SEW2871 stimulation in both S1P1 and S1P3 CHO cell
`lines (Table I). Substantial ligand concentration-dependent
`
`pAKT and pERK1 signals were induced by SEW2871 in S1P1
`but not S1P3 CHO cells, whereas modest phosphorylation of
`pERK2 was also seen. In contrast, S1P activated kinases in
`both cell lines equally (not shown).
`The multiple signals induced by SEW2871 are sufficient to
`replicate complex functional responses of S1P through S1P1. In
`a Transwell migration assay, SEW2871 (Fig. 3B) and S1P (Fig.
`3E) induced equivalent cell migration in vivo in S1P1-CHO
`cells with obvious morphology for stimulation of cytoskeletal
`rearrangements. Minimal cell migration or cytoskeletal reor-
`ganization occurred in response to either S1P or SEW2871 in
`untransfected CHO cells (⬍0.01% of cell migrated) (Fig. 3, A
`and D), whereas S1P3 CHO cells migrated and changed shape
`in response to S1P (Fig. 3C) but not SEW2871 (Fig. 3F), con-
`firming the selectivity of SEW2871. Despite its structural dis-
`similarities to S1P, and lack of headgroups, SEW2871 is a
`selective low nanomolar full agonist of S1P1 in all biochemical
`parameters and one complex cellular behavior tested, and
`could potentially be usefully studied in vivo.
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`13843
`
`pERK1
`
`S1P1-CHO
`
`S1P3-CHO
`
`S1P1-CHO
`
`S1P3-CHO
`
`S1P1-CHO
`
`S1P3-CHO
`
`nM
`5
`50
`500
`a ND, not detected.
`
`4.7⫻
`12.1⫻
`13.8⫻
`
`1.9⫻
`1.7⫻
`1.7⫻
`
`1.5⫻
`1.8⫻
`1.8⫻
`
`1.1⫻
`0.6⫻
`1.1⫻
`
`4.2⫻
`4.7⫻
`5.8⫻
`
`NDa
`ND
`ND
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`cline in lymphocyte numbers with a correlative decline in the
`percentage of lymphocytes within the leukocyte differential
`count (Fig. 4C).
`The plasma EC50 of SEW2871 for lymphopenia was ⬃2 ␮M
`(Fig. 4B). There was a dose-response relationship between
`plasma SEW2871 levels and the number of blood lymphocytes.
`We showed the relationship between plasma levels of S1P1
`receptor agonist and the maintenance of circulating blood lym-
`phocyte numbers in the duration of action study (Fig. 4D),
`where 20 mg/kg SEW2871 was gavaged and blood lymphocyte
`numbers and compound plasma concentrations were measured
`for the first 42 h. The curves for plasma levels of SEW2871 and
`the induction and maintenance of lymphopenia were mirror
`images of each other. Induction of lymphopenia was as rapid
`for SEW2871, as for non-selective S1P receptor agonists such
`as AAL(R) (9–11), and full lymphopenia was maintained for
`more than the first 12 h. As SEW2871 concentrations in plasma
`decline, the lymphopenia reverses, suggesting that the contin-
`uous presence of S1P1 receptor agonist is necessary for the
`maintenance of lymphopenia.
`SEW2871 Inhibits Lymphocyte Migration into Murine Lym-
`phatic Sinuses—S1P receptor agonists inhibit egress of lym-
`phocytes into lymphatic sinus in peripheral and mesenteric
`lymph nodes and Peyer’s patch but not spleen (7, 9). Effects are
`easily seen histologically within 6–15 h of a single dose of
`agonist. SEW2871 (Fig. 5B) but not vehicle (Fig. 5A) induced
`clearing of lymphatic sinuses (arrows) and the log-jamming of
`lymphocytes immediately subjacent to sinus-lining endothe-
`lium in lymph nodes. SEW2871 histological changes were in-
`distinguishable from those seen with the non-selective S1P
`receptor agonist control AAL-(R) (not shown) and those pub-
`lished for FTY720 (9). Both SEW2871 and the non-selective
`S1P agonists inhibit the egress of lymphocytes across sinus
`lining endothelium supporting the conclusion that activation of
`S1P1 alone is sufficient to shut down entry of lymphocytes into
`lymph.
`Freshly Isolated Lymphocytes from Spleen or Lymph Node Do
`Not Respond to SEW2871 with a Ligand-evoked Calcium
`Flux—SEW2871 has rapid effects upon the bulk trafficking of
`lymphocytes in vivo, although these effects are confined to
`lymph node and thymus but not spleen, despite the facts that
`naı¨ve lymphocyte populations in lymph node and spleen show
`no distinguishing characteristics. Inhibition of
`lymphocyte
`egress from lymph node and thymus but not spleen suggests
`that this mechanism may therefore depend upon non-lympho-
`cytic stromal cell effects in addition perhaps to direct effects
`upon lymphocytes. To assess whether SEW2871 mediated its
`effects upon lymphocytes directly or indirectly, we looked for
`evidence of S1P1 activation and expression on freshly isolated
`murine lymphocytes that had not been cultured at all.
`Spleen adherent cells, but not lymphocytes freshly isolated
`from spleen, lymph node, or thymus responded to SEW2871
`with a ligand-induced calcium flux in FLIPR format assays
`(Fig. 6A). These data were confirmed by flow cytometry, where
`freshly isolated CD4⫹ (shown) or CD8⫹ (data not shown) T
`
`FIG. 3. Migration assay on S1P1- and S1P3-expressing cells in
`response to S1P and SEW2871. CHO, CHO-S1P1, and CHO-S1P3
`cells were assayed in a cell migration assay on Transwell membranes
`upon stimulation with 100 nM S1P (A–C) or 1 ␮M SEW2871 (D–F). Only
`cells passing through the membranes are stained with propidium iodide
`and appeared red. Although SEW2871 (B) and S1P (E) induced equiv-
`alent cell migration in vivo in S1P1-CHO cells, S1P3-CHO cells mi-
`grated only in response to S1P (C) but not SEW2871 (F), confirming the
`selectivity of SEW2871. Minimal cell migration or cytoskeletal reorga-
`nization occurred in response to either S1P or SEW2871 in untrans-
`fected CHO cells (⬍0.01% of cell migrated) (A and D).
`
`SEW2871 Induces and Maintains Lymphopenia in Mice,
`Which Correlates with Plasma Agonist Levels—Relatively non-
`selective S1P receptor agonists, such as FTY720-P and its
`phosphonate, produce rapid lymphopenia in peripheral blood
`that
`is the basis of
`their immunosuppression. Because
`SEW2871 was a selective agonist of S1P1 alone, we tested its
`efficacy for the induction and maintenance of lymphopenia in
`vivo. We gavaged mice with SEW2871 (1.25 to 20 mg/kg) and
`measured compound plasma levels and circulating blood lym-
`phocyte numbers at 5 h.
`Plasma levels of SEW2871, measured at 5 h by GC-MS were
`linear with oral dose in the range of 0–30 mg/kg (Fig. 4A) in
`mice. SEW2871 produced a rapid and dose-dependent periph-
`eral blood lymphopenia after 5 h (ED50 ⫽ 5.5 ⫾ 1.04 mg/kg
`(mean ⫾ S.E.; n ⫽ 4; Fig. 4B) when tested at doses up to 150
`mg/kg. Because S1P agonists induce lymphopenia without af-
`fecting peripheral blood myelomonocytic cells, there was a de-
`
`Discrete Functions for S1P1 and S1P3 Receptors
`TABLE I
`S1P1-mediated Akt and ERK1/2 phosphorylation
`The -fold increases from the control Me2SO-treated cells are shown.
`pAkt
`
`pERK2
`
`SEW2871
`
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`Discrete Functions for S1P1 and S1P3 Receptors
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`FIG. 4. Pharmacokinetics and pharmacodynamics of SEW2871 in mice. A, gavage of SEW2871 from 1.25 to 30 mg/kg in a Tween
`20-Me2SO vehicle gave dose-linear plasma concentrations of SEW2871 at 5 h, the time for routine assay of maximal lymphopenia. SEW2871
`concentrations in plasma (in nanomoles/liter) were measured by GC-MS and the means ⫾ S.D. (n ⫽ 4) are shown. Non-compartmental analysis
`of GC-MS plasma levels over 24 h following single oral dosing at 10 mg/kg derived the following pharmacokinetic parameters: Cmax 2.0 ␮g/ml, Tmax
`6 h; t1⁄2 7.1 h; plasma clearance 30.7 ml/kg/h; Vd 313 ml/kg. B, blood lymphocyte numbers measured by autoanalyzer at 5 h were related to plasma
`concentrations of SEW2871. Data from 41 individual mice are shown; R2 ⫽ 0.9811. C, reduction in blood lymphocyte numbers as well as in the
`percentage of lymphocytes within a differential white cell count was seen, as selective lymphopenia with relatively unaltered monocytes and
`neutrophils was induced by SEW2871. D, SEW2871 induced and maintained lymphopenia. 20 mg/kg of SEW2871 was administered by gavage and
`mice (n ⫽ 4) harvested at each time point up to 42 h. Mean ⫾ S.D. are shown for SEW2871 concentrations (micromolar) and lymphocyte numbers
`per milliliter of blood collected by cardiac puncture. Plasma levels of SEW2871 correlated with the maintenance of lymphopenia. Peak lymphopenia
`was maintained for ⬎12 h and gradually declined as plasma concentrations of SEW2871 declined.
`
`lymphocytes did not undergo a calcium flux in response to
`either SEW2871 (at concentrations up to 10 ␮M) or S1P (1 ␮M),
`but did respond to ionomycin (Fig. 6B).
`We also examined phosphorylation of Akt and ERK1 in
`freshly isolated lymph node lymphocytes. Neither S1P nor
`SEW2871 at both 50 and 500 nM induced phosphorylation of
`Akt and ERK in lymphocytes, whereas stimulation with PMA
`induced a 9.2-fold increase in ERK phosphorylation of the same
`cells (data not shown).
`In addition, we were not able to identify the presence of
`mS1P1 upon freshly isolated lymphocytes by Western blotting
`or immunohistology, whereas both transfected cells and lymph-
`oid organ stroma can be shown to express S1P1 by these meth-
`ods.3 Further work to identify functional S1P receptors on
`lymphocytes in vivo is required.
`
`3 C. Alfonso and H. Rosen, manuscript in preparation.
`
`S1P3 Regulates Heart Rate and Is Not Required for the In-
`duction of Lymphopenia—S1P1 and S1P3 are coexpressed in
`some cells, especially endothelium (7, 9). The association of a
`dose-dependent bradycardia with administration of the rela-
`tively non-selective receptor FTY720 in humans (27) led us to
`study the lymphopenic and heart rate responses that associ-
`ated with S1P1 and S1P3. Induction of lymphopenia (Fig. 7A) in
`homozygous S1P3⫺/⫺ mice was indistinguishable from wild-
`type mice, with no statistically significant difference in the
`depth of lymphopenia at 5 h between the S1P1-selective agonist
`SEW2871 and the S1P1, S1P3, S1P4, and S1P5 active prodrug
`AAL-(R), which is phosphorylated to its active form AFD-(R) (10,
`11). Deletion of S1P3 therefore did not affect the S1P receptor
`agonist-induced inhibition of lymphocyte recirculation.
`Consistent with previous observations that S1P3-active com-
`pounds were associated with toxicity and heart rate changes in
`rodents (44), we observed that the acute heart rate changes
`
`

`

`Discrete Functions for S1P1 and S1P3 Receptors
`activation of S1P1 is sufficient to control lymphocyte numbers
`and plays no discernable role in control of sinus rhythm,
`whereas S1P3 regulates sinus rhythm and not lymphocyte
`recirculation.
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`DISCUSSION
`Establishing the relationships between receptor subtype us-
`age and discrete physiological p