of April 13, 2017.This information is current as

3Modulators Independently of S1PSphingosine-1-Phosphate Receptor Circulating Monocytes Are Reduced by

Maryanne L. Brown and Louise K. ModisSusan M. Lukas, Kelli R. Ryan, Anthony J. Slavin, Prathima Adusumalli, Sudha N. Desai, Lori A. Patnaude,Anderson, Dimitria E. Stefanopoulos, Steven E. Fogal, Nuruddeen D. Lewis, Sokol A. Haxhinasto, Shawn M.

http://www.jimmunol.org/content/190/7/3533doi: 10.4049/jimmunol.1201810February 2013;

2013; 190:3533-3540; Prepublished online 22J Immunol 

Referenceshttp://www.jimmunol.org/content/190/7/3533.full#ref-list-1

, 13 of which you can access for free at: cites 36 articlesThis article

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2013 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on April 13, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

by guest on April 13, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

The Journal of Immunology

Circulating Monocytes Are Reduced bySphingosine-1-Phosphate Receptor ModulatorsIndependently of S1P3

Nuruddeen D. Lewis, Sokol A. Haxhinasto, Shawn M. Anderson, Dimitria E. Stefanopoulos,

Steven E. Fogal, Prathima Adusumalli, Sudha N. Desai, Lori A. Patnaude, Susan M. Lukas,

Kelli R. Ryan, Anthony J. Slavin, Maryanne L. Brown, and Louise K. Modis

Sphingosine-1-phosphate (S1P) receptors are critical for lymphocyte egress from secondary lymphoid organs, and S1P receptor

modulators suppress lymphocyte circulation. However, the role of S1P receptors on monocytes is less clear. To elucidate this, we

systematically evaluated monocytes in rats and mice, both in naive and inflammatory conditions, with S1P receptor modulators

FTY720 and BAF312. We demonstrate that S1P receptor modulators reduce circulating monocytes in a similar time course

as lymphocytes. Furthermore, total monocyte numbers were increased in the spleen and bone marrow, suggesting that S1P receptor

modulation restricts egress from hematopoietic organs. Monocytes treated ex vivo with FTY720 had reduced CD40 expression and

TNF-a production, suggesting a direct effect on monocyte activation. Similar reductions in protein expression and cytokine

production were also found in vivo. Suppression of experimental autoimmune encephalomyelitis in mice and rats by FTY720

correlated with reduced numbers of lymphocytes and monocytes. These effects on monocytes were independent of S1P3, as

treatment with BAF312, a S1P1,4,5 modulator, led to similar results. These data reveal a novel role for S1P receptors on monocytes

and offer additional insights on the mechanism of action of S1P receptor modulators in disease. The Journal of Immunology,

2013, 190: 3533–3540.

Sphingosine-1-phosphate (S1P) is a bioactive lipid mediatorthat regulates multiple cellular processes, including immunecell trafficking (1). A gradient of S1P is recognized by S1P

receptors on immune cells, leading to their entry into circulation(2). The biological relevance of this pathway was highlightedby the development of FTY720, a potent S1P1,3,4,5 modulator, asa therapy for multiple sclerosis (3). FTY720 leads to internalizationand degradation of S1P receptors on lymphocytes; consequently,lymphocyte egress from secondary lymphoid organs is inhibited,resulting in a significant decrease in circulating lymphocyte levels(4) and reduced lymphocyte infiltration into the CNS (5).S1P receptors may also be involved in innate immune cell

trafficking (2). In vitro, mast cells (6), dendritic cells (7), eosin-ophils (8), NK cells (9), macrophages (10), and monocytes (11)have been shown to migrate toward S1P, albeit weakly. In vivo,NK cell trafficking is dependent upon S1P (12); and similar tolymphocytes, FTY720 treatment restricts NK cell egress fromlymph nodes and bone marrow (13). Additionally, migration ofin vitro generated, mature S1P1

2/2 or S1P32/2 dendritic cells is

defective following injection into mice (14).The role of S1P receptors on monocytes in vivo is less clear.

Several reports suggest that monocyte trafficking may be restricted

by modulation of S1P receptors based on reduced numbers of mac-rophages in tissue, such as the following: the peritoneal cavity fol-lowing thioglycolate-induced peritonitis (10), peripheral nerves inexperimental autoimmune neuritis (15), the CNS in experimentalautoimmune encephalomyelitis (EAE) (16), renal glomeruli inexperimental mesangioproliferative glomerulonephritis (17), ath-erosclerotic plaques (18), and heart allografts (19). However, aneffect on circulating monocytes has not been reported. Moreover,FTY720 has been shown to increase the number of macrophagesin mesenteric lymph nodes, suggesting that S1P receptors may beimportant for monocyte egress from lymph nodes in a mannersimilar to lymphocytes (20). However, it is not clear whether thisis a direct or indirect effect on either the numbers or trafficking ofcirculating monocytes.In this work, we examined the effect of S1P receptor modulators

on monocytes in vivo and observed reduced circulating monocytenumbers in mice and rats, in both normal and inflammatory con-ditions. We demonstrate that this effect is independent of S1P3 byusing S1P receptor modulators with different selectivities. Fur-thermore, we provide evidence that FTY720 restricts monocyteegress from the spleen and bone marrow, as monocyte cell num-bers are significantly increased at these sites. We also show thatmonocytes express S1P receptors and that FTY720 can act di-rectly upon monocytes to alter their activation, as demonstrated byreduced surface protein expression and cytokine production.These findings demonstrate an important role for S1P receptors onmonocytes in rodents and suggest that additional studies on thefunctions of S1P receptor modulators on humans may be merited.

Materials and MethodsAnimals

Female C57BL/6 mice (6–10 wk of age) were purchased from The JacksonLaboratory. Female Lewis rats (150–200 g) were purchased from Charles

Department of Immunology and Inflammation, Boehringer Ingelheim Pharmaceuti-cals, Ridgefield, CT 06877

Received for publication June 29, 2012. Accepted for publication January 22, 2013.

Address correspondence and reprint requests to Dr. Louise K. Modis, Department ofImmunology and Inflammation, Research and Development 1461, Boehringer Ingel-heim Pharmaceuticals, 900 Ridgebury Road, P.O. Box 368, Ridgefield, CT 06877.E-mail address: louise-kelly.modis@boehringer-ingelheim.com

Abbreviations used in this article: AUC, area under the curve; EAE, experimentalautoimmune encephalomyelitis; EdU, 5-ethynyl-29-deoxyuridine; S1P, sphingosine-1-phosphate.

Copyright� 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201810

by guest on April 13, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

River Laboratories. Animals were fed and watered ad libitum under 12-hlight/dark cycles. All studies were performed in accordance with Institu-tional Animal Care and Use Committee guidelines.

Chemical synthesis and dosing

FTY720 was purchased from Cayman Chemical. BAF312 (21) was pre-pared as the zwitterion, according to the synthetic procedures described(22), and crystallized, as described (23). Compounds were dosed in a ve-hicle containing 0.5% methylcellulose (Spectrum Chemical) and 0.2%Tween 80 (EMD Chemicals). Mice and rats were dosed once daily by oralgavage. Prophylactic dosing of mice began 1 d before immunization, andtherapeutic dosing of mice began once mice had a clinical score .1 ora score of 1 for at least 2 d. For in vitro studies, cell culture medium wasused as the vehicle.

Whole-blood differential analysis

Whole blood was collected from animals by retro-orbital bleeding andanalyzed immediately using the Hemavet 1700FS (Drew Scientific).

Tissue preparations and flow cytometry

Whole blood was isolated retro-orbitally, and 100 ml was used for staining.Spleens were smashed in 2 ml Cell Staining Buffer (BioLegend) over a 40-mm strainer, and 30 ml was used for staining. Cell Staining Buffer wasadded to make a final volume of 100 ml. The right tibia was used to isolatebone marrow. Bone marrow was resuspended in 800 ml Cell StainingBuffer, and 100 ml was used for staining. TruStain fcX (10 mg/ml; Bio-Legend) was added directly to the samples and incubated on ice for 10–15min to block FcRs. The following Abs in a total volume of 50 ml were thenadded at the concentration suggested by the manufacturer. Abs werepurchased from BioLegend (Ly6C [Ag], HK1.4 [clone]; CD115, AFS98;CD86, GL-1; I-A/I-E [MHC II], M5/114.15.2; CD274 [PD-L1], 10F.9G2;CD40, 3/23; CD69, H1.2F3; CD80, 16-10A1; CD44, IM7; CD62L, MEL-14; B220, RA3-6B2; CD4, RM4-5; CD3, 145-2C11; CD8, 53-6.7; Gr-1,RB6-8C5; NK1.1, PK136), Becton Dickinson Biosciences (CD45, 30-F11;CD11b, M1/70), and eBioscience (CD335 [NKp46], 29A1.4). Isotypecontrol Abs were used at the same protein concentrations as their corre-sponding markers. Cells were stained in the dark on ice for 20 min, andthen 1.6 ml One-Step Fix/Lyse Solution (eBioscience) was added, mixed,and incubated with the cells for 30 min at room temperature. The cellsolution was then centrifuged, and the supernatant was discarded. The cellswere then washed twice with Cell Staining Buffer, resuspended in 200 mlCell Staining Buffer, run on the FACS Canto II (Becton Dickinson Bio-sciences), and analyzed using Flow Jo 7.6.3.

The total cell numbers shown in Fig. 1B were obtained by multiplyingthe percentage positive of each cell type (as determined by flow cytometry)by the whole blood cell count obtained from the Hemavet, then dividingthat number by 100. For Fig. 2B, the total cell counts were obtained bymultiplying the percentage of monocytes by the total cell concentrations.The cell concentrations were derived by taking the total number of cellsrun through the flow cytometer for each sample, divided by the run time ofthe sample to get cells/time. This number was then divided by the speed ofthe cytometer to get the cell concentration. The cell counts were thenmultiplied by their respective dilutions and the total volume of the cellsuspension. Mice were assumed to have a total blood volume of 58.5 ml/kgbody weight.

S1P receptor expression analysis

S1P receptor expression data were obtained from publicly available micro-array datasets using NextBio [www.nextbio.com (24)]. The data were pro-cessed and normalized by NextBio to compare between different datasets, asdescribed. Expression levels of S1P receptors were obtained for CD8+ T cells(GSE11446), B220+ B cells (GSE6980), NK1.1+ NK cells (GSE6506),granulocytes (Mac-1+ Gr-1+; GSE10246), Ly6C high and low monocytes(GSE17256), and corticotectal neurons (GSE17784). All cell types wereobtained from C57BL/6 mice. Data are presented as the mean 6 SD.

Monocyte enrichment and stimulation

Blood was isolated from C57BL/6 mice and lysed using RBC lysis buffer(BioLegend), according to the manufacturer’s instructions. The EasySepMouse Monocyte Enrichment kit (Stemcell Technologies) was used toenrich for monocytes. This was performed according to the manufacturer’sinstructions. Cell purity was assessed by CD11b+ Ly6G2 according to themanufacturer’s instructions and was on average 90.2% pure.

Monocytes were cultured at 37˚C in RPMI 1640 medium containing10% FBS, 10% L929-conditioned medium, 13 penicillin/streptomycin,

2 mM L-glutamine, and 10 mM HEPES. FTY720 was dissolved in thismedium and was used at 10 nM. LPS was used at 10 ng/ml. After 2 h, cellswere centrifuged and supernatants were collected and frozen until needed.Cytokine levels of TNF-a were assessed using a sandwich immunoassay(Meso Scale Discovery). Cells were also stained for CD40 and analyzed byflow cytometry.

In vivo LPS challenge and cytokine measurement

C57BL/6 mice were dosed with either 0.3 mg/kg FTY720, BAF312, orvehicle (0.5% methylcellulose and 0.2% Tween 80). After 22 h, mice re-ceived an i.p. injection of 200 ng LPS or vehicle (saline). Two hours later,whole blood was collected. The blood was then centrifuged, and the serumwas stored at 220˚C until assessed for cytokine levels. Serum levels ofCCL2 (MCP-1), TNF-a, IL-6, and IL-10 were measured using a multiplexsandwich immunoassay (Meso Scale Discovery).

EAE induction

Mice were s.c. injected with 100 mg myelin oligodendrocyte glycopro-tein35–55 (AnaSpec) in IFA (Difco) containing 250 mg Mycobacteriumtuberculosis (Difco). On the same day and again 48 h later, mice receivedan i.p. injection of 200 ng pertussis toxin (List Biological Laboratories).Mice were scored as follows: 0, no symptoms; 1, limp tail; 2, limp tail andinability to right itself when placed on its back; 3, partial hind limb pa-ralysis; 4, complete hind limb paralysis; 5, complete hind limb paralysiswith forelimb involvement or moribund. EAE was induced in rats by s.c.injection of 100 mg guinea pig myelin basic protein68–86 (AnaSpec) in IFAcontaining 800 mg M. tuberculosis. Rats were scored as follows: 0, nosymptoms; 1, limp tail; 2, ataxia and/or altered gait; 3, partial hind limbparalysis; 4, complete hind limb paralysis; 5, complete hind limb paralysiswith forelimb involvement.

Statistics

For the EAE clinical score data, area under the curve (AUC) was calculatedand the Kruskal-Wallis test was performed, followed by a Dunn’s posttest todetermine significance between multiple groups. For other data, either anunpaired t test was used when comparing two groups or a one-wayANOVA followed by the Newman-Keuls posttest was used for compar-ison between multiple groups. These calculations were performed usingGraphPad Prism 5.04.

ResultsFTY720 reduces circulating levels of monocytes and NK cellswithout affecting neutrophil levels

To determine the effects of FTY720 on innate immune cells, wedosed mice with FTY720 at 0.03, 0.3, and 3 mg/kg and performeda differential analysis of peripheral blood collected at 4 and 24 hafter dosing. In agreement with previous reports, using size-basedanalysis (Hemavet), we found a significant, dose-dependent de-crease in circulating lymphocytes at both time points when com-pared with vehicle alone (up to an 87% reduction).We also observeda significant reduction in circulating monocytes. Monocyte levelswere decreased up to 78% in a dose-dependent manner withFTY720 treatment (Fig. 1A). We also monitored circulatingneutrophil levels and found that there was no significant effect inresponse to FTY720 treatment (Fig. 1A).Using flow cytometry, we confirmed the lymphocyte and mono-

cyte lowering in mice and extended our analysis to additional celltypes. Mice were dosed with 0.03, 0.1, 0.3, and 3 mg/kg FTY720,and whole blood immune cell counts were assessed by flowcytometry. We confirmed that CD4+ (97% reduction) and CD8+

T cells (91% reduction) along with B220+ B cells (60% reduction)were decreased in peripheral blood 24 h after dosing with all testeddoses of FTY720 (Fig. 1B). Significant decreases in monocytes(CD11b+ CD115+; 56% reduction) and NK cells (NK1.1+ NKp46+;43% reduction) were also detected; however, no effect on neu-trophils (CD11b+ Gr-1+) was observed (Fig. 1B). Thus, the numberof circulating monocytes and NK cells is reduced followingFTY720 with a similar time course as lymphocytes.We dosed rats with FTY720 at 0.03, 0.1, and 0.3 mg/kg and

performed a differential analysis of peripheral blood collected at 4

3534 FTY720 REDUCES MONOCYTE NUMBERS IN THE CIRCULATION

by guest on April 13, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

and 24 h after dosing. Similar to our results in mice, we founda significant, dose-dependent decrease in circulating lymphocytes atboth time points when compared with vehicle alone (up to an 88%reduction; Fig. 1C). Monocyte levels were also decreased in a dose-dependent manner with FTY720 treatment up to an 80% reduction(Fig. 1C). We also monitored circulating neutrophil levels byHemavet, and, although a decrease in response to FTY720 treat-ment was observed, it was not a significant effect (Fig. 1C).

FTY720 restricts monocyte egress from the spleen and bonemarrow

Due to the decreased levels of circulating monocytes observedupon administration of FTY720, we wanted to determine whether

monocyte levels were also altered in lymphoid tissues. We dosedmice with FTY720 at 0.3 mg/kg and 2 mg 5-ethynyl-29-deoxy-uridine (EdU) (a 5-bromo-29-deoxyuridine alternative) and iso-lated the blood, spleen, and bone marrow 4 h later and performedflow cytometry to assess monocyte levels in these tissues. Inaddition to our observation that FTY720 reduced circulatingmonocyte levels, we found that FTY720 causes a significant ac-cumulation of monocytes in both the spleen and bone marrow(Fig. 2B). This effect was observed for both Ly6Chigh and Ly6Clow

monocytes. Additionally, we calculated the sum of the totalmonocytes in these tissues and found that there were significantlymore Ly6Chigh monocytes, but not Ly6Clow monocytes (Fig. 2C).These data suggest that for the Ly6Clow monocytes, the changes

FIGURE 1. FTY720 reduces circulating lymphocyte, monocyte, and NK cell levels, but not neutrophils. (A) C57BL/6 mice were dosed with vehicle, 0.03

mg/kg, 0.3 mg/kg, or 3 mg/kg FTY720 (n = 7 mice per group). Whole blood was taken at 0, 4, and 24 h after the administration of the dose. Lymphocyte,

neutrophil, and monocyte cell counts were assessed using the Hemavet. (B) C57BL/6 mice were dosed with vehicle, 0.03, 0.1, 0.3, or 3 mg/kg FTY720.

After 24 h, whole blood was collected and CD4+ T cells, CD8+ T cells, B220+ B cells, CD11b+ CD115+ monocytes, NK1.1+ NKp46+ NK cells, and CD11b+

Gr-1+ neutrophil cell counts were assessed by flow cytometry (n = 29–30 mice in the untreated and vehicle groups; n = 10 in the 0.03, 0.1, and 0.3 mg/kg

groups; and n = 20 in the 3 mg/kg group). (C) Lewis rats were dosed with vehicle, 0.03 mg/kg, 0.1 mg/kg, or 0.3 mg/kg FTY720. Whole blood was taken at

0, 4, and 24 h after the administration of the dose. Lymphocyte, neutrophil, and monocyte cell counts were assessed using the Hemavet (n = 12 rats for the

0-h time point, and n = 5 rats per group for all other time points). *p , 0.05, **p , 0.01, ***p , 0.001 versus vehicle. Mean 6 SEM is shown.

The Journal of Immunology 3535

by guest on April 13, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

we observed were due to cell relocation. However, the observedchanges for Ly6Chigh monocytes could be due to increased pro-liferation or reduced cell turnover. We next analyzed the numberof EdU+ bone marrow monocytes and found that FTY720 in-creased their numbers, suggesting an increase in proliferation (Fig.2D). This effect, however, was restricted to the Ly6Chigh subset,thus explaining our results found in Fig. 2C. These results dem-onstrate that FTY720 causes monocytes to accumulate in thespleen and bone marrow while also causing increased proliferationin the bone marrow.

FTY720 directly restricts monocyte activation

Our previous results demonstrate that FTY720 changes monocytelevels by reducing their numbers in the circulation and increasingtheir numbers in the spleen and bone marrow. These data, however,do not address whether this effect is direct or indirect. The first stepwe took to address this was to analyze S1P receptor expression onmonocyte subsets and other immune cells by accessing and ana-lyzing publicly available microarray datasets using NextBio (Fig.3A). These data demonstrate that monocytes express S1P recep-tors with the highest levels observed for S1pr2 and S1pr5. Thesedata also confirm previous publications that lymphocytes expresshigh levels of S1pr1 and NK cells have high levels of S1pr5.Expression of S1pr4 was not included in these datasets; never-theless, other studies have shown low-level expression of S1pr4 onmonocytes as compared with other immune cells (25).To further determine whether FTY720 acts directly upon

monocytes, we enriched monocytes from the blood (∼90.2% pure)and cultured them in the presence of FTY720 (10 nM) and LPS(10 ng/ml). After 2 h, we found FTY720 reduced CD40 proteinlevels on monocytes (Fig. 3B). In the presence of LPS, there wasan induction of CD40 levels that was significantly reduced withFTY720. Furthermore, we analyzed cytokine production andfound LPS induced the production of TNF-a, which was signifi-cantly reduced in the presence of FTY720 (Fig. 3C). These data

demonstrate that FTY720 acts directly on monocytes to altersurface protein expression and cytokine production.

S1P receptor modulation in vivo alters monocyte surfaceprotein expression and cytokine production

To assess whether S1P receptor modulation alters monocyte ac-tivation in vivo, mice were dosed with 0.3 mg/kg FTY720. Twenty-four hours later, we collected peripheral blood and assessed thecell surface expression of multiple proteins on monocyte subsets.We found that on Ly6Chigh monocytes, PD-L1, CD40, and CD69were decreased by FTY720, whereas CD80 was increased (Fig.4A). On Ly6Clow monocytes, CD40 and CD69 were also decreasedby FTY720 compared with control. We also monitored cell apo-ptosis and necrosis using annexin V and propidium iodide andfound no significant difference between the groups (data notshown). These results suggest that FTY720 alters monocyte sur-face protein expression in vivo and may affect monocyte function.Because we demonstrated that FTY720 reduced circulating

levels of monocytes and altered their surface protein expression andcytokine production, we wanted to determine whether S1P receptormodulation would have an impact on serum cytokine levels after aninflammatory stimulus in vivo. To test this, we dosed mice with 0.3mg/kg FTY720 and BAF312, a S1P1,4,5 modulator, for 22 h andchallenged them with 200 ng LPS. After 2 h, we collected wholeblood and assessed the levels of proinflammatory cytokines. Wefound that induction of IL-6, TNF-a, CCL2, and IL-10 by LPSwas significantly decreased by FTY720 and BAF312 (Fig. 4B).These results demonstrate that S1P receptor modulation decreasescytokine production after an LPS challenge.

FTY720 reduces monocytes during EAE

To determine whether the impact of FTY720 on monocytes istransient or sustained in a long-term model of inflammation, weinduced EAE in both mice and rats and measured the effects of 0.03and 0.3 mg/kg FTY720 on clinical disease and circulating levels

FIGURE 2. FTY720 restricts monocyte egress

from the spleen and bone marrow while enhancing

proliferation. C57BL/6 mice were orally dosed

with vehicle or 0.3 mg/kg FTY720 and simulta-

neously given an i.p. injection of 2 mg EdU. After

4 h, the blood, spleen, and bone marrow were

isolated. Single-cell suspensions were made and

analyzed by flow cytometry. (A) Monocyte gating

is shown for each tissue. (B) Total cell counts are

shown for total monocytes, Ly6Chigh monocytes,

and Ly6Clow monocytes in the blood, spleen, and

bone marrow. (C) The sum of the cell counts is

shown for Ly6Chigh and Ly6Clow monocytes. (D)

Gating of bone marrow EdU+ monocytes and total

cell counts are shown. n = 8–10 mice per group.

*p , 0.05, **p , 0.01, ***p , 0.001 versus

vehicle.

3536 FTY720 REDUCES MONOCYTE NUMBERS IN THE CIRCULATION

by guest on April 13, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

of lymphocytes, monocytes, and neutrophils. When treated pro-phylactically, FTY720 suppressed clinical disease in mice at 0.3mg/kg (Fig. 5A). Peripheral blood was analyzed at day 14 post-immunization, and the levels of lymphocytes, monocytes, andneutrophils were significantly reduced in mice at both testeddoses, as follows: 0.03 mg/kg (reductions of 56, 61, and 56%,respectively) and 0.3 mg/kg (reductions of 86, 82, 74, respec-tively; Fig. 5A).We also treated EAE mice with a therapeutic regimen of FTY720

(i.e., daily dosing began after mice developed disease; Fig. 5B).Although disease was not completely abolished at 0.3 mg/kg, theclinical score was significantly decreased, which correlated withdecreased levels of circulating monocytes and lymphocytes. Theseresults indicate that FTY720 also restricts circulating levels ofmonocytes under sustained inflammatory conditions.In rats, prophylactic treatment with 0.03 and 0.3 mg/kg FTY720

significantly reduced clinical disease. Peripheral blood was ana-lyzed at the end of the study, and lymphocytes, monocytes, andneutrophils were also significantly reduced at doses of 0.03 mg/kg

(reductions of 89, 66, and 47%, respectively) and 0.3 mg/kg (88, 70,and 64%, respectively; Fig. 5C).

Monocyte trafficking is not dependent on S1P3

S1P3 has been implicated in controlling monocyte trafficking (10);therefore, we wanted to assess whether decreased circulatingmonocytes could be achieved with a S1P receptor modulator thatdoes not agonize S1P3. We synthesized BAF312, a S1P1,4,5 agonist,confirmed its potency (EC50) in vitro with GTPgS assays for S1Preceptors, and demonstrated potencies of 0.2 nM for S1P1,.10 mMfor S1P3, and 0.5 nM for S1P5. FTY720 was assayed in paralleland demonstrated potencies of 2 nM for S1P1, 175 nM for S1P3,and 2.5 nM for S1P5 (L. Patnaude, S. Lukas, S. Haxhinasto, andL. Modis, manuscript in preparation). We tested BAF312 for po-tency in naive (0.03, 0.3, and 3 mg/kg) and EAE (0.03 and 0.3mg/kg) conditions. In naive mice, similar to FTY720, we observedsignificant decreases in lymphocytes and monocytes at 4 h thatcontinued through 24 h (reductions of 83 and 86%, respectively;Fig. 6A), and we did not observe any effects on neutrophils. In

FIGURE 3. FTY720 acts directly on monocytes

to alter cell surface expression and cytokine pro-

duction. (A) S1P receptor mRNA expression is

shown for the indicated cell types. Data were ob-

tained from publicly available microarray datasets

using NextBio, as described in Materials and

Methods. (B and C) Monocytes were isolated from

peripheral blood and cultured for 2 h in the presence

of 10 nM FTY720 and 10 ng/ml LPS. (B) CD40

levels measured by flow cytometry (n = 8). (C) TNF-

a cytokine levels measured by sandwich immuno-

assay (n = 14). *p , 0.05 versus vehicle and ###p ,0.001 versus LPS.

FIGURE 4. S1P receptor modulation al-

tered monocyte protein expression and re-

duced serum cytokine levels after a LPS

challenge. (A) C57BL/6 mice were dosed

with 0.3 mg/kg FTY720 or vehicle, and,

after 24 h, peripheral blood was isolated.

Monocyte subsets (Ly6Chigh and Ly6Clow)

were assessed by flow cytometry for cell

surface expression of several proteins. Mean

fluorescence intensity data are shown (n = 5–

6 mice per group). *p , 0.05, **p , 0.1

versus vehicle. (B) C57BL/6 mice were

dosed with 0.3 mg/ml FTY720, BAF312, or

vehicle. After 22 h, mice received an i.p.

injection of 200 ng LPS or vehicle. Two

hours later, whole blood was collected, and

IL-6, TNF-a, IL-10, and CCL2 were as-

sessed by a sandwich immunoassay. *p ,0.05, **p , 0.01, ***p , 0.001.

The Journal of Immunology 3537

by guest on April 13, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

EAE, we demonstrated that BAF312 significantly reduced clinicalscores when dosed prophylactically or therapeutically in mice at0.3 mg/kg (Fig. 6B, 6C). As expected, lymphocyte levels weresignificantly reduced when assessed near the peak of disease(i.e., days 14 and 16 postimmunization for the prophylactic [73%reduction] and therapeutic [37% reduction] regimens, respec-tively). We also observed significant decreases in monocyte lev-els, up to a 61% reduction with the prophylactic regimen, and upto a 57% reduction with the therapeutic regimen. BAF312 alsoinhibited clinical disease in rats in a dose-dependent manner,which correlated with reduced levels of circulating lymphocytesand monocytes (92 and 66% reductions, respectively; Fig. 6D).These results indicate that direct S1P3 agonism is not necessaryfor an impact on monocyte numbers in circulation.

DiscussionIn this study, we demonstrate in mice and rats using two differentS1P receptor modulators (FTY720 and BAF312) that S1P receptorsare involved in maintaining monocyte numbers in the circulationunder normal and inflammatory conditions.We further demonstratethat FTY720 causes monocytes to accumulate in the spleen andbone marrow. These observed effects on monocytes are similar tothe effects observed on lymphocytes, that S1P receptor modulatorsrestrict egress from hematopoietic organs.We also demonstrate thatFTY720 acts directly upon isolated monocytes to alter their ac-tivation, as demonstrated by reduced surface protein expression andcytokine production. Similar results were also observed in vivo.Thus, our findings reveal that S1P receptors play a significant rolein monocyte trafficking and may also alter monocyte function.A reduction in the number of macrophages has been observed in

several tissues of FTY720-treated mice, including the following:

atherosclerotic plaques in ApoE-deficient mice (18), peritonealcavity with thioglycolate-induced peritonitis (10), peripheralnerves in experimental autoimmune neuritis (15), CNS in EAE(16), renal glomeruli in experimental mesangioproliferative glo-merulonephritis (17), and heart allografts (19). However, nomechanism has been demonstrated. Given that tissue macrophagestypically derive from circulating monocytes, and under conditionsof tissue injury there is increased monocyte trafficking into dis-eased tissues (26, 27), our results demonstrating decreased cir-culating monocyte levels upon FTY720 administration provide anexplanation for these findings.Our observation that FTY720 altered several cell surface

markers on monocytes suggests that FTY720 may alter monocytefunction. In both the Ly6Chigh and Ly6Clow monocyte subset, weobserved significant decreases in CD69 expression. CD69 hasbeen demonstrated to interact with S1P1 in T cells and inhibitT cell responsiveness to S1P (28). Because FTY720 causes in-ternalization and degradation of S1P1, CD69 may be internalizedalso due to their interaction. Additionally, ligation of CD69 onmonocytes triggers monocyte activation, which may be reducedby FTY720 (29). Similarly, CD40 was also reduced on bothmonocyte subsets, further suggesting that FTY720 may decreasemonocyte activation. These data are also substantiated by ourobservation that FTY720 reduced monocyte cytokine productionand serum cytokine levels after an in vivo challenge with LPS. Wehypothesize that this reduced serum response can be mediated byboth reduced monocyte numbers and activation state.Pathogenesis in EAE models can be blocked by either reducing

T cells (30) or monocytes, as illustrated by treating mice and ratswith clodronate liposomes (31). Similarly, GM-CSF2/2 mice,which have a monocytic defect, are protected from EAE (30–32).

FIGURE 5. FTY720 suppresses clinical signs of disease in mouse and rat EAE while decreasing lymphocytes, monocytes, and neutrophils. EAE was

induced in both C57BL/6 mice and Lewis rats, as described in Materials and Methods. Animals with EAE were either untreated, or treated daily with

a vehicle, 0.03 mg/kg, or 0.3 mg/kg FTY720. (A) Prophylactic dosing of mice began 1 d before immunization, and clinical scores were evaluated daily

beginning on day 7 postimmunization. Whole blood was assessed for circulating lymphocytes, monocytes, and neutrophils at day 14 postimmunization. For

clinical score data, n = 9–10 mice per group. For cell counts, n = 4–5 mice per group. (B) Therapeutic dosing of mice began once mice had a clinical score

.1 or a score of 1 for at least 2 d. Whole-blood assessment of circulating lymphocytes, monocytes, and neutrophils was performed at day 16 post-

immunization. For clinical score data, n = 16–21 mice per group. For cell counts, n = 4–5 mice per group. (C) Prophylactic dosing of rats began 1 d before

immunization, and clinical scores were evaluated daily beginning on day 7 postimmunization. Whole-blood assessment of circulating lymphocytes,

monocytes, and neutrophils was performed at day 20 postimmunization. For clinical score data, n = 10 rats per group. For cell counts, n = 5 rats per group.

Mean 6 SEM is shown. Significant differences in the clinical score data were assessed using the AUC data from individual mice. #p , 0.05 for 0.03 mg/kg

versus vehicle, **p , 0.01, and ***p , 0.001 for 0.3 mg/kg versus vehicle.

3538 FTY720 REDUCES MONOCYTE NUMBERS IN THE CIRCULATION

by guest on April 13, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

It has also been shown that inhibition of the M-CSF receptor(CD115) reduces clinical disease during EAE (33). Additionally,monocytes from CCR22/2 mice are reduced in number and un-able to traffic into the CNS (34); consequently, these mice are alsoprotected from EAE (35). Our data are consistent with theseobservations, as we see reductions in the proinflammatory Ly6Chigh

monocyte subset, which would be expected to directly affect thelevels of macrophages at sites of inflammation. Furthermore, thereduction in monocyte numbers parallels that of lymphocytes, bothin timing and with respect to the PK/PD effect (both are maximallyreduced at 0.3 mg/kg in healthy mice). Therefore, the reduction inthe number of circulating monocytes may be partially responsiblefor the observed efficacy of S1P receptor modulators in EAE.We demonstrated that BAF312, which does not modulate S1P3,

reduced circulating monocyte levels in a dose-dependent mannersimilar to FTY720, suggesting that monocytes traffic indepen-dently of S1P3. This is in contrast to a report that monocyte

trafficking is dependent upon S1P3 (10). However, when the authorscompared wild-type mice containing S1P3

2/2 bone marrow withS1P3

2/2 mice containing wild-type bone marrow, no difference wasobserved in macrophage infiltration into the peritoneum. This ob-servation, along with our data utilizing BAF312, suggests thatmonocyte expression of S1P3 is not solely responsible for monocytetrafficking.Although other groups have mentioned that they did not observe

reductions in circulating monocytes with FTY720 treatment (15,18, 20, 36, 37), monocytes were not the focus of these studies,monocyte levels were assessed in a disease model in whichmonocytes are already affected (18), or surface markers were usedthat are not specific for monocytes (36). Our studies, however,provide an in-depth analysis of the effects of FTY720 and BAF312on monocytes at several doses under multiple conditions andwere consistent in both mice and rats. To confirm that the role ofFTY720 on monocytes trafficking is direct, a future step using

FIGURE 6. BAF312 restricts lymphocyte and monocyte circulation and suppresses clinical signs of disease in mouse and rat EAE. (A) C57BL/6 naive

mice were dosed with vehicle, 0.03 mg/kg, 0.3 mg/kg, or 3 mg/kg BAF312. Whole blood was taken at 0, 4, and 24 h after the administration of the dose.

Lymphocyte, neutrophil, and monocyte cell counts were assessed using the Hemavet (n = 7 mice per group). *p , 0.05, **p , 0.01, ***p , 0.001 versus

vehicle. (B–D) EAE was induced in both C57BL/6 mice and Lewis rats, as described in Materials and Methods. Animals with EAE were either untreated,

or treated daily with a vehicle, 0.03 mg/kg, or 0.3 mg/kg BAF312. (B) Prophylactic dosing of mice began 1 d before immunization, and clinical scores were

evaluated daily beginning on day 7 postimmunization. Whole-blood assessment of circulating lymphocytes, monocytes, and neutrophils was performed at

day 14 postimmunization. For clinical score data, n = 9–10 mice per group. For cell counts, n = 4–5 mice per group. (C) Therapeutic dosing of mice began

once mice had a clinical score .1 or a score of 1 for at least 2 d. Whole-blood assessment of circulating lymphocytes, monocytes, and neutrophils was

performed at day 16 postimmunization. For clinical score data, n = 16–21 mice per group. For cell counts, n = 4–5 mice per group. (D) Prophylactic dosing

of rats began 1 d before immunization, and clinical scores were evaluated daily beginning on day 7 postimmunization. Whole-blood assessment of cir-

culating lymphocytes, monocytes, and neutrophils was performed at day 20 postimmunization. For clinical score data, n = 10 rats per group. For cell counts,

n = 5 rats per group. The untreated and vehicle-treated groups shown in (A–C) are the same data as shown in Figs. 1A, 5A, and 5B because the compounds

FTY720 and BAF312 were given during the same experiment. Mean6 SEM is shown. Significant differences in the clinical score data were assessed using

the AUC data from individual mice. *p , 0.05, **p , 0.01, ***p , 0.001 for 0.3 mg/kg versus vehicle.

The Journal of Immunology 3539

by guest on April 13, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from

mice in which S1P receptors are specifically deleted from myeloidcells in adult mice in an inducible manner is necessary.In summary, our data indicate that S1P receptor modulators

lead to a significant reduction in circulating monocyte levels inmice and rats. Although an impact of FTY720 on monocytes inhumans at therapeutic doses (0.5 mg in multiple sclerosis and upto 5 mg in trials) has not been reported, cynomologous monkeystreated with FTY720 at 0.5 mg/kg (∼2 mg per animal) showed –a reduction in circulating monocyte levels (38). Given the expres-sion of S1P receptors on human monocytes and macrophages (11,39), additional studies on monocyte levels and functional markers inhumans may be warranted. In conclusion, we have performed asystematic, carefully controlled analysis and uncovered novelroles of S1P receptor modulation on monocytes in rodents.

DisclosuresThe authors are employed by Boehringer Ingelheim Pharmaceuticals.

References1. Cyster, J. G., and S. R. Schwab. 2012. Sphingosine-1-phosphate and lymphocyte

egress from lymphoid organs. Annu. Rev. Immunol. 30: 69–94.2. Rivera, J., R. L. Proia, and A. Olivera. 2008. The alliance of sphingosine-1-

phosphate and its receptors in immunity. Nat. Rev. Immunol. 8: 753–763.3. Brinkmann, V., A. Billich, T. Baumruker, P. Heining, R. Schmouder, G. Francis,

S. Aradhye, and P. Burtin. 2010. Fingolimod (FTY720): discovery and devel-opment of an oral drug to treat multiple sclerosis. Nat. Rev. Drug Discov. 9: 883–897.

4. Cyster, J. G. 2005. Chemokines, sphingosine-1-phosphate, and cell migration insecondary lymphoid organs. Annu. Rev. Immunol. 23: 127–159.

5. Kappos, L., J. Antel, G. Comi, X. Montalban, P. O’Connor, C. H. Polman,T. Haas, A. A. Korn, G. Karlsson, and E. W. Radue; FTY720 D2201 StudyGroup. 2006. Oral fingolimod (FTY720) for relapsing multiple sclerosis.N. Engl. J. Med. 355: 1124–1140.

6. Jolly, P. S., M. Bektas, A. Olivera, C. Gonzalez-Espinosa, R. L. Proia, J. Rivera,S. Milstien, and S. Spiegel. 2004. Transactivation of sphingosine-1-phosphatereceptors by FcepsilonRI triggering is required for normal mast cell degranu-lation and chemotaxis. J. Exp. Med. 199: 959–970.

7. Maeda, Y., H. Matsuyuki, K. Shimano, H. Kataoka, K. Sugahara, and K. Chiba.2007. Migration of CD4 T cells and dendritic cells toward sphingosine 1-phosphate (S1P) is mediated by different receptor subtypes: S1P regulates thefunctions of murine mature dendritic cells via S1P receptor type 3. J. Immunol.178: 3437–3446.

8. Roviezzo, F., F. Del Galdo, G. Abbate, M. Bucci, B. D’Agostino, E. Antunes,G. De Dominicis, L. Parente, F. Rossi, G. Cirino, and R. De Palma. 2004. Humaneosinophil chemotaxis and selective in vivo recruitment by sphingosine 1-phosphate. Proc. Natl. Acad. Sci. USA 101: 11170–11175.

9. Kveberg, L., Y. Bryceson, M. Inngjerdingen, B. Rolstad, and A. A. Maghazachi.2002. Sphingosine 1 phosphate induces the chemotaxis of human natural killercells: role for heterotrimeric G proteins and phosphoinositide 3 kinases. Eur. J.Immunol. 32: 1856–1864.

10. Keul, P., S. Lucke, K. von Wnuck Lipinski, C. Bode, M. Graler, G. Heusch, andB. Levkau. 2011. Sphingosine-1-phosphate receptor 3 promotes recruitment ofmonocyte/macrophages in inflammation and atherosclerosis. Circ. Res. 108:314–323.

11. Ulrych, T., A. Bohm, A. Polzin, G. Daum, R. M. Nusing, G. Geisslinger,T. Hohlfeld, K. Schror, and B. H. Rauch. 2011. Release of sphingosine-1-phosphate from human platelets is dependent on thromboxane formation. J.Thromb. Haemost. 9: 790–798.

12. Walzer, T., L. Chiossone, J. Chaix, A. Calver, C. Carozzo, L. Garrigue-Antar,Y. Jacques, M. Baratin, E. Tomasello, and E. Vivier. 2007. Natural killer celltrafficking in vivo requires a dedicated sphingosine 1-phosphate receptor. Nat.Immunol. 8: 1337–1344.

13. Jenne, C. N., A. Enders, R. Rivera, S. R. Watson, A. J. Bankovich, J. P. Pereira,Y. Xu, C. M. Roots, J. N. Beilke, A. Banerjee, et al. 2009. T-bet-dependent S1P5expression in NK cells promotes egress from lymph nodes and bone marrow.J. Exp. Med. 206: 2469–2481.

14. Rathinasamy, A., N. Czeloth, O. Pabst, R. Forster, and G. Bernhardt. 2010. Theorigin and maturity of dendritic cells determine the pattern of sphingosine 1-phosphate receptors expressed and required for efficient migration. J. Immunol.185: 4072–4081.

15. Zhang, Z., Z.-Y. Zhang, U. Fauser, and H. J. Schluesener. 2008. FTY720 ame-liorates experimental autoimmune neuritis by inhibition of lymphocyte andmonocyte infiltration into peripheral nerves. Exp. Neurol. 210: 681–690.

16. Rausch, M., P. Hiestand, C. A. Foster, D. R. Baumann, C. Cannet, and M. Rudin.2004. Predictability of FTY720 efficacy in experimental autoimmune encepha-lomyelitis by in vivo macrophage tracking: clinical implications for ultrasmallsuperparamagnetic iron oxide-enhanced magnetic resonance imaging. J. Magn.Reson. Imaging 20: 16–24.

17. Martini, S., S. Kramer, T. Loof, Y. Wang-Rosenke, U. Daig, K. Budde,H.-H. Neumayer, and H. Peters. 2007. S1P modulator FTY720 limits matrixexpansion in acute anti-thy1 mesangioproliferative glomerulonephritis. Am. J.Physiol. Renal Physiol. 292: F1761–F1770.

18. Keul, P., M. Tolle, S. Lucke, K. von Wnuck Lipinski, G. Heusch, M. Schuchardt,M. van der Giet, and B. Levkau. 2007. The sphingosine-1-phosphate analogueFTY720 reduces atherosclerosis in apolipoprotein E-deficient mice. Arterioscler.Thromb. Vasc. Biol. 27: 607–613.

19. Ogawa, R., M. Takahashi, S. Hirose, H. Morimoto, H. Ise, T. Murakami,T. Yasue, K. Kuriyama, M. Hongo, E. Kobayashi, and U. Ikeda. 2007. A novelsphingosine-1-phosphate receptor agonist KRP-203 attenuates rat autoimmunemyocarditis. Biochem. Biophys. Res. Commun. 361: 621–628.

20. Singer, I. I., M. Tian, L. A. Wickham, J. Lin, S. S. Matheravidathu,M. J. Forrest, S. Mandala, and E. J. Quackenbush. 2005. Sphingosine-1-phosphate agonists increase macrophage homing, lymphocyte contacts, andendothelial junctional complex formation in murine lymph nodes. J. Immunol.175: 7151–7161.

21. Gergely, P., B. Nuesslein-Hildesheim, D. Guerini, V. Brinkmann, M. Traebert,C. Bruns, S. Pan, N. S. Gray, K. Hinterding, N. G. Cooke, et al. 2012. The se-lective sphingosine 1-phosphate receptor modulator BAF312 redirects lymphocytedistribution and has species-specific effects on heart rate. Br. J. Pharmacol. 167:1035–1047.

22. Pan, S., W. Gao, N. Gray, Y. Mi, and Y. Fan, and The Genomics Institute of theNovartis Research Foundation. 2004. Immunosuppressant compounds and com-positions. World Intellectual Property Organization, Geneva, Switzerland.

23. Liu, Y., D. Papoutsakis, and E. Roddy, and Novartis Pharma AG. 2010New polymorphic form of 1-(4-{ L-[ (E)-4-cyclohexyl–3-trifluoromethyl-ben-zyloxyimino]-ethyl)-2-ethyl-bezy L)-azetidine-3-carboxylic. World IntellectualProperty Organization, Geneva, Switzerland.

24. Kupershmidt, I., Q. J. Su, A. Grewal, S. Sundaresh, I. Halperin, J. Flynn,M. Shekar, H. Wang, J. Park, W. Cui, et al. 2010. Ontology-based meta-analysisof global collections of high-throughput public data. PLoS One 5: e13066.

25. Gautier, E. L., T. Shay, J. Miller, M. Greter, C. Jakubzick, S. Ivanov, J. Helft,A. Chow, K. G. Elpek, S. Gordonov, et al; Immunological Genome Consortium.2012. Gene-expression profiles and transcriptional regulatory pathways thatunderlie the identity and diversity of mouse tissue macrophages. Nat. Immunol.13: 1118–1128.

26. Iijima, N., L. M. Mattei, and A. Iwasaki. 2011. Recruited inflammatory mono-cytes stimulate antiviral Th1 immunity in infected tissue. Proc. Natl. Acad. Sci.USA 108: 284–289.

27. Shi, C., and E. G. Pamer. 2011. Monocyte recruitment during infection andinflammation. Nat. Rev. Immunol. 11: 762–774.

28. Shiow, L. R., D. B. Rosen, N. Brdickova, Y. Xu, J. An, L. L. Lanier, J. G. Cyster,and M. Matloubian. 2006. CD69 acts downstream of interferon-a/b to inhibitS1P1 and lymphocyte egress from lymphoid organs. Nature 440: 540–544.

29. De Maria, R., M. G. Cifone, R. Trotta, M. R. Rippo, C. Festuccia, A. Santoni,and R. Testi. 1994. Triggering of human monocyte activation through CD69,a member of the natural killer cell gene complex family of signal transducingreceptors. J. Exp. Med. 180: 1999–2004.

30. Codarri, L., G. Gyulveszi, V. Tosevski, L. Hesske, A. Fontana, L. Magnenat,T. Suter, and B. Becher. 2011. RORgt drives production of the cytokine GM-CSFin helper T cells, which is essential for the effector phase of autoimmune neu-roinflammation. Nat. Immunol. 12: 560–567.

31. King, I. L., T. L. Dickendesher, and B. M. Segal. 2009. Circulating Ly-6C+myeloid precursors migrate to the CNS and play a pathogenic role during au-toimmune demyelinating disease. Blood 113: 3190–3197.

32. El-Behi, M., B. Ciric, H. Dai, Y. Yan, M. Cullimore, F. Safavi, G.-X. Zhang,B. N. Dittel, and A. Rostami. 2011. The encephalitogenicity of T(H)17 cells isdependent on IL-1- and IL-23-induced production of the cytokine GM-CSF. Nat.Immunol. 12: 568–575.

33. Uemura, Y., H. Ohno, Y. Ohzeki, H. Takanashi, H. Murooka, K. Kubo, andI. Serizawa. 2008. The selective M-CSF receptor tyrosine kinase inhibitorKi20227 suppresses experimental autoimmune encephalomyelitis. J. Neuro-immunol. 195: 73–80.

34. Mildner, A., H. Schmidt, M. Nitsche, D. Merkler, U.-K. Hanisch, M. Mack,M. Heikenwalder, W. Bruck, J. Priller, and M. Prinz. 2007. Microglia in the adultbrain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions.Nat. Neurosci. 10: 1544–1553.

35. Fife, B. T., G. B. Huffnagle, W. A. Kuziel, and W. J. Karpus. 2000. CC che-mokine receptor 2 is critical for induction of experimental autoimmune en-cephalomyelitis. J. Exp. Med. 192: 899–905.

36. Ishii, M., J. G. Egen, F. Klauschen, M. Meier-Schellersheim, Y. Saeki, J. Vacher,R. L. Proia, and R. N. Germain. 2009. Sphingosine-1-phosphate mobilizes os-teoclast precursors and regulates bone homeostasis. Nature 458: 524–528.

37. Liesz, A., L. Sun, W. Zhou, S. Schwarting, E. Mracsko, M. Zorn, H. Bauer,C. Sommer, andR.Veltkamp. 2011. FTY720 reduces post-ischemic brain lymphocyteinflux but does not improve outcome in permanent murine cerebral ischemia. PLoSOne 6: e21312.

38. Food and Drug Administration Peripheral and Central Nervous SystemDrugs Advisory Committee. Fingolimod (NDA 22-527) Background Package.June 10, 2010. Available at: http://www.fda.gov/downloads/advisorycommittees/committeesmeetingmaterials/drudr/peripheralandcentralnervoussystemdrugsadvisorycommittee/ucm214670.pdf. Accessed: March 5, 2012.

39. Duong, C. Q., S. M. Bared, A. Abu-Khader, C. Buechler, A. Schmitz, andG. Schmitz. 2004. Expression of the lysophospholipid receptor family and in-vestigation of lysophospholipid-mediated responses in human macrophages.Biochim. Biophys. Acta 1682: 112–119.

3540 FTY720 REDUCES MONOCYTE NUMBERS IN THE CIRCULATION

by guest on April 13, 2017

http://ww

w.jim

munol.org/

Dow

nloaded from