Pharmacology & Pharmacy, 2013, 4, 638-646
Published Online November 2013 (http://www.scirp.org/journal/pp)
http://dx.doi.org/10.4236/pp.2013.48090
Open Access PP
Role of Sphingosine 1-Phosphate (S1P) Receptor 1 in
Experimental Autoimmune Encephalomyelitis
—II. S1P-S1P1 Axis Induces Pro-Inflammatory Cytokine Production in Astrocytes
Noriyasu Seki1, Hirotoshi Kataoka1, Kunio Sugahara1, Atsushi Fukunari2, Kenji Chiba1,2*
1Pharmacology Research Laboratories I, Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Japan; 2Advanced
Medical Research Laboratories, Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama, Japan.
Received October 11th, 2013; revised November 5th, 2013; accepted November 12th, 2013
Copyright © 2013 Noriyasu Seki et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Therapeutic administration of fingolimod hydrochloride (FTY720), the functional antagonist at sphingosine 1-phos-
phate (S1P) receptor 1 (S1P1) shows a marked improving effect on experimental autoimmune encephalomyelitis (EAE)
induced by myelin oligodendrocyte glycoprotein (MOG) in C57BL/6 mice. However, this treatment showed an only
partial inhibition of Th1/Th17 cell infiltration into the central nervous system (CNS), suggesting that down-regulation
of lymphocytic S1P1 is insufficient to explain the therapeutic effect of FTY720 on EAE. On the other hand, the thera-
peutic administration of FTY720 reduced the mRNA expressions of IL-6, CCL2, and glial fibrillary acidic protein, an
activation marker of astrocytes, in the CNS of EAE mice. In human astrocytic glyoma, U373MG cells, mRNA expres-
sion of S1P1 was higher as compared with those of the other S1P receptor subtypes and phosphorylation of Akt was
induced by S1P, FTY720-phosphate (FTY720-P), or an S1P1-selective agonist, SEW2871. FTY720-P appeared to in-
duce down-regulation of S1P1 in U373MG cells, implying a functional antagonism at S1P1 on astrocytes. S1P but not
FTY720-P induced production of IL-6, IL-8, and CCL2 significantly and treatment with FTY720-P or SEW2871 inhib-
ited production of these pro-inflammatory cytokines from U373MG cells stimulated with S1P. These results suggest
that S1P-S1P1 axis induces production of pro-inflammatory cytokines by astrocytes. Consequently, it is highly probable
that the therapeutic effects of FTY720 on EAE are caused by inhibiting not only egress of myelin-specific Th cells from
the draining lymph nodes but also activation of astrocytes in the CNS.
Keywords: Sphingosine 1-Phosphosphate Receptor 1; Fingolimod Hydrochloride (FTY720); Experimental
Autoimmune Encephalomyelitis; Astrocytes; Pro-Inflammatory Cytokines
1. Introduction
A phospholipid mediator, sphingosine 1-phospahte (S1P)
and its receptor 1 (S1P1) play an essential role in lym-
phocyte egress from the secondary lymphoid organs
[1-5]. Fingolimod hydrochloride (FTY720) is an orally
active S1P receptor modulator with a structure closely
related to sphingosine [6,7]. Phosphorylated FTY720
(FTY720-P, (S)-enantiomer) by sphingosine kinases shows
an agonistic activity at four types of S1P receptors (S1P1,
S1P3, S1P4, and S1P5) [8]. Notably, FTY720-P induces
down-regulation of S1P1 by internalization and subse-
quent degradation to reduce S1P responsiveness and acts
as a functional antagonist at S1P1 [9-13].
Experimental autoimmune encephalomyelitis (EAE) is
a CD4 T cell-dependent animal models for human multi-
ple sclerosis (MS) [14-17]. There are several reports that
FTY720 is highly effective in EAE in rats and mice
[18-23]. As reported previously, prophylactic adminis-
tration of FTY720 (0.3 mg/kg, orally) almost completely
inhibited development of EAE induced by immunization
with myelin proteolipid protein peptide in SJL/J mice
[20]. In addition, prophylactic FTY720 administration
markedly reduced the infiltration of Th1 and Th17 cells
into the central nervous system (CNS) of EAE mice
whereas this treatment increased the frequency of Th1
and Th17 cells in the inguinal lymph nodes, implying
sequestration of myelin antigen-specific Th1/Th17 cells
into draining lymph nodes [24]. From these results, it is
*Corresponding author.

Role of Sphingosine 1-Phosphate (S1P) Receptor 1 in Experimental Autoimmune Encephalomyelitis 639
suggested that prophylactic effects of FTY720 on EAE
are predominantly caused by inhibiting lymphocyte eg-
ress from draining lymph nodes.
On the other hand, therapeutic administration of
FTY720 after establishment of EAE showed significant
improvement of EAE symptoms on chronic-progressing
EAE induced by immunization with myelin oligodendro-
cyte glycoprotein (MOG) in C57BL/6 mice [20,23].
However, we confirmed that therapeutic treatment with
FTY720 only showed a partial inhibition of Th1/Th17
cell infiltration into the spinal cord of EAE mice,
suggesting involvement of different mechanisms not
mediated via lymphocytic S1P1. Recently, Choi et al.
have reported that FTY720 efficacy was lost in CNS
mutants lacking S1P1 on glial fibrillaly acidic protein
(GFAP)-expressing astrocytes but not on neurons [25].
Their studies on S1P1 receptor rescue and pharmacolo-
gical experiments revealed the loss of S1P1 on astrocytes
through functional antagonism by FTY720-P.
In the present study, we found that S1P can induce
production of interleukin (IL)-6, IL-8 and CC-chemokine
ligand 2 (CCL2) in human astrocytic glyoma, U373MG
cells via S1P1. Furthermore, we demonstrated that
FTY720-P inhibits S1P-induced pro-inflammatory cyto-
kine production in U373MG cells, suggesting a func-
tional antagonism at S1P1 on astrocytes.
2. Materials and Methods
2.1. Mice
Inbred strains of female C57BL/6 mice were purchased
from Charles River Japan and were used at 7 to 12 weeks
of age. All animal experiments were performed under an
experimental protocol approved the ethics review com-
mittee for animal experimentation of Research Division,
Mitsubishi Tanabe Pharma Corporation.
2.2. Agents, Cell Lines, and Antibodies
FTY720 was synthesized according to the method
described previously [6] and was dissolved in distilled
water for oral administration. The (S)-enantiomer of
FTY720-P (>99.5% enantio excess) synthesized was
dissolved in ethanol for in vitro experiments [7].
SEW2871, an S1P1-selective agonist was synthesized
according to the method reported previously [26]. S1P
was purchased from Sigma-Aldrich. IL-1β and tumor
necrosis factor (TNF)-α were purchased from Peprotech.
MOG35-55 (MEVGWYRSPFSRVVHLYRNGK) peptide
was obtained from Peptide Institute. RPMI 1640 medium
was supplemented with 10% fetal calf serum (FCS) which
was pretreated with charcoal, 10 mM HEPES, 50 U/ml
penicillin, 50 μg/ml streptomycin, 0.1 mM non-essential
amino acids, 1 mM sodium pyruvate, and 50 μM 2-mer-
captoethanol. Human astrocytic glioma, U373MG cells
obtained from American Type Culture Collection were
maintained by culturing at 37˚C in 5% CO2 in Dul-
becco’s modified eagle medium (DMEM, low glucose)
supplemented with 10% FCS pretreated with charcoal,
50 U/ml penicillin, and 50 μg/ml streptomycin.
Cy-Chrome-conjugated rat anti-mouse CD4 mono-
clonal antibody (mAb) (GK1.5), fluorescein isothocya-
nate (FITC)-conjugated rat anti-mouse interferon (IFN)-γ
mAb (XMG1.2), phycoerythrin (PE)-conjugated rat anti-
mouse IL-17 mAb (TC11-18H10.1), and polyclonal anti-
bodies (pAbs) against ionized calcium binding adaptor
molecule 1 (Iba1) and GFAP were purchased from BD
Bioscience. Mouse anti-human S1P1 pAb (H-60) and
rabbit anti-phosphorylated Akt (Thr308) pAb were ob-
tained from Santa Cruz and Cell Signaling Technology,
respectively. Alexa 594-conjugated anti-rabbit immuno-
globulin (Ig) G (Molecular Probes) and horse radish per-
oxidase (HRP)-conjugated anti-rabbit IgG (GE Health-
care Bioscience) were used as secondary antibodies.
2.3. EAE Induction
For induction of EAE, C57BL/6 mice received immuni-
zation of MOG35-55 in Freund’s complete adjuvant sub-
cutaneously on day 0, followed by intravenous injection
with 200 ng of pertussis toxin (List Biological Labor-
atories) on day 0 and 2 [20]. Individual mice were scored
for clinical signs of EAE using the following criteria: 0,
no paralysis; 0.5, stiff tail; 1, limp tail; 1.5, limp tail with
inability to right; 2, paralysis of one limb; 2.5, paralysis
of one limb and weakness of one other limb; 3, complete
paralysis of both hind limbs; 4, moribund state; 5, death.
2.4. Intracellular Cytokines Staining and Flow
Cytometry Analysis
Lymphocytes prepared from the inguinal lymph nodes or
spinal cords were stimulated with 50 ng/ml phorbol-12-
myristate-13-acetate and 500 nM ionomycin in the
presence of 2 μM monensin for 5 h in RPMI 1640
medium containing 10% FCS at 37˚C in 5% CO2. After
blocking with anti-mouse CD16/32 mAb, the cells were
stained with Cy-Chrome-conjugated anti-mouse CD4
mAb and permeabilized with 0.5% Triton X-100. The
intracellular cytokine staining was performed by using
FITC-conjugated anti-mouse IFN-γ mAb and PE-con-
jugated anti-mouse IL-17 mAb and flow cytometry
analysis was conducted using FACScan (BD Bio-
sciences).
2.5. Real Time Polymerase Chain Reaction
Total RNA was extracted using TRIZOL (Invitrogen)
and a tow-step quantitative revers transcriptation-poly-
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Role of Sphingosine 1-Phosphate (S1P) Receptor 1 in Experimental Autoimmune Encephalomyelitis
640
merase chain reaction (RT-PCR) was performed to deter-
mine various molecules mRNA expression using a re-
lative standard curve method with cellular housekeeping
enzyme (glyceraldehyde 3-phosphate dehydrogenase:
GAPDH or β-actin) as normalization control. Comple-
mentary DNA was synthesized with TaqMan Reverse
Transcription Reagents (Applied Biosystems) using ran-
dom hexamers and 0.5 μg of total RNA. Complementary
DNA was amplified with various molecules TaqMan
probe (6-carboxy-fluorecein label)/primer, cellular house-
keeping enzyme TaqMan probe (VIC label)/primer, and
TaqMan Universal PCR Master Mix in an ABI PRISM
7900 Sequence Detection System (Applied Biosystems).
For analyses of mRNA expressions in mouse tissues,
following TaqMan probes/primers were used: CD3
(Mm00599683_m1), IFN-γ (Mm00801778_m1), TNF-α
(Mm00443258_m1), T-bet (Mm00450960_m1), a IL-6
(Mm00446190_m1), IL-17 (Mm00439619_m1), CCL2
(Mm00441242_m1), CCL5 (Mm01302427_m1), C-X-C
chemokine ligand 10 (CXCL10) (Mm00445235_m1),
and GFAP (Mm01253033_m1). GAPDH TaqMan probe/
primer was used as normalization control. For quantifica-
tion, standard curves were generated for various molecu-
les using serially diluted cDNA samples from the spinal
cords in EAE mice. For every sample, a level of mRNA
normalized by calculating the ratio of each target mole-
cule/GAPDH level. To analyze mRNA expression of
retinoic acid receptor-related orphan receptor (ROR) γt in
mouse tissues, real time PCR was performed with an
ABI PRISM 7700 sequence detector using SYBR Green
PCR master mix (Applied Biosystems). The following
primer pairs were used (5’→3’): RORγt forward, CCG
CTG AGA GGG CTT CAC; RORγt reverse, TGC AGG
AGT AGG CCA CAT TAC A [27]. The reaction was
incubated for 2 min at 50˚C, denatured for 10 min at
95˚C and subjected to 40 step amplification cycles with
annealing/extension at 60˚C for 1 min followed by
denaturation at 95˚C for 15 s. Data were analyzed using
Sequence Detector software (Applied Biosystems). For
analyses of mRNA expressions of human S1P receptors,
following the TaqMan probes/ primers were used: S1P1
(Hs00173499_m1), S1P2 (Hs01003373_m1), S1P3
(Hs01019574_m1), S1P4 (Hs00269446_s1), S1P5
(Hs00258220_s1). β-actin TaqMan probe/primer was
used as normalization control. For every sample, a level
of mRNA normalized by calculating the ratio of each
target molecule/β-actin level.
2.6. Immunohistochemical Staining
The spinal cord samples were fixed in 10% formalin and
embedded in paraffin. Six μm-thickness sections of the
spinal cord were stained with hematoxylin and eosin
(H&E). For immunochemical staining, 6 μm-thickness
frozen sections of the spinal cord were immediately fixed
in cold-acetone and incubated with polyclonal antibody
against Iba1 or GFAP. The sections were then incubated
with secondary antibodies conjugated with amino acid
polymer and peroxidase (Histofine Simple Stain MAX-
PO kit; Nichirei), colorized with diaminobenzidine in the
presence of hydrogen peroxide, and counterstained with
hematoxylin.
2.7. Western Blotting Analysis for Akt
Phosphorylation
After serum starvation for 24 h, U373MG cells (106 cells)
were stimulated with S1P or FTY720-P for 10 min and
then the cells were lysed. Proteins in the cell lysate were
separated by SDS-PAGE and transferred to polyviny-
lidene difluoride membrane (Millipore). After blocking,
the membrane was incubated with anti-phosphorylated
Akt (Thr308) antibody and subsequently with HRP-
conjugated secondary antibody. Phosphorylated Akt was
visualized with ECL Plus Kit (GE Healthcare Bio-
science).
2.8. Immunofluorescence Microscopy
U373MG cells were stimulated with FTY720-P at 100
nM for 30 min, and then the cells were washed with
phosphate buffered saline (PBS) and fixed with 3.7%
formaldehyde in PBS at room temperature for 20 min.
Permeabilization was performed in PBS containing 0.5%
Triton X-100 and 1 mg/ml bovine serum albumin for 5
min. Immunofluorescence staining was performed by
incubation with anti-human S1P1 antibody for 1 h, fol-
lowed by detected with alexa-594-conjugated anti-rabbit
IgG at room temperature for 30 min. Immunofluo-
rescence was digitally captured under a confocal laser
microscope (Carl Zeiss).
2.9. Cytokine Production
U373MG cells (104 cells) were cultured in the presence
of S1P or FTY720-P in DMEM containing with 10%
FCS for 48 h at 37˚C in 5% CO2. In other experiments,
U373MG cells were pretreated with FTY720-P or
SEW2871 for 2 h under serum-free condition, and then
the cells were cultured in the presence of 100 nM S1P for
48 h. Concentrations of IL-6, IL-8, and CCL2 in the cul-
ture supernatants were determined by BDTM Cytometric
Beads Array (BD Biosciences).
2.10. Statistical Analyses
Results were express as the mean ± standard error mean
(SEM). Statistical differences of EAE clinical scores
were analyzed by Mann-Whitney U test. In other experi-
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Role of Sphingosine 1-Phosphate (S1P) Receptor 1 in Experimental Autoimmune Encephalomyelitis 641
ments, statistical differences were calculated by Stu-
dent’s t-test or Dunnett’s multi-comparison test. Dif-
ferences between groups were considered significant
when p < 0.05 (*, #: p < 0.05, **, ##: p < 0.01).
3. Results
3.1. Therapeutic Administration of FTY720
Improves Progressing EAE Symptoms
When C57BL/6 mice were immunized with MOG35-55
peptide, chronic-progressing EAE was developed at 10 to
11 days after immunization. Mean clinical scores of EAE
symptoms reached a maximal level (approximately score
3.0) on day 16 and were maintained around 2.5 to 3.5
during administration period (until 42 days after immuni-
zation) in vehicle-treated control mice. Therapeutic ad-
ministration of FTY720 (0.3 mg/kg, orally) significantly
improved progressing EAE symptoms (Figure 1(A)). On
(C) Spinal cordDLN
Control FTY720
Counts
CD4
IL-17
IFN - γ
Count s
IL-17
Control FTY720
CD4
IFN - γ
5.1 1.4
22.4
4.0 0.7
39.0
0.1
3.9
2.6 0.1
4.7
3.0
32.7 17.128.730.0
(A)
(B)
IL-17
*
RORγt
*
0
0.5
1.0
1.5
2.0
0
0.2
0.4
0.6 TNF-α
*
IFN - γ
0
0.1
0.2
0.3
0
0.02
0.04
0.06
0.08
T-bet
**
mRNA / GAPDH
In the spinal cord (n=3)
0
0.5
1.0
1.5
0
0.5
1.0
1.5 CD3
**
14 1618 2022 2426 2830 3234 3638 4042
1.0
1.5
2.0
2.5
3.0
3.5
Clinical score
Days after immunization
***
** **** ** ** ****
Control
FTY720 0.3 mg/kg p.o.
(n=12)
Figure 1. Therapeutic administration of FTY720 (0.3 mg/kg
p.o.) improves EAE but partially reduc es infiltration of Th1
and Th17 cells into the spinal cord of EAE mice.
day 28, although the therapeutic treatment with FTY720
significantly reduced mRNA expressions of IFN-γ, TNF-
α, T-bet, IL-17, and RORγt in the spinal cord (Figure
1(B)), this treatment induced only partial reduction in the
numbers of infiltrated CD4 T cells (48% reduction), Th1
cells (9% reduction), and Th17 cells (59% of reduction)
into the spinal cord of EAE mice (Figure 1(C)).
3.2. Therapeutic Administration of FTY720
Down-Modulates Astrocyte Activation in
EAE
Histological examinations (H&E staining) revealed infil-
tration of inflammatory cells, demyelination, and atrophy
in the spinal cord of EAE mice on day 42 after immuni-
zation (Figure 2(A)). Furthermore, we observed accumu-
lation of Iba1-expressing microglial cells and GFAP-ex-
pressing activated astrocytes into the area of tissue dam-
ages in the spinal cord of EAE mice, indicating astrocytic
gliosis. No atrophy or no astrocytic gliosis was found in
the spinal cord by therapeutic treatment with FTY720 at
0.3 mg/kg (Figure 2(B)).
The mRNA expressions of IL-6 and CCL2 were
significantly increased in the spinal cord of EAE mice (3
to 14 days after vehicle administration) as compared with
normal mice (Figure 3(A)). Furthermore, mRNA expres-
sions of GFAP, CXCL10, and CCL5 were markedly in-
creased in the spinal cord of EAE mice, suggesting astro-
Iba1 GFAPH&E
EAE controlFTY720 0.3 mg/kg p.o.
200 μm
50 μm
50 μm
200 μm
(
A)
(
B) Iba1 GFAPH&E
Figure 2. Therapeutic administration of FTY720 (0.3 mg/kg
p.o.) improves astrocytic gliosis in the spinal cord of EAE
mice (Iba1, microglia; GFAP, astrocytes).
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Role of Sphingosine 1-Phosphate (S1P) Receptor 1 in Experimental Autoimmune Encephalomyelitis
642
(B)
Day 3
CCL2
mRNA / GAPDH
in the spinal cord (n=3)
IL-6
CCL5CXCL10GFAP
*
p=0.07
0
0.05
0.10
0.15
0.20
0
0.5
1.0
1.5
0
0.05
0.10
0.15
0
0.1
0.2
0.3
0.4
0
0.1
0.2
0.3
0.4
0
0.05
0.10
0.15
0.20
0.2
0.4
0.6
0
0.2
0.4
0.6
0.8
0
0.5
1.0
1.5
0
*
(A)
Day 7Day 14
Day 14Day 14Day 14
mRNA / GAPDH
in the spinal cord (n=3)
Day 3Day 7Day 14
Figure 3. Therapeutic administration of FTY720 (0.3 mg/kg
p.o.) decreases mRNA expressions of pro-inflammatory
cytokines and GFAP in the spinal cord of EAE mice.
cyte activation (Figure 3(B)). Therapeutic administration
of FTY720 at 0.3 mg/kg reduced mRNA expressions of
IL-6 (40% to 58% reductions) and CCL2 (37% and 48%
reduction on day 3 and day 7, respectively) (Figure 3(A)).
Similarly, mRNA expressions of GFAP, CXCL10, and
CCL5 were lower in the spinal cord of EAE mice after
FTY720 administration, suggesting down-modulation of
astrocytes activation (Figure 3(B)).
3.3. FTY720-P Induces Down-Regulation of
S1P1 on Human Astrocytic Glioma Cells
Because our results of mouse EAE revealed that FTY720
may down-modulate astrocyte activation, we focused to
analyze astrocyte function using human astrocytic glioma,
U373MG cells and found that the expression level of
S1P1 mRNA was higher as compared with those of the
other S1P receptor subtypes in U373MG cells (Figure
4(A)). Furthermore, we demonstrated that phosphoryla-
tion of Akt was induced by treatment with S1P (0.1 nM
or higher), FTY720-P (0.1 nM or higher), or S1P1-selec-
tive agonist, SEW2871 (100 nM or higher) in U373MG
cells, suggesting that S1P and FTY720-P induce S1P1-
dependent Akt phosphorylation (Figure 4(B)). There was
no change in total Akt protein contents in U373MG cells
in the presence or absence of these compounds (data not
shown). By immunofluorescence using anti-human S1P1
pAb under con-focal microscopy, we confirmed that
S1P1 is abundant in cell surface and cytoplasm of
S1PR mRNA / β-actin
(n=3)
S1P1 S1P2 S1P3 S1P4 S1P5
0.008
0.010
0
0.002
0.004
0.006
S1P
FTY720-P
SEW2871
(A) mRNA expression of S1P receptors
0nM 101001000 10000
00.1110100 nM
(B) Akt phosphorylation in U373MG cells
(C) S1P1 expression in U373MG cells
Control FTY720-P
U373MG cells
Figure 4. The expression level of S1P1 mRNA is higher in
U373MG cells and FTY720-P induces Akt phosphorylation
and down-regulation of S1P1.
U373MG cells (Figure 4(C), left). By contrast, S1P1
expression was markedly reduced by treatment with 100
nM FTY720-P, indicating internalization (down-regula-
tion) of S1P1 in U373MG cells (Figure 4(C), right).
3.4. FTY720-P Inhibits Pro-Inflammatory
Cytokine Production Induced by S1P in
Astrocytic Glyoma, U373MG Cells
Because U373MG cells are reported to produce high
level of IL-6 by stimulation with IL-1β [28], we
examined the ability of S1P to induce production of pro-
inflammatory cytokines including IL-6 in this cell line.
We found that S1P at 100 nM or higher markedly induce
production of IL-6, IL-8, and CCL2 cells whereas
FTY720-P up to 1000 nM showed no clear effect in
U373MG cells (Figure 5(A)). Treatment with FTY720-P
(100 nM or higher) significantly inhibited S1P-induced
production of IL-6, IL-8, and CCL2 in a dose-dependent
manner (Figure 5(B)). Similar results were obtained
when SEW2781 at 10000 nM was treated, suggesting
that S1P induces production of IL-6, IL-8 and CCL2 via
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Role of Sphingosine 1-Phosphate (S1P) Receptor 1 in Experimental Autoimmune Encephalomyelitis 643
Pro-inflammatory cytokine (ng/mL)
0
1
2
3
4
5
6
CCL2
10 10
3
FTY720-P
10
2
S1P
- 10 10
3
10
2
(nM)
0
5
10
15
20
IL-8
0
0.5
1.0
1.5
2.0
2.5
(A)
IL-6
0
5
10
15
20
0
0.2
0.4
0.6
0.8
1.0
1.2
0
1
2
3
4
5
-
010 10
3
10
4
FTY720-P SEW
10
4
10
2
S1P (100 nM)
CCL2
*** ** ##
IL-6
**
**
**
##
IL- 8
**
**
**
#
(nM)
Pro-inflammatory cytokine (ng/mL)
(B)
Figure 5. S1P induces IL-6, IL-8 and CCL2 produc tion and
FTY720-P inhibits production of these pro-inflammatory
cytokines induced by S1P in U373MG cells.
S1P1 (Figure 5(B)). On the other hand, FTY720-P did
not affect IL-1β- or TNF-α-induced IL-6 production in
U373 MG cells (data not shown).
4. Discussion
In this study, we demonstrated that therapeutic adminis-
tration of FTY720 showed a significant improvement of
chronic EAE induced by MOG in C57BL/6 mice;
however this treatment partially reduced infiltration of
Th1 and Th17 cells into the CNS. On the other hand, the
therapeutic treatment with FTY720 resulted in no atro-
phy of the spinal cord or no astrocytic gliosis, and re-
duced mRNA expressions of GFAP and several pro-
inflammatory cytokines such as IL-6 and CCL2 in the
CNS of EAE mice. Based on these results, it is thought
that down-regulation of lymphocytic S1P1 is insufficient
to explain the therapeutic effect of FTY720 on EAE.
Namely, this means that the improvement of chronic
EAE by FTY720 is likely due to not only a functional
antagonism at lymphocytic S1P1 but also other me-
chanisms.
Foster et al. reported that in chronic EAE induced by
MOG in DA rats, therapeutic treatment with FTY720
after establishment of the diseases markedly improves
EAE symptoms [21]. They proposed the possibility that
FTY720-P directly acts on neural cells in the CNS as
well as lymphocytes because the concentrations of
FTY720 and FTY720-P in the brain was higher 10 times
than in peripheral blood [21]. Recently, Choi et al.
demonstrated that FTY720 efficacy was lost in CNS
mutants lacking S1P1 on neural cells, particularly GFAP-
expressing astrocytes but not on neurons [25]. Their studies
on S1P1 receptor rescue and pharmacological experi-
ments revealed the loss of S1P1 on astrocytes through
functional antagonism by FTY720-P as one of primary
mechanism of FTY720. From these findings, it is highly
probable that the functional antagonism at S1P1 on
astrocytes by FTY720-P partly attribute the improvement
of EAE symptoms by therapeutic treatment with FTY720
after disease establishment.
To examine the direct effect of FTY720-P on astrocyte
functions via S1P1, we used human astrocytic glioma,
U373MG cells because this cell line expressed higher
mRNA level of S1P1 rather than the other S1P receptors.
Therefore, U373MG cells are thought to be useful for
analysis of astrocyte functions mediated by S1P-S1P1
axis. In this study, we confirmed that phosphorylation of
Akt was induced by S1P, FTY720-P, or SEW2871 in
U373MG cells, suggesting that S1P and FTY720-P act as
an agonist at S1P1 [26,29]. Consistent with the results
using human S1P1-stably expressing CHO cells [8,30] or
human astrocytes [25], our data clearly demonstrated that
FTY720-P induces and maintains down-regulation of
S1P1 in U373MG cells, suggesting a functional anta-
gonism at S1P1 on astrocytes. Similar results have been
reported that FTY720 desensitizes cell surface S1P1 on
astrocytes derived from human fetal CNS specimens for
more than 24 h [31].
Pro-inflammatory cytokines such as IL-6, CCL2, and
CXCL10 are considered to play an important role in
progression of EAE based on the studies using knockout
mice of these molecules [32-35]. Furthermore, it has
been shown that IL-6 derived from astrocytes can induce
EAE [36] and that CXCL10 mRNA is expressed mainly
in astrocytes in MOG-induced EAE mice [37]. Based on
these findings, it is considerably meaningful to elucidate
the role of S1P1-S1P axis in pro-inflammatory cytokine/
chemokine productions by astrocytes. Doorn et al. re-
ported that TNF-α can induce CCL2 production by
primary human astrocytes or U373MG cells [38]. How-
ever, there is no report revealing whether S1P at physiol-
ogical concentrations can induce production of pro-in-
flammatory cytokines by astrocytes.
In this study, we found that S1P 100 nM or higher can
induce production of IL-6, IL-8, and CCL2 significantly
in U373MG cells. Furthermore, pretreatment with
FTY720-P (100 nM or higher) or SEW2871 (10000 nM)
inhibited production of IL-6, IL-8, and CCL2 by S1P-
activated U373MG cells, implying the involvement of
S1P-S1P1 axis in astrocyte activation. Trough concen-
trations of FTY720-P in the brain reached to approxi-
mately 900 nM in rodents when FTY720 (0.3 mg/kg,
Open Access PP

Role of Sphingosine 1-Phosphate (S1P) Receptor 1 in Experimental Autoimmune Encephalomyelitis
644
orally) was administrated daily for 3 weeks [21]. There-
fore, it is highly possible that FTY720-P inhibits pro-
duction of IL-6, IL-8 and CCL2 by S1P-activated astro-
cytes in EAE mice if S1P concentrations are elevated to
more than 100 nM by neuroinflammation [39].
Although it was reported that FTY720 (10 to 100 μM)
partially reduced TNF-α-induced CCL2 production in
human astrocytes or U373 MG cells [38], this effect may
be unlikely because of its extremely high concentrations.
A mechanism underlying S1P-induced production of
IL-6, IL-8, and CCL2 in U373MG cells is probably
mediated through a signal transduction pathway via NF-
κB because NF-κB is shown to be activated by down-
stream signaling of S1P1 [40,41]. Unlike S1P, FTY720-P
did not induce IL-6, IL-8, or CCL2 by U373MG cells.
FTY720-P may not efficiently bring about production of
these cytokines because of down-regulation of cell sur-
face S1P1 expression [9-13].
In conclusion, our findings suggest that FTY720-P
down-modulates production of pro-inflammatory cyto-
kines by S1P-activated astrocytes via S1P1. Conse-
quently, it is highly probable that the therapeutic effects
of FTY720 on EAE are caused by inhibiting not only
egress of myelin-specific Th cells from the draining
lymph nodes but also activation of astrocytes in the CNS.
5. Acknowledgements
The authors are grateful thank for their excellent assis-
tance of Mamoru Koyama, Yasuko Ogawa, and Dr.
Hiroyuki Utsumi in Advanced Medical Research Labora-
tories, and Dr. Hirofumi Matsuyuki in Pharamacology
Research Laboratories. We also thank Dr. Kunitomo
Adachi at Medicinal Chemistry Laboratories I, Mitsu-
bishi Tanabe Pharma Corporation for synthesizing (S)-
enantiomer of FTY720-P and SEW2871.
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