Pharmacology & Pharmacy, 2013, 4, 628-637
Published Online November 2013 (http://www.scirp.org/journal/pp)
http://dx.doi.org/10.4236/pp.2013.48089
Open Access PP
Role of Sphingosine 1-Phosphate (S1P) Receptor 1 in
Experimental Autoimmune Encephalomyelitis
—I. S1P-S1P1 Axis Induces Migration of Th1 and Th17 Cells
Noriyasu Seki1, Yasuhiro Maeda1, Hirotoshi Kataoka1, Kunio Sugahara1, Kenji Chi ba 1,2*
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 2nd, 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
Infiltration of myelin-specific helper T (Th) cells into the central nervous system (CNS) plays a key role in pathogenesis
of experimental autoimmune encephalomyelitis (EAE). In this study, we investigated the involvement of sphingosine
1-phosphate (S1P)-S1P receptor 1 (S1P1) axis in lymphocytes for EAE development when C57BL/6 mice were immu-
nized with myelin oliogodendrocyte glycoprotein (MOG). The expression of S1P1 mRNA and S1P responsiveness of
lymphocytes in draining lymph nodes (DLN) were down-regulated markedly after MOG immunization until onset of
EAE. Accompanying with reacquisition of down-regulated S1P1 transcript and S1P responsiveness in DLN lympho-
cytes, MOG-immunized mice developed EAE symptoms with significant infiltration of Th1 and Th17 cells into the
CNS and a marked elevation of IFN-γ, T-bet, IL-17, and RORγt mRNA expressions. Prophylactic administration of an
S1P1 functional antagonist, fingolimod hydrochloride (FTY720, 0.3 mg/kg, orally) significantly inhibited EAE devel-
opment and almost completely prevented infiltration of Th1 and Th17 cells into the CNS with a marked reduction of
IFN-γ, T-bet, IL-17, and RORγt mRNA expressions. Similar results were obtained by treatment with an S1P1-selective
agonist, SEW2871 or an S1P lyase inhibitor, 2-acetyl-4-tetrahydroxybutylimidazole. Moreover, FTY720-phosphate and
SEW2871 inhibited in vitro migration of Th1 and Th17 cells toward S1P but did not affect cytokine production or gen-
eration of Th1 or Th17 cells. These results suggest that reacquisition of S1P1 expression in DLN lymphocytes plays a
major role in trafficking of myelin antigen-specific Th1/Th17 cells from DLN to the CNS in EAE and that prophylactic
effect of FTY720 on EAE is predominantly caused by functional antagonism via lymphocytic S1P1.
Keywords: Sphingosine 1-Phosphosphate Receptor 1; Fingolimod Hydrochloride (FTY720); Experimental
Autoimmune Encephalomyelitis; Th1 Cells; Th17 Cells
1. Introduction
Experimental autoimmune encephalomyelitis (EAE) is a
CD4 T cell-mediated inflammatory disease of the central
nervous system (CNS) which clinically manifests as as-
cending paralysis [1,2]. It can be induced in susceptible
animals by immunization with myelin proteins or pep-
tides or by adoptive transfer of myelin-specific CD4 T
cells. EAE shares many clinical and histopathological
features with multiple sclerosis (MS) and is a commonly
used animal model of this human autoimmune disease
[3,4]. The generation of effector CD4 T cells is a critical
event for the progression of EAE. EAE was believed to
be a Th1-induced autoimmune disease because of the
increased expression of Th1 cytokines in the affected
CNS and because injection of myelin specific CD4 Th1
but not Th2 cells into immune-competent mice is suffi-
cient to induce EAE [5-9]. On the other hand, interleukin
17 (IL-17)-producing Th17 cells have been implicated in
pathogenesis of EAE [10-12]. It is currently thought that
IL-17 up-regulates the expression of pro-inflammatory
cytokines/chemokines that mediate inflammation in the
CNS to promote EAE [13,14]. In contrast, Th2 cells and
regulatory T cells play a protective role in EAE [15-17].
A lipid mediator, sphingosine 1-phosphate (S1P) and
its receptor 1 (S1P1) play an essential role in lympho-
cyte egress from the secondary lymphoid organs (SLO)
*Corresponding author.

Role of Sphingosine 1-Phosphate (S1P) Receptor 1 in Experimental Autoimmune Encephalomyelitis 629
and thymus [18-22]. Fingolimod hydrochloride (FTY720)
is a first-in-class, orally active S1P1 receptor functional
antagonist [23-28], and has been used as a first oral drug
for relapsing MS in more than 70 countries, because of
its superior efficacy compared with interferon (IFN)-β-1a
injection in relapsing-remitting MS [29-33]. FTY720 is
phosphorylated to FTY720-phosphate (FTY720-P) by
sphingosine kinases and FTY720-P down-regulates S1P
responsiveness of lymphocytes by internalization and
degradation of S1P1 [27,34-36]. Consequently, FTY720-
P converted from FTY720 acts as a functional antagonist
at lymphocytic S1P1 and inhibits S1P-S1P1 axis-medi-
ated lymphocyte egress from the SLO and thymus [19].
Several reports have been demonstrated that FTY720
shows marked prophylactic and therapeutic effects on
EAE in mice and rats [37-41]. As reported previously,
oral administration of FTY720 (0.3 to 1 mg/kg) after
establishment of EAE showed almost complete prevent-
ing effects on relapsing of EAE induced by myelin pro-
teolipid protein (PLP) in SJLJ/ mice [39]. In this EAE
model, treatment with FTY720 markedly reduced infil-
tration of PLP-specific Th17 and Th1 cells into the spinal
cord of EAE mice. On the contrary, FTY720 increased
the frequency of PLP-specific Th17 and Th1 cells to ap-
proximately 3-fold in inguinal lymph nodes. These find-
ings suggest that FTY720 shows preventing effects on
relapsing of EAE by inhibiting egress of myelin anti-
gen-specific Th17 and Th1 cells from draining lymph
nodes (DLN).
In this study, we investigated the role of S1P-S1P1
axis in DLN lymphocytes for EAE development when
C57BL/6 mice were immunized with myelin oliogoden-
drocyte glycoprotein (MOG). Our findings suggest that
S1P1 expression in DLN lymphocytes was down-regu-
lated immediately after antigen immunization and that
the reacquisition of S1P1 expression in DLN lympho-
cytes triggers the trafficking of Th1 and Th17 cells into
the CNS in EAE. We also demonstrate that prophylactic
effect of FTY720 on EAE is likely due to sequestration
of myelin-specific Th1/Th17 cells into DLN by down-
regulation of lymphocytic S1P1.
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 9 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 and Antibodies
FTY720 was synthesized according to the method de-
scribed previously [23] and was dissolved in distilled
water for oral administration. The (S)-enantiomer of
FTY720-P (>99.5% enantio excess) was synthesized
according to the previous method [26] and was dissolved
in ethanol for in vitro experiments. An S1P1-selective
agonist, SEW2871 and an S1P lyase inhibitor, 2-acetyl-
4-tetrahydroxybutylimidazole (THI) were synthesized
and used according to the respective methods reported
previously [42,43]. S1P was purchased from Sigma-Al-
drich.
MOG35-55 (MEVGWYRSPFSRVVHLYRNGK) 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. Cy-Chrome-conjugated rat anti-mouse CD4
monoclonal antibody (mAb) (GK1.5), fluorescein iso-
thiocyanate (FITC)-conjugated rat anti-mouse IFN-γ
mAb (XMG1.2), phycoerythrin (PE)-conjugated rat anti-
mouse IL-17 mAb (TC11-18H10.1), PE-conjugated rat
anti-mouse CD3 mAb (RA3-6B2), and Cy-Chrome-con-
jugated rat anti-mouse CD8 mAb (53 - 6.7) were pur-
chased from BD Bioscience. Hamster anti-mouse CD3
mAb (145-2C11), FITC-conjugated rat anti-mouse CD4
mAb (RM4-5), hamster anti-mouse CD28 mAb (37.51),
and rat anti-mouse CD16/32 mAb (93, FcR block) were
purchased from eBiosciences. Rat anti-mouse IFN-γ
mAb (RMMG-1) and rat anti-mouse IL-4 mAb (11B11)
were obtained from Biosource. IL-2, IL-6, and trans-
forming growth factor (TGF)-β1 were purchased from
Peprotech. IL-23 was obtained from R&D Systems.
2.3. EAE Induction
For the induction of EAE, C57BL/6 mice received a sin-
gle immunization of MOG35-55 in Freund’s complete ad-
juvant containing killed Mycobacterium tuberculosis
H37Ra subcutaneously on day 0, followed by intrave-
nous injection with 200 ng of pertussis toxin (List Bio-
logical Laboratories) on day 0 and 2 [39]. 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. Real Time Polymerase Chain Reaction
Total RNA was extracted using TRIZOL (Invitrogen)
and concentrations of total RNA were measured spec-
trophotometrically. A two-step quantitative reversetran-
scriptation-polymerase chain reaction (RT-PCR) was
performed to determine various molecules mRNA ex-
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Role of Sphingosine 1-Phosphate (S1P) Receptor 1 in Experimental Autoimmune Encephalomyelitis
630
pression using the relative standard curve method, with
cellular housekeeping enzyme, GAPDH as the normali-
zation control. Complementary DNA was synthesized
with TaqMan Reverse Transcription Reagents (Applied
Biosystems) using random hexamers and 0.5 μg of total
RNA. Real-time PCR was performed with an ABI
PRISM 7900 Sequence Detection System (Applied Bio-
systems). In the real-time PCR, following TaqMan
probes (6-carboxy-fluorecein label)/primers were used:
CD3 (Mm00599683_m1), IFN-γ (Mm00801778_m1),
TNF-α (Mm00443258_m1), T-bet (Mm00450960_m1),
IL-17 (Mm00439619_m1), TGF-β1 (Mm00441724_m1),
IL-6 (Mm00446190_m1), and IL-23 (Mm00518984_m1)
in the presence of GAPDH TaqMan probe (VIC label)/
primer. To measure the level of mRNA of S1P1 and
RORγt, real time PCR was performed with an ABI
PRISM 7900 sequence detector using SYBR Green PCR
master mix (Applied Biosystems). The following primer
pairs were used (5’→3’): S1P1 forward, AAA TGC CCC
AAC GGA GAC T; S1P1 reverse, CTG ATT TGC TGC
GGC TAA ATT C [34], RORγt forward, CCG CTG
AGA GGG CTT CAC; RORγt reverse, TGC AGG AGT
AGG CCA CAT TAC A [44]. The reaction was incu-
bated for 2 min at 50˚C denatured for 10 min at 95˚C and
subjected to 40 step amplification cycles with anneal-
ing/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 quantifica-
tion, standard curves were generated for various mole-
cules using serially diluted cDNA samples from the spi-
nal cord in EAE mice (day 14 after immunization). For
every sample, the level of mRNA normalized by calcu-
lating the ratio of each target molecule/GAPDH level.
2.5. Intracellular Cytokines Staining
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 pres-
ence of 2 μM monensin for 5 h in RPMI 1640 medium
containing 10% FCS at 37˚C in 5% CO2. After blocking
with rat 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 carried out by using FITC-conju-
gated anti-mouse IFN-γ mAb and PE-conjugated anti-
mouse IL-17 mAb. Flow cytometry analysis was con-
ducted using FACScan with CellQuest software (Becton
Dickinson).
2.6. Migration Assay
Migration assays were conducted according to the me-
thod described previously [34]. After serum starvation,
mouse inguinal lymph node lymphocytes (5 × 105 cells)
were added to the upper wells of 5-μm pore, polycarbon-
ate 24-well tissue culture inserts (Costar) in 100 μl, with
600 μl of 10 nM S1P solution or medium in the bottom
wells. All migration assays were conducted in RPMI
1640 medium containing 0.5% fatty acid free bovine
serum albumin for 3 h at 37˚C in 5% CO2. The migrated
cells recovered from each well were counted using com-
parison to a known number of beads as an internal stan-
dard of Flow-Count fluorosphere (Beckman Coulter),
and were stained with appropriate mAbs to identify CD4
T cells, Th1 cells, and Th17 cells. The number of the
cells in the starting population and the migrated popula-
tion was determined by flow cytometry with an Epics-
XL (Beckman Coulter), calculated for each phenotype,
and the percent migration was determined from these
values.
2.7. Generation of Th1 and Th17 Cells in Vitro
Mouse spleen cells were purified to >95% of CD4 T cell
by passing through mouse CD4 subset enrichment col-
umns (R&D Systems). CD4 T cells (106 cells) were sti-
mulated with anti-CD3 mAb (10 μg/ml) and anti-CD28
mAb (1 μg/ml) in RPMI 1640 medium containing 10%
FCS for 48 h under Th1 condition: anti-IL-4 mAb (20
μg/mL); or Th17 condition: IL-6 (20 ng/ml), TGF-β1 (10
ng/ml), anti-IFN-γ mAb (10 μg/ml), and anti-IL-4 mAb
(20 μg/ml) [11,45]. Then the cells under Th1 or Th17
condition were added IL-2 (10 ng/ml) or IL-23 (5 ng/ml),
respectively and cultured for additional 96 h. Proportions
of Th1/Th17 cells were analyzed by intracellular cyto-
kine staining according to the method as described above.
The recovered cells (2.5 × 104 cells) were re-stimulated
with anti-CD3mAb plus anti-CD28 mAb for 24 h and
amounts of IFN-γ and IL-17 in the culture supernatants
were determined by ELISA.
2.8. 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 ex-
periments, statistical differences were calculated by Stu-
dent’s t-test. Differences between groups were consid-
ered significant when p < 0.05 (*: p < 0.05, **: p < 0.01).
3. Results
3.1. Reacquisition of S1P1 Expression in DLN
Lymphocytes Triggers Infiltration of
Th1/Th17 Cells into the CNS
When C57BL/6 mice were immunized with MOG35-55
peptide on day 0, one mouse developed EAE on day 11
and more than 80% of mice showed EAE associated
symptoms on day 14. To investigate the involvement of
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Role of Sphingosine 1-Phosphate (S1P) Receptor 1 in Experimental Autoimmune Encephalomyelitis 631
S1P-S1P1 axis in EAE development, we analyzed S1P1
mRNA expression in the inguinal DLN by real time
RT-PCR and S1P responsiveness of DLN lymphocytes
by migration assay toward 10 nM S1P. As shown in
Figure 1(A), mean clinical scores were significantly
elevated on day 14 to 28. The S1P1 mRNA expression in
DLN down-regulated markedly on day 3, was maintained
low until onset of EAE (day 9), and thereafter recovered
gradually along with EAE development. Similarly, S1P
responsiveness of DLN lymphocytes decreased markedly
on day 3, was kept low until day 11, and thereafter re-
turned to the level comparable with that before immuni-
zation. The mRNA expressions of IFN-γ and IL-17 in
DLN increased significantly on day 3 to 6 and then de-
creased to the level before immunization (Figure 1(B)).
On the other hand, the mRNA expressions of IFN-γ and
IL-17 in the spinal cord elevated siginificantly on day 14
to 28 whereas no detectable level of IFN-γ/IL-17 mRNA
was found before EAE onset (day 6) (Figure 1(C)).
Next, we analyzed infiltration of Th1 and Th17 cells
into the spinal cord of EAE mice. As shown in Figure
2(A), proportions of Th1 cells and Th17 cells in DLN
began to increase on day 3, peaked on day 6 to 9, and
decreased gradually along with EAE development. On
the other hand, significant numbers of Th1 cells (22.4%
to 52.3% of CD4 T cells) and Th17 cells (4.4% to 5.1%
of CD4 T cells) were infiltrated into the spinal cord of
EAE mice on day 14 to 28 (Figure 2(B)). We also con-
firmed no infiltration of Th1/Th17 cells in the spinal cord
before the onset of EAE (data not shown). These results
(A)
Clinical score
0
2
4
6
8
10
S1P1 mRNA in DLN (n=3)
S1P1/GAPDH
0
2
4
6
8
10
S1P responsiveness (n=3)
Migration toward
S1P (% of input)
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Clinical score (n=10)
(B)
0
1
2
3
4
5
6
IFN - γ/GAPDH
0
5
10
15
20
IL-17/GAPDH
IFN - γ mRNA
IL-17 mRNA
DLN
IFN - γ/GAPDH
IL-17/GAPDH
(C)
036911 14 21 28
Days after immunization
0
0.5
1.0
1.5
2.0
0
0.5
1.0
1.5
2.0
Spinal cord
IFN - γmRNA
IL-17 mRNA
(n=3)
(n=3)
Figure 1. S1P1 transcript and S1P responsiveness in DLN,
and IFN-γ/IL-17 mRNA expression in the DLN and spinal
cord of EAE mice.
IL-17
IFN - γ
Day 11Day 14Day 21Day 28
Day 0Day 3Day 6Day 9
CD4 T cells in DLN
(A)
20.8 4.84.6 3.5
0.10.0 2.00.2
0.7 1.026.213.9
4.65.2 7.4 6.4
2.44.3 1.40.1 1.50.02.30.2
4.43.6 4.52.94.52.55.1
(B)
IL-17
IFN - γ
CD4 T cells in the spinal cord
Day 14Day 16Day 21Day 28
1.45.1
51.252.342.3 22.4
Figure 2. Frequency of Th1 and Th17 cells in the DLN and
spinal cord of EAE mice.
suggest that reacquisition of S1P1 expression in DLN
lymphocytes triggers infiltration of Th1/Th17 cells into
the CNS and EAE development.
3.2. FTY720 Inhibits EAE by Reducing
Infiltration of Th1/Th17 Cells into the CNS
To clarify the involvement of S1P1 on trafficking of
Th1/Th17 cells from DLN to the CNS, we administered
FTY720 (0.3 mg/kg, orally once a day) from day 0 to 16
to MOG35-55-immunized mice. Consistent with previous
studies, the prophylactic administration of FTY720
resulted in a marked delay in the onset of EAE with a
significant decrease of mean clinical scores (Figure
3(A)). The mRNA expressions of CD3, IFN-γ, TNF-α,
T-bet, IL-17, and RORγt in the spinal cords were signi-
ficantly reduced to less than 10% of vehicle-treated EAE
control on day 16 (Figure 3(B)). We also analyzed the
infiltration of Th1 and Th17 cells into the spinal cords by
intracellular cytokine staining on day 16. The number of
lymphocytes prepared from the spinal cords was ex-
tremely decreased to less than 1% of control by treatment
with FTY720 (lymphocytes in the spinal cords: EAE
control, 9.4 × 105 cells/5mice; FTY720, 0.05 × 105
cells/5mice). Flow cytometry analyses revealed that
FTY720 markedly decreased the numbers of Th1/Th17
cells in the spinal cord of EAE mice (Figure 3(C)).
There were no clear change in mRNA levels of CD3,
IFN-γ, TNF-α, and T-bet whereas those of IL-17 and
RORγt were increased to approximately 1.5-fold in DLN
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4.7 3.7
49.6
7.0 7.0
33.3
(A)
0
0.5
1.0
1.5
2.0
0
0.5
1.0
1.5
2.0
0
0.5
1.0
1.5
2.0
0
0.5
1.0
1.5
2.0
0
0.5
1.0
1.5
2.0
0
1
2
3
4
(B)
mRNA / GAPDH
in the spinal cord (n=3)
CD3 IFN-γTNF-α
T-betIL-17 RORγt
** **
** ***
(C) Control FTY720
Counts
CD4
IL-17
IFN - γ
30.8 9.8
CD4 T cells in the spinal cord
810121416
0
0.5
1.0
1.5
2.0
2.5
3.0 Control
FTY720
Clinical score
9111315
***
**
** **
Days after immunization
0.3 mg/kg
(n=15)
Figure 3. Prophylactic administration of FTY720 (0.3
mg/kg p.o.) inhibits EAE and decreases infiltration of Th1
and Th17 cells into the spinal cord of EAE mice.
of EAE mice given FTY720 (Figure 4(A)). In addition,
no change was seen in mRNA level of IL-6, TGF-β1, or
IL-23 (data not shown). Flow cytomery analyses demon-
strated that the proportions of Th1 and Th17 cells in
DLN are increased to 2- to 4-fold by FTY720 treatment
(Figure 4(B)). These findings suggest that prophylactic
FTY720 inhibits EAE development by sequestrating
Th1/Th17 cells into the DLN and reducing infiltration of
these Th cells to the CNS.
3.3. S1P-S1P1 Axis Regulates Trafficking of
Th1/Th17 Cells from DLN to the CNS in
EAE
An S1P1-selective agonist, SEW2871 and an S1P lyase
inhibitor, THI have shown to reduce circulating lympho-
cytes in the blood by inhibiting S1P1-dependent lym-
1.4 0.2
5.6
1.0
13.3
5.5
(A)
0
0.5
1.0
1.5
2.0
0
0.5
1.0
1.5
2.0
0
1
2
3
4
0
1
2
3
0
0.5
1.0
1.5
0
1
2
3
mRNA / GAPDH
in DLN (n=3)
CD3 IFN-γTNF-α
T-betIL-17 RORγt
(B)
IL-17
Control FTY720
CD4
IFN -γ
22.7 27.9
Counts
CD4 T cells in DLN
Figure 4. Prophylactic administration of FTY720 (0.3
mg/kg p.o.) increases proportions of Th1 and Th17 ells in
DLN of EAE mice.
phocyte egress from the SLO [24,42,46,47]. Prophylactic
administration of SEW2871(30 mg/kg, orally twice a day)
or THI (50 mg/kg, orally twice a day) significantly inhi-
bited development of EAE (Figure 5(A)) with a signi-
ficant reduction of mRNA expressions of CD3, IFN-γ,
TNF-α, T-bet, IL-17, and RORγt (Figure 5(B)). More-
over, SEW2871 and THI markedly decreased the number
of CD4 T cells and the proportions of Th1 and Th17 cells
in the spinal cord of EAE mice (Figure 5(C)).
3.4. S1P-S1P1 Axis Induces Migration of Th1
and Th17 Cells
It has been well documented that S1P-S1P1 axis plays an
essential role in lymphocyte egress from the SLO. Indeed,
S1P at 10 to 100 nM induces migration of murine naïve
CD4 T cells in vitro; however it remains unclear whether
S1P can induce migration of Th1 or Th17 cells.
In this study, we found that S1P (10 nM) significantly
induces in vitro migration of both Th1 and Th17 cells
from DLN lymphocytes of EAE-established mice (Fig-
ure 6(A)). Migration of Th1 cells or Th17 cells toward
S1P was almost completely inhibited by pretreatment with
FTY720-P at 1 nM or higher (Figure 6(A)). Similar re-
sults were obtained by pretreatment with 300 nM SEW
2871 (data not shown). On the other hand, FTY720-P
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Role of Sphingosine 1-Phosphate (S1P) Receptor 1 in Experimental Autoimmune Encephalomyelitis 633
Control SEW2871THI
(C)
Counts
CD4
IL- 1 7
IFN - γ
3.6 2.9
49.9
2.1
17.6
0.8 0.8
11.9
39.27.3 6.8
3.5
CD3 IFN - γTNF-α
0
0.5
1.0
1.5
0
0.5
1.0
1.5
2.0 T-be t
0
0.5
1.0
1.5
2.0
0
0.5
1.0
1.5
2.0 IL- 1 7
0
0.5
1.0
1.5
2.0
2.5
0
0.2
0.4
0.6
0.8
1.0 RORγt
mRNA / GAPDH
in the spinal cord (n=3)
(B)
CD4 T cells in the spinal cord
(A) Control
SEW2871
Clinical score
Days after immunization
Control
THI
89 1011121314151617
0
0.5
1.0
1.5
2.0
2.5
3.0
89 1011121314151617
0
0.5
1.0
1.5
2.0
2.5
3.0
*
** ** *** **
** ** ******
** **
*
*
*** *
**
(n=12)
30 mg/kg bid
(n=12)
50 mg/kg bid
Figure 5. SEW2871 (30 mg/kg p.o. bid) and THI (50 mg/kg
p.o. bid) inhibit EAE development and decrease infiltration
of Th1 and Th17 cells into the spinal cord of EAE mice.
up to 100 nM did not affect either IFN-γ production by
Th1 cells or IL-17 production by Th17 cells (Figure
6(B)). Furthermore, FTY720-P showed no effect on gen-
eration of either Th1 or Th17 cells in vitro (Figure 6(C)).
4. Discussion
EAE is a CD4 T cell-dependent animal model for human
MS and development of EAE is likely due to infiltration
of encephalitogenic, myelin antigen-specific Th1 cells
and/or Th17 cells into the CNS [3,4,6]. Since the patho-
genic Th1/Th17 cells are originally generated from naïve
CD4 T cells after myelin antigen presentation in the DLN
[10-12,48-50], trafficking of these Th1/Th17 cells from
DLN to CNS is thought to play a key role in EAE devel-
opment. On the other hand, it is strongly suggested that
S1P-S1P1 axis plays an important role in lymphocyte
10 10 10 10
20
30
40
50
60
70
80
Migration of Th1 cells
(% of input, n=3)
0
5
10
15
20
25
S1P (nM)
FTY720-P (nM)0.3 13
Migration of Th17 cells
(% of input, n=3)
0
10
20
30
40
50
IL-17 production
(ng/mL, n=3)
0
20
40
60
80
100
120
IFN-γproduction
(ng/mL, n=3)
0.1110 100FTY720-P (nM)
(B)
(A)
*
****
***
IFN-γ
IL-17
Th1
Th1 7
IFN-γIL-17
CD4 CD4
49.031.047.131.269.05.2 61.15.1
(C) Control FTY720-PControlFTY720-P
Figure 6. FTY720-P inhibits migration of Th1 and Th17
cells toward S1P but not cy tokine production or generation
of Th1/Th17 cells.
trafficking between DLN and inflammatory cites because
lymphocyte egress from the SLO depends on lympho-
cytic S1P1 [19]. However it remains unclear relation
between S1P-S1P1 axis in DLN lymphocytes including
pathogenic Th1/Th17 cells and EAE development.
In the present study, we demonstrated that level of
S1P1 mRNA expression in DLN was markedly down-
regulated after immunization with MOG antigen and was
maintained low for several days until EAE development.
Similarly, S1P responsiveness of DLN lymphocytes stri-
kingly reduced after antigen exposure and was kept low
until the onset of EAE. Our flow cytometry data revealed
that the numbers of Th1 cells and Th17 cells increased in
DLN before EAE development, suggesting generation of
Th1/Th17 cells by antigen stimulation. On the contrary,
the number of Th1/Th17 cells decreased in DLN accom-
panying with the recovery from down-regulated S1P1
transcript and reacquisition of S1P responsiveness in
DLN lymphocytes. In parallel, significant numbers of
infiltrated Th1/Th17 cells were found in the CNS and
most of MOG-immunized mice showed EAE associated
symptoms. Namely, our results imply that encephalito-
genic Th1/Th17 cells begin to infiltrate into the CNS
when down-regulated lymphocytic S1P1 expression is
recovered to a homeostatic level in DLN.
During immune response, antigen-specific T cells are
transiently retained within DLN, undergoing activation,
and clonal expansion and then exiting as effector cells.
This retention mechanism is believed to involve transient
Open Access PP

Role of Sphingosine 1-Phosphate (S1P) Receptor 1 in Experimental Autoimmune Encephalomyelitis
634
loss of S1P responsiveness by down-regulation of S1P1
receptor on activated T cells. Matloubian et al. have been
reported using OVA-specific DO11.10 TCR transgenic T
cells that at one day after antigen exposure, the activated
antigen-specific T cells in DLN had lost their respon-
siveness to S1P and they had down-regulated S1P1 ex-
pression in mRNA level markedly [19]. On the contrary,
3 days after antigen immunization, antigen-specific T
cells appeared in circulation, and lymphocytes in DLN
restored S1P responsiveness and increased S1P1 mRNA
expression. Therefore, antigen-induced down-regulation
of S1P1 transcript and S1P responsiveness is associated
with the initial retention of antigen-specific activated T
cells in DLN, and reacquisition of S1P responsiveness is
associated with their exit. However, there is no report on
lymphocyte retention into DLN by down-regulation of
S1P1 transcript after antigen exposure in mouse EAE
induced by MOG.
Our findings in MOG-induced mouse EAE clearly
demonstrated that S1P1 mRNA expression and S1P re-
sponsiveness in DLN were markedly down-regulated
after MOG immunization until the onset of EAE whereas
the reacquisition of S1P1 mRNA expression and S1P
responsiveness in DLN was observed in parallel with the
development of EAE. On the other hand, the mRNA ex-
pressions of both IFN-γ and IL-17 and the frequency of
Th1 and Th17 cells in DLN were increased markedly
after MOG antigen exposure, suggesting retention of
antigen-activated CD4 T cells in DLN for their matura-
tion to Th1 and Th17 cells. Indeed, CD4 T cells in DLN
on day 3 to 9 after MOG antigen immunization showed
no detectable migratory response toward S1P.
Prophylactic administration of FTY720, SEW2871, or
THI inhibited the development of EAE and decreased the
infiltration of Th1 and Th17 cells into the CNS markedly.
As reported previously, FTY720-P (0.1 nM or higher)
and SEW2871 (300 nM or higher) can induce down-
regulation of S1P1 in S1P1 stably-expressing Chinese
hamster ovary cells and CD4 T cells [34]. On the other
hand, THI inhibits S1P lyase activity in vivo and gener-
ated high S1P condition down-regulates S1P1 expression
[42]. From these results, it is presumed that prophylactic
administration of these compounds in vivo induces down-
regulation of S1P1 and loss of S1P responsiveness in
lymphocytes and inhibits egress of pathogenic Th1 and
Th17 cells from DLN in EAE mice.
Although it has been reported that naïve murine CD4
T cells migrate 10 to 100 nM S1P [19], it remained un-
clear whether Th1 or Th17 cells can migrate toward S1P.
Our results clearly demonstrated that S1P (10 nM) can
induce migratory response of both Th1 and Th17 cells
prepared from EAE mice. Furthermore, pretreatment
with FTY720-P or SEW2871 almost completely inhib-
ited the migration of Th1 and Th17 cells toward S1P.
These results suggest that S1P at physiological concen-
tration induces migration of encephalitogenic Th1 and
Th17 cells via S1P1 and that FTY720-P leads to loss of
S1P responsiveness of these Th cells by internalization
and subsequent degradation of S1P1.
Liao et al. have been reported that like IL-23, S1P at
1000 nM can enhance generation of Th17 cells when
CD4 T cells were stimulated with anti-CD3 mAb plus
anti-CD28 mAb and a mixture of TGF-β1, IL-1, and IL-6
[51]. Since FTY720-P at 100 nM inhibits IL-17 produc-
tion from CD4 T cells by stimulation with 1000 nM S1P,
it is proposed that immunosuppression by FTY720 may
be attributable partly to inhibition of Th17-mediated in-
flammation. On other hand, our results indicate that
FTY720-P up to 100 nM showed no effect on generation
or cytokine production of Th1 and Th17 cells without
adding exogenous S1P. Furthermore, when THI is ad-
ministered to mice, S1P concentrations increased to ap-
proximately 10 to 100-fold in the blood, lymph, and SLO
[42,46]; however no enhancement of frequency of Th17
cells in DLN lymphocytes was seen in THI-treated EAE
mice. Because S1P concentrations are kept low in the
SLO [42,46], it is likely that low S1P condition in DLN
does not affect the generation of Th17 cells.
In conclusion, our results in this study imply that
S1P-S1P1 axis induces migration of Th1 and Th17 cells
and plays a regulatory role in the trafficking of patho-
genic Th1 and Th17 cells from DLN to the CNS in EAE
development.
5. Acknowledgements
We thank Dr. Kunitomo Adachi in Medicinal Chemistry
Laboratories I, Mitsubishi Tanabe Parma Corporation for
synthesizing (S)-enantiomer of FTY720-P, SEW2871
and THI.
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