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`0022-3565/03/3051-70–77$7.00
`THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
`Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics
`JPET 305:70–77, 2003
`
`Vol. 305, No. 1
`45658/1054066
`Printed in U.S.A.
`
`Amelioration of Experimental Autoimmune Encephalomyelitis in
`Lewis Rats by FTY720 Treatment
`
`MASAYUKI FUJINO, NAOKO FUNESHIMA, YUSUKE KITAZAWA, HIROMITSU KIMURA, HIROSHI AMEMIYA,
`SEIICHI SUZUKI, and XIAO-KANG LI
`Laboratory of Transplantation Immunology, Department of Innovative Surgery, National Research Institute for Child Health and Development,
`Tokyo, Japan
`Received October 18, 2002; accepted January 8, 2003
`
`ABSTRACT
`Experimental autoimmune encephalomyelitis (EAE) is a T-cell-
`dependent autoimmune disease that reproduces the inflamma-
`tory demyelinating pathology of multiple sclerosis (MS). We
`investigated the efficacy and mechanism of immunosuppres-
`sion against EAE by administering 2-amino-[2-(4-octylphenyl)
`ethyl]-1,3-propanediol hydrochloride (FTY720) in Lewis rats im-
`munized with myelin basic protein together with complete
`Freund’s adjuvant. FTY720 treatment almost completely pro-
`tected the rats against disease. The protection by FTY720 was
`associated with a dramatic reduction in the number of lympho-
`cytes staining for T-cell receptors in the spinal cord as exam-
`ined by immunohistochemistry. The mRNA expression of Th1
`
`cytokines interleukin (IL)-2, IL-6, and interferon-␥ in the spinal
`cord was also reduced dramatically as assessed by reverse-
`transcription polymerase chain reaction. Furthermore, lympho-
`cytes isolated from the spleen of FTY720-treated rats were
`transferred into naive recipient rats against EAE manifestation
`by reducing both disease incidence and clinical score. These
`results suggested that the protective anti-inflammatory effect of
`treatment with FTY720 was, to a large extent, due to the
`inhibition of encephalitogenic T-cell responses and/or their mi-
`gration into the central nervous system and may be a potential
`candidate for use in treating patients with MS.
`
`Multiple sclerosis (MS) is a common and often disabling
`disease of the central nervous system (CNS). The early active
`MS lesions are characterized by the presence of mononuclear
`cell infiltrates around venules and small veins, followed by
`myelin breakdown and astrogliosis, resulting in irreversible
`disability. The etiology of the disease remains uncertain but
`is widely considered to involve organ-specific autoimmune
`destruction of CNS myelin.
`Acute experimental autoimmune encephalomyelitis (EAE),
`an inflammatory disease of the CNS, has been widely used as
`an animal model for testing novel therapeutic approaches for
`MS. The disease can be induced in different species of labo-
`
`This study was supported by research grants from the Ministry of Health,
`Labor, and Welfare of Japan (12-KO-2, Millennium Project H12-Saisei-016)
`and a grant-in-aid (10307030) and a grant for Organized Research Combina-
`tion System from the Ministry of Education, Culture, Sports, Science, and
`Technology of Japan.
`M.F. and N.F. contributed equally to this work.
`Article, publication date, and citation information can be found at
`http://jpet.aspetjournals.org.
`DOI: 10.1124/jpet.102.045658.
`
`ratory animals by injecting central nervous tissue antigens
`emulsified in an appropriate adjuvant, e.g., complete
`Freund’s adjuvant (CFA). In Lewis rats, a susceptible strain,
`EAE is manifested by a paralytic attack that affects the tail
`and hind limbs 11 to 14 days after injection of guinea pig
`myelin basic protein (MBP) as an encephalitogenic antigen.
`Consistent with this, EAE can also be induced in naive ani-
`mals by transferring MBP-activated T cells. The initial ob-
`servation by Paterson (1960) that the autoimmune disease
`EAE could be induced by transferring lymphocytes from ac-
`tivate-sensitized rats to naive histocompatible recipients con-
`firmed the condition to be principally an immune cell-medi-
`ated phenomenon. Others also reported that relatively small
`numbers of spleen cells have transferred full clinical signs of
`EAE if cultured with mitogen concanavalin A (Con A)
`(Panitch and McFarlin, 1977) or with the antigen MBP before
`transfer (Richert et al., 1979).
`Clinically, the disease follows an acute and monophasic
`course. The main pathological event is the appearance of
`inflammatory cell infiltrates forming perivascular cuffs. The
`
`ABBREVIATIONS: MS, multiple sclerosis; CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; CFA, complete
`Freund’s adjuvant; MBP, myelin basic protein; Con A, concanavalin A; FTY720, 2-amino-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride;
`CsA, cyclosporin A; FK506, tacrolimus; IL, interleukin; INF-␥, interferon-␥; PBS, phosphate-buffered saline; TdT, terminal deoxynucleotidyl
`transferase; RT-PCR, Reverse-transcription polymerase chain reaction; bp, base pair; TUNUL, terminal deoxynucleotidyl transferase dUTP
`nick-end labeling; S1P, sphingosine 1-phosphate; ISP-1, ((2S,3R,4R)-(E)-2-3,4-dihydroxymethyl-14-oxoeicos-6-enoic acid, myriocin ⫽ thermozy-
`mocidin).
`
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`inflammatory infiltrates in acute EAE and MS contain pre-
`dominantly a diverse accumulation of T cells, macrophages,
`and some B cells. Pharmacological studies using both the
`active and adoptive models of EAE have provided useful
`information on the mechanisms by which steroid and non-
`steroid immunomodulatory drugs may act and be of potential
`value in treating MS.
`A potent immunosuppressive compound, ISP-1, and its
`derivatives, mycestericins, were isolated from the culture
`broth of Isaria sinclairii, a species of vegetative wasp (Fujita
`et al., 1994a). Chemical modification of ISP-1 led to a novel
`synthetic immunosuppressant, FTY720, which has more po-
`tent immunosuppressive activity and less toxicity than ISP-1
`(Fujita et al., 1994b). FTY720 administered at 0.1 mg/kg or
`more significantly prolonged the survival of skin, cardiac,
`liver, renal, pancreas, lung, and small bowel allografts in rats
`(Brinkmann et al., 2001). Furthermore, FTY720 combined
`with cyclosporin A (CsA) or tacrolimus (FK506) produced
`synergistic immunosuppressive effects (Yanagawa et al.,
`1998).
`A striking feature of FTY720 is that it induces a marked
`decrease in the number of peripheral blood lymphocytes,
`especially T cells, at doses that prolong allograft survival
`(Hoshino et al., 1996). A recent article showed that FTY720
`selectively induces cell death in mature T-lymphocyte, espe-
`cially CD4-positive cells, in peripheral blood without depress-
`ing bone marrow (Enosawa et al., 1996). It has been hypoth-
`esized that apoptotic
`cell death of
`lymphocytes and
`acceleration of lymphocyte homing decrease the number of
`lymphocytes (Suzuki et al., 1996b; Chiba et al., 1998; Yana-
`gawa et al., 1998).
`Recently, Brinkmann et al. (2002) reported that FTY720
`prevented development of EAE in Wister rats. We attempted
`to confirm and extend their findings by evaluating the sup-
`pressive effects of FTY720 on EAE in Lewis rats, which have
`presented evidence that the immune and neuroendocrine
`system can contribute to susceptibility to inflammatory au-
`toimmune disease (MacPhee and Mason, 1988). In the
`present study, we show that oral administration of FTY720
`almost completely protected rats immunized with MBP/CFA
`against EAE, resulting in a dramatic reduction of leukocyte
`infiltration into the CNS and decreased expression of IL-2,
`IL-6, and INF-␥ in the CNS. Furthermore, the capacity to
`generate disease could be inhibited when isolated spleen cells
`were transferred from FTY720-treated rats to naive Lewis
`rats.
`
`Materials and Methods
`Animals. We purchased 250- to 280-g, 10-week-old, male inbred
`Lewis rats from Shizuoka Laboratory Animal Center (Shizuoka,
`Japan). All animals were provided water and food ad libitum and
`were housed in accordance with institutional animal care policies.
`Induction of Acute EAE. The methods of acute EAE induction
`were similar to those published previously (Schmitz et al., 1991). We
`emulsified MBP (kindly provided by Dr. W. F. Hickey; Department of
`Pathology, Dartmouth Medical School, Dartmouth Hitchcock Medi-
`cal Center, Lebanon, NH) in 0.9% saline in an equal volume of
`complete Freund’s adjuvant (ICN Biomedicals, Inc., Aurora, OH)
`containing 4 mg/ml of heat-inactivated Mycobacterium butyricum
`(Difco, Detroit, MI) and then immunized male Lewis rats with 0.1 ml
`of emulsion subcutaneously on the dorsum of two sides of the tail.
`The total dose of MBP was 50 to 75 ␮g/rat.
`
`Amelioration of EAE by FTY720 Treatment
`
`71
`
`Induction of Adoptive Transferred EAE. For adoptive trans-
`ferred EAE, we immunized rats with MBP, as described above.
`Fourteen days later, we prepared spleen cell suspensions from the
`actively EAE-induced Lewis rats with FTY720- and saline-treated
`control by Ficoll Isopaque (Lympholyte-Rat, CEDARLANE LABO-
`RATORIES Ltd., ON, Canada) density-gradient centrifugation. We
`harvested interface layer cells, washed them twice in PBS, and then
`used these cells, consisting of lymphocytes, for the following proce-
`dure. We cultured isolated erythrocyte-free lymphocyte suspensions
`from the immunized rats for 2 days with 50 ␮g/ml Con A (Wako Pure
`Chemicals, Osaka, Japan). After washing them with RPMI 1640
`(Sigma-Aldrich, St. Louis, MO), we injected 6 ⫻ 106 cells into naive
`Lewis rats.
`Chemical Compound. FTY720, a gift from Yoshitomi Pharma-
`ceutical Industries (Osaka, Japan), was dissolved in physiological
`saline.
`Treatment Schedule of Rats. The rats were treated with either
`FTY720 (1 mg/kg/day) or saline. The drug was given orally once a day
`on days 0 to 14 after immunization with MBP.
`Specimens. Three animals from each group were sacrificed under
`ether anesthesia on days 7, 14, 21, and 28 after sensitization. The
`spinal cord and spleen were removed quickly. Blocks up to 1 cm3
`were embedded in optimal cutting temperature compound (Tissue-
`Tek, Elkhart, IN) and snap frozen in isopentane, which was pre-
`cooled in acetone and dry ice, and 6-␮m frozen sections were cut in a
`cryostat for DNA fragmentation analysis and immunohistology. A
`second portion of the spinal cord and spleen was immediately snap-
`frozen for subsequent molecular analyses, and a third portion of the
`samples was fixed in 10% neutral buffered formalin for neuropathol-
`ogy.
`Clinical Grading of EAE. Rats were evaluated daily and graded
`by a blinded investigator according to the following scale: grade 0 ⫽
`no signs; grade 1 ⫽ limp tail; grade 2 ⫽ hind limb weakness suffi-
`cient to impair righting; grade 3 ⫽ paraplegia; and grade 4 ⫽ para-
`plegia with forelimb weakness, moribund condition.
`In Situ Assay for DNA Fragmentation. As previously de-
`scribed (Li et al., 2001), we used Apop Tag Plus Kit (Oncor, Gaith-
`ersburg, MD), which uses certain reagents for nonisotopic DNA
`end-extension in situ and other reagents for immunohistochemical
`staining of the extended DNA technique, to detect DNA fragmenta-
`tion. Briefly, we cut cryosections (6 ␮m), fixed them in 10% neutral
`buffered formalin in a Coplin jar, and quenched them in 0.5 to 1%
`hydrogen peroxide in PBS for 5 min at room temperature. We then
`incubated each section with a working strength terminal deoxynu-
`cleotidyl transferase (TdT) reaction mixture consisting of 38 ␮l of
`reaction buffer and 16 ␮l of TdT enzyme in a humidified chamber at
`37°C for 1 h and terminated the reaction with a prewarmed working
`strength stop/wash buffer for 30 min at 37°C. To visualize incorpo-
`rated TdT, we incubated sections with peroxidase-conjugated anti-
`digoxigenin antibody for 30 min at room temperature, washed them
`three times in a Coplin jar, and incubated them with 4-dimethylami-
`noazobenzene substrate working solution for 3 to 6 min at room
`temperature. The reaction was terminated by washing with H2O,
`and sections were counterstained with methyl green and mounted.
`Negative controls were prepared by substituting PBS for the TdT
`enzyme in the reaction mixture.
`Reverse-Transcription Polymerase Chain Reaction (RT-
`PCR). We extracted total cellular RNA from frozen spinal cord and
`spleen tissue by using ISOGEN (Nippon Gene, Tokyo, Japan), as
`described previously (Li et al., 2001), and confirmed the RNA quality
`on formaldehyde-agarose gels. One microgram of total RNA was used
`for first-strand cDNA synthesis in 20 ␮l of 100 mM Tris-HCl, 500
`mM KCl, 5 mM MgCl2, 1 mM dNTP, 1 U/␮l RNase inhibitor, random
`9-mer primer, and 0.25 U/␮l avian myeloblastosis virus reverse
`transcriptase (Takara, Shiga, Japan). We performed PCR amplifica-
`tion in a 100-␮l reaction mixture containing 200 ␮M of each of the
`regular dNTPs, 10 pmol of each primer, and 2.5 U of TaqDNA
`polymerase (TaKaRa) using primers IL-2 (300 base pairs; bp), 5⬘-
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`Fig. 1. Suppression of acute EAE in the Lewis rat by FTY720. Rats were
`immunized with MBP/CFA to induce EAE, as described under Materials
`and Methods. FTY720 (squares) or control (circles) saline was given daily
`by oral administration at 1 mg/kg/day. A, survival (n ⫽ 6/FTY720; n ⫽
`6/control); B, maximum clinical score (n ⫽ 12/FTY720; n ⫽ 19/control); C,
`mean clinical score (n ⫽ 23/FTY720; n ⫽ 16/control); D, body weight loss
`(n ⫽ 23/FTY720; n ⫽ 16/control). Data complied from four separate
`experiments.
`
`72
`
`Fujino et al.
`
`CAGCTGTTGCT GGACTTACAGG-3⬘ and 5⬘-CACAGTTGATGGCT-
`CATCATCG-3⬘; IL-6 (294 bp), 5⬘-GACTTCACAGAGGATACCC-3⬘
`and 5⬘-TAAGTTGTTCTTCACAAACTCC-3⬘; INF-␥ (310 bp), 5⬘-
`GGATATCTGGAGGAACTGGCAAAAG-3⬘ and 5⬘-GCTAGATT CTG-
`GTGACAGCTGGTG-3⬘; ␤-actin (461 bp) 5⬘-CATCGTGGGCCGCT-
`CTAGGCA-3⬘ and 5⬘-CCGGCCAGCCAAGTCCAGACGC-3⬘. We used
`the TaKaRa Thermal Cycler 480 PCR system (Takara, Shiga, Japan)
`and the “hot start” technique to increase specificity. The thermal
`cycling parameters were denaturation at 94°C for 30 s, annealing at
`60°C for 30 s, and extension for 90 s at 72°C (40 cycles). PCR products
`(10 ␮l) were analyzed on 1 to 1.8% agarose gels. We visualized
`prominent bands of the correct size with ethidium bromide staining
`and measured the intensity of each band by a compact digital camera
`(DC40) with analysis software (BioMax 1D image analysis software;
`Eastman Kodak Co., Rochester, NY), as described in a previous
`article (Li et al., 2001). The relative quantities of genes are presented
`as the ratio between the intensity of IL-2, IL-6, or INF-␥ band and
`that of the housekeeping gene ␤-actin.
`Immunohistology and Neuropathology. We characterized the
`cells with monoclonal antibodies R73 (␣/␤T-cell receptor) to evaluate
`T-cell infiltrates (Serotic, Oxford, UK), air-dried the slides, fixed
`them in acetone at ⫺20°C overnight, and then air-dried them for 1 h.
`The primary antibody consisted of mouse IgG1 isotype diluted to 1:50
`in a solution containing 2% bovine serum albumin and 0.1% sodium
`azide in PBS. The second antibody consisted of goat antibody to
`mouse IgG conjugated to alkaline phosphatase (Santa Cruz Bio-
`chemicals, Santa Cruz, CA), diluted at 1:100 in the above working
`solution. Color development was performed with an alkaline phos-
`phatase substrate kit (Vector Laboratories, Inc., Burlingame, CA).
`We obtained an optimum section morphology when the sections were
`air dried for 1 h before counterstaining in hematoxylin (Sigma-
`Aldrich).
`Statistical Analyses. Recipient survival times were compared
`among the groups by Gehan’s generalized Wilcoxon test. Compari-
`sons of the mean day of onset of disease and mean peak disease
`severity between any two groups of rats were analyzed by the Stu-
`dent’s t test; P values less than 0.05 were considered significant.
`
`Results
`Effect of FTY720 on Lewis EAE Rats. A total of 51 rats
`(22 FTY720-treated and 29 saline-treated) were used in these
`studies. The treated and control groups were compared with
`regard to maximal clinical score and time to clinical onset of
`EAE. The results showed that 40% of the rats died after EAE
`induction. FTY720 administration, however, almost com-
`pletely prevented EAE-induced rat death (Fig. 1A). The dif-
`ference in maximum clinical score between FTY720 and con-
`trol groups was significant, with P ⬍ 0.0001 using the
`Student’s t test. FTY720-treated rats were less subject to
`EAE induction than saline-treated rats (Fig. 1, B and C).
`Furthermore, FTY720 treatment also prevented the decrease
`of body weight in EAE rats (Fig. 1D) in addition to reducing
`the clinical score.
`Effect of FTY720 on the Formation of Inflammatory
`Lesions in the CNS. We performed histological studies of
`spinal cords to investigate the effect of FTY720 blockade on
`the formation of inflammatory lesions in the CNS. As shown
`in Fig. 2, A–D, inflammatory lesions were readily detectable
`in control rats, whereas the spinal cords from rats adminis-
`tered FTY720 exhibited a complete absence of inflammatory-
`cell infiltrates.
`Effect of FTY720 on the Infiltration of T Lympho-
`cytes. In EAE, MBP-specific T lymphocytes attack the my-
`elinated tissue of the CNS. EAE in Lewis rats generally has
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`Amelioration of EAE by FTY720 Treatment
`
`73
`
`Fig. 2. Histological findings of the spinal cord in
`the EAE Lewis rat. Histological findings of the
`MBP/CFA-immunized rats treated with FTY720
`or control saline. In HE staining in the spinal
`cords of rats, inflammatory lesions were readily
`detectable in control rats, whereas the spinal
`cords from rats administered FTY720 exhibited
`a complete absence of inflammatory-cell infil-
`trates (A–H). Influx of inflammatory T cells in
`the spinal cords of rats was shown in the control
`rats, whereas administration of FTY720 dramat-
`ically decreased infiltration of T lymphocytes (I–
`P). Detection of apoptotic cells in the spinal cords
`of rats by the use of the TUNEL method was
`shown in control rats, whereas treatment of
`FTY720 decreased TUNEL-positive cells in the
`spinal cords of rats (Q–X). Original magnifica-
`tion 100⫻. Data are representative of three sep-
`arate experiments.
`
`an acute, monophasic course. We identified the expression of
`T-cell receptors in CNS to investigate T lymphocyte infiltra-
`tion and the effect of FTY720 on those cells. As shown in Fig.
`2, J–L, infiltration of T lymphocytes was found in the spinal
`cords of saline-treated rats. Administration of FTY720 dra-
`matically decreased infiltration of T lymphocytes (Fig. 2,
`N–P), however. By day 14, this difference was more marked;
`there were also more T cells in the portal tracts of the control
`group than in that of the FTY720-treated group.
`Effect of FTY720 on the Induction of Apoptosis in the
`CNS. Apoptosis related to EAE is well known in Lewis rats
`(Pender et al., 1991). To identify apoptosis in the CNS, we
`performed TUNEL staining of the spinal cords of Lewis rats
`with EAE. We observed many apoptotic cells in the spinal
`cords of control saline-treated rats on days 21 and 28 with the
`TUNEL method but none in FTY720-treated rats (Fig. 2, S
`and T versus W and X).
`Activation of Infiltrating Cells and Suppression of
`Cytokine Production. The development of clinical EAE
`has been associated with the production of various inflam-
`matory cytokines associated with the Th1 phenotype, includ-
`ing IL-2, IL-6, and IFN-␥(Ando et al., 1989; Samoilova et al.,
`1998). The mRNA levels of these inflammatory products have
`been identified and quantified in the spinal cords and spleen
`both of FTY720-administered and control saline-adminis-
`tered rats by the RT-PCR method. Expression of these cyto-
`
`kines was dramatically reduced in the spinal cords in
`FTY720-treated rats (Fig. 3, A and B), whereas very little
`reduction was seen in spleens of the FTY720- and control
`saline-administrated rats (Fig. 4, A and B).
`Adoptive Transfer of Protection against EAE. To un-
`derstand the mechanism involved in suppressing EAE by
`administering FTY720, we tested whether this lack of re-
`sponse could be adoptively transferred by spleen cells from
`the FTY720-treated donors. As shown in Fig. 5, the results
`demonstrated that Con A-activated splenocytes from rats
`administered saline successfully transferred EAE to naive
`recipient rats. In contrast, Con A-stimulated spleen cells
`from FTY720-treated donors transferred into naive recipient
`rats against EAE manifestation by reducing both disease
`incidence and clinical score (Fig. 5).
`
`Discussion
`Despite numerous advances in the past decade, the cause
`and pathogenesis of the inflammatory CNS demyelinating
`disorder MS remain unknown. EAE, an inflammatory CNS
`demyelinating disorder that serves as the prime animal
`model for MS, can be induced in a number of species by
`immunization with myelin components or injection of auto-
`immune T lymphocytes and has been used to study immune
`tolerance (Zamvil and Steinman, 1990). Recently, EAE re-
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`74
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`Fujino et al.
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`Fig. 3. RT-PCR for IL-2, IL-6, and INF-␥mRNAs
`in the spinal cords of the EAE Lewis rat. A,
`representative data show the intensity of RT-
`PCR products obtained from spinal cord samples
`in each group. Compared with control rats,
`mRNA expression for IL-2, IL-6, and INF-␥ de-
`creased in the FTY720-treated rats. B, the inten-
`sity of each band was calculated using Kodak
`Digital Science 1D image analysis software. The
`relative quantities of the genes are presented as
`the ratio of intensities of IL-2, IL-6, and INF-␥
`bands against those of the housekeeping gene
`␤-actin. Data are representative of three inde-
`pendent experiments and indicate the mean ra-
`tio of triplicate results in each experiment.
`
`Fig. 4. RT-PCR for IL-2, IL-6, and INF-␥mRNAs
`in the spleen of the EAE Lewis rat. A, represen-
`tative data show the intensity of RT-PCR prod-
`ucts obtained from spleen samples in each group.
`Compared with control rats, no decrease of
`mRNA expression for IL-2, IL-6, and INF-␥ was
`shown in the FTY720-treated rats. B, the rela-
`tive quantities of the genes in spleen were plot-
`ted against those of control as described in Fig. 3
`
`search has reached a stage on which a considerable range of
`new therapeutic strategies has emerged, and some of them
`may be very close to clinical application. A common thread in
`these strategies is that they could become useful for treating
`many different cell-mediated autoimmune diseases.
`CsA and FK506 are well known immunosuppressants and
`have contributed to preventing EAE. For instance, actively
`induced EAE can be inhibited by administering CsA orally at
`1 mg/kg/day (Bolton et al., 1982b; Deguchi et al., 1991);
`adoptive transfer-induced EAE can also be inhibited (Bolton
`et al., 1982a). Inamura et al. (1988) demonstrated that
`FK506, like CsA, also inhibited actively induced EAE. Bolton
`(1992) showed that inhibition of adoptive transfer-induce
`EAE using the drug. These immunosuppressants are known
`to exert their immunosuppressive activity by inhibiting the
`production of Th1-associated cytokines in Ag-stimulated T
`cells (Borel, 1990). Although CsA binds to cyclophilin and
`FK506 to FK506-binding protein, both cyclophilin/CsA and
`FK506-binding protein/FK506 complexes inhibit the phos-
`phatase activity of calcineurin that activates the nuclear
`factor of activated T cell involved in promoting IL-2 gene
`transcription (Liu et al., 1991). Because CsA and FK506
`affect the same process of T-cell activation, they exhibit quite
`
`similar side effects, such as renal and liver toxicities (Platz et
`al., 1994). Thus, CsA- or FK506-based multiple drug therapy
`with steroids or other immunosuppressants has been widely
`used to reduce the side effects of individual immunosuppres-
`sants in clinical situations (McWhinnie and Morris, 1991).
`These immunosuppressants also cause metabolic derange-
`ments and organ toxicities at therapeutic doses. Therefore,
`drug therapies to disable or eliminate only T cells that are
`involved in a particular disease would potentially be very
`useful. The latest progress of immunosuppressive therapy
`has brought enormous advantages not only in the field of
`organ transplantation but also in the treatment of allergic
`and autoimmune diseases.
`There is thus great interest in the recently characterized
`and potent immunosuppressant FTY720. FTY720 is a syn-
`thetic drug produced by modifying ISP-1 purified from cul-
`ture filtrates of I. sincrailii, an ascomycete. FTY720 has
`demonstrated a unique mechanism to trigger rat spleen cells
`and several cell lines undergoing apoptosis in in vitro sys-
`tems and in animal organ transplantation models and has an
`effective immunosuppressive activity for preventing allograft
`rejection without toxic side effects (Suzuki et al., 1996a,b).
`Through a mechanism completely different
`from CsA,
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`Amelioration of EAE by FTY720 Treatment
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`75
`
`FK506, DSG, and other conventional immunosuppressants,
`FTY720 prevents allograft rejection by inducing apoptosis
`cell death in peripheral lymphocytes (Suzuki et al., 1996b)
`and accelerating lymphocyte homing (Chiba et al., 1998;
`Yanagawa et al., 1998)
`The present study was undertaken to investigate effects of
`FTY720 upon the course and pathology of EAE, a T-cell-
`mediated demyelinating disease of the central nervous sys-
`tem. Kitabayashi et al. (2000) demonstrated that FTY720
`prevents development of experimental autoimmune myocar-
`ditis. Furthermore, similar autoimmune diseases such as
`experimental autoimmune thyroiditis (Hozumi et al., 1999),
`experimental autoimmune uveoretinitis (Kurose et al., 2000),
`autoimmune type I diabetes (Yan et al., 1998), and systemic
`lupus erythematosus (Okazaki et al., 2002) were prevented
`in FTY720-treated animals. Consistent with the above stud-
`ies, administration of FTY720 improved clinical scores dra-
`matically in EAE in Lewis rats (Fig. 1). As demonstrated in
`a previous study, the expression of adhesion molecules re-
`lated to T-cell trafficking is enhanced in spinal cords, and
`monoclonal antibodies of these molecules inhibit EAE dis-
`ease (Lee and Benveniste, 1999). Furthermore, increased
`T-cell infiltration of spinal cords has, in fact, been described
`in various reports (Sun et al., 2000). Therefore, infiltrated T
`cells were thought to be closely involved in the development
`of EAE (Hickey et al., 1991). The present study also con-
`firmed T-cell infiltration by immunohistochemical staining
`anti-T-cell receptor monoclonal antibody (Fig. 2). Other stud-
`ies using FTY720 and autoimmune disease models found
`T-cell elimination in the inflammation lesion (Yan et al.,
`1998; Hozumi et al., 1999; Kitabayashi et al., 2000; Kurose et
`al., 2000; Okazaki et al., 2002). Consistent with the above
`studies, we demonstrated a marked reduction in central ner-
`vous system damage and infiltrating cells in FTY720-treated
`rats compared with control rats (Fig. 2). Therefore, FTY720
`administration might inhibit EAE development by inhibiting
`encephalitogenic T-cell responses and/or their migration into
`the CNS. These findings have identified FTY720 as a possi-
`ble therapeutic agent for human MS.
`As previously reported (Bonetti et al., 1997), a number of
`apoptotic cells were invariably associated with clinical dis-
`ease in saline-treated control EAE rats. Our study found
`apoptotic cell death in the spinal cords of EAE rats but not in
`rats treated with FTY720. This observation was correlated
`with the lack of infiltration cells in the spinal cord.
`Patterns of cytokine expression in spinal cords of EAE
`Lewis rats have been reported previously, and the elevation
`of cytokine expressions in such tissues is believed to contrib-
`ute to pathology (Sun et al., 2000). These reports suggested
`that autoreactive T cells in spinal cords were activated by
`Th1-associated cytokines (IL-2, IL-6, and IFN-␥) but not Th2-
`associated cytokines (IL-4 and IL-10). Therefore, elevation of
`cytokine expressions was thought to be an important compo-
`nent of EAE disease in addition to the T-cell infiltration into
`the spinal cord. In our present EAE model, mRNA expres-
`sions of Th1-associated cytokines (IL-2, IL-6, and IFN-␥) in
`the spinal cords were markedly decreased in rats that had
`been administered FTY720 compared with control saline-
`treated rats (Fig. 3). It seems that a lack of infiltration in the
`spinal cord in EAE rats treated with FTY720 resulted from a
`lack of inflammation; so, cytokines were not up-regulated in
`the spinal cord. Therefore, these data indicated that FTY720
`
`Fig. 5. Prevention of adoptively transferred EAE by treatment with
`FTY720. Rats were adoptively transferred spleen cells from MBP/CFA-
`immunized rats treated with FTY720 (squares) or control saline (circles).
`A, survival; B, maximum clinical score; C, mean clinical score; D, body
`weight loss. Data are from two separate experiments.
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`Fujino et al.
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`inhibits the induction of at least three inflammatory cyto-
`kines in vivo by preventing T cells from infiltrating spinal
`cords. In contrast to spinal cords, the expression of cytokines
`was not inhibited in spleen with FTY720 treatment (Fig. 4).
`This is consistent with a previous article by Yanagawa et al.
`(1998). In that study, FTY720 significantly reduced the num-
`ber of peripheral blood T cells in skin-allografted rats. Fur-
`thermore, FTY720 markedly decreased T-cell infiltration
`into allografts while, in contrast to CsA, had little effect on
`IL-2 and IFN-␥ mRNA in expression in allografts.
`Pinschewer et al. (2000) reported that FTY720 impairs the
`circulation and homing of effect T cells to peripheral lesions
`without affecting the induction and expansion of immune
`responses in secondary lymphoid organs. To verify whether
`the above mechanism is involved in our model, we attempted
`to adoptively transfer to naive Lewis rat using spleen cells
`isolated from actively EAE-induced Lewis rats with FTY720
`treatment. Clinical EAE was adoptively transferred to cell
`recipients using the lymphocytes with saline-treated control
`rats, whereas EAE was not induced in spleen cells isolated
`from rats treated with FTY720 (Fig. 5). We therefore specu-
`lated that the MBP-specific spleen cells might not be in-
`cluded in cells transferred from FTY720-treated rats, al-
`though there is some possibility of inhibiting the induction of
`encephalitogenic T cells due to its inhibition of the encepha-
`litogenic T-cell migration and homing to peripheral organs
`including the spleen. Further studies are needed to clarify
`this.
`More recent studies found that FTY720 targets sphin-
`gosine 1-phosphate (S1P) receptors (Brinkmann et al., 2002;
`Mandala et al., 2002). Those studies demonstrated that
`FTY720 was phosphorylated by sphingosine kinase and that
`the phosphorylated compound is a potent agonist at four
`sphingosine 1-phosphate receptors, and the effects of FTY720
`are actively induced. Furthermore, the studies speculated
`that EAE might relate to a direct effect on neuronal cells
`and/or oligodendrocytes expressing S1P receptors. Because
`activation of S1P receptors can antagonize apoptotic pro-
`cesses, which are associated with early stages of progressive
`neurodegenerative and demyelinating diseases (Brinkmann
`et al., 2002). Consistent with the above reports, FTY720 both
`prevented active induction of EAE and adoptively trans-
`ferred EAE, a principally immune cell-mediated phenome-
`non. These data indicated that FTY720 administration might
`inhibit EAE development by inhibiting encephalitogenic T-
`cell responses and/or their migration into the CNS.
`In conclusion, our findings suggest that administration of
`FTY720 effectively prevents development of EAE in rat mod-
`els. Although this study did not precisely examine the ad-
`verse effects of the drug, none of the FTY720-treated rats
`died during the therapy, and the drug-treated rats gained
`body weight during therapy. The data suggested that
`FTY720 may be safety for the clinical situation. FTY720
`might be a candidate for treating patients with MS because
`of its strong capacity to suppress EAE and because of its
`therapeutic effects.
`
`Acknowledgments
`We gratefully acknowledge Chie Komatsu for expert technical
`assistance.
`
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