FTY720, a Novel Immunosuppressant, Induces Sequestration
`of Circulating Mature Lymphocytes by Acceleration of
`Lymphocyte Homing in Rats. I. FTY720 Selectively Decreases
`the Number of Circulating Mature Lymphocytes by
`Acceleration of Lymphocyte Homing
`
`Kenji Chiba,1 Yoshiki Yanagawa, Yumi Masubuchi, Hirotoshi Kataoka, Takafumi Kawaguchi,
`Makio Ohtsuki, and Yukio Hoshino
`FTY720, given i.v. or orally at 0.03 mg/kg or more, significantly prolonged skin allograft survival in a dose-dependent manner and
`showed more potent immunosuppressive activity than cyclosporin A (CsA) or tacrolimus (FK506) in MHC-incompatible rat
`strains of WKAH donors and F344 recipients. However, unlike CsA or FK506, FTY720 up to 1000 nM did not affect IL-2
`production in allogeneic MLC. Within 3 to 24 h after a single oral administration of FTY720 at 0.1 to 1 mg/kg, the number of
`lymphocytes in the rats was markedly decreased in the peripheral blood and thoracic duct lymph and partially in spleen. By
`contrast, the number of lymphocytes in peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN), and Peyer’s patches (PP)
`was significantly increased at the same time. Intravenous transfusion of calcein-labeled rat lymphocytes into rats revealed that
`FTY720 significantly accelerated lymphocyte homing to PLN, MLN, and PP, dose dependently. Since FTY720-induced lymphocyte
`homing was completely blocked by simultaneous treatment of the calcein-labeled lymphocytes with mAbs against CD62L, CD49d,
`and CD11a before the transfusion, the acceleration of lymphocyte homing by FTY720 appears to be mediated by lymphocyte-
`homing receptors. These findings indicate that FTY720 sequesters circulating mature lymphocytes into PLN, MLN, and PP by
`acceleration of lymphocyte homing and thereby decreases the number of lymphocytes in peripheral blood, thoracic duct lymph,
`and spleen. Based on these observations, sequestration of circulating mature-lymphocytes is presumed to be a main mechanism
`of the immunosuppressive activity of FTY720. The Journal of Immunology, 1998, 160: 5037–5044.
`
`widely used to reduce the side effects of individual immunosup-
`pressants in clinical organ transplantation (7, 8).
`We previously reported that a potent immunosuppressive com-
`pound, ISP-I, and its derivatives, mycestericins, were isolated from
`the culture broth of Isaria sinclairii, a species of vegetative wasp
`(9, 10). Chemical modification of ISP-I led to a novel synthetic
`immunosuppressant, 2-amino-2-[2-(4-octylphenyl)ethyl]propane-
`1,3-diol hydrochloride (FTY720), which has more potent immu-
`nosuppressive activity and less toxicity than ISP-I
`(11–14).
`FTY720, at 0.1 mg/kg or more, significantly prolonged skin or
`cardiac allograft survival and host survival in lethal graft-vs-host
`reaction in rats (15–17). In addition, combination treatment with
`FTY720 and a subtherapeutic dose of CsA resulted in a synergistic
`effect on canine renal allografts as well as rat skin or cardiac al-
`lografts (15, 16, 18, 19). A striking feature of FTY720 is induction
`of a marked decrease in the number of PBL, especially T cells, at
`doses that prolong allograft survival (15, 16). It has been hypoth-
`esized that the decrease in lymphocyte number is caused by apo-
`ptotic cell death of lymphocytes, because FTY720 at 4 ␮M (1.4
`␮g/ml) or more induces apoptosis of rat spleen cells and human
`peripheral blood cells in vitro (20, 21). However, the trough level
`of the blood concentration of FTY720 in dogs given 5 mg/kg is
`less than 200 ng/ml (21). In addition, the blood concentration
`range of FTY720 is 0.2 to 20 ng/ml when given to rats at 0.1
`mg/kg to 1 mg/kg (unpublished data from our laboratories). Thus,
`the hypothesis concerning FTY720-induced apoptosis is insuffi-
`cient to explain the intrinsic mechanism of the decreasing effect on
`
`C yclosporin A (CsA)2 and tacrolimus (FK506) have made
`
`great contributions to the prevention of acute rejection in
`human organ transplantations (1, 2). Both of these two
`immunosuppressants are known to exert their immunosuppressive
`activity by inhibiting the production of Th1-associated cytokines
`in Ag-stimulated helper T cells (3–5). Although CsA and FK506
`bind to different proteins, cyclophilin and FK506 binding protein
`(FKBP), respectively, both cyclophilin/CsA and FKBP/FK506
`complexes inhibit the phosphatase activity of calcineurin, which
`activates NF-AT involved in the promotion of IL-2 gene transcrip-
`tion (6). Because CsA and FK506 affect the same process of T cell
`activation, they show quite similar side effects, such as renal and
`liver toxicities (1, 2). Thus, CsA- or FK506-based multiple drug
`therapy with steroids or other immunosuppressants has been
`
`Industries, Limited,Iruma,
`
`Research Laboratories, Yoshitomi Pharmaceutical
`Saitama, Japan
`Received for publication October 17, 1997 Accepted for publication January
`12, 1998
`The costs of publication of this article were defrayed in part by the payment of page
`charges This article must therefore be hereby marked advertisement in accordance
`with 18 U S C Section 1734 solely to indicate this fact
`1 Address correspondence and reprint requests to Dr Kenji Chiba, Research Labo-
`ratories, Yoshitomi Pharmaceutical Industries, Ltd 3–7–25, Koyata, Iruma, Saitama,
`358-0026 Japan E-mail address: chiba@yoshitomi co jp
`2 Abbreviations used in this paper: CsA, cyclosporin A; FK506, tacrolimus; TDL,
`thoracic duct lymph; PLN, peripheral lymph nodes; MLN, mesenteric lymph nodes;
`PP, Peyer’s patches; HEV, high endothelial venules; FTY720, 2-amino-2-[2-(4-oc-
`tylphenyl)ethyl]propane-1,3-diol hydrochloride; p o , by mouth; PE, phycoerythrin
`
`Copyright © 1998 by The American Association of Immunologists
`
`0022-1767/98/$02 00
`
`Apotex v. Novartis
`IPR2017-00854
`NOVARTIS 2034
`
`

`

`5038
`
`FTY720 ACCELERATES LYMPHOCYTE HOMING IN RATS
`
`FIGURE 1. The chemical structure of FTY720
`
`PBL number by FTY720, because it is clearly impossible for
`FTY720 to induce apoptotic cell death of lymphocytes at a dose
`range of 0.1 to 1 mg/kg in vivo.
`The immunologically mature lymphocytes are known to contin-
`uously recirculate in the peripheral blood, spleen, lymphatic ves-
`sels, TDL, PLN, MLN, and PP (22). It has also been reported that
`lymphocyte recirculation is regulated by lymphocyte trafficking
`via lymphocyte-homing receptors, including CD62L, CD49d/␤7
`integrin, and CD11a/CD18 (22–26). We focused on lymphocyte
`recirculation in vivo and hypothesized that, if lymphocyte homing
`is modulated by FTY720, the number of PBL would decrease
`without death of lymphocytes. In this paper, we will describe that
`the marked decrease in the number of PBL induced by FTY720 is
`due to acceleration of lymphocyte homing to PLN, MLN, and PP
`via lymphocyte-homing receptors.
`
`Materials and Methods
`Animals
`Inbred male F344 rats (RT1lv1) and WKAH rats (RT1k) were pur-
`chased from Japan Charles River (Atsugi, Kanagawa, Japan) and
`Japan SLC (Hamamatsu, Shizuoka, Japan), respectively. All rats
`were used at 4 to 12 wk of age.
`
`Cell lines
`A mouse IL-2-dependent cytotoxic T cell line, CTLL-2 (27), was obtained
`from the American Type Culture Collection (ATCC, Rockville, MD) and
`maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine,
`10 mM HEPES, 100 U/ml penicillin, 60 ␮g/ml kanamycin sulfonate, 50
`␮M 2-ME, 10% FCS (Boehringer Mannheim, Mannheim, Germany) and
`20% Con A-stimulated rat spleen cell culture supernatant prepared by the
`method previously described (28)
`Reagents
`FTY720 was synthesized according to the method previously described
`(13) The chemical structure of FTY720 is shown in Figure 1 For i v
`injection, FTY720 was dissolved in 1% ␣-cyclodextrin and 5% mannitol
`solution CsA (Sandimmun, for i v injection; Sandoz, Basel, Switzerland)
`and FK506 (Prograf, for i v injection, Fujisawa Pharmaceutical, Osaka,
`Japan) was diluted with saline For oral administration, FTY720 was dis-
`solved in distilled water CsA (Sandimmun, for oral solution) and FK506
`(Prograf, for i v injection) were diluted with olive oil (Sigma Chemicals,
`St Louis, MO) and with distilled water, respectively Control animals re-
`ceived the vehicle only For in vitro treatments, FTY720, CsA (Sandim-
`mun, for i v injection) and FK506 (Prograf, for i v injection) were dis-
`solved in saline and diluted to the appropriate concentrations with RPMI
`1640 medium containing 10% FCS
`Monoclonal Abs
`FITC-conjugated anti-rat CD3 mAb (1F4; Ref 29) was obtained from
`Caltag Laboratories (South San Francisco, CA) Biotinylated anti-rat CD3
`mAb (G4 18; Ref
`30) and phycoerythrin (PE)-conjugated anti-rat
`CD45RA or A/B mAb (OX-33; Ref 31), FITC-conjugated anti-rat CD4
`mAb (OX-38; Ref 32), PE-conjugated anti rat CD8 mAb (OX-8; Ref 33)
`and streptavidin-Cy-chrome conjugate were obtained from PharMingen
`(La Jolla, CA) Hamster anti-rat CD62L mAb (HRL3; Ref 34), mouse
`anti-rat CD49d mAb (TA-2; Ref 35), mouse anti-rat CD11a mAb (clone
`WT 1; Ref 36), and biotinylated TA-2 were purchased from Seikagaku-
`kogyo (Tokyo, Japan) PE-conjugated HRL-3, PE-conjugated WT 1, and
`isotype-matched control IgGs were obtained from PharMingen
`
`Rat skin allograft
`MHC-incompatible rat skin allograft was performed by the method de-
`scribed previously, with WKAH rats (RT1k) as donors and F344 rats
`(RT1lv1) as recipients (15) Briefly, full-thickness skin grafts (2 0 ⫻ 2 0
`cm2 pieces) from donor rats were transplanted to the lateral thorax of the
`recipient rats and covered with sterile bactericidal gauze The entire chest
`was then wrapped with an elastic bandage The dressings were removed on
`day 5 and the grafts were inspected daily until rejection, which was defined
`as more than 90% necrosis of the graft epithelium FTY720, CsA, or
`FK506 was administered daily to the allografted recipients for 14 days
`from the day of transplantation
`IL-2 production in allogeneic MLC in rats
`Effects on IL-2 production in rat allogeneic MLC were evaluated according
`to the method previously reported (28) Allogeneic MLC was performed by
`using nylon nonadherent splenic lymphocytes from F344 rats as responder
`cells and mitomycin C (Kyowa Hakko, Tokyo, Japan)-pretreated spleen
`cells from WKAH rats as stimulator cells Responder cells at 5 ⫻ 106
`cells/well were cocultured with an equal number of stimulator cells in 2 0
`ml of RPMI 1640 medium containing 50 ␮M 2-ME and 10% FCS After
`culturing for 48 h at 37°C in 5% CO2, the culture supernatants were col-
`lected, and their IL-2 activities were determined by CTLL-2 proliferation
`assay (27) Briefly, CTLL-2 cells (104 cells/well) were cultured in the
`presence of serial twofold dilution of culture supernatants for 20 h at 37°C,
`pulsed with 0 5 ␮Ci of [3H]thymidine (TdR) (Amersham, Tokyo, Japan)
`for 4 h at 37°C in 5% CO2 and then harvested onto glass fiber filters using
`an automatic cell harvester The radioactivity incorporated into the cells
`was determined by a scintillation counter (1450 MicroBeta; Pharmacia
`Biotech, Uppsala, Sweden) IL-2 activity in the supernatants is expressed
`as U/ml in comparison with recombinant rat IL-2 (Genzyme, Cambridge
`MA) as a standard (27)
`Flow cytometry
`Peripheral blood was collected from the tail veins of F344 rats Spleen,
`PLN (axillary lymph nodes were used as PLN in this study), MLN, and PP
`were removed from rats, and single cell suspensions were prepared by
`mincing and passing through stainless mesh Lymphocytes in TDL were
`collected by cannulation of thoracic duct under anesthesia according to the
`method described previously (37) Flow cytometry analysis was performed
`by using EPICS XL-MCL (Coulter, Miami, FL) Lymphocytes from rat
`peripheral blood, TDL, spleen, PLN, MLN, or PP were stained with FITC-
`anti-rat CD3 mAb (1F4) and PE-anti-rat CD45RA or A/B mAb (OX-33),
`which is reported to bind B cells only (31) The number of total lympho-
`cytes was determined by the lymphocyte gating method The numbers of T
`
`FIGURE 2. Dose-response relationship between FTY720, CsA, and
`FK506 and skin allograft survival in an MHC-incompatible rat strain sys-
`tem A, i v administration; B, oral administration MHC-incompatible rat
`skin allograft was performed using WKAH rats (RT1k) as donors and F344
`rats (RT1lv1) as recipients Full-thickness skin grafts (2 0 ⫻ 2 0 cm2) from
`donor rats were transplanted to the lateral thorax of recipient rats The
`grafts were inspected daily until rejection, which was defined as more than
`90% necrosis of the graft epithelium FTY720, CsA, or FK506 was ad-
`ministered to the allografted recipients for 14 days after the transplantation
`Each symbol represents the mean ⫾ SE of eight animals The statistical
`differences in allograft survival time compared with vehicle-treated control
`group were calculated by the generalized Wilcoxon test with Hommel’s
`multiple comparison test (* p ⬍ 0 05)
`
`

`

`The Journal of Immunology
`
`5039
`
`Results
`Effects of FTY720, CsA, and FK506 on skin allograft survival in
`MHC-incompatible strain combination in rats
`To clarify the efficacy and potency of the immunosuppressive ac-
`tivity of FTY720, the prolonging effect of FTY720, CsA, and
`FK506 on rat skin allograft survival was examined in MHC-in-
`compatible rat strains of WKAH donors and F344 recipients. The
`immunosuppressants were administered i.v. or orally for 14 days
`from the day of the transplantation. In this skin allograft models,
`all grafts in the control (vehicle-treated) group were rejected 6 to
`7 days after transplantation. As shown in Figure 2, FTY720 at 0.03
`mg/kg or higher doses significantly prolonged allograft survival in
`a dose-dependent manner by either i.v. or oral administration. CsA
`and FK506 were also effective at doses of 3 mg/kg or more and 0.3
`mg/kg or more, respectively, in this model. These results indicate
`that FTY720 possesses more potent immunosuppressive activity
`than CsA or FK506 on allograft rejection in an MHC-incompatible
`combination.
`
`Effects of FTY720, CsA, and FK506 on IL-2 production in rat
`allogeneic MLC
`The effect of FTY720 on IL-2 production in allogeneic MLC in
`rats was examined in comparison with that of CsA and FK506. The
`results are shown in Figure 3. CsA and FK506 dose-dependently
`inhibited IL-2 production in rat allogeneic MLC, consistent with
`the results of previous studies in mice and humans (3–5). The IC50
`values (concentrations that inhibit 50%) of CsA and FK506 for
`IL-2 production were 3.5 nM and 0.043 nM, respectively. By con-
`trast, FTY720 up to 1000 nM did not affect IL-2 production in rat
`allogeneic MLC (Fig. 3) and failed to inhibit T cell proliferation by
`
`FIGURE 4. Time course of numbers of lymphocytes, T cells, and B
`cells in blood, TDL, and spleen in rats following a single oral administra-
`tion of FTY720 FTY720 was administered orally to F344 rats, and pe-
`ripheral blood, TDL, and spleen were periodically collected Lymphocytes
`of blood, TDL, and spleen were stained with FITC-anti-rat CD3 mAb
`(1F4) and PE-anti-rat CD45RA or A/B mAb (OX-33) The numbers of
`total lymphocytes, T cells, and B cells were determined by two-color flow
`cytometry 䢇, FTY720 at 0 1 mg/kg p o ; Œ, FTY720 at 1 mg/kg p o Each
`symbol represents the mean ⫾ SE of four animals Statistical significance
`was calculated by Dunnett’s test (*: p ⬍ 0 05, **: p ⬍ 0 01 vs vehicle-
`treated control group)
`
`FIGURE 3. Effect of FTY720, CsA, and FK506 on IL-2 production in
`rat allogeneic MLC Allogeneic MLC was performed by using nylon non-
`adherent splenic lymphocytes from F344 rats (RT1lv1) as responder cells
`and mitomycin C-pretreated spleen cells from WKAH rats (RT1k) as stim-
`ulator cells Responder cells (5 ⫻ 106 cells/well) were cocultured with an
`equal number of stimulator cells in 2 0 ml of RPMI 1640 medium con-
`taining 50 ␮M 2-ME and 10% FCS After culturing for 48 h, the super-
`natants were collected and were assessed for IL-2 activity by CTLL-2
`proliferation assay IL-2 activity is expressed as U/ml (mean ⫾ SE of
`triplicate determinations), with recombinant rat IL-2 as a standard Statis-
`tical differences were calculated by Dunnett’s test (**: p ⬍ 0 01 vs culture
`with medium alone)
`
`cells and B cells were determined by two-color flow cytometry The pro-
`portions of CD4⫹ T cells and CD8⫹ T cells were determined by three-color
`flow cytometry using biotinylated-anti-rat CD3 mAb (G4 18), FITC-anti-
`rat CD4 (OX-38), PE-anti-rat CD8 (OX-8), and streptavidin-Cy-chrome
`conjugate Expression of lymphocyte-homing receptors on rat T cells was
`determined by two-color flow cytometry using FITC-anti-rat CD3 (IF4),
`PE-anti-rat CD62L mAb (HRL3), biotinylated-anti-rat CD49d mAb (TA-
`2), PE-anti-rat CD11a mAb (WT 1), and streptavidin-Cy-Chrome
`conjugate
`
`Analysis of lymphocyte homing with calcein-labeled lymphocytes
`Lymphocytes (1 ⫻ 108 cells) from PLN and MLN of F344 rats were
`labeled by incubation for 30 min on ice in 10 ml of RPMI 1640 medium
`containing 0 2 ␮M calcein-AM (Molecular Probes, Eugene, OR) (38) Af-
`ter labeling with calcein, the viability of lymphocytes was more than 94%
`by trypan blue dye exclusion test The calcein-labeled lymphocytes (5 ⫻
`107 cells/0 5 ml) were i v transfused at 2 5 h after FTY720 administration
`to F344 rats Then, the rats were sacrificed 30 min after the transfusion, and
`the peripheral blood, spleen, PLN, MLN, and PP were collected The num-
`ber of calcein-labeled lymphocytes in these tissues was determined by flow
`cytometry To examine the effect of mAbs against lymphocyte-homing
`receptors, the calcein-labeled lymphocytes were treated with 60 ␮g/ml of
`hamster anti-rat CD62L mAb (HRL3), mouse anti-rat CD49d mAb (TA-2),
`mouse anti-rat CD11a mAb (WT 1), or all three mAbs at 4°C for 30 min
`As a control, isotype-matched irrelevant IgGs were used under the same
`conditions After the treatment of mAbs, the calcein-labeled lymphocytes
`were transfused i v to the rats, and their tissue distribution was determined
`as described above
`
`Statistical analysis
`The statistical differences in allograft survival time compared with vehicle-
`treated control group were calculated by the generalized Wilcoxon test
`with Hommel’s multiple comparison test In other experiments, statistical
`differences compared with the vehicle-treated control were calculated by
`Dunnett’s test Differences between groups were considered significant at
`p ⬍ 0 05
`
`

`

`5040
`
`FTY720 ACCELERATES LYMPHOCYTE HOMING IN RATS
`
`plete recovery within 2 wk (data not shown). By contrast, the num-
`bers of lymphocytes in PLN, MLN, and PP were significantly in-
`creased in a dose-dependent manner after administration of
`FTY720 (Fig. 5). The number of PLN lymphocytes reached a max-
`imum at 12 h after FTY720 administration and returned to the
`control level by 24 h. Lymphocytes in MLN and PP also increased
`to 180% and 300%, respectively, of the controls at 24 h after
`FTY720 administration and then returned to the control level
`within 5 days. The time courses of the numbers of T cells and B
`cells in PLN, MLN, and PP were similar to that of the total lym-
`phocyte number. The increase in numbers of T cells was especially
`marked in PLN, MLN, and PP. These findings indicate that
`FTY720 modulates the tissue distribution of lymphocytes in blood,
`TDL, spleen, PLN, MLN, and PP in rats. Figure 6, A and B, shows
`the proportions of T cells, B cells, or T cell subsets (CD4⫹ or
`CD8⫹ T cells) in these lymphoid tissues 12 h after administration
`of FTY720. The numbers of T cells, B cells, CD4⫹ T cells or
`CD8⫹ T cells showed changes similar to the total numbers of
`lymphocytes in all tissues tested. By contrast, FTY720 did not
`
`FIGURE 5. Time course of numbers of lymphocytes, T cells, and B
`cells in PLN, MLN, and PP in rats following a single oral administration
`of FTY720 FTY720 was administered orally to F344 rats, and PLN, MLN,
`and PP were periodically collected Lymphocytes of PLN, MLN, and PP
`were stained with FITC-anti-rat CD3 mAb (1F4) and PE-anti-rat CD45RA
`or A/B mAb (OX-33) The numbers of total lymphocytes, T cells, and B
`cells were determined by two-color flow cytometry 䢇, FTY720 at 0 1
`mg/kg ; Œ, FTY720 at 1 mg/kg Each symbol represents the mean ⫾ SE of
`four animals Statistical significance was calculated by Dunnett’s test (*:
`p ⬍ 0 05, **: p ⬍ 0 01 vs vehicle-treated control group)
`
`alloantigen stimulation or IL-2-dependent proliferation of CTLL-2
`cells (data not shown). These findings suggest that FTY720 exerts
`a potent immunosuppressive effect by a mechanism distinct from
`that of CsA and FK506.
`Lymphocytes were selectively decreased by FTY720 in blood,
`TDL, and spleen, but increased in PLN, MLN, and PP
`As described in our previous papers, FTY720 significantly de-
`creases the number of PBLs in allografted rats, especially the num-
`ber of T cells (15, 16). To clarify the mechanism of the decrease
`in the number of lymphocytes by FTY720, the tissue distribution
`of lymphocytes was analyzed in the peripheral blood, TDL, spleen,
`PLN, MLN, and PP of F344 rats, following a single oral admin-
`istration of FTY720 (0.1 and 1 mg/kg). The proportions of lym-
`phocytes, T cells, T cell subsets (CD4⫹ or CD8⫹ T cells), and B
`cells were determined by flow cytometry. Figure 4 shows the time
`course of lymphocyte, T cell, and B cell numbers in blood, TDL,
`and spleen after a single oral administration of FTY720. The num-
`bers of total lymphocytes, T cells, and B cells in blood and TDL
`dramatically decreased to less than 10% of the control values
`within 3 to 24 h after administration. The decrease in the number
`of lymphocytes by FTY720 was more marked in TDL than in
`peripheral blood. The numbers of splenic lymphocytes, T cells,
`and B cells were also significantly decreased by FTY720 treatment
`to 40% to 80% of the control with a time course similar to that of
`the PBLs. Lymphocyte numbers in blood and spleen had recovered
`to the control level on day 7 after administration. Although the
`number of lymphocytes in TDL was still decreased to 20% to 40%
`of the control by FTY720 administration, there was almost com-
`
`FIGURE 6. Effect of FTY720 on the numbers of various cells in
`blood and lymphoid tissues in rats 12 h after a single oral administration
`of FTY720 A, T cells and B cells in blood, TDL, spleen, PLN, MLN,
`and PP; B, CD4⫹ T cells and CD8⫹ T cells in blood, spleen, PLN,
`MLN, and PP; C, red blood cells (RBC) in blood, thymocyte subpopu-
`lations, and bone marrow (BM) cells Each column represents the mean
`of four animals
`
`

`

`The Journal of Immunology
`
`5041
`
`anti-CD49d or CD11a mAb. Unlike normal lymphocyte hom-
`ing, CD62L mAb inhibited the FTY720-induced lymphocyte
`homing by 85% in PP, 70% in MLN, and 50% in PLN. In
`addition, FTY720-induced lymphocyte homing is almost com-
`pletely blocked (⬎90% inhibition) by simultaneous treatment
`with mAbs against CD49d, CD62L, and CD11a in PLN, MLN,
`and PP. These results suggest that FTY720-induced accelera-
`tion of lymphocyte homing, as well as normal lymphocyte hom-
`ing, is mediated by lymphocyte-homing receptors, including
`CD62L, CD49d/␤7 integrin, and CD11a/CD18.
`Effect of FTY720 on expression of CD62L, CD49d, and CD11a
`on lymphocytes in rats following single oral administration
`Since the acceleration of lymphocyte homing by FTY720 appears
`to be mediated by lymphocyte-homing receptors, there is a possi-
`bility that FTY720 up-regulates the expression of lymphocyte-
`homing receptors on lymphocytes. To determine whether FTY720
`induces up-regulation, the expression of CD62L, CD49d, and
`CD11a on T cells was analyzed in rats at 1 h in blood and at 3 h
`in MLN after single oral administration of FTY720. The expres-
`sion of these receptors could not be determined in blood at 3 h after
`
`FIGURE 8. The influence of anti-lymphocyte homing-receptor mAbs
`on FTY720-induced lymphocyte homing in rats The calcein-labeled
`lymphocytes were treated with 60 ␮g/ml of hamster anti-rat CD62L
`mAb (HRL3), mouse anti-rat CD49d mAb (TA-2), mouse anti-rat
`CD11a mAb (WT 1), or all three mAbs at 4°C for 30 min As a control,
`isotype-matched irrelevant IgGs were used under the same conditions
`After treatment with mAbs, calcein-labeled lymphocytes were trans-
`fused i v into the rats, and tissue distribution of lymphocytes was de-
`termined by flow cytometry A, normal lymphocyte homing; B, FTY720
`(1 mg/kg p o )-induced lymphocyte homing Each column represents
`the mean ⫾ SE of four animals
`
`FIGURE 7. Effect of FTY720 on lymphocyte homing of calcein-labeled
`lymphocytes in rats The calcein-labeled lymphocytes (5 ⫻ 107 cells/0 5
`ml) were transfused i v into F344 rats 2 5 h after oral administration of
`FTY720 The rats were sacrificed 30 min later, and peripheral blood,
`spleen, PLN, MLN, and PP were collected The number of calcein-labeled
`lymphocytes in these tissues was determined by flow cytometry Each col-
`umn represents the mean ⫾ SE of four animals Statistical significance was
`calculated by Dunnett’s test (*: p ⬍ 0 05, **: p ⬍ 0 01 vs vehicle-treated
`control group)
`
`cause any clear changes in the number of red blood cells, thymo-
`cytes (CD4⫹, CD8⫹ or CD4⫹CD8⫹ subpopulation), or bone mar-
`row cells 12 h after administration of FTY720 (Fig. 6C). Thus, the
`changes in lymphocyte distribution induced by FTY720 appear to
`be specific for mature lymphocytes but do lack selectivity for T
`cells, B cells, or T cell subsets.
`
`FTY720 accelerates lymphocyte homing to PLN, MLN, and PP
`To determine the effect of FTY720 on lymphocyte trafficking
`between blood to various lymphoid tissues in rats, calcein-la-
`beled lymphocytes were i.v. transfused to the strain- and sex-
`matched F344 rats 2.5 h after administration of FTY720. The
`rats were sacrificed 30 min later, and the tissue distribution of
`calcein-labeled lymphocytes was determined by flow cytom-
`etry. Figure 7 shows the number of calcein-labeled lymphocytes
`found in the PLN, MLN, PP, spleen, and blood of rats given
`FTY720 (0.1 and 1 mg/kg) orally as compared with vehicle-
`treated control rats. FTY720 significantly increased the number
`of calcein-labeled lymphocytes in PLN, MLN, and PP in a dose-
`dependent manner but decreased in spleen and blood. These
`results indicate that FTY720 accelerates lymphocyte homing
`from blood or spleen to PLN, MLN, and PP. In addition, the
`involvement of lymphocyte-homing receptors in FTY720-in-
`duced acceleration of lymphocyte homing was assessed by pre-
`treatment with mAb against CD62L, CD49d, or CD11a with
`calcein-labeled lymphocytes. With a similarity to the results in
`previous studies (39, 40), anti-CD62L mAb and CD49d mAb
`prevented normal
`lymphocyte homing to PLN and MLN,
`whereas CD11a mAb partially inhibited homing to PLN, MLN,
`and PP (Fig. 8A). Anti-CD62 mAb inhibited normal lympho-
`cyte homing by 90% in PLN, 70% in MLN, and 50% in PP.
`Treatment with CD49d mAb, on the other hand, resulted in
`inhibition of normal lymphocyte homing by 20% in PLN, 60%
`in MLN, and 80% in PP. Treatment with anti-CD11a mAb dis-
`played a partial inhibition of normal lymphocyte homing by
`35% to 60% in these lymphoid tissues. Simultaneous treatment
`with these mAbs resulted in marked inhibition (⬎90%) of nor-
`mal lymphocyte homing in these lymphoid tissues. As shown in
`Figure 8B, FTY720-induced lymphocyte homing, as well as
`normal lymphocyte homing, was prevented by treatment with
`
`

`

`5042
`
`FTY720 ACCELERATES LYMPHOCYTE HOMING IN RATS
`
`ever, steroids and cyclophosphamide markedly decrease the
`number of PBLs and immunologically incompetent thymocytes
`associating atrophy of thymic cortex. By contrast, FTY720 did
`not have any clear effect on the numbers of thymocytes and
`bone marrow cells in rats (Fig. 6C). We also confirmed that
`FTY720 did not affect the corticosteriod levels in peripheral
`blood (our unpublished data). From these findings, we presume
`that the decrease in the number of PBLs by FTY720 is not due
`to inhibition of intrathymic differentiation of T cells or corti-
`costeriod induction in vivo. Thus, the decrease in the number of
`lymphocytes by FTY720 is likely to be selective for immuno-
`logically mature lymphocytes, which have a capability of lym-
`phocyte trafficking between blood and lymphoid tissues, recog-
`nizing foreign antigens, and inducing both cell-mediated and
`humoral immune responses.
`The time course studies of lymphocyte number in blood and
`lymphoid tissues revealed that lymphocytes decreased in blood,
`TDL, and spleen but increased in PLN, MLN, and PP within 3
`to 24 h after FTY720 administration to rats (Figs. 4 and 5). In
`addition, the results of lymphocyte-trafficking studies by trans-
`fusion of calcein-labeled lymphocytes confirmed that the traf-
`ficking of circulating lymphocytes to PLN, MLN, and PP in rats
`was accelerated at 3 h after FTY720 administration (Fig. 7).
`From these findings, we conclude that FTY720 sequesters cir-
`culating mature lymphocytes into PLN, MLN, and PP by ac-
`celeration of lymphocyte homing and thereby decreases the
`number of lymphocytes in blood, TDL, and spleen.
`Lymphocyte trafficking between blood and lymphoid tissues,
`including PLN, MLN, and PP is known to be regulated and
`dependent on the expression of specific cell surface adhesion
`molecules (22–26, 39, 40). Recirculation of lymphocytes con-
`sists of trafficking from blood to PLN, MLN, and PP and re-
`turning to peripheral blood via lymphatic vessels and TDL. The
`homing of circulating lymphocytes to PLN, MLN, and PP was
`reported to be mediated by the attachment of lymphocytes to
`HEV in these lymphoid tissues (22–26). The attachment of lym-
`phocytes to HEV is involved in adhesion between lymphocyte-
`homing receptors, including CD62L (L-selectin), CD49d/␤7 in-
`tegrin (lymphocyte PP adhesion molecule-1 (LPAM-1)), and
`CD11a/CD18 (LFA-1), and their ligands mucosal addressin cell
`adhesion molecule-1 (MAdCAM-1), glycosylation-dependent
`cell adhesion molecule-1 (GlyCAM-1), and ICAM-1 (22–26,
`39, 40). In the present study, FTY720-induced lymphocyte
`homing to PLN, MLN, and PP was completely blocked by si-
`multaneous treatment with mAbs against CD49d, CD62L, and
`CD11a (Fig. 8). These findings indicate that FTY720 acceler-
`ates lymphocyte homing mediated by lymphocyte-homing re-
`ceptors,
`including CD62L, CD49d/␤7 integrin, and CD11a/
`CD18. However, anti-CD62L-mAb treatment
`resulted in
`different patterns between normal and FTY720-induced lym-
`phocyte homings to PLN, MLN, and PP in rats. Normal lym-
`phocyte homing to PP is predominantly mediated by CD49d/␤7
`integrin, and partially by CD62L. In contrast, involvement of
`CD62L appeared to be more dominant in FTY720-induced lym-
`phocyte homing than in normal lymphocyte homing to PP. In
`other experiments, expression of CD62L, CD49d, and CD11a
`was unaffected by FTY720. Based on these results, there is a
`possibility that FTY720 promotes adhesion between lympho-
`cytes and HEV by enhancing avidity/affinity between adhesion
`molecules and ligands, including activation of integrins. Since
`GlyCAM-1 (43) and macrophage inflammatory protein-1 (MIP-
`1␤) (44) have been reported to be triggering molecules for in-
`tegrin activation, FTY720 may promote adhesion between lym-
`phocytes and HEV by induction of these triggering molecules.
`
`FIGURE 9. Effect of FTY720 on expression of CD62L, CD49d, and
`CD11a on rat T cells Lymphocytes from blood and MLN were stained
`with FITC-anti-rat CD3 (IF4), PE-anti-rat CD62L mAb (HRL3), bioti-
`nylated-anti-rat CD49d mAb (TA-2), PE-anti-rat CD11a mAb (WT 1),
`and streptavidin-Cy-chrome conjugate Expression of lymphocyte-hom-
`ing receptors on T cells was determined by two-color flow cytometry at
`1 h in blood or at 3 h in MLN after a single oral administration of
`FTY720 to rats
`
`administration, because of the disappearance of T cells from the
`peripheral blood. Figure 9 shows the typical CD62L, CD49d, and
`CD11a levels on T cells of rats given FTY720 at 1 mg/kg orally.
`FTY720 did not have any up-regulating effect on CD62L, CD49d,
`or CD11a expression on T cells in blood or MLN.
`
`Discussion
`In this present study, we documented that FTY720 shows a
`powerful immunosuppressive effect in MHC-incompatible rat
`skin allografts and has more potent immunosuppressive activity
`than CsA and FK506, whether administered i.v. or orally (Fig.
`2). CsA and FK506 are known to exert immunosuppressive ac-
`tivity by inhibiting the production of Th1 cell-associated cyto-
`kines in Ag-stimulated helper T cells (3, 4). As reported pre-
`viously, FTY720, unlike CsA, did not inhibit IL-2 production
`by Con A-stimulated T cells in rats (15). Consistent with the
`results of mitogen-stimulated IL-2 production, FTY720 did not
`affect IL-2 production by alloantigen stimulation in rats (Fig.
`3). Since FTY720 did not affect the process of T cell activation,
`including IL-2 production or IL-2-dependent T cell prolifera-
`tion, FTY720 presumably possesses a unique immunosuppres-
`sive mechanism of action distinct from that of CsA and FK506
`and thus shows a synergistic effect on allograft survival when
`combined with CsA (15, 16).
`The most striking feature of FTY720 is the induction of a
`dramatic decrease in number of lymphocytes in periphera