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The significance of timing of FTY720 administration on the immunosuppressive effect to prolong rat skin allograft survival
International Journal of Immunopharmacology 22 (2000) 597±602 www.elsevier.com/locate/ijimmpharm
The signi®cance of timing of FTY720 administration on the immunosuppressive eect to prolong rat skin allograft survival Yoshiki Yanagawa, Yukio Hoshino, Kenji Chiba* Tokyo Laboratories, Yoshitomi Pharmaceutical Industries Ltd., 3-7-25 Koyata, Iruma, Saitama 358-0026, Japan Received 3 December 1999; accepted 15 February 2000
Abstract FTY720, a potent immunosuppressant, dramatically decreases the number of peripheral blood lymphocytes within a few hours after administration. The current study assessed the signi®cance of timing of FTY720 administration on the immunosuppressive eect to prolong rat skin allograft survival (WKAH donor to F344 recipient). The median survival time of allografts was 7 days in the control recipients. FTY720 (1 mg/kg/day) signi®cantly prolonged allograft survival when administered from days 0 and 3, but failed to exert an immunosuppressive eect when administered from day 4. Intragraft T cells, especially CD8+ T cells, were markedly increased in number from day 4 to 6, peaking on day 5 in control recipients. FTY720 markedly decreased the number of intragraft CD8+ T cells on day 5 when administered from days 0 and 3. In recipients administered with FTY720 from day 4, the number of intragraft CD8+ T cells were only partially decreased on day 5. Intragraft CD8+ T-cell number in those recipients on day 5 was almost the same as that in control recipients on day 4. In addition, FTY720 did not aect the increase in frequency of CD25+ cells in the CD8+ T-cell subset in allografts. It is likely that recipients treated with FTY720 from day 4 reject allografts by intragraft immune responses involved in CD8+ T cells which had in®ltrated before day 4, similar to control recipients. These ®ndings suggest that FTY720 should be administered before increase in T cell in®ltration into grafts to inhibit acute allograft rejection. 7 2000 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. Keywords: FTY720; Immunosuppressant; Skin allograft; T cell in®ltration
1. Introduction 2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-
* Corresponding author. Tel.: +81-42-963-3121; fax: +8142-964-1906.
diol hydrochloride (FTY720), a novel immunosuppressant, is a synthetic structural analog of ISP-1, a metabolite of the ascomycete Isaria sinclairii [1±3]. FTY720 at 0.1±1 mg/kg shows more potent immunosuppressive activity than cyclosporin A and FK506, and exerts a synergistic eect with cyclosporin A to prolong allograft
0192-0561/00/$20.00 7 2000 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 2 - 0 5 6 1 ( 0 0 ) 0 0 0 2 2 - 9
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Y. Yanagawa et al. / International Journal of Immunopharmacology 22 (2000) 597±602
survival in skin, cardiac, and renal transplant models in rodent or primate [4±8]. FTY720 does not aect lymphocyte proliferation and IL-2 production in rat allogeneic mixed lymphocyte culture in vitro, suggesting that this chemical exerts its potent immunosuppressive eect by a mechanism distinct from cyclosporin A and FK506 [6,9]. In contrast to all established immunosuppressants, a striking feature of FTY720 is that it markedly decreases the number of peripheral blood lymphocytes, especially T cells, at the doses which prolong allograft survival [6±8]. Circulating lymphocytes in the blood home into lymph nodes and Peyer's patches through interaction of lymphocyte-homing receptors to their ligands on high endothelial venules, and then subsequently return to the blood again [10,11]. In earlier studies, we showed that FTY720-induced disappearance of peripheral blood lymphocytes is due to sequestration of circulating lymphocytes within lymph nodes and Peyer's patches by accelerating lymphocyte-homing [6]. The acceleration of lymphocyte homing and the subsequent sequestration of lymphocytes appears to be related to the immunosuppressive activity of FTY720. T cell in®ltration into grafted organs is thought to be an important component of graft rejection. T cell in®ltration into grafts has, in fact, been described in various graft models [12,13]. In the current study, we assessed the signi®cance of timing of FTY720 administration on the immunosuppressive eect and T cell in®ltration into grafts in rat skin allografts.
2. Experimental procedures
2.1. Animals Inbred strains of male F344 and WKAH rats were purchased from Japan Charles River (Atsugi, Kanagawa, Japan) and Japan SLC (Hamamatsu, Shizuoka, Japan), respectively. All the rats used were at the age of 4±6 weeks.
2.2. Agents FTY720 was synthesized by Toito Co. [2], dissolved in distilled water, and given orally by gavage. Control animals received the vehicle only. 2.3. Rat skin allograft MHC-incompatible rat skin allografts were performed by the method described previously with WKAH rats RT1k as donors and F344 rats RT1lv1 as recipients [6,9]. Brie¯y, fullthickness skin grafts (2.0 2.0 cm square 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 de®ned as more than 90% necrosis of the graft epithelium. FTY720 was orally administered at 1 mg/kg daily to the transplanted animals. 2.4. Measurement of T cell number in peripheral blood and skin grafts Peripheral blood was collected from the tail vein of allografted rats. Single cell suspensions of skin grafts were prepared as previously described [9]. The skin graft removed from transplanted recipients was chopped into small fragments, then incubated for 1 h at 378C on a rocker in 4 ml of RPMI 1640 containing 10% FCS, 2 mg/ml collagenase type IV (Worthington Biochemical, San Luis Obispo, CA) and 20 mg/ml DNase I (Boehringer Mannheim, Mannheim, Germany). The digested skin graft was passed through a stainless steel mesh, and skin parenchymal cells were sedimented over Ficoll buer, density 1.090 g/cm3 (Immuno-Biological Laboratories, Fujioka, Japan), at 1700 g for 20 min. The cells were stained with biotin-conjugated anti-rat CD3 monoclonal antibody (mAb) (G418 [14]), ¯uorescein isothiocyanate (FITC)-conjugated anti-rat CD4 mAb (OX-38 [15]), phycoerythrin (PE)-conjugated anti-rat CD8a mAb (OX-8 [16]), and
Y. Yanagawa et al. / International Journal of Immunopharmacology 22 (2000) 597±602
streptavidin-Cy-Chrome2. For analysis of frequency of CD25+ cells in CD8+ T cells, the cells were stained with FITC-conjugated G418, PEconjugated OX-8, biotin-conjugated anti-rat CD25 mAb (OX-39 [17]), and streptavidin-CyChrome1. The numbers of T cell subsets were determined by ¯ow cytometry with EPICS1 XL (Coulter Co. Maiami, FL). All mAbs and streptavidin-Cy-Chrome were purchased from PharMingen (La Jolla, CA). 2.5. Statistical analysis The statistical dierences 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 dierences were calculated by an unpaired Student's t-test. 3. Results 3.1. Eect of timing of FTY720 administration on prolongation of rat skin allograft survival Fig. 1 shows the eect of timing of FTY720
Fig. 1. Eects of timing of FTY720 administration on prolonging rat skin allograft survival. WKAH skin was transplanted to MHC-incompatible F344 rats. FTY720 at 1 mg/kg was administered orally on consecutive days from day 0, 3, or 4 after transplantation. Each group consists of eight animals.
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administration on prolongation of rat skin allograft survival. WKAH skin was transplanted to F344 rats, and FTY720 at 1 mg/kg was orally administered to recipients on consecutive days from days 0, 3, and 4 after transplantation. In this allograft model, the median survival time of control allografts was 7 days. When administered from days 0 and 3, FTY720 signi®cantly prolonged allograft survival with median survival times of 11 and 13.5 days, respectively ( p < 0.01 versus control group). In contrast, FTY720 failed to prolong allograft survival when administered from day 4. 3.2. Eect of FTY720 on T cell number in blood of recipients FTY720 at 1 mg/kg was orally administered to recipients on consecutive days from days 0, 3, and 4 after transplantation, and numbers of CD4+ T cells and CD8+ T cells in the blood were temporally analyzed by ¯ow cytometry (Fig. 2). Both CD4+ T cells and CD8+ T cells in blood were completely depleted on the next day of FTY720 administration ( p < 0.01 versus control group). 3.3. Time course change of intragraft T-cell number in rat skin allograft Rat skin allografts were digested by collagenase, and numbers of CD4+ T cells and CD8+ T cells in allografts were analyzed by ¯ow cytometry. WKAH skin was transplanted to MHCincompatible F344 rats, and then intragraft T cell number was determined on days 2, 3, 4, 5 and 6 (Fig. 3). Isografts (F344 donor to F344 recipient) were analyzed on day 5 to control the nonspeci®c in¯ammatory response associated with the transplantation procedure. The numbers of intragraft CD8+ T cells and CD4+ T cells were slightly increased until day 3, and then increased from day 4 to 6, reaching the peak on day 5. Both CD8+ T cells and CD4+ T cells in allografts were signi®cantly increased in number when compared with isograft control on day 5 ( p < 0.01). Intragraft CD8+ T cells were markedly increased in number when compared with intragraft CD4+
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Fig. 2. Kinetics of numbers of CD4+ and CD8+ T cells in peripheral blood of allografted rats. WKAH skin was transplanted to MHC-incompatible F344 rats. FTY720 at 1 mg/kg was administered orally on consecutive days from days 0, 3, and 4 after transplantation. Numbers of CD4+ and CD8+ T cells in peripheral blood were determined by ¯ow cytometry. Each symbol represents the mean2SE of three animals.
T cells. The numbers of CD8+ T cells and CD4+ T cells in allografts on day 5 were 16.5-
fold and 4.4-fold of those in isografts, respectively. 3.4. Eect of FTY720 on in®ltrating CD8+ T-cell number in rat skin allografts
Fig. 3. Kinetics of T cell in®ltration into rat skin allografts. WKAH skin was transplanted to MHC-incompatible F344 rats. Allografts were digested by collagenase, then numbers of CD8+ T cells and CD4+ T cells in the allografts were determined by ¯ow cytometry. Isografts (F344 donor to F344 recipient) were analyzed on day 5. Each symbol represents the mean2SE of three animals.
WKAH skin was transplanted to F344 rats, and FTY720 at 1 mg/kg was orally administered to recipients on consecutive days from days 0, 3, and 4 after transplantation. On day 5, allografts were digested by collagenase, then the number of CD8+ T cells in the allografts was determined by ¯ow cytometry (Fig. 4). CD8+ T cells in the allografts of recipients administered FTY720 from day 0 and 3 were decreased to 20% and 25%, respectively. By contrast, in recipients administered with FTY720 from days 4, the number of CD8+ T cells in the allografts was partially decreased to 53%, and was almost the same as that of control allografts on day 4. On the other hand, FTY720 did not aect the increase in frequency of CD25+ cells in the CD8+ T cell subset in allografts on day 5 (Fig. 5)
Y. Yanagawa et al. / International Journal of Immunopharmacology 22 (2000) 597±602
Fig. 4. Eect of FTY720 on the number of in®ltrated CD8+ T cells in rat skin allografts on day 5. WKAH skin was transplanted to MHC-incompatible F344 rats. FTY720 at 1 mg/kg was administered orally on consecutive days from days 0, 3, and 4 after transplantation. Allografts were digested by collagenase on day 5, then the numbers of CD8+ T cells in the allografts were determined by ¯ow cytometry. Each column represents the mean2 SE of three animals (: p < 0.05, : p < 0.01, t-test).
4. Discussion FTY720 dramatically decreases the number of peripheral blood lymphocytes, especially T cells, within a few hours after administration at doses which prolong allograft survival [6,7]. The lymphocyte depletion in the blood appears to result in inhibiting T cell recruitment into in¯ammatory sites. FTY720, in fact, decreases intragraft T cell number in rat skin allografts [9]. In the current study, we determined the eect of timing of FTY720 administration on prolonging skin allo-
Fig. 5. Eect of FTY720 on intragraft activation of CD8+ T cells in rat skin allografts on day 5. WKAH skin was transplanted to MHC-incompatible F344 rats. FTY720 at 1 mg/kg was administered orally on day 4. Allografts were digested by collagenase on day 5, then CD25+ expression on CD8+ T cells in the allografts were determined by ¯ow cytometry. Each column represents the mean 2SE of three animals (NS: not signi®cant, : p < 0.05, t-test).
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graft survival in MHC-incompatible rat strains of WKAH donors and F344 recipients. FTY720 signi®cantly prolonged allograft survival when administered from day 3, but failed to show immunosuppressive eect when administered from day 4 (Fig. 1). FTY720 almost entirely depleted peripheral blood T cells on day 5±8 when administered from day 4 (Fig. 2). In this case, T cell tracking from peripheral blood into allografts seems to be markedly inhibited after day 5. Therefore, T cell tracking into allografts on day 5±7 may be dispensable for allograft rejection in our present model. T cell in®ltration into grafts and cytotoxic dierentiation of intragraft CD8+ T cells appear to be an important component in the process of allograft rejection [12,18,19]. In our present model, intragraft T cells, especially CD8+ T cells, were markedly increased in number from day 4 in the control group (Fig. 3). CD8+ T cells in the blood of recipients administered FTY720 from day 4 were almost entirely depleted on day 5 (Fig. 2). In contrast, the number of CD8+ T cells in allografts exposed to FTY720 from day 4 was only partially decreased on day 5, and were almost the same as that of control allografts on day 4 (Fig. 4). In addition, FTY720 did not aect the increase in frequency of CD25+ cells in CD8+ T cell subset in the allografts (Fig. 5).These ®ndings suggest that FTY720 fails to suciently eliminate intragraft CD8+ T-cells which had already in®ltrated, and does not aect intragraft activation of CD8+ T cells. The number of CD4+ T cells as well as CD8+ T cells in allografts was signi®cantly increased on day 5 (Fig. 3). Therefore, in®ltration of not only CD8+ T cells but also CD4+ T cells may be an important component of the graft rejection in the present model. In the current study, we focused on intragraft CD8+ T cells, since the number of intragraft CD8+ T cells was more markedly increased than that of CD4+ T cells. Although data were not shown, our preliminary data indicate that FTY720 tends to decrease the number of intragraft CD4+ T cells, but do not aect CD25 expression on intragraft CD4+ T cells as well as CD8+ T cells. It is likely that recipients treated with FTY720 from day 4 reject allografts by intragraft immune re-
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sponses involved in T cells which had in®ltrated before day 4, in the same manner as the control recipients. From the above observation, we presumed that FTY720 has to be administered before increase in T cell in®ltration into grafts to inhibit acute allograft rejection. References [1] Adachi K, Kohara T, Nakao N, Arita M, Chiba K, et al. Design, synthesis, and structure activity relationships of 2-substituted-2-amino-1,3-propanediols: discovery of a novel immunosuppressant, FTY720. BioMed Chem Lett 1995;5:853±936. [2] Fujita T, Hirose R, Yoneta M, Sasaki S, Inoue K, et al. Potent immunosuppressants, 2-alkyl-2-aminopropane1,3-diols. J Med Chem 1996;9:4451±9. [3] Kiuchi M, Adachi K, Kohara T, Teshima K, Masubuchi Y, et al. Synthesis and biological evaluation of 2,2-disubstituted 2-aminoethanols: analogues of FTY720. BioMed Chem Let 1998;8:101±6. [4] Chiba K, Hoshino Y, Suzuki C, Masubuchi Y, Yanagawa Y, et al. FTY720, a novel immunosuppressant possessing unique mechanisms. I. Prolongation of skin allograft survival and synergistic eect in combination with cyclosporine in rat. Transplant Proc 1996;28:1056±9. [5] Hoshino Y, Suzuki C, Ohtsuki M, Masubuchi Y, Amano Y, Chiba K. FTY720, a novel immunosuppressant possessing unique mechanisms. Long-term graft survival induction in rat heterotopic cardiac allograft and synergistic eect in combination with cyclosporine A. Transplant Proc 1996;28:1060±1. [6] Chiba K, Yanagawa Y, Masubuchi Y, Kataoka H, Kawaguchi T, et al. 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. J Immunol 1998;160:5037±44. [7] Wang ME, Tejpal N, Qu X, Yu J, Okamoto M, et al. Immunosuppressive eects of FTY720 alone or in combination with cyclosporine and/or sirolimus. Transplantation 1998;65:899±905.
[8] Troncoso P, Stepkowski SM, Wang ME, Qu X, Chueh SC, et al. Prophylaxis of acute renal allograft rejection using FTY720 in combination with subtherapeutic doses of cyclosporine. Transplantation 1999;67:145±51. [9] Yanagawa Y, Sugahara K, Kataoka H, Kawaguchi T, Masubuchi Y, Chiba K. FTY720, a novel immunosuppressant, induces sequestration of circulating mature-lymphocytes by acceleration of lymphocyte homing in rats. II. FTY720 prolongs skin allograft survival by decreasing T cell in®ltration into grafts but not cytokine production in vivo. J Immunol 1998;160:5493±9. [10] Ford WL, Gowans JL. The trac of lymphocytes. Semin Hematol 1969;6:67±83. [11] Butcher EC, Picker LJ. Lymphocyte homing and homeostasis. Science 1996;272:60±6. [12] O'Connell PJ, Pacheco-Silva A, Nickerson PW, Muggia RA, Bastos M, et al. Unmodi®ed pancreatic islet allograft rejection results in the preferential expression of certain T cell activation transcripts. J Immunol 1993;150:1093±104. [13] Carolyn FM, Simeonovic CJ, Fung MC, Wilson JD, Hapel AJ. Intragraft expression of cytokine transcripts during pig proislet xenograft rejection and tolerance in mice. J Immunol 1995;154:2470±82. [14] Nicolls MR, Aversa GG, Pearce NW, Spinelli A, Berger MF, et al. Induction of long-term speci®c tolerance to allografts in rats by therapy with an anti-CD3-like monoclonal antibody. Transplantation 1993;55:459±68. [15] Jeeries WA, Green JR, Williams AF. Authentic T helper CD4 (W3/25) antigen on rat peritoneal macrophages. J Exp Med 1985;162:117±27. [16] Nagel NT, Kraus E, Brown MH, Tiefenthaler G, Mitnacht R, et al. Dierential thymus dependence of rat CD8 isoform expression. Eur J Immunol 1992;22:2841±8. [17] Mouzaki A, Diamantstein T. Four epitopes on the rat 55-kDa subunit of the interleukin 2 receptor as de®ned by newly developed mouse anti-rat interleukin 2 receptor monoclonal antibodies. Eur J Immunol 1987;17:1661±4. [18] Rosenberg AS, Munitz TI, Maniero TG, Singer A. Cellular basis of skin allograft rejection across a class I major histocompatibility barrier in mice depleted of CD8+ T cells in vivo. J Exp Med 1991;173:1463±71. [19] Lipman ML, Stevens AC, Strom TB. Heightened intragraft CTL gene expression in acutely rejecting renal allografts. J Immunol 1994;152:5120±7.
The signi®cance of timing of FTY720 administration on the immunosuppressive eect to prolong rat skin allograft survival Yoshiki Yanagawa, Yukio Hoshino, Kenji Chiba* Tokyo Laboratories, Yoshitomi Pharmaceutical Industries Ltd., 3-7-25 Koyata, Iruma, Saitama 358-0026, Japan Received 3 December 1999; accepted 15 February 2000
Abstract FTY720, a potent immunosuppressant, dramatically decreases the number of peripheral blood lymphocytes within a few hours after administration. The current study assessed the signi®cance of timing of FTY720 administration on the immunosuppressive eect to prolong rat skin allograft survival (WKAH donor to F344 recipient). The median survival time of allografts was 7 days in the control recipients. FTY720 (1 mg/kg/day) signi®cantly prolonged allograft survival when administered from days 0 and 3, but failed to exert an immunosuppressive eect when administered from day 4. Intragraft T cells, especially CD8+ T cells, were markedly increased in number from day 4 to 6, peaking on day 5 in control recipients. FTY720 markedly decreased the number of intragraft CD8+ T cells on day 5 when administered from days 0 and 3. In recipients administered with FTY720 from day 4, the number of intragraft CD8+ T cells were only partially decreased on day 5. Intragraft CD8+ T-cell number in those recipients on day 5 was almost the same as that in control recipients on day 4. In addition, FTY720 did not aect the increase in frequency of CD25+ cells in the CD8+ T-cell subset in allografts. It is likely that recipients treated with FTY720 from day 4 reject allografts by intragraft immune responses involved in CD8+ T cells which had in®ltrated before day 4, similar to control recipients. These ®ndings suggest that FTY720 should be administered before increase in T cell in®ltration into grafts to inhibit acute allograft rejection. 7 2000 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. Keywords: FTY720; Immunosuppressant; Skin allograft; T cell in®ltration
1. Introduction 2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-
* Corresponding author. Tel.: +81-42-963-3121; fax: +8142-964-1906.
diol hydrochloride (FTY720), a novel immunosuppressant, is a synthetic structural analog of ISP-1, a metabolite of the ascomycete Isaria sinclairii [1±3]. FTY720 at 0.1±1 mg/kg shows more potent immunosuppressive activity than cyclosporin A and FK506, and exerts a synergistic eect with cyclosporin A to prolong allograft
0192-0561/00/$20.00 7 2000 International Society for Immunopharmacology. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 2 - 0 5 6 1 ( 0 0 ) 0 0 0 2 2 - 9
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Y. Yanagawa et al. / International Journal of Immunopharmacology 22 (2000) 597±602
survival in skin, cardiac, and renal transplant models in rodent or primate [4±8]. FTY720 does not aect lymphocyte proliferation and IL-2 production in rat allogeneic mixed lymphocyte culture in vitro, suggesting that this chemical exerts its potent immunosuppressive eect by a mechanism distinct from cyclosporin A and FK506 [6,9]. In contrast to all established immunosuppressants, a striking feature of FTY720 is that it markedly decreases the number of peripheral blood lymphocytes, especially T cells, at the doses which prolong allograft survival [6±8]. Circulating lymphocytes in the blood home into lymph nodes and Peyer's patches through interaction of lymphocyte-homing receptors to their ligands on high endothelial venules, and then subsequently return to the blood again [10,11]. In earlier studies, we showed that FTY720-induced disappearance of peripheral blood lymphocytes is due to sequestration of circulating lymphocytes within lymph nodes and Peyer's patches by accelerating lymphocyte-homing [6]. The acceleration of lymphocyte homing and the subsequent sequestration of lymphocytes appears to be related to the immunosuppressive activity of FTY720. T cell in®ltration into grafted organs is thought to be an important component of graft rejection. T cell in®ltration into grafts has, in fact, been described in various graft models [12,13]. In the current study, we assessed the signi®cance of timing of FTY720 administration on the immunosuppressive eect and T cell in®ltration into grafts in rat skin allografts.
2. Experimental procedures
2.1. Animals Inbred strains of male F344 and WKAH rats were purchased from Japan Charles River (Atsugi, Kanagawa, Japan) and Japan SLC (Hamamatsu, Shizuoka, Japan), respectively. All the rats used were at the age of 4±6 weeks.
2.2. Agents FTY720 was synthesized by Toito Co. [2], dissolved in distilled water, and given orally by gavage. Control animals received the vehicle only. 2.3. Rat skin allograft MHC-incompatible rat skin allografts were performed by the method described previously with WKAH rats RT1k as donors and F344 rats RT1lv1 as recipients [6,9]. Brie¯y, fullthickness skin grafts (2.0 2.0 cm square 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 de®ned as more than 90% necrosis of the graft epithelium. FTY720 was orally administered at 1 mg/kg daily to the transplanted animals. 2.4. Measurement of T cell number in peripheral blood and skin grafts Peripheral blood was collected from the tail vein of allografted rats. Single cell suspensions of skin grafts were prepared as previously described [9]. The skin graft removed from transplanted recipients was chopped into small fragments, then incubated for 1 h at 378C on a rocker in 4 ml of RPMI 1640 containing 10% FCS, 2 mg/ml collagenase type IV (Worthington Biochemical, San Luis Obispo, CA) and 20 mg/ml DNase I (Boehringer Mannheim, Mannheim, Germany). The digested skin graft was passed through a stainless steel mesh, and skin parenchymal cells were sedimented over Ficoll buer, density 1.090 g/cm3 (Immuno-Biological Laboratories, Fujioka, Japan), at 1700 g for 20 min. The cells were stained with biotin-conjugated anti-rat CD3 monoclonal antibody (mAb) (G418 [14]), ¯uorescein isothiocyanate (FITC)-conjugated anti-rat CD4 mAb (OX-38 [15]), phycoerythrin (PE)-conjugated anti-rat CD8a mAb (OX-8 [16]), and
Y. Yanagawa et al. / International Journal of Immunopharmacology 22 (2000) 597±602
streptavidin-Cy-Chrome2. For analysis of frequency of CD25+ cells in CD8+ T cells, the cells were stained with FITC-conjugated G418, PEconjugated OX-8, biotin-conjugated anti-rat CD25 mAb (OX-39 [17]), and streptavidin-CyChrome1. The numbers of T cell subsets were determined by ¯ow cytometry with EPICS1 XL (Coulter Co. Maiami, FL). All mAbs and streptavidin-Cy-Chrome were purchased from PharMingen (La Jolla, CA). 2.5. Statistical analysis The statistical dierences 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 dierences were calculated by an unpaired Student's t-test. 3. Results 3.1. Eect of timing of FTY720 administration on prolongation of rat skin allograft survival Fig. 1 shows the eect of timing of FTY720
Fig. 1. Eects of timing of FTY720 administration on prolonging rat skin allograft survival. WKAH skin was transplanted to MHC-incompatible F344 rats. FTY720 at 1 mg/kg was administered orally on consecutive days from day 0, 3, or 4 after transplantation. Each group consists of eight animals.
599
administration on prolongation of rat skin allograft survival. WKAH skin was transplanted to F344 rats, and FTY720 at 1 mg/kg was orally administered to recipients on consecutive days from days 0, 3, and 4 after transplantation. In this allograft model, the median survival time of control allografts was 7 days. When administered from days 0 and 3, FTY720 signi®cantly prolonged allograft survival with median survival times of 11 and 13.5 days, respectively ( p < 0.01 versus control group). In contrast, FTY720 failed to prolong allograft survival when administered from day 4. 3.2. Eect of FTY720 on T cell number in blood of recipients FTY720 at 1 mg/kg was orally administered to recipients on consecutive days from days 0, 3, and 4 after transplantation, and numbers of CD4+ T cells and CD8+ T cells in the blood were temporally analyzed by ¯ow cytometry (Fig. 2). Both CD4+ T cells and CD8+ T cells in blood were completely depleted on the next day of FTY720 administration ( p < 0.01 versus control group). 3.3. Time course change of intragraft T-cell number in rat skin allograft Rat skin allografts were digested by collagenase, and numbers of CD4+ T cells and CD8+ T cells in allografts were analyzed by ¯ow cytometry. WKAH skin was transplanted to MHCincompatible F344 rats, and then intragraft T cell number was determined on days 2, 3, 4, 5 and 6 (Fig. 3). Isografts (F344 donor to F344 recipient) were analyzed on day 5 to control the nonspeci®c in¯ammatory response associated with the transplantation procedure. The numbers of intragraft CD8+ T cells and CD4+ T cells were slightly increased until day 3, and then increased from day 4 to 6, reaching the peak on day 5. Both CD8+ T cells and CD4+ T cells in allografts were signi®cantly increased in number when compared with isograft control on day 5 ( p < 0.01). Intragraft CD8+ T cells were markedly increased in number when compared with intragraft CD4+
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Fig. 2. Kinetics of numbers of CD4+ and CD8+ T cells in peripheral blood of allografted rats. WKAH skin was transplanted to MHC-incompatible F344 rats. FTY720 at 1 mg/kg was administered orally on consecutive days from days 0, 3, and 4 after transplantation. Numbers of CD4+ and CD8+ T cells in peripheral blood were determined by ¯ow cytometry. Each symbol represents the mean2SE of three animals.
T cells. The numbers of CD8+ T cells and CD4+ T cells in allografts on day 5 were 16.5-
fold and 4.4-fold of those in isografts, respectively. 3.4. Eect of FTY720 on in®ltrating CD8+ T-cell number in rat skin allografts
Fig. 3. Kinetics of T cell in®ltration into rat skin allografts. WKAH skin was transplanted to MHC-incompatible F344 rats. Allografts were digested by collagenase, then numbers of CD8+ T cells and CD4+ T cells in the allografts were determined by ¯ow cytometry. Isografts (F344 donor to F344 recipient) were analyzed on day 5. Each symbol represents the mean2SE of three animals.
WKAH skin was transplanted to F344 rats, and FTY720 at 1 mg/kg was orally administered to recipients on consecutive days from days 0, 3, and 4 after transplantation. On day 5, allografts were digested by collagenase, then the number of CD8+ T cells in the allografts was determined by ¯ow cytometry (Fig. 4). CD8+ T cells in the allografts of recipients administered FTY720 from day 0 and 3 were decreased to 20% and 25%, respectively. By contrast, in recipients administered with FTY720 from days 4, the number of CD8+ T cells in the allografts was partially decreased to 53%, and was almost the same as that of control allografts on day 4. On the other hand, FTY720 did not aect the increase in frequency of CD25+ cells in the CD8+ T cell subset in allografts on day 5 (Fig. 5)
Y. Yanagawa et al. / International Journal of Immunopharmacology 22 (2000) 597±602
Fig. 4. Eect of FTY720 on the number of in®ltrated CD8+ T cells in rat skin allografts on day 5. WKAH skin was transplanted to MHC-incompatible F344 rats. FTY720 at 1 mg/kg was administered orally on consecutive days from days 0, 3, and 4 after transplantation. Allografts were digested by collagenase on day 5, then the numbers of CD8+ T cells in the allografts were determined by ¯ow cytometry. Each column represents the mean2 SE of three animals (: p < 0.05, : p < 0.01, t-test).
4. Discussion FTY720 dramatically decreases the number of peripheral blood lymphocytes, especially T cells, within a few hours after administration at doses which prolong allograft survival [6,7]. The lymphocyte depletion in the blood appears to result in inhibiting T cell recruitment into in¯ammatory sites. FTY720, in fact, decreases intragraft T cell number in rat skin allografts [9]. In the current study, we determined the eect of timing of FTY720 administration on prolonging skin allo-
Fig. 5. Eect of FTY720 on intragraft activation of CD8+ T cells in rat skin allografts on day 5. WKAH skin was transplanted to MHC-incompatible F344 rats. FTY720 at 1 mg/kg was administered orally on day 4. Allografts were digested by collagenase on day 5, then CD25+ expression on CD8+ T cells in the allografts were determined by ¯ow cytometry. Each column represents the mean 2SE of three animals (NS: not signi®cant, : p < 0.05, t-test).
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graft survival in MHC-incompatible rat strains of WKAH donors and F344 recipients. FTY720 signi®cantly prolonged allograft survival when administered from day 3, but failed to show immunosuppressive eect when administered from day 4 (Fig. 1). FTY720 almost entirely depleted peripheral blood T cells on day 5±8 when administered from day 4 (Fig. 2). In this case, T cell tracking from peripheral blood into allografts seems to be markedly inhibited after day 5. Therefore, T cell tracking into allografts on day 5±7 may be dispensable for allograft rejection in our present model. T cell in®ltration into grafts and cytotoxic dierentiation of intragraft CD8+ T cells appear to be an important component in the process of allograft rejection [12,18,19]. In our present model, intragraft T cells, especially CD8+ T cells, were markedly increased in number from day 4 in the control group (Fig. 3). CD8+ T cells in the blood of recipients administered FTY720 from day 4 were almost entirely depleted on day 5 (Fig. 2). In contrast, the number of CD8+ T cells in allografts exposed to FTY720 from day 4 was only partially decreased on day 5, and were almost the same as that of control allografts on day 4 (Fig. 4). In addition, FTY720 did not aect the increase in frequency of CD25+ cells in CD8+ T cell subset in the allografts (Fig. 5).These ®ndings suggest that FTY720 fails to suciently eliminate intragraft CD8+ T-cells which had already in®ltrated, and does not aect intragraft activation of CD8+ T cells. The number of CD4+ T cells as well as CD8+ T cells in allografts was signi®cantly increased on day 5 (Fig. 3). Therefore, in®ltration of not only CD8+ T cells but also CD4+ T cells may be an important component of the graft rejection in the present model. In the current study, we focused on intragraft CD8+ T cells, since the number of intragraft CD8+ T cells was more markedly increased than that of CD4+ T cells. Although data were not shown, our preliminary data indicate that FTY720 tends to decrease the number of intragraft CD4+ T cells, but do not aect CD25 expression on intragraft CD4+ T cells as well as CD8+ T cells. It is likely that recipients treated with FTY720 from day 4 reject allografts by intragraft immune re-
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sponses involved in T cells which had in®ltrated before day 4, in the same manner as the control recipients. From the above observation, we presumed that FTY720 has to be administered before increase in T cell in®ltration into grafts to inhibit acute allograft rejection. References [1] Adachi K, Kohara T, Nakao N, Arita M, Chiba K, et al. Design, synthesis, and structure activity relationships of 2-substituted-2-amino-1,3-propanediols: discovery of a novel immunosuppressant, FTY720. BioMed Chem Lett 1995;5:853±936. [2] Fujita T, Hirose R, Yoneta M, Sasaki S, Inoue K, et al. Potent immunosuppressants, 2-alkyl-2-aminopropane1,3-diols. J Med Chem 1996;9:4451±9. [3] Kiuchi M, Adachi K, Kohara T, Teshima K, Masubuchi Y, et al. Synthesis and biological evaluation of 2,2-disubstituted 2-aminoethanols: analogues of FTY720. BioMed Chem Let 1998;8:101±6. [4] Chiba K, Hoshino Y, Suzuki C, Masubuchi Y, Yanagawa Y, et al. FTY720, a novel immunosuppressant possessing unique mechanisms. I. Prolongation of skin allograft survival and synergistic eect in combination with cyclosporine in rat. Transplant Proc 1996;28:1056±9. [5] Hoshino Y, Suzuki C, Ohtsuki M, Masubuchi Y, Amano Y, Chiba K. FTY720, a novel immunosuppressant possessing unique mechanisms. Long-term graft survival induction in rat heterotopic cardiac allograft and synergistic eect in combination with cyclosporine A. Transplant Proc 1996;28:1060±1. [6] Chiba K, Yanagawa Y, Masubuchi Y, Kataoka H, Kawaguchi T, et al. 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. J Immunol 1998;160:5037±44. [7] Wang ME, Tejpal N, Qu X, Yu J, Okamoto M, et al. Immunosuppressive eects of FTY720 alone or in combination with cyclosporine and/or sirolimus. Transplantation 1998;65:899±905.
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