- Email: [email protected]
FTY720 Inhibits TH1-Mediated Allogeneic Humoral Immune Response
FTY720 Inhibits TH1-Mediated Allogeneic Humoral Immune Response G. Peñuelas-Rivas, R. Domı´nguez-Perles, V. Brinkmann, M.L. del Rio, A. Muñoz-Luna, P. Ramírez-Romero, P. Parrilla-Paricio, and J.I. Rodrı´guez-Barbosa ABSTRACT Phosphorylated FTY720 is an analog of Sphingosine 1 Phosphate (S1P) with immunosuppressive activity that negatively regulates the expression of S1P-Receptor 1. It also inhibits the migration of CD4 and CD8 single-positive T cells from the thymus to the periphery, sequesters peripheral blood lymphocytes in lymph nodes and Peyer’s patches, and delays the exit of effector T cells toward the graft. The aim of our work was to study the effect of FTY720 on the kinetics of skin allograft rejection in a fully mismatched model; euthymic (Euthy) versus thymectomized (ATX) C57BL/6 mice (haplotype H-2b) recipients of BALB/c mice (haplotype H-2d) donor cells. The animals were injected daily with FTY720 (1 mg/kg) intraperitoneally for 2 weeks. To monitor the humoral immune response, serum samples collected at day 0 (pre-immune) and at day 23 after skin graft rejection were examined using BALB/c thymocytes as antigens in flow cytometry. To confirm the effect of FTY720 on peripheral lymphocytes, peripheral blood was analyzed by flow cytometry. Euthy and ATX FTY720-treated mice showed prolongation of skin allograft survival when compared with nontreated Euthy and ATX controls (P ⬍ .005). Unexpectedly, FTY720-treated Euthy mice showed significantly delayed graft rejection when compared to similarly treated ATX mice (P ⬍ .005). The delayed graft rejection in FTY720-treated Euthy mice correlated with a reduced content of Th1mediated IgG2a and IgG2b antibodies when compared with FTY720-treated ATX mice (P ⬍ .05). In conclusion, FTY720 delays the kinetics of allograft rejection in a fully mismatched model by inhibiting Th1-mediated humoral immune responses. The presence of the host thymus appears to be required for this phenomenon.
T
HE PHOSPHORYLATED FORM of FTY720 is an analogous immune modulator to sphingosine 1 phosphate (S1P). The active originator compound was initially isolated from Isaria sinclarii.1– 4 FTY720 treatment increases the survival of vascularized solid allografts in animals.5 It causes egress of T cells from peripheral blood to secondary lymphoid organs, but does not affect the activation of T cells or interfere with the response to viral infections.4,5 FTY720 phosphorylated by sphingosine kinase in vivo becomes the biologically active compound FTY720-P, which has similarities to S1P,6,7 and binds four of five receptors of S1P (S1PR) involved in lymphocyte migration.4,8,9 MATERIALS AND METHODS Animals Eight- to 12-week-old C57BL/6 (H2b) female mice (n ⫽ 20) were used as recipients of fully mismatched skin allografts from female BALB/c (H2d). The four experimental groups were: untreated and FTY720-treated ATX and Euthy mice.
Skin Graft Transplantation Skin graft beds were prepared on the posterolateral thorax of recipient B6 mice under anesthesia. Skin grafts harvested from the tail of female BALB/c mice were sutured in the upper part of the dorsal thoracic region. Skin grafts were covered with Vaseline gauze and protected with an adhesive bandage for the first week after surgery. Skin graft survival was assessed daily. Time of
From the Unit of Transplantation Research, Experimental Surgery, Arrixaca University Hospital, Murcia, Spain (G.P-R., R.D-P., M.L.dR., A.M-L., P.R-R., P.P-P., J.I.R-B.); and Transplantation & Immunology, Novartis Institutes for Biomedical Research, Basel, Switzerland (V.B.). Supported by Fondo de Investigaciones Sanitarias, FIS 01/ 3026 and the Consejeria de Sanidad de la Comunidad de Murcia (Fundación Séneca, Transplantation Program). Address reprint requests to Dr J.I. Rodrı´guez-Barbosa, Unit of Transplantation Research, Experimental Surgery, Carretera Madrid-Cartagena s/n, Arrixaca University Hospital, 30120 - El Palmar, Murcia, Spain. E-mail: [email protected]
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.09.184
© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
4124
Transplantation Proceedings, 37, 4124 – 4126 (2005)
FTY720 INHIBITS TH1 IMMUNE RESPONSE
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Fig 1. rejection was defined as the first day on which more than the 75% of the graft was necrotic.
FTY720 Treatment FTY720, kindly provided by Novartis Pharma AG (Basel, Switzerland), was dissolved in sterile distilled water. FTY720 (1 mg/kg) was intraperitoneally administered daily for 15 days. The treatment began on the day of transplantation.
Measurement of Anti-Donor Serum Antibodies Serum samples were collected on day 0 and day 23 after allograft rejection to analyze the humoral immune response by flow cytometry using thymocytes of the same haplotype as the donor allograft. Donor thymocytes were incubated with pretransplant and posttransplant recipient serum. The staining was developed with the use of the following antibodies: biotinylated anti-mouse IgM, IgG1, IgG2a, and IgG2b followed by Streptavidin-CyChrome.
Statistical Analysis
Flow Cytometry To monitor the effect of FTY720 on peripheral blood lymphocytes, samples were collected from the retroorbital venous plexus. PBMCs were isolated with Ficoll and samples analyzed on a FACScan Flow cytometer (Becton Dickinson Immunocytometry Systems). Cells were stained with the following antibodies: Biotin-labeled anti-mouse CD11b-BIO, FITC-labeled anti-mouse B220, FITC-labeled antimouse CD3, PE-labeled anti-mouse CD4, PE-labeled anti-mouse CD8, and biotin-labeled anti-mouse CD44. Streptavidin-CyChrome conjugate was used to develop antibodies bound to biotin. All the reagents were purchased from BD PharMingen.
Fig 2.
Skin graft survival curves were calculated using Kaplan-Meier life tables and analyzed by the log-rank test. A P value less than .05 was considered significant. Student’s t test and Mann-Whitney test were used to determine the significance of differences in means and medians, respectively.
RESULTS
Euthy and ATX FTY720-treated mice showed prolonged skin allograft survival when compared with nontreated Euthy and ATX controls (P ⬍ .05). Unexpectedly, FTY720-treated Eu-
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PEÑUELAS-RIVAS, DOMINGUEZ-PERLES, BRINKMANN ET AL
Fig 3.
thy mice showed significantly delayed graft rejection when compared to similarly treated ATX mice (P ⬍ .005; Fig 1). The delay in graft rejection among FTY720-treated Euthy mice correlated with a reduced Th1-mediated IgG2a immune response when compared with FTY720-treated ATX mice (P ⬍ .05; Fig 2), but no differences were found for the IgG2bmediated humoral immune response (Fig 3). IgM and IgG1 anti-donor humoral immune response was similar in all the groups studied (data not shown). DISCUSSION
In FTY720-treated mice, the presence of the thymus was required to delay rejection of a fully mismatched skin allograft.
REFERENCES 1. Calne R: FTY: not just a homely drug. Transplantation 77:1327, 2004 2. Rosen H, Sanna G, Alfonso C: Egress: a receptor-regulated step in lymphocyte trafficking. Immunol Rev 195:160, 2003 3. Sanchez T, Hla T: Structural and functional characteristics of S1P receptors. J Cell Biochem 92:913, 2004 4. Yopp AC, Fu S, Honig SM, et al: FTY720-enhanced T cell homing is dependent on CCR2, CCR5 and CXCR4: evidence for distintic chemokine compartments. J Immunol 173:855, 2004 5. Yopp AC, Krieger NR, Ochando JC, et al: Therapeutic manipulation of T cell chemotaxis in transplantation. Curr Opin Immunol 16:1, 2004 6. Suzuki S: FTY720: mechanism of action and its effect on organ transplantation (review). Transplant Proc 31:2779, 1999 7. Le Moine A, Goldman M, Abramowicz D: Multiple pathways to allograft rejection. Transplantation 73:1373, 2002
T
HE PHOSPHORYLATED FORM of FTY720 is an analogous immune modulator to sphingosine 1 phosphate (S1P). The active originator compound was initially isolated from Isaria sinclarii.1– 4 FTY720 treatment increases the survival of vascularized solid allografts in animals.5 It causes egress of T cells from peripheral blood to secondary lymphoid organs, but does not affect the activation of T cells or interfere with the response to viral infections.4,5 FTY720 phosphorylated by sphingosine kinase in vivo becomes the biologically active compound FTY720-P, which has similarities to S1P,6,7 and binds four of five receptors of S1P (S1PR) involved in lymphocyte migration.4,8,9 MATERIALS AND METHODS Animals Eight- to 12-week-old C57BL/6 (H2b) female mice (n ⫽ 20) were used as recipients of fully mismatched skin allografts from female BALB/c (H2d). The four experimental groups were: untreated and FTY720-treated ATX and Euthy mice.
Skin Graft Transplantation Skin graft beds were prepared on the posterolateral thorax of recipient B6 mice under anesthesia. Skin grafts harvested from the tail of female BALB/c mice were sutured in the upper part of the dorsal thoracic region. Skin grafts were covered with Vaseline gauze and protected with an adhesive bandage for the first week after surgery. Skin graft survival was assessed daily. Time of
From the Unit of Transplantation Research, Experimental Surgery, Arrixaca University Hospital, Murcia, Spain (G.P-R., R.D-P., M.L.dR., A.M-L., P.R-R., P.P-P., J.I.R-B.); and Transplantation & Immunology, Novartis Institutes for Biomedical Research, Basel, Switzerland (V.B.). Supported by Fondo de Investigaciones Sanitarias, FIS 01/ 3026 and the Consejeria de Sanidad de la Comunidad de Murcia (Fundación Séneca, Transplantation Program). Address reprint requests to Dr J.I. Rodrı´guez-Barbosa, Unit of Transplantation Research, Experimental Surgery, Carretera Madrid-Cartagena s/n, Arrixaca University Hospital, 30120 - El Palmar, Murcia, Spain. E-mail: [email protected]
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.09.184
© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
4124
Transplantation Proceedings, 37, 4124 – 4126 (2005)
FTY720 INHIBITS TH1 IMMUNE RESPONSE
4125
Fig 1. rejection was defined as the first day on which more than the 75% of the graft was necrotic.
FTY720 Treatment FTY720, kindly provided by Novartis Pharma AG (Basel, Switzerland), was dissolved in sterile distilled water. FTY720 (1 mg/kg) was intraperitoneally administered daily for 15 days. The treatment began on the day of transplantation.
Measurement of Anti-Donor Serum Antibodies Serum samples were collected on day 0 and day 23 after allograft rejection to analyze the humoral immune response by flow cytometry using thymocytes of the same haplotype as the donor allograft. Donor thymocytes were incubated with pretransplant and posttransplant recipient serum. The staining was developed with the use of the following antibodies: biotinylated anti-mouse IgM, IgG1, IgG2a, and IgG2b followed by Streptavidin-CyChrome.
Statistical Analysis
Flow Cytometry To monitor the effect of FTY720 on peripheral blood lymphocytes, samples were collected from the retroorbital venous plexus. PBMCs were isolated with Ficoll and samples analyzed on a FACScan Flow cytometer (Becton Dickinson Immunocytometry Systems). Cells were stained with the following antibodies: Biotin-labeled anti-mouse CD11b-BIO, FITC-labeled anti-mouse B220, FITC-labeled antimouse CD3, PE-labeled anti-mouse CD4, PE-labeled anti-mouse CD8, and biotin-labeled anti-mouse CD44. Streptavidin-CyChrome conjugate was used to develop antibodies bound to biotin. All the reagents were purchased from BD PharMingen.
Fig 2.
Skin graft survival curves were calculated using Kaplan-Meier life tables and analyzed by the log-rank test. A P value less than .05 was considered significant. Student’s t test and Mann-Whitney test were used to determine the significance of differences in means and medians, respectively.
RESULTS
Euthy and ATX FTY720-treated mice showed prolonged skin allograft survival when compared with nontreated Euthy and ATX controls (P ⬍ .05). Unexpectedly, FTY720-treated Eu-
4126
PEÑUELAS-RIVAS, DOMINGUEZ-PERLES, BRINKMANN ET AL
Fig 3.
thy mice showed significantly delayed graft rejection when compared to similarly treated ATX mice (P ⬍ .005; Fig 1). The delay in graft rejection among FTY720-treated Euthy mice correlated with a reduced Th1-mediated IgG2a immune response when compared with FTY720-treated ATX mice (P ⬍ .05; Fig 2), but no differences were found for the IgG2bmediated humoral immune response (Fig 3). IgM and IgG1 anti-donor humoral immune response was similar in all the groups studied (data not shown). DISCUSSION
In FTY720-treated mice, the presence of the thymus was required to delay rejection of a fully mismatched skin allograft.
REFERENCES 1. Calne R: FTY: not just a homely drug. Transplantation 77:1327, 2004 2. Rosen H, Sanna G, Alfonso C: Egress: a receptor-regulated step in lymphocyte trafficking. Immunol Rev 195:160, 2003 3. Sanchez T, Hla T: Structural and functional characteristics of S1P receptors. J Cell Biochem 92:913, 2004 4. Yopp AC, Fu S, Honig SM, et al: FTY720-enhanced T cell homing is dependent on CCR2, CCR5 and CXCR4: evidence for distintic chemokine compartments. J Immunol 173:855, 2004 5. Yopp AC, Krieger NR, Ochando JC, et al: Therapeutic manipulation of T cell chemotaxis in transplantation. Curr Opin Immunol 16:1, 2004 6. Suzuki S: FTY720: mechanism of action and its effect on organ transplantation (review). Transplant Proc 31:2779, 1999 7. Le Moine A, Goldman M, Abramowicz D: Multiple pathways to allograft rejection. Transplantation 73:1373, 2002