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Influence of CsA treatment on adoptive transfer of immunity after allogeneic kidney transplantation in rats
Influence of CsA Treatment on Adoptive Transfer of Immunity After Allogeneic Kidney Transplantation in Rats Y.L. Gu, U. Dahmen, L. Doebel, J. Li, O. Dirsch, S. Polywka, and C.E. Broelsch
P
ATIENTS with end-stage renal disease as well as kidney transplantation recipients have a high risk of acquiring hepatitis B infection due to chronic disease, blood transfusion, as well as immunosuppressive therapy. Response rate to active vaccination is very low in dialysis patients before transplantation as well as in immunosuppressed patients after kidney transplantation. Previous experiments have shown that donor immunity can be transferred to a transplant recipient via liver transplantation in rats. This study aims to explore the efficacy of adoptive transfer of immunity by allogeneic kidney transplantation in rats and the influence of immunosuppressive treatment on the adoptive immune transfer.
MATERIALS AND METHODS Animals Male inbred ACI (RT11) rats weighing 200 to 300 g were used as kidney donors. Male inbred Lewis (LEW, RT1l) rats weighing 200 to 300 g were used as recipients. All animals were obtained from Harlan-Winkelmann Company. The rats received a standard laboratory diet and were kept under standardized conditions (automatic controlled light was maintained from 7 AM to 7 PM; environmental temperature was regulated to 20 to 24°C at a relative humidity of 55 ⫾ 5%; four rats were kept in one cage). Animal housing and procedures were carried out according to the German Animal Welfare Legislation. Prior to operation, the animals had free access to food and water. The animals were monitored daily, and each rat was sacrificed 12 weeks after transplantation or when its general condition deteriorated.
Experimental Design Kidney donors were immunized twice with HBV vaccine prior to organ donation. Kidneys from these vaccinated donors were transplanted into fully MHC-mismatched recipients. The experimental group (n ⫽ 5) was treated daily with a standard dose of Cyclosporine A (CsA); whereas, the control group (n ⫽ 5) did not receive any immunosuppressive treatment. Donors and recipients were monitored weekly for anti-HBs titers.
Vaccination Protocol (Donors) ACI rats weighing 80 to 100 g were chosen as donors. Each rat was first anaesthetized by inhalation of methoxyflurane (Metofane, Janssen GmbH, Neuss, Germany). After drawing blood (0.3 to 0.5 mL) from the tail, 0.2 mL recombinant hepatitis B vaccine (Engerix, SmithKline Beecham) was injected intramuscularly. The serum was separated and stored at ⫺20°C until anti-HBs titer 0041-1345/01/$–see front matter PII S0041-1345(00)02064-9
measurement by microparticle-enzyme-immunoassay (MEIA). A response was considered positive if the anti-HBs level was greater than 10 mIU/mL.1 Four weeks later all rats were boosted with the same hepatitis B vaccine in the same way.
Renal Transplantation Under methoxyflurane anesthesia orthotopic left renal transplantation was performed according to the technique described by Oesterwitz.2 The renal artery was anastomosed by end-to-end suture technique with 10-0 Ethilon suture using eight interrupted stitches. The end-to-end anastomosis for the vein was performed running suture technique. Ureter anastomosis was performed end-to-end close to the renal pelvis with 10-0 Ethilon suture and four interrupted stitches were required. The abdominal wound was closed in two layers. The rats had free access to standard laboratory chow before and after transplantation.
Immunosuppressive Therapy The recipients in the immunosuppressive therapy group were treated with 5 mg/kg per day CsA3 (Sandimmune, Sandoz Ltd., Basel, Switzerland) by intramuscular injection once a day after kidney transplantation. All the recipients were followed for more than 10 weeks after kidney transplantation. Blood sample was drawn weekly from the tail vein of each recipient after renal transplantation under Metofane anaesthesia. The serum was separated and stored at ⫺20°C until anti-HBs titer measurement.
Microparticle Enzyme Immunoassay A fully automated microparticle enzyme immunoassay was used for detection and quantification of rat serum antibody against hepatitis B surface antigen (anti-HBs), which is described by Abbott Laboratories.4 First, microparticles coated with rHBsAg are added to the specimen. An aliquot of the reaction mixture is transferred to the glass fiber matrix. The microparticles bind irreversibly to the matrix and unbound material is washed through the matrix. Biotinylated rHBsAg is then added to the matrix to react with From the Department of General and Transplantation Surgery (Y.L.G., U.D., L.D., J.L., C.E.B.), Institute of Pathology (O.D.), University Hospital of Essen, Essen, Institute of Microbiology and Virology (S.P.), University Hospital of Hamburg, Hamburg, Germany. Address reprint requests to Dr U. Dahmen, University Hospital Essen, Department of General and Transplantation Surgery, Hufelandstrasse 55, 45122 Essen, Germany. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
398
Transplantation Proceedings, 33, 398–400 (2001)
INFLUENCE OF CSA TREATMENT
399
captured anti-HBs. Anti-biotin/alkaline phosphates conjugate is added to the matrix to react with the biotinylated rHBsAg/captured latex microparticle complex. Unbound conjugate is removed by washing the matrix. The bound conjugate complex is detected by incubation with the fluorogenic substrate 4-methylumbeliferyl phosphate. The rate of fluorescence signal generation is proportional to the amount of anti-HBs bound to the microparticle/matrix solid phase. Anti-HBs concentrations in specimens are calculated automatically by comparison of the specimen rate to value determined from a stored standard curve.
Statistical Analysis All measurements and calculations were analyzed using the Microsoft Excel Statistics Program. All the data were expressed as mean ⫾ standard deviation (SD). The significance of differences between the groups was assessed using the Student t test. The result was considered significant when the P value was below .05.
RESULTS
All donor animals developed a high titer after vaccination with no statistically significant difference between the two groups (135896.92 ⫾ 84389.42 mIU/mL) in the treatment group, respectively 144345.92 ⫾ 62240.98 mIU/mL in the untreated group; P ⬎ .05). Kidney recipients under immunosuppressive treatment with CsA developed a much higher anti-HBs titer at postoperative day (POD) 7 than without treatment (135.70 ⫾ 69.07 mIU/mL at POD 7 compared to 63.73 ⫾ 57.43 mIU/mL; P ⫽ .027, Fig 1a). Higher titer development in the first postoperative week was accompanied by a longer persistence of the effective titer; which could be measured for 5.20 ⫾ 1.64 weeks (4, 4, 6, 6, 7 weeks) in the treated group as compared to (3.60 ⫾ 1.67) weeks (1, 3, 4, 5, 5, weeks) in the untreated rats (Fig 1b). DISCUSSION
Kidney graft recipients with liver disease due to viral hepatitis B (HBV) have a significant morbidity and mortality. Safe and effective means of treating HBV are quite limited and are far from satisfactory. Adoptive transfer of immunologically active cells is a new approach in the therapy of HBV.5 Few trials using this approach have been reported. Nevertheless, there have been intriguing reports of incidental adoptive transfer of immunity to HBV occurring in rare cases after bone marrow transplantation. Furthermore, there have been several case reports regarding the clearance of HBsAg in patients with chronic HBV who underwent bone marrow transplantation for leukaemia or multiple myeloma from donors who happened to have pre-existing immunity to HBV.6 Lok et al7 reported on a series of 14 patients who were HBsAg-positive and underwent bone marrow transplantation.7 Three of the recipients who all received a bone marrow graft from donors with pre-existing anti-HBs antibody (HBsAb) and anti-HBc antibody cleared HBsAg within 1 to 3 years of transplantation. In contrast, none of the 11 recipients receiving marrow from donors without antibody to HBV cleared HBsAg
Fig 1A. All donor animals developed a high titer after vaccination with no statistically significant differences between the two groups, whereas kidney recipients under immunosuppressive treatment with CsA developed a much higher anti-Hbs titer at POD 7 than without treatment (P ⫽ .027). (B) Higher titer development in the first postoperative week was accompanied by a longer persistance of the effective titer (defined as being above 10 mIU/mL), which could be measured for 5.20 ⫾ 1.64 weeks in the CsA treated group as compared to 3.60 ⫾ 1.67 weeks in the untreated rats.
during the follow-up period. Shouval and coworkers have demonstrated the feasibility of immunizing bone marrow transplant donors with HBV vaccine before transplantation, and they reported on successful transfer of HBsAb immunity to the recipient.8 However, bone marrow transplantation is associated with a significant number of adverse effects and risk. Despite positive single center experience, bone marrow augmented organ transplantation is not yet an accepted therapeutic strategy although it might prove useful for this purpose. Nevertheless, a small number of donor cells are always transferred from donor to recipient during organ transplantation and can even survive for prolonged periods of time.9 Previous experiments have demonstrated that donor specific immunity can be transferred successfully from either a skin-sensitized donor10 or a HBV vaccinated donor11 to a recipient via liver transplantation in rats. This denotes that donor immunity can also be transferred by a solid organ graft. The current experiment was based on this hypothesis. The results indicated that kidney transplantation from a vaccinated donor resulted in effective antibody production in the recipient and the anti-HBs titer lasted for about 6 weeks. Several mechanisms of this transfer may be envis-
400
aged. Pure passive transfer of donor anti-HBs is unlikely because the half-life of anti-HBs is only a few days. However, it cannot be ruled out that donor antibodies remaining in the kidney graft were transferred to the recipient. The transfer of active donor lymphocytes is a more likely possibility. Solid organ grafts contain passenger leukocytes,12,13 the number and lineage of which vary considerably among different organs. In a mouse heart transplantation model, donor leukocytes migrating from the transplanted heart into the recipient were only detectable for a few days within the spleen unless the animal received immunosuppression.14 It was shown in the ongoing experiment that animals under immunosuppressive treatment did develop higher titer levels than untreated recipients which persisted for a longer period of time. This effect is most likely due to the continuous secretion of antibodies by plasma cells which was unaffected by the immunosuppression; whereas prolongation of the titer persistence might be explained by the prolonged presence of plasma cells being protected from rejection. Moreover, this represents an indirect proof that functional cells of the donor were transferred into the recipient via kidney transplantation. Therefore, the primed passenger lymphocytes within the kidney graft are supposed to play a major role in this adoptive immune transfer through kidney transplantation.
GU, DAHMEN, DOEBEL ET AL
REFERENCES 1. Centers for Disease Control and Prevention: MMWR 36:353, 1987 2. Oesterwitz H, Althaus P: Urol Res 10:149, 1982 3. Okamura R, Tanaka K, Asonuma K, et al: Eur Surg Res 21:235, 1989 4. Ostrow DH, Edwards B, Kimes D, et al: J Virol Methods 32:265, 1991 5. Hoofnagle JH, Lau D: J Viral Hepat 4(suppl 1):41, 1997 6. Ilan Y, Nagler A, Adler R, et al: Gastroenterology 104:1818, 1993 7. Lok ASF, Liang RHS, Hung H.-T.: Ann Intern Med 116:957, 1982 8. Shouval D, Ilan Y: J Hepatol 23:98, 1995 9. Starzl TE, Demetris AJ, Murase N, et al: Immunol Today 17:577, 1996 10. Dahmen U, Tanigawa T, Doebel L, et al: Transplant Proc 29:1123, 1997 11. Dahmen U, Tanigava T, Doebel L: Presented at the I.H.P.B.A. Congress, Hamburg, Germany, 1997 12. Hart DN, Fabre JW: J Exp Med 154:347, 1981 13. Schlitt HJ, Raddatz G, Steinhoff G, et al: Transplantation 56:951, 1993 14. Lechler RI, Batchelor JR: J Exp Med 155:31, 1982
P
ATIENTS with end-stage renal disease as well as kidney transplantation recipients have a high risk of acquiring hepatitis B infection due to chronic disease, blood transfusion, as well as immunosuppressive therapy. Response rate to active vaccination is very low in dialysis patients before transplantation as well as in immunosuppressed patients after kidney transplantation. Previous experiments have shown that donor immunity can be transferred to a transplant recipient via liver transplantation in rats. This study aims to explore the efficacy of adoptive transfer of immunity by allogeneic kidney transplantation in rats and the influence of immunosuppressive treatment on the adoptive immune transfer.
MATERIALS AND METHODS Animals Male inbred ACI (RT11) rats weighing 200 to 300 g were used as kidney donors. Male inbred Lewis (LEW, RT1l) rats weighing 200 to 300 g were used as recipients. All animals were obtained from Harlan-Winkelmann Company. The rats received a standard laboratory diet and were kept under standardized conditions (automatic controlled light was maintained from 7 AM to 7 PM; environmental temperature was regulated to 20 to 24°C at a relative humidity of 55 ⫾ 5%; four rats were kept in one cage). Animal housing and procedures were carried out according to the German Animal Welfare Legislation. Prior to operation, the animals had free access to food and water. The animals were monitored daily, and each rat was sacrificed 12 weeks after transplantation or when its general condition deteriorated.
Experimental Design Kidney donors were immunized twice with HBV vaccine prior to organ donation. Kidneys from these vaccinated donors were transplanted into fully MHC-mismatched recipients. The experimental group (n ⫽ 5) was treated daily with a standard dose of Cyclosporine A (CsA); whereas, the control group (n ⫽ 5) did not receive any immunosuppressive treatment. Donors and recipients were monitored weekly for anti-HBs titers.
Vaccination Protocol (Donors) ACI rats weighing 80 to 100 g were chosen as donors. Each rat was first anaesthetized by inhalation of methoxyflurane (Metofane, Janssen GmbH, Neuss, Germany). After drawing blood (0.3 to 0.5 mL) from the tail, 0.2 mL recombinant hepatitis B vaccine (Engerix, SmithKline Beecham) was injected intramuscularly. The serum was separated and stored at ⫺20°C until anti-HBs titer 0041-1345/01/$–see front matter PII S0041-1345(00)02064-9
measurement by microparticle-enzyme-immunoassay (MEIA). A response was considered positive if the anti-HBs level was greater than 10 mIU/mL.1 Four weeks later all rats were boosted with the same hepatitis B vaccine in the same way.
Renal Transplantation Under methoxyflurane anesthesia orthotopic left renal transplantation was performed according to the technique described by Oesterwitz.2 The renal artery was anastomosed by end-to-end suture technique with 10-0 Ethilon suture using eight interrupted stitches. The end-to-end anastomosis for the vein was performed running suture technique. Ureter anastomosis was performed end-to-end close to the renal pelvis with 10-0 Ethilon suture and four interrupted stitches were required. The abdominal wound was closed in two layers. The rats had free access to standard laboratory chow before and after transplantation.
Immunosuppressive Therapy The recipients in the immunosuppressive therapy group were treated with 5 mg/kg per day CsA3 (Sandimmune, Sandoz Ltd., Basel, Switzerland) by intramuscular injection once a day after kidney transplantation. All the recipients were followed for more than 10 weeks after kidney transplantation. Blood sample was drawn weekly from the tail vein of each recipient after renal transplantation under Metofane anaesthesia. The serum was separated and stored at ⫺20°C until anti-HBs titer measurement.
Microparticle Enzyme Immunoassay A fully automated microparticle enzyme immunoassay was used for detection and quantification of rat serum antibody against hepatitis B surface antigen (anti-HBs), which is described by Abbott Laboratories.4 First, microparticles coated with rHBsAg are added to the specimen. An aliquot of the reaction mixture is transferred to the glass fiber matrix. The microparticles bind irreversibly to the matrix and unbound material is washed through the matrix. Biotinylated rHBsAg is then added to the matrix to react with From the Department of General and Transplantation Surgery (Y.L.G., U.D., L.D., J.L., C.E.B.), Institute of Pathology (O.D.), University Hospital of Essen, Essen, Institute of Microbiology and Virology (S.P.), University Hospital of Hamburg, Hamburg, Germany. Address reprint requests to Dr U. Dahmen, University Hospital Essen, Department of General and Transplantation Surgery, Hufelandstrasse 55, 45122 Essen, Germany. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
398
Transplantation Proceedings, 33, 398–400 (2001)
INFLUENCE OF CSA TREATMENT
399
captured anti-HBs. Anti-biotin/alkaline phosphates conjugate is added to the matrix to react with the biotinylated rHBsAg/captured latex microparticle complex. Unbound conjugate is removed by washing the matrix. The bound conjugate complex is detected by incubation with the fluorogenic substrate 4-methylumbeliferyl phosphate. The rate of fluorescence signal generation is proportional to the amount of anti-HBs bound to the microparticle/matrix solid phase. Anti-HBs concentrations in specimens are calculated automatically by comparison of the specimen rate to value determined from a stored standard curve.
Statistical Analysis All measurements and calculations were analyzed using the Microsoft Excel Statistics Program. All the data were expressed as mean ⫾ standard deviation (SD). The significance of differences between the groups was assessed using the Student t test. The result was considered significant when the P value was below .05.
RESULTS
All donor animals developed a high titer after vaccination with no statistically significant difference between the two groups (135896.92 ⫾ 84389.42 mIU/mL) in the treatment group, respectively 144345.92 ⫾ 62240.98 mIU/mL in the untreated group; P ⬎ .05). Kidney recipients under immunosuppressive treatment with CsA developed a much higher anti-HBs titer at postoperative day (POD) 7 than without treatment (135.70 ⫾ 69.07 mIU/mL at POD 7 compared to 63.73 ⫾ 57.43 mIU/mL; P ⫽ .027, Fig 1a). Higher titer development in the first postoperative week was accompanied by a longer persistence of the effective titer; which could be measured for 5.20 ⫾ 1.64 weeks (4, 4, 6, 6, 7 weeks) in the treated group as compared to (3.60 ⫾ 1.67) weeks (1, 3, 4, 5, 5, weeks) in the untreated rats (Fig 1b). DISCUSSION
Kidney graft recipients with liver disease due to viral hepatitis B (HBV) have a significant morbidity and mortality. Safe and effective means of treating HBV are quite limited and are far from satisfactory. Adoptive transfer of immunologically active cells is a new approach in the therapy of HBV.5 Few trials using this approach have been reported. Nevertheless, there have been intriguing reports of incidental adoptive transfer of immunity to HBV occurring in rare cases after bone marrow transplantation. Furthermore, there have been several case reports regarding the clearance of HBsAg in patients with chronic HBV who underwent bone marrow transplantation for leukaemia or multiple myeloma from donors who happened to have pre-existing immunity to HBV.6 Lok et al7 reported on a series of 14 patients who were HBsAg-positive and underwent bone marrow transplantation.7 Three of the recipients who all received a bone marrow graft from donors with pre-existing anti-HBs antibody (HBsAb) and anti-HBc antibody cleared HBsAg within 1 to 3 years of transplantation. In contrast, none of the 11 recipients receiving marrow from donors without antibody to HBV cleared HBsAg
Fig 1A. All donor animals developed a high titer after vaccination with no statistically significant differences between the two groups, whereas kidney recipients under immunosuppressive treatment with CsA developed a much higher anti-Hbs titer at POD 7 than without treatment (P ⫽ .027). (B) Higher titer development in the first postoperative week was accompanied by a longer persistance of the effective titer (defined as being above 10 mIU/mL), which could be measured for 5.20 ⫾ 1.64 weeks in the CsA treated group as compared to 3.60 ⫾ 1.67 weeks in the untreated rats.
during the follow-up period. Shouval and coworkers have demonstrated the feasibility of immunizing bone marrow transplant donors with HBV vaccine before transplantation, and they reported on successful transfer of HBsAb immunity to the recipient.8 However, bone marrow transplantation is associated with a significant number of adverse effects and risk. Despite positive single center experience, bone marrow augmented organ transplantation is not yet an accepted therapeutic strategy although it might prove useful for this purpose. Nevertheless, a small number of donor cells are always transferred from donor to recipient during organ transplantation and can even survive for prolonged periods of time.9 Previous experiments have demonstrated that donor specific immunity can be transferred successfully from either a skin-sensitized donor10 or a HBV vaccinated donor11 to a recipient via liver transplantation in rats. This denotes that donor immunity can also be transferred by a solid organ graft. The current experiment was based on this hypothesis. The results indicated that kidney transplantation from a vaccinated donor resulted in effective antibody production in the recipient and the anti-HBs titer lasted for about 6 weeks. Several mechanisms of this transfer may be envis-
400
aged. Pure passive transfer of donor anti-HBs is unlikely because the half-life of anti-HBs is only a few days. However, it cannot be ruled out that donor antibodies remaining in the kidney graft were transferred to the recipient. The transfer of active donor lymphocytes is a more likely possibility. Solid organ grafts contain passenger leukocytes,12,13 the number and lineage of which vary considerably among different organs. In a mouse heart transplantation model, donor leukocytes migrating from the transplanted heart into the recipient were only detectable for a few days within the spleen unless the animal received immunosuppression.14 It was shown in the ongoing experiment that animals under immunosuppressive treatment did develop higher titer levels than untreated recipients which persisted for a longer period of time. This effect is most likely due to the continuous secretion of antibodies by plasma cells which was unaffected by the immunosuppression; whereas prolongation of the titer persistence might be explained by the prolonged presence of plasma cells being protected from rejection. Moreover, this represents an indirect proof that functional cells of the donor were transferred into the recipient via kidney transplantation. Therefore, the primed passenger lymphocytes within the kidney graft are supposed to play a major role in this adoptive immune transfer through kidney transplantation.
GU, DAHMEN, DOEBEL ET AL
REFERENCES 1. Centers for Disease Control and Prevention: MMWR 36:353, 1987 2. Oesterwitz H, Althaus P: Urol Res 10:149, 1982 3. Okamura R, Tanaka K, Asonuma K, et al: Eur Surg Res 21:235, 1989 4. Ostrow DH, Edwards B, Kimes D, et al: J Virol Methods 32:265, 1991 5. Hoofnagle JH, Lau D: J Viral Hepat 4(suppl 1):41, 1997 6. Ilan Y, Nagler A, Adler R, et al: Gastroenterology 104:1818, 1993 7. Lok ASF, Liang RHS, Hung H.-T.: Ann Intern Med 116:957, 1982 8. Shouval D, Ilan Y: J Hepatol 23:98, 1995 9. Starzl TE, Demetris AJ, Murase N, et al: Immunol Today 17:577, 1996 10. Dahmen U, Tanigawa T, Doebel L, et al: Transplant Proc 29:1123, 1997 11. Dahmen U, Tanigava T, Doebel L: Presented at the I.H.P.B.A. Congress, Hamburg, Germany, 1997 12. Hart DN, Fabre JW: J Exp Med 154:347, 1981 13. Schlitt HJ, Raddatz G, Steinhoff G, et al: Transplantation 56:951, 1993 14. Lechler RI, Batchelor JR: J Exp Med 155:31, 1982