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Effect of donor-specific transfusion in combination with FK 506 in rat cardiac allotransplantation
Effect of Donor-Specific Transfusion in Combination With FK 506 in Rat Cardiac Allotransplantation E.A. Antoniou, M. Drayson, A.J. Howie, P. McMaster, and M. D’Silva
T
RANSPLANTATION of solid organs has been established as an important therapeutic alternative for end-stage disease of these organs since the introduction of effective immunosuppressive agents. However, the level of immunosuppression needed to block rejection with current immunosuppressive agents is associated, in some degree, with decreased ability to mount necessary immune responses against infection and neoplasia. In addition, each drug presents toxic side effects specific to the agent. Combination immunosuppression therapy has been used in solid organ transplantation to reduce drug toxicity.1,2 The introduction of donor-specific immunosuppression since Medawar’s experiments,3 has been a major goal in transplantation research. Multiple blood transfusions prior to transplantation can improve organ allograft survival.4,5 A step forward involved studies comparing relative contributions of class I MHC antigen-bearing cells such as platelets,6 – 8 erythrocytes,9,10 T cells11 or even hepatocytes,12 and cells expressing both class I and II antigens such as lymphocytes13–15 or dendritic cells16 all prolonged graft survival but with variable success. Furthermore, attempts of using donor-specific transfusion (DST) in combination with shortterm cyclosporine (CyA) therapy have shown a prolongation of allograft survival in adult animal models,17–19 validating this effect with clinical relevance in cadaveric transplantation.20 The aim of this study was to investigate whether FK 506,21,22 a potent immunosuppressive agent, used in low-dose 1 mg/kg in combination with a perioperative adjunctive DST could prolong allograft survival in a stringent rat model of cardiac allotransplantation. MATERIALS AND METHODS Heterotopic abdominal heart grafting was performed from DA(RT1av1) to Lew(RT11) rats, weighing 200 to 250 g each, using a previously described technique.1 Donor DA rats also provided lymphocytes for DST to Lew recipients. The subjects were commercially obtained from Charles River Ltd (United Kingdom) and cared for humanely during the course of the study according to prevailing Home Office Guidelines in the United Kingdom. Rats were allowed unrestricted access to food and water and were housed in filter-top barrier housing cages in light and temperaturecontrolled quarters. Prior to experimentation, animals were allowed to acclimatize to lab conditions for 1 week. The procedures performed in this study were licensed by the UK Home Office governing body. Surgical procedures were performed aseptically
and each animal was monitored a minimum of twice daily for its postoperative condition. Transplanted hearts were graded daily for rejection using a previously validated heart beat scoring system, ranging from four (normal) to zero (rejection), described by Morrissey et al.23 In this study, DST, 108 lymphocytes pooled single cell suspension from spleen and lymph nodes from DA donor rats, was administered intravenously (IV) to Lew recipients rats: (a) 14 days before transplantation (pretreatment, day 14), (b) on the day of transplantation (day 0), and (c) in two doses, on the day of transplantation and on the fourth postoperative day (day 0, day 4). FK 506 (1 or 2 mg/kg) was used as monotherapy or combination therapy, added to DST schedule. FK 506 was administered orally by gavage for 10 days, starting the day of grafting until day 9 posttransplantation. Rejection was defined as lack of contractions of the heart graft clinically, confirmed at necropsy and by histologic examination. Monotherapy groups served as positive controls. Survival data were analyzed using Student’s t test and one-way analysis of variance (ANOVA). Results were expressed as (SD). A two-sided P value ⬍ .05 was considered significant.
RESULTS
Table 1 shows the results. The DA-to-LEW allocombination is strongly MHC incompatible at both major and minor loci. The mean graft survival time for the control group, without any treatment, was 4.8 days (range 4 to 5). All grafts had clinical features of acute rejection (AR) that were histologically proved. DST pretreatment (day 14) did not prolong heart allograft survival significantly beyond the survival of the control group (mean survival 5.2 days). When DST was administered on the day of transplant (day 0), there was a significant survival prolongation of the grafts compared to the control group (mean survival 6.2 days, P: .043). FK 506 1 mg/kg (low dose) monotherapy, given for 10 days posttransplant, prolonged graft survival in this allocombination from 4.8 (controls) to 18.5 days mean survival time. When the dose was increased to 2 mg/kg, there was a further prolongation of the graft survival (mean survival From the Renal Transplant Unit (E.A.A.) and The Liver Unit (P.M., M.D’S.), Queen Elizabeth Hospital, and the Departments of Immunology (M.Dr.) and Pathology (A.J.H.), University of Birmingham, Birmingham, B15 2TH, United Kingdom, Address reprint requests to Mr E.A. Antoniou, MD, PhD, SR, Department of Nephrology, Renal Transplant Unit, Queen Elizabeth Hospital, Edgbaston, Birmingham, B15 2TH, United Kingdom.
0041-1345/99/$–see front matter PII S0041-1345(99)00572-2
© 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2796
Transplantation Proceedings, 31, 2796–2798 (1999)
COMBINATION TREATMENT
2797
Table 1. Graft Survival Under DST Alone or in Combination With FK 506 (FK)
1 2 3 4 5 6 7 8
Groups
Treatment*
Survival (d)
Mean (SD)
⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽
Control (none) DST(d ⫺ 14) DST(d 0) FK1(d0 – d9) FK1(d0 – d9)⫹DST(d0) FK1(d0 – d9)⫹DST(d0,d4) FK2(d0 – d9) FK2(d0 – d9)⫹DST(d0)
4 –5 5– 6 5– 8 11–29 20 –24 13–36 18 –26 24 –32
4.8 5.2 6.2 18.5 21.7 25.4 22 26.2
(n (n (n (n (n (n (n (n
5) 5) 10) 6) 4) 5) 6) 5)
P
— NS vs group 1 .043 vs group 1 S vs groups 1 and 3 NS vs group 4 NS vs group 4 NS vs group 4 .05 vs group 4
*DST dose: 2 mL suspension of donor lymphocytes (108) given IV. FK dose: 1 or 2 mg/kg given orally by gavage.
time 22 days, range 18 to 26 days, group 7) although the P value was not significant compared with the group of 1 mg/kg (group 4). Combining DST with 1 mg/kg FK 506 was not effective whether DST was given in two doses or a single dose at grafting (mean survival time 25.4 and 21.7 days, respectively). Raising the dose of FK 506 to 2 mg/kg elicits a survival benefit when combined with DST at transplant (mean survival time 26.2 days, range 24 to 32). DISCUSSION
Our experiments showed that DST alone provides a slight improvement in control allograft survival when it is given on day 0. Fabre and Morris24 originally and Opelz et al25 later had reported that destruction of the graft had been prevented by giving the recipients a DST 2 weeks before cardiac allotransplantation. Although this is an old observation made prior to the CyA era, it has been recently supported by other investigators.26 However, our results showed that when DST was given to this strong allogeneic rat model 14 days prior to transplantation (day 14), there was no significant improvement of graft survival beyond the control graft survival. Surprisingly, the grafts did not manifest a gradual decrease in the strength of ventricular contractions; all grafts were graded either grade ⫹4 or ⫹3 (strong heart beat) the day before rejection. FK 506 is a novel immunosuppressive agent that has been used effectively as a primary immunosuppressant or as rescue therapy both in experimental1,2 and clinical transplantation.22,27,28 It was isolated from Streptomyces tsukubaensis29 and is a macrolide molecule that inhibits expression of interleukin-2 via T lymphocytes.30,31 Ten doses of 1 mg/kg/d FK 506 given alone posttransplantation to prevent rejection significantly prolonged allograft survival beyond that found with DST. DST administered on day 0, or given as two doses on days 0 and 4 in the presence of 1 mg/kg FK 506, did not further enhance graft survival obtained under FK 506 alone. There was a nonsignificant trend to prolong graft survival when an increased dose of 2 mg/kg FK 506 was combined with DST on day 0. We have not demonstrated a significant benefit to graft survival by the addition of DST to therapy with FK 506. On the other hand, there has been no antagonistic effect from the addition of DST to FK 506 therapy. The results we obtained in this study differ from those published by other investigators15,19 who found
a beneficial effect on graft survival by adding DST to CyA therapy. These investigators used different allocombinations in their experiments, which is an important factor in allograft survival and could partially explain the discrepancy between their results and ours. With respect to the efficacy of DST in combination with different immunosuppressive agents, in a previous study,32 the combination of DST with FTY720 (a new immunosuppressive agent) showed similar results to those in this study. ACKNOWLEDGMENTS The authors express their sincere thanks to Dr M. Xu (Transplant Microsurgery Laboratory) and Ms K. Shimel (Biomedical Services Unit) for the excellent technical support provided.
REFERENCES 1. Antoniou EA, Xu M, Howie AJ, et al: Transplant Proc 29:2888, 1997 2. Antoniou EA, DeRoover A, Howie AJ, et al: Microsurgery (in press) 3. Billingham RE, Medawar PB: Nature 172:603, 1953 4. Opelz G, Terasaki PI: N Engl J Med 299:799, 1978 5. Salvatiera O, Vincenti F, Amend W, et al: Ann Surg 192:543, 1980 6. Hibberd AD, Morris PJ: Transplant Proc 17:1655, 1985 7. Touraine JL, Pouteil-Noble C, Raffaele P, et al: Transplantation 51:777, 1991 8. Borleffs JC, Neuchaus P, Van Rood JJ, et al: Lancet 1:1117, 1982 9. Wood KJ, Morris PJ: Transplantation 39:56, 1985 10. Baird MA, Heslop BF: Transplantation 45:66, 1987 11. Odorico JS, Naji A: Surg Forum XLII:411, 1992 12. Foster S, Cranston D, Wood KJ, et al: Transplantation 45:228, 1988 13. Cranston D, Wood KJ, Carter MP, et al: Transplantation 43:809, 1987 14. Lauchart W, Alkans B, Davies DA: Transplantation 29:259, 1980 15. Oluwole SF, Rashid AF, Reemtsma K, et al: Transplantation 45:1131, 1988 16. El-Malik F, Malik ST, Varghese Z, et al: Transplantation 38:213, 1984 17. Martinelli GP, Rhune-Loy R, Sher I, et al: Transplantation 39:1, 1985 18. Yasumura T, Kahan BD: Transplantation 36:603, 1983 19. Ahmed SM, Baslaim G, Eshelby D, et al: Transplantation 57:634, 1994
2798 20. Brunson ME, Tchervenkov JI, Alexander JW: Transplantation 52:545, 1991 21. Ochiai T, Nagata M, Nakajima K, et al: Transplantation 44:729, 1987 22. European Tacrolimus Multicentre Renal Study Group: Transplantation 64:436, 1997 23. Morrisey PE, Gollin G, Brusett K, et al: Transplantation 57:631, 1994 24. Fabre JW, Morris PJ: Transplantation 14:608, 1972 25. Opelz G, Sengar DPS, Mickey MR, et al: Transplant Proc 5:253, 1973 26. Bradley JA: Immunol Lett 29:127, 1991
ANTONIOU, DRAYSON, HOWIE ET AL 27. The US Multicenter FK 506 Liver Study Group: N Engl J Med 331:1110, 1994 28. Williams R, Neuhaus P, Bismuth H, et al: Transpl Int 9(suppl 1):144S, 1996 29. Tanaka H, Kuroda A, Marusawa H, et al: J Am Chem Soc 109:5031, 1987 30. Goto T, Kino T, Hatanaka M, et al: Transplant Proc 23:2713, 1991 31. Peters DH, Fitton A, Plosker GL, et al: Drugs 46:764, 1993 32. Antoniou EA, Drayson M, Howie AJ, et al: Transplant Proc 30:1042, 1998
T
RANSPLANTATION of solid organs has been established as an important therapeutic alternative for end-stage disease of these organs since the introduction of effective immunosuppressive agents. However, the level of immunosuppression needed to block rejection with current immunosuppressive agents is associated, in some degree, with decreased ability to mount necessary immune responses against infection and neoplasia. In addition, each drug presents toxic side effects specific to the agent. Combination immunosuppression therapy has been used in solid organ transplantation to reduce drug toxicity.1,2 The introduction of donor-specific immunosuppression since Medawar’s experiments,3 has been a major goal in transplantation research. Multiple blood transfusions prior to transplantation can improve organ allograft survival.4,5 A step forward involved studies comparing relative contributions of class I MHC antigen-bearing cells such as platelets,6 – 8 erythrocytes,9,10 T cells11 or even hepatocytes,12 and cells expressing both class I and II antigens such as lymphocytes13–15 or dendritic cells16 all prolonged graft survival but with variable success. Furthermore, attempts of using donor-specific transfusion (DST) in combination with shortterm cyclosporine (CyA) therapy have shown a prolongation of allograft survival in adult animal models,17–19 validating this effect with clinical relevance in cadaveric transplantation.20 The aim of this study was to investigate whether FK 506,21,22 a potent immunosuppressive agent, used in low-dose 1 mg/kg in combination with a perioperative adjunctive DST could prolong allograft survival in a stringent rat model of cardiac allotransplantation. MATERIALS AND METHODS Heterotopic abdominal heart grafting was performed from DA(RT1av1) to Lew(RT11) rats, weighing 200 to 250 g each, using a previously described technique.1 Donor DA rats also provided lymphocytes for DST to Lew recipients. The subjects were commercially obtained from Charles River Ltd (United Kingdom) and cared for humanely during the course of the study according to prevailing Home Office Guidelines in the United Kingdom. Rats were allowed unrestricted access to food and water and were housed in filter-top barrier housing cages in light and temperaturecontrolled quarters. Prior to experimentation, animals were allowed to acclimatize to lab conditions for 1 week. The procedures performed in this study were licensed by the UK Home Office governing body. Surgical procedures were performed aseptically
and each animal was monitored a minimum of twice daily for its postoperative condition. Transplanted hearts were graded daily for rejection using a previously validated heart beat scoring system, ranging from four (normal) to zero (rejection), described by Morrissey et al.23 In this study, DST, 108 lymphocytes pooled single cell suspension from spleen and lymph nodes from DA donor rats, was administered intravenously (IV) to Lew recipients rats: (a) 14 days before transplantation (pretreatment, day 14), (b) on the day of transplantation (day 0), and (c) in two doses, on the day of transplantation and on the fourth postoperative day (day 0, day 4). FK 506 (1 or 2 mg/kg) was used as monotherapy or combination therapy, added to DST schedule. FK 506 was administered orally by gavage for 10 days, starting the day of grafting until day 9 posttransplantation. Rejection was defined as lack of contractions of the heart graft clinically, confirmed at necropsy and by histologic examination. Monotherapy groups served as positive controls. Survival data were analyzed using Student’s t test and one-way analysis of variance (ANOVA). Results were expressed as (SD). A two-sided P value ⬍ .05 was considered significant.
RESULTS
Table 1 shows the results. The DA-to-LEW allocombination is strongly MHC incompatible at both major and minor loci. The mean graft survival time for the control group, without any treatment, was 4.8 days (range 4 to 5). All grafts had clinical features of acute rejection (AR) that were histologically proved. DST pretreatment (day 14) did not prolong heart allograft survival significantly beyond the survival of the control group (mean survival 5.2 days). When DST was administered on the day of transplant (day 0), there was a significant survival prolongation of the grafts compared to the control group (mean survival 6.2 days, P: .043). FK 506 1 mg/kg (low dose) monotherapy, given for 10 days posttransplant, prolonged graft survival in this allocombination from 4.8 (controls) to 18.5 days mean survival time. When the dose was increased to 2 mg/kg, there was a further prolongation of the graft survival (mean survival From the Renal Transplant Unit (E.A.A.) and The Liver Unit (P.M., M.D’S.), Queen Elizabeth Hospital, and the Departments of Immunology (M.Dr.) and Pathology (A.J.H.), University of Birmingham, Birmingham, B15 2TH, United Kingdom, Address reprint requests to Mr E.A. Antoniou, MD, PhD, SR, Department of Nephrology, Renal Transplant Unit, Queen Elizabeth Hospital, Edgbaston, Birmingham, B15 2TH, United Kingdom.
0041-1345/99/$–see front matter PII S0041-1345(99)00572-2
© 1999 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2796
Transplantation Proceedings, 31, 2796–2798 (1999)
COMBINATION TREATMENT
2797
Table 1. Graft Survival Under DST Alone or in Combination With FK 506 (FK)
1 2 3 4 5 6 7 8
Groups
Treatment*
Survival (d)
Mean (SD)
⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽ ⫽
Control (none) DST(d ⫺ 14) DST(d 0) FK1(d0 – d9) FK1(d0 – d9)⫹DST(d0) FK1(d0 – d9)⫹DST(d0,d4) FK2(d0 – d9) FK2(d0 – d9)⫹DST(d0)
4 –5 5– 6 5– 8 11–29 20 –24 13–36 18 –26 24 –32
4.8 5.2 6.2 18.5 21.7 25.4 22 26.2
(n (n (n (n (n (n (n (n
5) 5) 10) 6) 4) 5) 6) 5)
P
— NS vs group 1 .043 vs group 1 S vs groups 1 and 3 NS vs group 4 NS vs group 4 NS vs group 4 .05 vs group 4
*DST dose: 2 mL suspension of donor lymphocytes (108) given IV. FK dose: 1 or 2 mg/kg given orally by gavage.
time 22 days, range 18 to 26 days, group 7) although the P value was not significant compared with the group of 1 mg/kg (group 4). Combining DST with 1 mg/kg FK 506 was not effective whether DST was given in two doses or a single dose at grafting (mean survival time 25.4 and 21.7 days, respectively). Raising the dose of FK 506 to 2 mg/kg elicits a survival benefit when combined with DST at transplant (mean survival time 26.2 days, range 24 to 32). DISCUSSION
Our experiments showed that DST alone provides a slight improvement in control allograft survival when it is given on day 0. Fabre and Morris24 originally and Opelz et al25 later had reported that destruction of the graft had been prevented by giving the recipients a DST 2 weeks before cardiac allotransplantation. Although this is an old observation made prior to the CyA era, it has been recently supported by other investigators.26 However, our results showed that when DST was given to this strong allogeneic rat model 14 days prior to transplantation (day 14), there was no significant improvement of graft survival beyond the control graft survival. Surprisingly, the grafts did not manifest a gradual decrease in the strength of ventricular contractions; all grafts were graded either grade ⫹4 or ⫹3 (strong heart beat) the day before rejection. FK 506 is a novel immunosuppressive agent that has been used effectively as a primary immunosuppressant or as rescue therapy both in experimental1,2 and clinical transplantation.22,27,28 It was isolated from Streptomyces tsukubaensis29 and is a macrolide molecule that inhibits expression of interleukin-2 via T lymphocytes.30,31 Ten doses of 1 mg/kg/d FK 506 given alone posttransplantation to prevent rejection significantly prolonged allograft survival beyond that found with DST. DST administered on day 0, or given as two doses on days 0 and 4 in the presence of 1 mg/kg FK 506, did not further enhance graft survival obtained under FK 506 alone. There was a nonsignificant trend to prolong graft survival when an increased dose of 2 mg/kg FK 506 was combined with DST on day 0. We have not demonstrated a significant benefit to graft survival by the addition of DST to therapy with FK 506. On the other hand, there has been no antagonistic effect from the addition of DST to FK 506 therapy. The results we obtained in this study differ from those published by other investigators15,19 who found
a beneficial effect on graft survival by adding DST to CyA therapy. These investigators used different allocombinations in their experiments, which is an important factor in allograft survival and could partially explain the discrepancy between their results and ours. With respect to the efficacy of DST in combination with different immunosuppressive agents, in a previous study,32 the combination of DST with FTY720 (a new immunosuppressive agent) showed similar results to those in this study. ACKNOWLEDGMENTS The authors express their sincere thanks to Dr M. Xu (Transplant Microsurgery Laboratory) and Ms K. Shimel (Biomedical Services Unit) for the excellent technical support provided.
REFERENCES 1. Antoniou EA, Xu M, Howie AJ, et al: Transplant Proc 29:2888, 1997 2. Antoniou EA, DeRoover A, Howie AJ, et al: Microsurgery (in press) 3. Billingham RE, Medawar PB: Nature 172:603, 1953 4. Opelz G, Terasaki PI: N Engl J Med 299:799, 1978 5. Salvatiera O, Vincenti F, Amend W, et al: Ann Surg 192:543, 1980 6. Hibberd AD, Morris PJ: Transplant Proc 17:1655, 1985 7. Touraine JL, Pouteil-Noble C, Raffaele P, et al: Transplantation 51:777, 1991 8. Borleffs JC, Neuchaus P, Van Rood JJ, et al: Lancet 1:1117, 1982 9. Wood KJ, Morris PJ: Transplantation 39:56, 1985 10. Baird MA, Heslop BF: Transplantation 45:66, 1987 11. Odorico JS, Naji A: Surg Forum XLII:411, 1992 12. Foster S, Cranston D, Wood KJ, et al: Transplantation 45:228, 1988 13. Cranston D, Wood KJ, Carter MP, et al: Transplantation 43:809, 1987 14. Lauchart W, Alkans B, Davies DA: Transplantation 29:259, 1980 15. Oluwole SF, Rashid AF, Reemtsma K, et al: Transplantation 45:1131, 1988 16. El-Malik F, Malik ST, Varghese Z, et al: Transplantation 38:213, 1984 17. Martinelli GP, Rhune-Loy R, Sher I, et al: Transplantation 39:1, 1985 18. Yasumura T, Kahan BD: Transplantation 36:603, 1983 19. Ahmed SM, Baslaim G, Eshelby D, et al: Transplantation 57:634, 1994
2798 20. Brunson ME, Tchervenkov JI, Alexander JW: Transplantation 52:545, 1991 21. Ochiai T, Nagata M, Nakajima K, et al: Transplantation 44:729, 1987 22. European Tacrolimus Multicentre Renal Study Group: Transplantation 64:436, 1997 23. Morrisey PE, Gollin G, Brusett K, et al: Transplantation 57:631, 1994 24. Fabre JW, Morris PJ: Transplantation 14:608, 1972 25. Opelz G, Sengar DPS, Mickey MR, et al: Transplant Proc 5:253, 1973 26. Bradley JA: Immunol Lett 29:127, 1991
ANTONIOU, DRAYSON, HOWIE ET AL 27. The US Multicenter FK 506 Liver Study Group: N Engl J Med 331:1110, 1994 28. Williams R, Neuhaus P, Bismuth H, et al: Transpl Int 9(suppl 1):144S, 1996 29. Tanaka H, Kuroda A, Marusawa H, et al: J Am Chem Soc 109:5031, 1987 30. Goto T, Kino T, Hatanaka M, et al: Transplant Proc 23:2713, 1991 31. Peters DH, Fitton A, Plosker GL, et al: Drugs 46:764, 1993 32. Antoniou EA, Drayson M, Howie AJ, et al: Transplant Proc 30:1042, 1998