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Prolonged ischemia enhances acute rejection in rat kidney grafts
Prolonged Ischemia Enhances Acute Rejection in Rat Kidney Grafts E.A. Kouwenhoven, R.W.F. de Bruin, I.M. Bajema, R.L. Marquet, and J.N.M. IJzermans
A
CUTE rejection episodes shorten the lifespan of an organ graft.1 In unravelling the factors that influence acute rejection, delayed graft function has been suggested to exacerbate early acute rejection. Some clinical studies demonstrate that kidney grafts with prolonged cold ischemia or delayed graft function, a parameter that correlates with the ischemic period, more often experience early acute rejection than those with minimal ischemia.2– 4 The underlying mechanism is rather putative. It has been postulated that ischemia-induced reperfusion injury initiates an inflammatory response that provokes increased host immunological reactivity, possibly by upregulated MHC expression on graft tissue after ischemia.5 However, it has not been excluded that ischemia-induced injury is superimposed on allogenicity. Therefore, the aim of this study was to study the impact of ischemia on the immune response in rat kidney allografts compared with that in isografts. MATERIALS AND METHODS Animals and Kidney Transplantation Inbred male rats weighing 200 to 250 g were used. They were bred under specific pathogen-free conditions and purchased from Harlan (Horst, The Netherlands). Kidneys were transplanted heterotopically with end-to-end ureter anastomosis.
Experimental Groups To induce ischemic injury, donor BN-kidneys were perfused in situ and stored for 24 h in 4°C University of Wisconsin solution before transplantation to WAG recipients. No immunosuppression was administered. Nonischemic BN-WAG allografts, and ischemic and non-ischemic WAG-WAG isografts served as controls to determine the relationship between ischemia and allogenicity.
Histology and Immunohistology The histomorphology according to the BANFF criteria for acute rejection, and infiltrating cells (CD4⫹- and CD8⫹-cells, macrophages), MHC class II and adhesion molecule expression (Pselectin, and ICAM-1) were assessed. Grafts were examined at days 1, 2, 3, 4, 6, and 8. (n ⫽ 5 animals/group/timepoint for allografts, n ⫽ 3 animals/group/timepoint for isografts). For statistical analysis of ordinal data (histopathology and adhesionmolecule expression) the Kruskal-Wallis-one-way ANOVA followed by the Mann-Whitney test were performed. One-way ANOVA followed by a NewMan Keuls test were used for the data of infiltrating cells. The effects of ischemia between the © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
syngeneic and allogeneic groups were compared using two-way ANOVA interaction test. Statistical significance was established at P ⬍ .05.
RESULTS
In allografts, exposure of the kidney to 24 h of ischemia led to a significantly earlier onset of interstitial cell infiltration and tubulitis compared with non-ischemic allografts: BANFF score of interstitial cell infiltration was 1 ⫾ 0 vs 0.25 ⫾ 0.29 at day 3, and 2 ⫾ 0 vs 1.25 ⫾ 0.25 at day 4. In contrast, in isografts, the effect of ischemia on the histology was not significant. From day 6, the histologic differences between ischemic and nonischemic grafts disappeared. In both allo- and isografts, ischemia led to more intense expression of P-selectin (day 1), ICAM-1 (day 2), and MHC class II on endothelium and proximal tubular cells (day 2). Concurrently with the upregulated ICAM-1 and MHC expression, significantly more CD4⫹ cells and macrophages infiltrated the ischemic allografts at day 2 and 3, and the ischemic isografts at day 4. The influx of these cells after ischemia was significantly greater in allografts than in isografts. DISCUSSION
Prolonged cold ischemia results in an earlier onset of the histologic and immunologic features of acute rejection in kidney allografts. Our data indicate that ischemia enhances the alloimmune response, since the infiltration of MHC class II⫹ and CD4⫹ cells was significantly higher in allografts then in isografts at days 2 and 3 posttransplantation when compared with their nonischemic counterparts. Moreover, histology showed earlier cellular infiltration in the renal interstitium and tubulitis, whereas ischemic isografts only showed insignificant histologic changes. Therefore, the earlier onset of acute rejection in 24-hour cold preserved allografts may be prevented by better preservation, or by adequate immunosuppressive therapy. From the Departments of Surgery and Pathology, Erasmus University, Rotterdam, The Netherlands. Address reprint requests to R.W.F. de Bruin, PhD, Laboratory for Experimental Surgery, Room Ee102b, Erasmus University, PO Box 1738, 3000 DR, Rotterdam, The Netherlands. 0041-1345/01/$–see front matter PII S0041-1345(00)02797-4 361
Transplantation Proceedings, 33, 361–362 (2001)
362
REFERENCES 1. Matas AJ, Gillingham KJ, Payne WD, et al: Transplantation 57:857, 1994 2. Troppmann C, Gillingham KJ, Benedetti E, et al: Transplantation 58:962, 1995
KOUWENHOVEN, DE BRUIN, BAJEMA ET AL 3. Shoskes DA, Cecka JM: Transplantation 66:1697, 1998 4. Boom H, Mallat MJ, De Fijter JW, et al: Kidney Int 58:859, 2000 5. Shoskes DA, Parfrey NA, Halloran PF: Transplantation 49:201, 1990
A
CUTE rejection episodes shorten the lifespan of an organ graft.1 In unravelling the factors that influence acute rejection, delayed graft function has been suggested to exacerbate early acute rejection. Some clinical studies demonstrate that kidney grafts with prolonged cold ischemia or delayed graft function, a parameter that correlates with the ischemic period, more often experience early acute rejection than those with minimal ischemia.2– 4 The underlying mechanism is rather putative. It has been postulated that ischemia-induced reperfusion injury initiates an inflammatory response that provokes increased host immunological reactivity, possibly by upregulated MHC expression on graft tissue after ischemia.5 However, it has not been excluded that ischemia-induced injury is superimposed on allogenicity. Therefore, the aim of this study was to study the impact of ischemia on the immune response in rat kidney allografts compared with that in isografts. MATERIALS AND METHODS Animals and Kidney Transplantation Inbred male rats weighing 200 to 250 g were used. They were bred under specific pathogen-free conditions and purchased from Harlan (Horst, The Netherlands). Kidneys were transplanted heterotopically with end-to-end ureter anastomosis.
Experimental Groups To induce ischemic injury, donor BN-kidneys were perfused in situ and stored for 24 h in 4°C University of Wisconsin solution before transplantation to WAG recipients. No immunosuppression was administered. Nonischemic BN-WAG allografts, and ischemic and non-ischemic WAG-WAG isografts served as controls to determine the relationship between ischemia and allogenicity.
Histology and Immunohistology The histomorphology according to the BANFF criteria for acute rejection, and infiltrating cells (CD4⫹- and CD8⫹-cells, macrophages), MHC class II and adhesion molecule expression (Pselectin, and ICAM-1) were assessed. Grafts were examined at days 1, 2, 3, 4, 6, and 8. (n ⫽ 5 animals/group/timepoint for allografts, n ⫽ 3 animals/group/timepoint for isografts). For statistical analysis of ordinal data (histopathology and adhesionmolecule expression) the Kruskal-Wallis-one-way ANOVA followed by the Mann-Whitney test were performed. One-way ANOVA followed by a NewMan Keuls test were used for the data of infiltrating cells. The effects of ischemia between the © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
syngeneic and allogeneic groups were compared using two-way ANOVA interaction test. Statistical significance was established at P ⬍ .05.
RESULTS
In allografts, exposure of the kidney to 24 h of ischemia led to a significantly earlier onset of interstitial cell infiltration and tubulitis compared with non-ischemic allografts: BANFF score of interstitial cell infiltration was 1 ⫾ 0 vs 0.25 ⫾ 0.29 at day 3, and 2 ⫾ 0 vs 1.25 ⫾ 0.25 at day 4. In contrast, in isografts, the effect of ischemia on the histology was not significant. From day 6, the histologic differences between ischemic and nonischemic grafts disappeared. In both allo- and isografts, ischemia led to more intense expression of P-selectin (day 1), ICAM-1 (day 2), and MHC class II on endothelium and proximal tubular cells (day 2). Concurrently with the upregulated ICAM-1 and MHC expression, significantly more CD4⫹ cells and macrophages infiltrated the ischemic allografts at day 2 and 3, and the ischemic isografts at day 4. The influx of these cells after ischemia was significantly greater in allografts than in isografts. DISCUSSION
Prolonged cold ischemia results in an earlier onset of the histologic and immunologic features of acute rejection in kidney allografts. Our data indicate that ischemia enhances the alloimmune response, since the infiltration of MHC class II⫹ and CD4⫹ cells was significantly higher in allografts then in isografts at days 2 and 3 posttransplantation when compared with their nonischemic counterparts. Moreover, histology showed earlier cellular infiltration in the renal interstitium and tubulitis, whereas ischemic isografts only showed insignificant histologic changes. Therefore, the earlier onset of acute rejection in 24-hour cold preserved allografts may be prevented by better preservation, or by adequate immunosuppressive therapy. From the Departments of Surgery and Pathology, Erasmus University, Rotterdam, The Netherlands. Address reprint requests to R.W.F. de Bruin, PhD, Laboratory for Experimental Surgery, Room Ee102b, Erasmus University, PO Box 1738, 3000 DR, Rotterdam, The Netherlands. 0041-1345/01/$–see front matter PII S0041-1345(00)02797-4 361
Transplantation Proceedings, 33, 361–362 (2001)
362
REFERENCES 1. Matas AJ, Gillingham KJ, Payne WD, et al: Transplantation 57:857, 1994 2. Troppmann C, Gillingham KJ, Benedetti E, et al: Transplantation 58:962, 1995
KOUWENHOVEN, DE BRUIN, BAJEMA ET AL 3. Shoskes DA, Cecka JM: Transplantation 66:1697, 1998 4. Boom H, Mallat MJ, De Fijter JW, et al: Kidney Int 58:859, 2000 5. Shoskes DA, Parfrey NA, Halloran PF: Transplantation 49:201, 1990