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Rejection of cardiac xenografts by NK cells and macrophages
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Rejection of Cardiac Xenografts by NK Cells and Macrophages Y. Lin, J. Goebels, M. Vandeputte, and M. Waer
UE TO the nature of hyperacute rejection of xenografts transplanted between phylogenetically disparate species, cellular immune responses in xenotransplantation are less well understood. Recent advances in controlling hyperacute rejection have allowed the survival of vascularized xenografls to the point where these may elicit cellular immune responses. There is increasing evidence showing that, if hyperacute rejection is circUmvented, discordant xenografts may undergo rejection within a few days. This delayed xenograft rejection is immunopathologically characterized by a prominent cellular infiltration consisting of mainly natural killer (NK) cells, macrophages and T cellsJ '~ In the present study, the role played by NK cells and macrophages in xenograft rejection was further studied in athymic nude rats receiving concordant xenografts. As these nude rats were previously shown to have strong NK cell activity, we were interested to see how this would influence the capacity of these T-deficient rats to reject hamster heart xenografts.
D
MATERIALS AND METHODS
Four- to six-week old male athymic nude rats (rnu/rnu; Harlan, Zeist, The Netherlands), weighing approximately 200 g, were used as recipients. Outbred Golden Syrian hamsters, weighing approximately 100 g, were used as donors. Methods used for heterotopic heart transplantation and monitoring of xenoantibody formation by FACS have been described previously.3 Immunosuppression with Leflunomide (LF; Hoechst, Germany) was given at a daily dose of 20 mg/kg by gavage. A dose of 1 mg of an anti-NK cell serum, Asialo GM-1 (Wako, Neuss, Germany), was given intravenously (IV) every other day to inhibit NK cell activity. For transfer of sensitized splenocytes, 100 x 106 splenocytes taken from rats rejecting a hamster heart xenograft were injected IV to newly transplanted nude rats receiving the same therapy. The splenocytes were depleted of macrophages by repeated-plastic adhesion. RESULTS AND DISCUSSION
Despite complete inhibition of xenoantibody formation by the treatment with LF, nude rats rejected hamster xenografts at the same tempo as untreated nude controls (mean survival time (MST) = 3.2 - 0.6 d; n = 5 and MST = 3.2 -+ 0.5 d; n = 4, respectively). Histopathologically, rejected
0041 - 1345/97/$17.00 PII S0041-1345(97)00399-0 2354
xenografts showed a predominant mononuclear cell infiltration. Addition of the anti-NK cell serum, Asialo GM-1, to the LF treatment, significantly prolonged xenograft survival to 6.2 -+ 0.8 days (P < 0.001). The delayed rejection in LF+Asialo GM-1 treated rats appeared to be due to a mechanism not involving xenoantibodies or NK cells, as both were strongly inhibited even at the time of rejection. In order to explore the mechanism underlying the delayed xenograft rejection in LF+Asialo GM-1 treated rats, we performed a sensitized splenocyte transfer assay whereby 100 x 106 sensitized splenocytes taken from LF+Asialo GM-1 treated rats at the time of rejection were injected IV to naive nude rats receiving a xenograft under the same therapy. This procedure significantly accelerated the xenograft rejection from 6 days (n = 5) to 3 days (n = 3). This accelerated rejection did not occur if the sensitized splenocytes were depleted from macrophages prior to transfer, suggesting that macrophages which were activated after xenografting played an important role in xenograft rejection in LF+Asialo GM-1 treated rats. This study suggests that when humoral immune responses to a vascularized xenograft are blocked, NK cells may reject xenografts as well. After suppressing these NK cells, macrophages may be brought about to provoke xenograft rejection. REFERENCES
1. Leventhal JR, Matas AJ, Sun LH, et al: Transplantation 56:1, 1993 2. Fryer JP, Leventhal AP, Dalmasso AP, et al: Transplant Immunol 2:87, 1994 3. Lin Y, Vandeputte M, Waer M: Kidney lnt 48:23, 1995 From the Laboratory for Experimental Transplantation and Division of Nephrology, University of Leuven, Leuven, Belgium. This work was supported by a grant of the "ONDERZOEKS RAAD" from the University of Leuven and by the "National Fund for Scientific Research-NFWO." Address correspondence to Prof Dr M. Waer, Laboratory for Experimental Transplantation and Division of Nephrology, University of Leuven, Herestraat 49, B-3000, Leuven, Belgium.
© 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
Transplantation Proceedings, 29, 2354 (1997)
Rejection of Cardiac Xenografts by NK Cells and Macrophages Y. Lin, J. Goebels, M. Vandeputte, and M. Waer
UE TO the nature of hyperacute rejection of xenografts transplanted between phylogenetically disparate species, cellular immune responses in xenotransplantation are less well understood. Recent advances in controlling hyperacute rejection have allowed the survival of vascularized xenografls to the point where these may elicit cellular immune responses. There is increasing evidence showing that, if hyperacute rejection is circUmvented, discordant xenografts may undergo rejection within a few days. This delayed xenograft rejection is immunopathologically characterized by a prominent cellular infiltration consisting of mainly natural killer (NK) cells, macrophages and T cellsJ '~ In the present study, the role played by NK cells and macrophages in xenograft rejection was further studied in athymic nude rats receiving concordant xenografts. As these nude rats were previously shown to have strong NK cell activity, we were interested to see how this would influence the capacity of these T-deficient rats to reject hamster heart xenografts.
D
MATERIALS AND METHODS
Four- to six-week old male athymic nude rats (rnu/rnu; Harlan, Zeist, The Netherlands), weighing approximately 200 g, were used as recipients. Outbred Golden Syrian hamsters, weighing approximately 100 g, were used as donors. Methods used for heterotopic heart transplantation and monitoring of xenoantibody formation by FACS have been described previously.3 Immunosuppression with Leflunomide (LF; Hoechst, Germany) was given at a daily dose of 20 mg/kg by gavage. A dose of 1 mg of an anti-NK cell serum, Asialo GM-1 (Wako, Neuss, Germany), was given intravenously (IV) every other day to inhibit NK cell activity. For transfer of sensitized splenocytes, 100 x 106 splenocytes taken from rats rejecting a hamster heart xenograft were injected IV to newly transplanted nude rats receiving the same therapy. The splenocytes were depleted of macrophages by repeated-plastic adhesion. RESULTS AND DISCUSSION
Despite complete inhibition of xenoantibody formation by the treatment with LF, nude rats rejected hamster xenografts at the same tempo as untreated nude controls (mean survival time (MST) = 3.2 - 0.6 d; n = 5 and MST = 3.2 -+ 0.5 d; n = 4, respectively). Histopathologically, rejected
0041 - 1345/97/$17.00 PII S0041-1345(97)00399-0 2354
xenografts showed a predominant mononuclear cell infiltration. Addition of the anti-NK cell serum, Asialo GM-1, to the LF treatment, significantly prolonged xenograft survival to 6.2 -+ 0.8 days (P < 0.001). The delayed rejection in LF+Asialo GM-1 treated rats appeared to be due to a mechanism not involving xenoantibodies or NK cells, as both were strongly inhibited even at the time of rejection. In order to explore the mechanism underlying the delayed xenograft rejection in LF+Asialo GM-1 treated rats, we performed a sensitized splenocyte transfer assay whereby 100 x 106 sensitized splenocytes taken from LF+Asialo GM-1 treated rats at the time of rejection were injected IV to naive nude rats receiving a xenograft under the same therapy. This procedure significantly accelerated the xenograft rejection from 6 days (n = 5) to 3 days (n = 3). This accelerated rejection did not occur if the sensitized splenocytes were depleted from macrophages prior to transfer, suggesting that macrophages which were activated after xenografting played an important role in xenograft rejection in LF+Asialo GM-1 treated rats. This study suggests that when humoral immune responses to a vascularized xenograft are blocked, NK cells may reject xenografts as well. After suppressing these NK cells, macrophages may be brought about to provoke xenograft rejection. REFERENCES
1. Leventhal JR, Matas AJ, Sun LH, et al: Transplantation 56:1, 1993 2. Fryer JP, Leventhal AP, Dalmasso AP, et al: Transplant Immunol 2:87, 1994 3. Lin Y, Vandeputte M, Waer M: Kidney lnt 48:23, 1995 From the Laboratory for Experimental Transplantation and Division of Nephrology, University of Leuven, Leuven, Belgium. This work was supported by a grant of the "ONDERZOEKS RAAD" from the University of Leuven and by the "National Fund for Scientific Research-NFWO." Address correspondence to Prof Dr M. Waer, Laboratory for Experimental Transplantation and Division of Nephrology, University of Leuven, Herestraat 49, B-3000, Leuven, Belgium.
© 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
Transplantation Proceedings, 29, 2354 (1997)