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NK cells, macrophages, and humoral immune responses are dominant in primary nonfunction of islet grafts in the dog-to-rat xenotransplant model
ELSEVIER
NK Cells, Macrophages, and Humoral Immune Responses Are Dominant in Primary Nonfunction of Islet Grafts in the Dog-to-Rat Xenotransplant Model S. Deng, R.J. Ketchum,
T. Kucher,
M. Weber, A. Naji, and K.L. Brayman
X
ENOGENEIC islet transplantation has potential application for the treatment of diabetes. However, islet xenografts are rapidly destroyed by vigorous xenogeneic immune responses, which are considered to be fundamentally different from those of allogeneic rejection. In our previous study, a high rate of primary nonfunction (PNF) and early graft failure was observed in canine-to-rat islet transplantation.’ Administration of routine immunosuppressive drugs, such as cyclosporine, has little effect on prolongation of graft survival in this combination. Further understanding of the mechanisms of rejection in this stringent model may provide relevant information on the development of strategies of clinical immunosuppression. In this study, we first investigated the fate of islet xenografts in immunocompromised nude rats. Furthermore, we analyzed the detailed phenotypic characterization of infiltrating cells in the islet grafts and investigated the effect of inhibiting lymphocyte activity, nitric oxide (NO) production, or macrophage function on canine islet xenograft survival in rat recipients. MATERIALS
AND
METHODS
Canine pancreata were surgically obtained from adult beagle dogs (Marshall Farms, North Rose, NY) and digested by intraductal injection of collagenase (Boehringer Mannheim, Indianapolis, Ind) using the automated method. Islets were then purified by discontinuous Euro-Ficoll gradient (Ficoll [Sigma Chemical, St Louis, MO] in Euro-Collins [Fresenius, New Brunswick, NJ]) centrifugation (Cobe 2991 centrifuge, Cobe, Lakewood, Co]). Purified islets (purity >90%) were cultured overnight in Nutrient Mixture F12 (Life Technologies, Grand Island, NY) supplemented with 10% fetal calf serum [FCS; HyClone, Logan, Utah] and 1% PSF (Life Technologies) at 37°C in 5% CO,. Recipients, immunocompromised Nude rats, and immunocompetent Lewis rats (-200 to 250 g, Harlan, Indianapolis, Ind) were rendered diabetic with streptozotocin (Zanosar, 65 mgikg, Upjohn, Kalamazoo, Mich) 7 days prior to transplantation. Canine islets (-5000 islets) were transplanted into the liver of recipients by portal vein injection. Animals were divided into the untreated control group and the groups treated with antilymphocyte serum (ALS; 1 mL intraperitoneally, -1 day [Accurate Chemical, Westbury, NY]), aminoguanidine (AG; 50 mgikg intraperitoneally, bid [Sigma Chemical]), or gadolinium (Gd, 2 mg/kg intravenously, -2 d, -1 d, 0 d [Sigma Chemical]).‘-“ Functional islet graft survival was evaluated by daily blood glucose measurement, with
graft failure indicated by consecutive days of hyperglycemia (>200 mg/dL). Immunohistologic examination of islet grafts was performed at regular timepoints (24, 48, and 72 hours) posttransplantation. The phenotype of infiltrating cells was determined by using the following primary monoclonal antibodies: anti-CD4 (W3/25), anti-CD8 (0X8), anti-macrophage (EDl), and anti-NK cell (3.2.3). Biotinylated horse anti-mouse IgG antibody was used as the secondary antibody. After a final incubation with an avidin-biotin-peroxidase complex, the peroxidase reaction was developed with a carbazolecontaining (diaminobenzidene, DAB) buffer for 15 minutes. Control sections were analyzed by omitting the primary antibody. RESULTS
Without recipient treatment, neither immunocompromised Nude nor immunocompetent Lewis rats became normoglycemic when canine islets were transplanted into the liver of the recipients. Inhibition of NO production (by AG) or suppression of host macrophage function (by Gd), slightly, but significantly, improved early canine islet function in Lewis rats, evidenced by an increase in duration of normoglycemia from 0 day in the control group to 1 day in the AG (P < .02) or Gd-treated group (P < .02). Broad immunosuppression by lymphocyte depletion (ALS), which depletes the lymphocytes, as well as macrophages and NK cells, significantly prolonged islet xenograft survival to a mean graft survival of 2.4 days (P < .Ol). Routine histologic examination showed an apparent leukocyte infiltration of islet grafts early, within the first 24 hours posttransplantation. Immunohistologic analysis demonstrated that these early infiltrating cells were CD4+, with a few CD8+ cells. Furthermore, it was determined that the majority of CD4+ cells were macrophages and NK cells. DISCUSSION
In islet xenotransplantation, cell-mediated immune rejection, especially by CD4+ lymphocytes, has been suggested From the Department of Surgery, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania. Address reprint requests to Kenneth Brayman, Department of Surgery, University of Pennsylvania Medical Center, Philadelphia, PA 19104.
0041-l 345/97/$17.00 PII SOO41-1345(97)00232-7
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2062
Transplantation Proceedings, 29, 2062-2063
(1997)
2063
CANINE ISLET XENOGRAFT to play an important role in the destruction of islet xenografts4 However, recent studies have demonstrated a significant role for non-T-cell-mediated immune responses in islet xenotransplant models.2,3 In this study, long-term graft survival was obtained in dog-to-nude mouse islet transplantation (data not shown), indicating that isolated canine islets retained normal viability and function. However, normoglycemia was never restored even in immunocompromised Nude (athymic) rat recipients of intraportal canine islet xenografts. This result strongly suggests that nonspecific immune responses, rather than T-cell-mediated immune events, play a dominant role in PNF and early graft failure in this highly discordant combination. The roles of macrophages and NK cells have recently been investigated in the rejection of both whole organ and cellular xenotransplants.2*3 Although less than likely for xenogeneic organ transplantation or concordant islet xenotransplantation, macrophages and/or NK cells seem to play more important roles in discordant islet xenograft rejection. In this study, immunohistologic examination demonstrated an early infiltrating of canine islet grafts with CD4+ macrophages and NK cells, indicating that the macrophages and NK cells might be responsible for the early failure of canine islet xenografts in rat recipients. This hypothesis is supported by demonstration of a significant improvement of early islet function by interruption of host macrophage function or inhibition of NO production. Nevertheless, the significant
prolongation of islet xenografts by using a broad immunosuppression (ALS), which depletes the lymphocytes, as well as macrophages and NK cells, suggests that immune cells (including macrophages, NK cells, and/or T cells) are involved in the complex interactions leading to PNF or early graft failure of islet xenografts in this model. Taken together with our previous study showing a beneficial effect of complement inhibition5 we conclude that NK cells, macrophages, and humoral immune responses are dominant in PNF of islet grafts in the dog-to-rat xenotransplant model. The intervention in multiple pathways, directed at the relevant arms of the immune response, will be required to successfully prevent early islet destruction and improve graft survival in islet xenotransplantation.
REFERENCES 1. Brayman KL, Morel P, Field J, et al: Transplant Proc 24:651,
1992 2. Fryer JP, Leventhal JR, Dalmasso AP, et al: Transplantation 59:171, 1995 3. Wallgren AC, Karlsson-Parra A, Korgren 0: Transplantation 60:594, 1995 4. Falqui L, Finke EH, Care1 JC, et al: Transplantation
51:1322,
1991 5. Deng S, Ketchum 28:805, 1996
RJ,
Levy
MM,
et al: Transplant
Proc
NK Cells, Macrophages, and Humoral Immune Responses Are Dominant in Primary Nonfunction of Islet Grafts in the Dog-to-Rat Xenotransplant Model S. Deng, R.J. Ketchum,
T. Kucher,
M. Weber, A. Naji, and K.L. Brayman
X
ENOGENEIC islet transplantation has potential application for the treatment of diabetes. However, islet xenografts are rapidly destroyed by vigorous xenogeneic immune responses, which are considered to be fundamentally different from those of allogeneic rejection. In our previous study, a high rate of primary nonfunction (PNF) and early graft failure was observed in canine-to-rat islet transplantation.’ Administration of routine immunosuppressive drugs, such as cyclosporine, has little effect on prolongation of graft survival in this combination. Further understanding of the mechanisms of rejection in this stringent model may provide relevant information on the development of strategies of clinical immunosuppression. In this study, we first investigated the fate of islet xenografts in immunocompromised nude rats. Furthermore, we analyzed the detailed phenotypic characterization of infiltrating cells in the islet grafts and investigated the effect of inhibiting lymphocyte activity, nitric oxide (NO) production, or macrophage function on canine islet xenograft survival in rat recipients. MATERIALS
AND
METHODS
Canine pancreata were surgically obtained from adult beagle dogs (Marshall Farms, North Rose, NY) and digested by intraductal injection of collagenase (Boehringer Mannheim, Indianapolis, Ind) using the automated method. Islets were then purified by discontinuous Euro-Ficoll gradient (Ficoll [Sigma Chemical, St Louis, MO] in Euro-Collins [Fresenius, New Brunswick, NJ]) centrifugation (Cobe 2991 centrifuge, Cobe, Lakewood, Co]). Purified islets (purity >90%) were cultured overnight in Nutrient Mixture F12 (Life Technologies, Grand Island, NY) supplemented with 10% fetal calf serum [FCS; HyClone, Logan, Utah] and 1% PSF (Life Technologies) at 37°C in 5% CO,. Recipients, immunocompromised Nude rats, and immunocompetent Lewis rats (-200 to 250 g, Harlan, Indianapolis, Ind) were rendered diabetic with streptozotocin (Zanosar, 65 mgikg, Upjohn, Kalamazoo, Mich) 7 days prior to transplantation. Canine islets (-5000 islets) were transplanted into the liver of recipients by portal vein injection. Animals were divided into the untreated control group and the groups treated with antilymphocyte serum (ALS; 1 mL intraperitoneally, -1 day [Accurate Chemical, Westbury, NY]), aminoguanidine (AG; 50 mgikg intraperitoneally, bid [Sigma Chemical]), or gadolinium (Gd, 2 mg/kg intravenously, -2 d, -1 d, 0 d [Sigma Chemical]).‘-“ Functional islet graft survival was evaluated by daily blood glucose measurement, with
graft failure indicated by consecutive days of hyperglycemia (>200 mg/dL). Immunohistologic examination of islet grafts was performed at regular timepoints (24, 48, and 72 hours) posttransplantation. The phenotype of infiltrating cells was determined by using the following primary monoclonal antibodies: anti-CD4 (W3/25), anti-CD8 (0X8), anti-macrophage (EDl), and anti-NK cell (3.2.3). Biotinylated horse anti-mouse IgG antibody was used as the secondary antibody. After a final incubation with an avidin-biotin-peroxidase complex, the peroxidase reaction was developed with a carbazolecontaining (diaminobenzidene, DAB) buffer for 15 minutes. Control sections were analyzed by omitting the primary antibody. RESULTS
Without recipient treatment, neither immunocompromised Nude nor immunocompetent Lewis rats became normoglycemic when canine islets were transplanted into the liver of the recipients. Inhibition of NO production (by AG) or suppression of host macrophage function (by Gd), slightly, but significantly, improved early canine islet function in Lewis rats, evidenced by an increase in duration of normoglycemia from 0 day in the control group to 1 day in the AG (P < .02) or Gd-treated group (P < .02). Broad immunosuppression by lymphocyte depletion (ALS), which depletes the lymphocytes, as well as macrophages and NK cells, significantly prolonged islet xenograft survival to a mean graft survival of 2.4 days (P < .Ol). Routine histologic examination showed an apparent leukocyte infiltration of islet grafts early, within the first 24 hours posttransplantation. Immunohistologic analysis demonstrated that these early infiltrating cells were CD4+, with a few CD8+ cells. Furthermore, it was determined that the majority of CD4+ cells were macrophages and NK cells. DISCUSSION
In islet xenotransplantation, cell-mediated immune rejection, especially by CD4+ lymphocytes, has been suggested From the Department of Surgery, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania. Address reprint requests to Kenneth Brayman, Department of Surgery, University of Pennsylvania Medical Center, Philadelphia, PA 19104.
0041-l 345/97/$17.00 PII SOO41-1345(97)00232-7
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
2062
Transplantation Proceedings, 29, 2062-2063
(1997)
2063
CANINE ISLET XENOGRAFT to play an important role in the destruction of islet xenografts4 However, recent studies have demonstrated a significant role for non-T-cell-mediated immune responses in islet xenotransplant models.2,3 In this study, long-term graft survival was obtained in dog-to-nude mouse islet transplantation (data not shown), indicating that isolated canine islets retained normal viability and function. However, normoglycemia was never restored even in immunocompromised Nude (athymic) rat recipients of intraportal canine islet xenografts. This result strongly suggests that nonspecific immune responses, rather than T-cell-mediated immune events, play a dominant role in PNF and early graft failure in this highly discordant combination. The roles of macrophages and NK cells have recently been investigated in the rejection of both whole organ and cellular xenotransplants.2*3 Although less than likely for xenogeneic organ transplantation or concordant islet xenotransplantation, macrophages and/or NK cells seem to play more important roles in discordant islet xenograft rejection. In this study, immunohistologic examination demonstrated an early infiltrating of canine islet grafts with CD4+ macrophages and NK cells, indicating that the macrophages and NK cells might be responsible for the early failure of canine islet xenografts in rat recipients. This hypothesis is supported by demonstration of a significant improvement of early islet function by interruption of host macrophage function or inhibition of NO production. Nevertheless, the significant
prolongation of islet xenografts by using a broad immunosuppression (ALS), which depletes the lymphocytes, as well as macrophages and NK cells, suggests that immune cells (including macrophages, NK cells, and/or T cells) are involved in the complex interactions leading to PNF or early graft failure of islet xenografts in this model. Taken together with our previous study showing a beneficial effect of complement inhibition5 we conclude that NK cells, macrophages, and humoral immune responses are dominant in PNF of islet grafts in the dog-to-rat xenotransplant model. The intervention in multiple pathways, directed at the relevant arms of the immune response, will be required to successfully prevent early islet destruction and improve graft survival in islet xenotransplantation.
REFERENCES 1. Brayman KL, Morel P, Field J, et al: Transplant Proc 24:651,
1992 2. Fryer JP, Leventhal JR, Dalmasso AP, et al: Transplantation 59:171, 1995 3. Wallgren AC, Karlsson-Parra A, Korgren 0: Transplantation 60:594, 1995 4. Falqui L, Finke EH, Care1 JC, et al: Transplantation
51:1322,
1991 5. Deng S, Ketchum 28:805, 1996
RJ,
Levy
MM,
et al: Transplant
Proc