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Primary nonfunction of islet xenografts in rat recipients results from non-t-cell-mediated immune responses
ELSEVIER
Primary Nonfunction of Islet Xenografts in Rat Recipients From Non-T-Cell-Mediated Immune Responses S. Deng, R.J. Ketchum,
T. Kucher,
M. Weber, A. Naji, and K.L. Brayman
P
RIMARY nonfunction (PNF) and early graft failure affect the functional mass of islet tissue engrafted, and represent a major problem in clinical islet transplantation. Previous studies have shown that grafts of isolated canine pancreatic islets survive and function in immunocompetent murine recipients until graft rejection. In contrast, a very high rate of PNF has been demonstrated in rat recipients of canine islet xenografts.’ Routine immunosuppressive protocols, such as cyclosporin therapy, have little effect on prolongation of graft survival in this combination. A better understanding of the mechanisms involved in this strong, rapid immune response in the rat may provide focus in the development of strategies of immunosuppression which would be clinically relevant. To identify the effector mechanism of PNF in the dog-to-rat islet transplant model, we have shown that long-term culture, or inhibition of native complement, slightly improves early islet function in this model.* In this study, we further investigate the effect of inhibiting lymphocyte activity, nitric oxide (NO) production, or macrophage (M$) function on canine islet xenograft survival in rat recipients.
MATERIALS
AND
Results
METHODS
Canine islets were isolated and purified (>90% purity) from beagle (Marshall Farms, North Rose, NY) pancreata using an automated method of collagenase (Boehringer Mannheim, Indianapolis, IN) digestion, and discontinuous Euroficoll gradient (Ficoll [Sigma Chemical, St Louis, MO] in Eurocollins [Fresenius USA, New Brunswick, NJ]) centrifugation (Cobe 2991 centrifuge, Cobe, Lakewood, Colo). Recipient rats (immunocompetent LEW [Harlan, Indianapolis, Ind] or immunocompromised Nude rats [Harlan, Indianapolis, Ind], -200 to 250 g) were rendered diabetic with streptozotocin (Zanosar, 65 mgikg, Upjohn, Kalamazoo, Mich) 7 days before transplantation. Recipient animals received -5000 islets, injected into the recipient’s portal vein, following overnight islet culture (18 hours, Nutrient Mixture F12 [Life Technologies, Grand Island, NY], supplemented with 10% FCS [HyClone, Logan, Utah] and 1% PSF [Life Technologies, Grand Island, NY], at 37°C in 5% CO?). Recipient groups were: untreated controls, antilymphocyte serum (AL& 1 mL IP, -1 d [Accurate Chemical, Westbury, NY]), aminoguanidine (AG, 50 mgikg IP, bid [Sigma Chemical, St Louis, MO]) or gadolinium (Gd, 2 mg/kg IV, -2 d, - 1 d, 0 d [Sigma Chemical, St Louis, Mo]).~,~ Functional islet graft survival was evaluated by daily blood glucose measurement, with
graft failure indicated by consecutive days of hyperglycemia (> 200 mg/dL).
RESULTS
AND
DISCUSSION
Long-term graft survival was obtained in dog-to-nude mouse islet transplantation, 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 suggests that non-specific immune responses, rather than T-cell mediated immune events, play a dominant role in early graft failure and PNF. Inhibition of NO production (by aminoguanidine), or interruption of host macrophage function (by gadolinium), briefly, but significantly, improved early canine islet function in LEW rats with a normoglycemia from day 0 in control group to day 1 in AG (P < .02) or Gd-treated groups (P < .02). This slight prolongation of islet function suggests that macrophages or NO production might be partially responsible for the early failure of canine islet xenografts in rat recipients. Nevertheless, broad immunosuppression by lymphocyte depletion (ALS), which depletes the panorama of lymphocytes as well as macrophages and NK cells, significantly prolonged islet xenograft survival to a mean graft survival of 2.4 days (P < .Ol). The significant prolongation of canine islet xenograft survival by ALS therapy suggests that immune cells (including macrophages, NK cells and/or T-cells) are involved in the complex interactions leading to islet xenograft failure in the dog-to-rat strain combination. These results suggest that intervention in multiple pathways will be required to successfully overcome PNF and early islet graft failure. Further definition of the cellular interactions leading to islet xenograft PNF is necessary to
From the Department of Surgery, University of Pennsylvania Medical Center, Philadelphia, PA, USA. Address reprint requests to Shaoping Deng, Department of Surgery, University of Pennsylvania Medical Center, Philadelphia, PA 19104.
0041-1315/97/$17.00 PII so041 -1345(97)00032-8
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1726
Transplantation
Proceedings,
29, 1726-l 727 (1997)
PRIMARY
NONFUNCTION
OF ISLET XENOGRAFTS
allow development of precise treatment strategies at the relevant arms of the immune response.
1727
directed
2. Deng S, Ketchum 28:805, 1996
RJ, Levy MM, et al: Transplant
Proc
3. Perloff JR, Levy MM, Ketchum RJ, et al: Transplant Proc REFERENCES 1. Brayman KL, Morel P, Field J, et al: Transplant Proc 24:651,
1992
27:3399, 1995
4. Kamel T, Callery MP, Flye MW, et al: J Surg Res 48:393, 1990
Primary Nonfunction of Islet Xenografts in Rat Recipients From Non-T-Cell-Mediated Immune Responses S. Deng, R.J. Ketchum,
T. Kucher,
M. Weber, A. Naji, and K.L. Brayman
P
RIMARY nonfunction (PNF) and early graft failure affect the functional mass of islet tissue engrafted, and represent a major problem in clinical islet transplantation. Previous studies have shown that grafts of isolated canine pancreatic islets survive and function in immunocompetent murine recipients until graft rejection. In contrast, a very high rate of PNF has been demonstrated in rat recipients of canine islet xenografts.’ Routine immunosuppressive protocols, such as cyclosporin therapy, have little effect on prolongation of graft survival in this combination. A better understanding of the mechanisms involved in this strong, rapid immune response in the rat may provide focus in the development of strategies of immunosuppression which would be clinically relevant. To identify the effector mechanism of PNF in the dog-to-rat islet transplant model, we have shown that long-term culture, or inhibition of native complement, slightly improves early islet function in this model.* In this study, we further investigate the effect of inhibiting lymphocyte activity, nitric oxide (NO) production, or macrophage (M$) function on canine islet xenograft survival in rat recipients.
MATERIALS
AND
Results
METHODS
Canine islets were isolated and purified (>90% purity) from beagle (Marshall Farms, North Rose, NY) pancreata using an automated method of collagenase (Boehringer Mannheim, Indianapolis, IN) digestion, and discontinuous Euroficoll gradient (Ficoll [Sigma Chemical, St Louis, MO] in Eurocollins [Fresenius USA, New Brunswick, NJ]) centrifugation (Cobe 2991 centrifuge, Cobe, Lakewood, Colo). Recipient rats (immunocompetent LEW [Harlan, Indianapolis, Ind] or immunocompromised Nude rats [Harlan, Indianapolis, Ind], -200 to 250 g) were rendered diabetic with streptozotocin (Zanosar, 65 mgikg, Upjohn, Kalamazoo, Mich) 7 days before transplantation. Recipient animals received -5000 islets, injected into the recipient’s portal vein, following overnight islet culture (18 hours, Nutrient Mixture F12 [Life Technologies, Grand Island, NY], supplemented with 10% FCS [HyClone, Logan, Utah] and 1% PSF [Life Technologies, Grand Island, NY], at 37°C in 5% CO?). Recipient groups were: untreated controls, antilymphocyte serum (AL& 1 mL IP, -1 d [Accurate Chemical, Westbury, NY]), aminoguanidine (AG, 50 mgikg IP, bid [Sigma Chemical, St Louis, MO]) or gadolinium (Gd, 2 mg/kg IV, -2 d, - 1 d, 0 d [Sigma Chemical, St Louis, Mo]).~,~ Functional islet graft survival was evaluated by daily blood glucose measurement, with
graft failure indicated by consecutive days of hyperglycemia (> 200 mg/dL).
RESULTS
AND
DISCUSSION
Long-term graft survival was obtained in dog-to-nude mouse islet transplantation, 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 suggests that non-specific immune responses, rather than T-cell mediated immune events, play a dominant role in early graft failure and PNF. Inhibition of NO production (by aminoguanidine), or interruption of host macrophage function (by gadolinium), briefly, but significantly, improved early canine islet function in LEW rats with a normoglycemia from day 0 in control group to day 1 in AG (P < .02) or Gd-treated groups (P < .02). This slight prolongation of islet function suggests that macrophages or NO production might be partially responsible for the early failure of canine islet xenografts in rat recipients. Nevertheless, broad immunosuppression by lymphocyte depletion (ALS), which depletes the panorama of lymphocytes as well as macrophages and NK cells, significantly prolonged islet xenograft survival to a mean graft survival of 2.4 days (P < .Ol). The significant prolongation of canine islet xenograft survival by ALS therapy suggests that immune cells (including macrophages, NK cells and/or T-cells) are involved in the complex interactions leading to islet xenograft failure in the dog-to-rat strain combination. These results suggest that intervention in multiple pathways will be required to successfully overcome PNF and early islet graft failure. Further definition of the cellular interactions leading to islet xenograft PNF is necessary to
From the Department of Surgery, University of Pennsylvania Medical Center, Philadelphia, PA, USA. Address reprint requests to Shaoping Deng, Department of Surgery, University of Pennsylvania Medical Center, Philadelphia, PA 19104.
0041-1315/97/$17.00 PII so041 -1345(97)00032-8
0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
1726
Transplantation
Proceedings,
29, 1726-l 727 (1997)
PRIMARY
NONFUNCTION
OF ISLET XENOGRAFTS
allow development of precise treatment strategies at the relevant arms of the immune response.
1727
directed
2. Deng S, Ketchum 28:805, 1996
RJ, Levy MM, et al: Transplant
Proc
3. Perloff JR, Levy MM, Ketchum RJ, et al: Transplant Proc REFERENCES 1. Brayman KL, Morel P, Field J, et al: Transplant Proc 24:651,
1992
27:3399, 1995
4. Kamel T, Callery MP, Flye MW, et al: J Surg Res 48:393, 1990