Dissecting the instant blood-mediated inflammatory reaction in islet xenotransplantation

Abstract

Background: A massive destruction of transplanted tissue occurs immediately following transplantation of pancreatic islets from pig to non-human primates. The detrimental instant blood-mediated inflammatory reaction (IBMIR), triggered by the porcine islets, is a likely explanation for this tissue loss. This reaction may also be responsible for mediating an adaptive immune response in the recipient that requires a heavy immunosuppressive regimen.

Materials and methods: Low molecular weight dextran sulfate (LMW-DS) and the complement inhibitor Compstatin were used in a combination of in vitro and in vivo studies designed to dissect the xenogeneic IBMIR in a non-human primate model of pancreatic islet transplantation. Adult porcine islets (10,000 IEQs/kg) were transplanted intraportally into three pairs of cynomolgus monkeys that had been treated with LMW-DS or heparin (control), and the effects on the IBMIR were characterized. Porcine islets were also incubated in human blood plasma in vitro to assess complement inhibition by LMW-DS and Compstatin.

Results: Morphological scoring and immunohistochemical staining revealed that the severe islet destruction and macrophage, neutrophilic granulocyte, and T-cell infiltration observed in the control (heparin-treated) animals were abrogated in the LMW-DS-treated monkeys. Both coagulation and complement activation were significantly reduced in monkeys treated with LMW-DS, but IgM and complement fragments were still found on the islet surface. This residual complement activation could be inhibited by Compstatin in vitro.

Conclusions: The xenogeneic IBMIR in this non-human primate model is characterized by an immediate binding of antibodies that triggers deleterious complement activation and a subsequent clotting reaction that leads to further complement activation. The effectiveness of LMW-DS (in vivo and in vitro) and Compstatin (in vitro) in inhibiting this IBMIR provides the basis for a protocol that can be used to abrogate the IBMIR in pig-human clinical islet transplantation.

Fig. 1

Plasma APTT values in transplanted diabetic monkeys (M5, M7-M10) treated with heparin (squares) or LMW-DS (circles).

Fig. 2

EDTA blood was drawn from a femoral vein catheter of the transplanted monkeys treated with heparin (squares) or LMW-DS (circles) at varying time points after porcine islet xenotransplantation. TAT, C3a, and sC5b-9 levels were assessed and expressed as percentage of the pre-transplant values.

Fig. 3

Visual examples of the morphological scoring system used to quantify different aspects of the IBMIR. Hematoxylin eosin-stained porcine islet grafts retrieved 24 hours after intraportal xenotransplantation from diabetic monkeys treated with LMW-DS or heparin were used. A summary of all transplanted monkeys is presented in Table 1.

Fig. 4

Immunohistochemical staining of porcine islet grafts retrieved 24 hours after intraportal xenotransplantation from diabetic monkeys treated with LMW-DS or heparin. The figure shows representative expression of insulin and of CD41 (platelets), CD68 (macrophages), and CD3 (T-cells) in the grafts. A summary of all transplanted monkeys is presented in Table 1. Magnification x200.

Fig. 5

Porcine islets incubated in hirudin-treated plasma for 30 min. The islets were stained for IgG (n=5), IgM (n=5), C3b/iC3b (n=5), C1q (n=3), C4 (n=5), C9 (n=3), and MBL (n=3). As negative control, an antibody recognizing mouse IgG was used (n=5). The islets were analyzed by (A) large particle flow cytometry and (B) confocal microscopy. In C the deposition of C3b/iC3b on the islet in the absence and presence of Compstatin is presented after analysis by large particle flow cytometry (n=5; statistical evaluation was performed at 5 and 30 min where n=7) (*=p<0.05; **=p<0.01).

Fig. 6

Binding of C3b/iC3b to the surface of microtiter wells after incubation with 10% serum in the presence of increasing doses of Compstatin for 30 min at 37°C. 0 (cross), 10 (triangle), 100 (squares), 1000 (diamond) mg/L of LMW-DS was present in the wells.