Bilirubin protects grafts against nonspecific inflammation-induced injury in syngeneic intraportal islet transplantation

Abstract

Nonspecific inflammatory response is the major cause for failure of islet grafts at the early phase of intraportal islet transplantation (IPIT). Bilirubin, a natural product of heme catabolism, has displayed anti-oxidative and anti-inflammatory activities. The present study has demonstrated that bilirubin protected islet grafts by inhibiting nonspecific inflammatory response in a syngeneic rat model of IPIT. The inflammation-induced cell injury was mimicked by exposing cultured rat insulinoma INS-1 cells to cytokines (IL-1β, TNF-α and IFN-γ) in in vitro assays. At appropriate lower concentrations, bilirubin significantly attenuated the reduced cell viability and enhanced cell apoptosis induced by cytokines, and protected the insulin secretory function of INS-1 cells. Diabetic inbred male Lewis rats induced by streptozotocin underwent IPIT at different islet equivalents (IEQs) (optimal dose of 1000, and suboptimal doses of 750 or 500), and bilirubin was administered to the recipients every 12 h, starting from one day before transplantation until 5 days after transplantation. Administration of bilirubin improved glucose control and enhanced glucose tolerance in diabetic recipients, and reduced the serum levels of inflammatory mediators including IL-1β, TNF-α, soluble intercellular adhesion molecule 1, monocyte chemoattractant protein-1 and NO, and inhibited the infiltration of Kupffer cells into the islet grafts, and restored insulin-producing ability of transplanted islets.

Figure 1

Cell viability and apoptosis. (A) INS-1 cells were cultured in RPMI 1640 media containing murine IL-1β (100 U/ml), TNF-α (500 U/ml) and IFN-γ (100 U/ml) for 16, 24, 32, 40 or 48 h. (B) INS-1 cells were cultured in the same media containing 0, 5, 10, 20, 30 or 50 µM of bilirubin for 1 h, then replaced with cytokine-containing media as above, and cells were further cultured for 48 h. (A, B) Untreated cells served as controls, and the cell viability was measured by CCK-8 assays. ^*indicates a significant increase of cell viability, and ^‡a significant reduction, compared with cytokines only-treated cells. (C) INS-1 cells were cultured in cytokine-containing media as above for 8, 16, or 24 h. (D) INS-1 cells were cultured in media containing 20 or 50 µM BR (bilirubin) for 1 h, then replaced with cytokine-containing media as above, and cells were further cultured for 24 h. (C, D) Untreated cells served as controls, and flow cytometry was performed to measure apoptosis rates. The above experiments were repeated thrice, and results were expressed mean ± SD. ^*indicates a significant increase of apoptosis rates from control at P < 0.05, and ^**a highly significant increase from control at P < 0.001. ^‡indicates a significant reduction of apoptosis rates from cytokine only-treated cells at P < 0.05. (E) Representative dot plots were from cytometrically analyzed untreated (control) INS-1 cells or INS-1 cells receiving different treatments. The percentage of cell population is shown in each quadrant. (F) Illustrated are representative photographs (× 400 magnification) for untreated (control) INS-1 cells or INS-1 cells subjected to different treatments, stained with Annexin V/PI and examined under laser scanning confocal microscopy to detect apoptotic cells.

Figure 2

Insulin producing activity. The INS-1 cells from Figure 1 (D) were used to measure glucose-stimulated insulin secretion (A), insulin secretion index (B) and intracellular insulin contents (C). The above experiments were repeated thrice, and results were expressed mean ± SD. ^*indicates a significant decrease from respective control cells at P < 0.05, and ^‡indicates a significant increase from respective cells treated by cytokines only at P < 0.05.

Figure 3

Bilirubin promotes glucose control and enhances glucose tolerance after IPIT. Streptozotocin-induced diabetic recipients received an optimal islet dose (1000 IEQ/rat) (A) or suboptimal doses (either 750[B] or 500[C] IEQ/rat). Recipients were given bilirubin (1 ml/kg body weight) or vehicle every 12 h, starting one day before and until 5 days after IPIT. Non-fasting glucose levels were assessed at indicated time points. (D) Percentage of recipients that achieved normal glucose level (< 200 mg/dl) in each group in (A, B and C). (E) 30 days after transplantation, the recipients were subjected to glucose tolerance test by fasting overnight and receiving i.p. injection of dextrose (2 g/kg body weight). Blood glucose levels were measured just before injection and 30, 60, 90 and 120 min after the injection. n, the number of rats assessed. ^*indicates a significant decrease from the respective vehicle-treated groups at respective time points at P < 0.05.

Figure 4

Infiltration of Kupffer cells and insulin production by islets in situ. Representative illustrations were taken from livers of vehicle (A, C) or bilirubin (B, D)-treated diabetic rats receiving intraportal injection of 1000 IEQ islets/rat and harvested 24 h (A, B) or 10 days (C, D) later. Each group had 10 rats, five of which were randomly sacrificed 24 h or 10 days after transplantation. The liver sections were immunostained with an anti-CD68 Ab to detect Kupffer cells (A, B), or an anti-insulin Ab to detect insulin (C, D). Brown color indicates positive cells.