Early graft failure of xenogeneic islets in NOD mice is accompanied by high levels of interleukin-1 and low levels of transforming growth factor-beta mRNA in the grafts.

Abstract

Early graft failure, graft rejection, and autoimmune recurrence remain unresolved issues in islet xenotransplantation in type 1 diabetes. The first aim of this study was to examine the existence of early graft failure in spontaneously diabetic autoimmune NOD mice after rat islet transplantation under technically controlled circumstances. The second aim was to examine the mediators of this early xenograft dysfunction. First, we demonstrated a higher percentage of early xenograft failure (48%) in spontaneously diabetic NOD mice as compared with chemically diabetic old NOD (13%, P < 0.05) and C57Bl/6 (7%, P < 0.01) mice. In addition, in spontaneously diabetic NOD mice, xenogeneic islets displayed early graft failure more frequently than allogeneic (23%, P ≤ 0.05) or isogeneic islets (7%, P < 0.01). No early graft failure was observed in allotransplantation or isotransplantation in chemically diabetic mice. Reverse transcriptase-polymerase chain reaction analysis of cytokine mRNA in islet xenografts 8 h after transplantation showed higher levels of interleukin (IL)-1 mRNA in autoimmune diabetic mice compared with chemically diabetic old NOD mice (1.40 ± 0.32 vs. 0.90 ± 0.14 IL-1 copies/␤-actin copies, P < 0.05). In contrast, mRNA levels of transforming growth factor (TGF)-␤ were lower in spontaneously diabetic NOD mice than in chemically diabetic old NOD mice (0.67 ± 0.16 vs. 1.36 ± 0.50 TGF-␤ copies/␤-actin copies, P < 0.05). No differences in tumor necrosis factor-␣, IL-6, and inducible nitric oxide synthase were seen between autoimmune and nonautoimmune diabetic mice. T-cell cytokines (IL-2, IL-4, IL-10, and ␥-interferon) were absent in all mice until 48 h after transplantation. These data suggest that early islet xenograft failure is more common in spontaneously diabetic NOD mice and could be due to a nonspecific inflammatory reaction locally in the grafts.

IL-1β and IFN-γ induce the expression of diverse chemokines and IL-15 in human and rat pancreatic islet cells, and in islets from pre-diabetic NOD mice

“…We have previously shown [64] and confirmed in this study that a state of inflammation with high local expression of IL-1 is demonstrated immediately after (8-h) islet transplantation in autoimmune NOD mice. IL-1 expression was also increased in BALB/c mice isografts, suggesting that this is probably secondary to non-specific tissue damage associated to the early engraftment.…”

Interferon regulatory factor-1 is a key transcription factor in murine beta cells under immune attack

“…To date, the pathophysiology of islet primary non-function is incompletely understood, but we have previously shown that it is of great importance in autoimmune hosts (spontaneously diabetic NOD mice) and seems to be associated with nonspecific inflammation at the implantation site, with a major role being played by IL-1β and free radicals (nitric oxide) [23,34]. This role of IL-1β in primary non-function also explains the difference in primary non-function between Irf-1 −/− and Stat-1 −/− islets transplanted into NOD mice, as IRF-1 is not only controlled by IFN-γ, but is also partially regulated by IL-1β, in contrast to STAT-1.…”

“…C57BL/6 mice were used as controls and were obtained from stocks purchased from Harlan (Horst, the Netherlands). NOD mice, inbred in our animal facility (Proefdierencentrum Leuven, Leuven, Belgium) since 1989, were used as diabetes-prone animals, with diabetes detected and defined as described [23]. All mice were housed under semi-barrier conditions.…”

“…Islet isolation, culture and treatment To obtain pancreatic islets, pancreases from Irf-1 −/− or control C57BL/6 mice were removed and islets were isolated by collagenase digestion [23]. Batches of 100 islets were collected and cultured overnight in RPMI 1640 medium (with Gluta-MAX-I), containing 100 U/ml penicillin, 100µg/ml streptomycin and 10% FCS [vol./vol.]…”

“…Islet transplantation and evaluation of graft function Freshly isolated Irf-1 −/− or control C57BL/6 islets (n=500) were transplanted under the kidney capsule of overtly diabetic NOD mice as described previously [23]. Islet primary nonfunction was defined as blood glucose levels never reaching normoglycaemia within 48 h after islet transplantation, while graft rejection was defined as a return to hyperglycaemia (non-fasting glycaemic values ≥11.1 mmol/l in three consecutive readings after initial normoglycaemia).…”