Treatment of autoimmune diabetes recurrence in non-obese diabetic mice by mouse interferon-beta in combination with an analogue of 1alpha,25-dihydroxyvitamin-D3

Abstract

Autoimmune diabetes recurrence is in part responsible for islet graft destruction in type 1 diabetic individuals. The aim of the present study was to design treatment modalities able to prevent autoimmune diabetes recurrence after islet transplantation in spontaneously diabetic NOD mice. In order to avoid confusion between autoimmune diabetes recurrence and allograft rejection, we performed syngeneic islet transplantations in spontaneously diabetic NOD mice. Mice were treated with mouse interferon-beta (IFN-beta, 1 x 105 IU/day), a new 14-epi-1,25-(OH)2D3-analogue (TX 527, 5 microg/kg/day) and cyclosporin A (CsA, 7.5 mg/kg/day) as single substances and in combinations. Treatment was stopped either 20 days (IFN-beta and CsA) or 30 days (TX 527) after transplantation. Autoimmune diabetes recurred in 100% of control mice (MST 11 days). None of the mono-therapies significantly prolonged islet graft survival. Combining CsA with TX 527 maintained graft function in 67% of recipients as long as treatment was given (MST 31 days, P < 0.01 versus controls). Interestingly, 100% of the IFN-beta plus TX 527-treated mice had normal blood glucose levels during treatment, and even had a more pronounced prolongation of graft survival (MST 62 days, P < 0.005 versus controls). Cytokine mRNA analysis of the grafts 6 days after transplantation revealed a significant decrease in IL-2, IFN-gamma and IL-12 messages in both IFN-beta plus TX 527- and CsA plus TX 527-treated mice, while only in the IFN-beta with TX 527 group were higher levels of IL-10 transcripts observed. Therefore, we conclude that a combination of IFN-beta and TX 527 delays autoimmune diabetes recurrence in islet grafts in spontaneously diabetic NOD mice.

Fig. 1

Syngeneic islet graft survival in mono- (a) and combination-treated (b) spontaneously diabetic NOD mice. Control mice (no symbol) showed autoimmune diabetes recurrence within two weeks after transplantation. Mono-therapy with IFN-β (♦), CsA (▾) and TX 527 (□) did not lead to a significant inhibition of syngeneic islet graft destruction compared with control animals. Treatment with IFN-β + CsA (▴) did not result in a delay in autoimmune diabetes recurrence, whereas treatment with CsA + TX 527 (▪) and IFN-β + TX 527 (•) prolonged mean graft survival time significantly to 31 days and 62 days, respectively. Note that IFN-β and CsA treatment was stopped 20 days after transplantation; TX 527 was stopped 30 days after transplantation. Significance (*) is expressed compared with control animals. **P < 0·01; ***P < 0·005.

Fig. 2

Histological examination of NOD islets transplanted under the kidney capsule of spontaneously diabetic NOD mice and removed 15–17 days later. (a) Islet graft of untreated, (b) CsA + TX 527- and (c) IFN-β + TX 527-treated NOD mice. Islet grafts of untreated NOD mice were heavily infiltrated by mononuclear cells, with complete destruction of islet-cell architecture. On the other hand, islet grafts in mice treated with either CsA + TX 527 or IFN-β + TX 527 revealed only a few infiltrating lymphocytes.

Fig. 2

Histological examination of NOD islets transplanted under the kidney capsule of spontaneously diabetic NOD mice and removed 15–17 days later. (a) Islet graft of untreated, (b) CsA + TX 527- and (c) IFN-β + TX 527-treated NOD mice. Islet grafts of untreated NOD mice were heavily infiltrated by mononuclear cells, with complete destruction of islet-cell architecture. On the other hand, islet grafts in mice treated with either CsA + TX 527 or IFN-β + TX 527 revealed only a few infiltrating lymphocytes.

Fig. 2

Histological examination of NOD islets transplanted under the kidney capsule of spontaneously diabetic NOD mice and removed 15–17 days later. (a) Islet graft of untreated, (b) CsA + TX 527- and (c) IFN-β + TX 527-treated NOD mice. Islet grafts of untreated NOD mice were heavily infiltrated by mononuclear cells, with complete destruction of islet-cell architecture. On the other hand, islet grafts in mice treated with either CsA + TX 527 or IFN-β + TX 527 revealed only a few infiltrating lymphocytes.

Fig. 3

Effect of different treatments on cytokine mRNA levels in NOD islet grafts 6 days after transplantation. IL-2 (a), IFN-γ (b), IL-4 (c), IL-10 (d), IL-12 (e) and TGF-β (f) mRNA levels are expressed relative to β-actin PCR product amplified from the same sample (cytokine copies/β-actin copies). Significance (*) is expressed compared with mRNA expression levels in islet grafts of control mice. *P < 0·05.

Fig. 4

In vivo calcemic effects of different treatments. Body weight (a), serum calcium (b) and tibia calcium (c) levels were measured at the time of autoimmune diabetes recurrence. Significance (*) is expressed compared to control animals. *P < 0·05.