Dirk Valckx

Prevention of autoimmune destruction of syngeneic islet grafts in spontaneously diabetic nonobese diabetic mice by a combination of a vitamin D3 analog and cyclosporine.

BACKGROUND Type 1 diabetes is characterized by the presence of an autoimmune memory, responsible for the destruction of even syngeneic islet grafts. This recurrence of autoimmunity is partly responsible for the need of extensive immunosuppression in pancreas and islet transplantation in type 1 diabetic patients. The aim of the study was to evaluate the capacity of a 20-epi-analog of vitamin D3, KH1060, both alone and in combination with cyclosporine (CsA) to prevent diabetes recurrence in syngeneic islet grafts in nonobese diabetic (NOD) mice. METHODS Spontaneously diabetic NOD mice grafted with syngeneic islets (n=500) under the kidney capsule were treated with KH1060, CsA, or a combination of both drugs from the day before transplantation until recurrence or 60 days after transplantation. RESULTS Vehicle-treated mice showed a recurrence of diabetes in 100% of cases (n=17) within 4 weeks. Treatment with high doses of CsA (15 mg/kg/day) or KH1060 (1 microg/kg/2 days) significantly prolonged islet survival (60 days and 50 days, respectively, versus 9.5 days in controls; P<0.001 and P<0.0001). Mice treated with subtherapeutical doses of both drugs combined (KH1060 0.5 microg/kg/2 days + CsA 7.5 mg/kg/day) had significant prolongation of graft survival (48 days; P<0.001) and more importantly, four of five mice that were still normoglycemic 60 days after transplantation showed no recurrence after discontinuation of all treatment. Histology of the grafts of control and combination-treated mice demonstrated that graft infiltration and islet destruction were less severe in grafts of combination-treated mice. Cytokine mRNA analysis in the grafts 6 days after transplantation revealed a clear suppression of interleukin-12 and T helper 1 cytokines and higher levels of interleukin-4 in combination-treated mice. CONCLUSIONS KH1060, an analog of 1,25(OH)2D3, delays autoimmune disease recurrence after syngeneic islet transplantation in NOD mice, both alone and especially in combination with CsA, possibly restoring tolerance to beta cells in 30% of cases.

1,25-Dihydroxyvitamin D3 restores sensitivity to cyclophosphamide-induced apoptosis in non-obese diabetic (NOD) mice and protects against diabetes

The activated form of vitamin D, 1,25(OH)2D3, and its analogues can prevent type I diabetes in NOD mice. Protection is achieved without signs of systemic immunosuppression and is associated with a restoration of the defective immune regulator system of the NOD mice. The aim of the present study was to investigate whether this restoration of regulator cell function is the only mechanism in the prevention of diabetes by 1,25(OH)2D3. We tested therefore if 1,25(OH)2D3 could prevent cyclophosphamide‐induced diabetes, since diabetes occurring after cyclophosphamide injection is believed to be due to an elimination of suppresser cells. NOD mice treated with 1,25(OH)2D3 (5 μg/kg every 2 days) from the time of weaning were clearly protected against diabetes induced by cyclophosphamide (200 mg/kg body wt at 70 days old) (2/12 (17%) versus 36/53 (68%) in control mice, P < 0.005). By co‐transfer experiments it was demonstrated that cyclophosphamide had indeed eliminated the suppresser cells present in 1,25(OH)2D3‐treated mice. Since cyclophosphamide injection did not break the protection offered by 1,25(OH)2D3, it was clear that diabetogenic effector cells were affected by 1,25(OH)2D3 treatment as well. This was confirmed by the finding that splenocytes from 1,25(OH)2D3‐treated mice were less capable of transferring diabetes in young, irradiated NOD mice, and by the demonstration of lower Th1 cytokine levels in the pancreases of 1,25(OH)2D3‐treated, cyclophosphamide‐injected mice. This better elimination of effector cells in 1,25(OH)2D3‐treated mice could be explained by a restoration of the sensitivity to cyclophosphamide‐induced apoptosis in both thymocytes and splenocytes, in normally apoptosis‐resistant NOD mice. Altogether, these data indicate that the protection against diabetes offered by 1,25(OH)2D3 may be independent of the presence of suppresser cells, and may involve increased apoptosis of Th1 autoimmune effector cells.

Increased mortality and impaired clonal deletion after staphylococcal enterotoxin B injection in old mice: Relation to cytokines and nitric oxide production

In the present study peripheral T cell tolerance and the occurrence of shock were evaluated in young and old mice after injection of Staphylococcal enterotoxin B (SEB). In young mice SEB immunization leads to tolerance based on deletion and anergy of SEB‐reactive Vβ8+ T cells. With aging, mice developed resistance to SEB‐induced deletion of Vβ8+ T cells as well as a high sensitivity to toxic shock. Compared to young mice, older mice injected with SEB showed increased serum levels of interferon‐γ (IFN‐γ), interleukin‐2 (IL‐2) and IL‐4. These results were confirmed by reverse transcription‐polymerase chain reaction (RT‐PCR), as splenic mRNA levels taken 2 h after SEB injection showed higher values of IL‐2, IL‐4, and IFN‐γ in old mice. In contrast, mRNA levels for FasL and tumour necrosis factor‐α (TNF‐α) were lower. No difference in IL‐10 mRNA was found. Compared to young mice, old mice showed a high, but statistically not significantly different (P = 0.20), production of nitric oxide (NO). Blocking of IFN‐γ with antibodies or reducing IFN‐γ by depletion of natural killer (NK) cells resulted, respectively, in a complete or partial protection against mortality in aged mice. Suppressing the NO production by the NO synthase inhibitor N‐nitro‐L‐arginine methylester (L‐NAME) increased the mortality in both young and old mice, and abrogated clonal deletion in the surviving young mice. In conclusion, in young mice NO production is a key factor in the protection against mortality and the development of clonal deletion after SEB injection. The higher mortality seen in older mice is mainly related to the elevated production of IFN‐γ and occurs despite a sufficient production of NO. The decreased clonal deletion of old mice may be related to their decreased expression of Fas ligand or TNF‐α after SEB injection.

Effect of treatments with cyclosporin A and anti-interferon-gamma antibodies on the mechanisms of immune tolerance in staphylococcal enterotoxin B primed mice

The authors were interested to investigate the effect of Cyclosporin A (CsA), known to block interleukin‐2 (IL‐2) production, or of anti‐interferon‐γ antibodies (anti‐IFN‐γ Abs) in a model of T cell tolerance induced by the injection of the superantigen Staphylococcal Enterotoxin B (SEB) in BALB/c mice. After SEB immunization, tolerance was mainly achieved through deletion and anergy of SEB‐reactive Vβ8+ T cells. Association of CsA treatment with SEB led to a greater decrease of the percentage of Vβ8+ CD4+ lymphocytes in the spleen and an abolition of clonal anergy. In contrast, treatment of SEB primed mice with anti‐IFN‐γ Abs resulted in an increased percentage of Vβ8+ CD4+ cells without affecting the induction of clonal anergy. The authors found that 1–2 h after SEB priming, splenic mRNA levels of IFN‐γ and IL‐4 were decreased by either CsA and anti‐IFN‐γ Abs, whereas FasL, Bcl‐2, p. 53, and c‐myc levels were not influenced by either treatment. However, SEB‐induced IL‐2 and IL‐10 mRNA expression was suppressed only by CsA, whereas tumour necrosis factor‐α (TNF‐α) was decreased only by anti‐IFN‐γ Abs. To investigate whether the effect of CsA on the tolerance mechanisms was related to suppression of IL‐2, CsA was administered together with recombinant IL‐2. Whereas anergy was not influenced, the decreased percentage of Vβ8+ CD4+ cells seen in CsA‐treated animals in the second week after SEB injection was partially corrected by the administration of IL‐2. Experiments involving bromodeoxiuridine incorporation revealed that the latter effect of IL‐2 was mainly due to a correction of the defective proliferation of Vβ8+ T cells after SEB injection in CsA‐treated mice. These results suggest that the effect of CsA and anti‐IFN‐γ Abs on tolerance mechanisms are in part explained by their action on cytokines.