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Eric P. K. Mensah-Brown et al.
Eur. J. Immunol. 2006. 36: 216–223
IL-23 leads to diabetes induction after subdiabetogenic treatment with multiple low doses of streptozotocin Eric P. K. Mensah-Brown1, Allen Shahin2, Mariam Al-Shamisi2, Xiaoging Wei3 and Miodrag L. Lukic2,4 1
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Department of Anatomy, Faculty of Medicine and Health Sciences, UAE University, Al Ain, United Arab Emirates Department of Microbiology and Immunology, Faculty of Medicine and Health Sciences, UAE University, Al Ain, United Arab Emirates Division of Immunology, Infection and inflammation, University of Glasgow, Glasgow, UK Faculty of Medicine, University of Kragujevac, Kragujevac, Serbia
IL-23, a proximal regulator of IL-17, may be a major driving force in the induction of autoimmune inflammation. We have used a model of subdiabetogenic treatment with multiple low doses of streptozotocin (MLD-STZ; 4 40 mg/kg body weight) in male C57BL/6 mice to study the effect of IL-23 on immune-mediated b cell damage and the development of diabetes, as evaluated by blood glucose, quantitative histology, immunohistochemistry and expression of relevant cytokines in the islets. Ten daily injections of 400 ng IL-23, starting on the first day of MLD-STZ administration led to significant and sustained hyperglycemia along with weight loss compared with controls (no IL-23), and a significant increase in the number of infiltrating cells, a lower insulin content, enhanced apoptosis, expression of IFN-c and IL-17 (not seen in the controls) and a significant increase in the expression of TNF-a and IL-18 in the pancreatic islets. IL-23 treatment started 5 days prior to MLD-STZ administration had no effect on diabetogenesis or cytokines expression in the pancreatic islets. We provide the first evidence in an animal model that IL-23 is involved in the development of type-1 diabetes, by inducing IL-17 and possibly IFN-c production in the target tissue.
Introduction Streptozotocin (STZ), a D-glucopyranose derivative of N-methyl–N-nitrosourea endowed with potent alkylating properties [1], acts also as a producer of reactive oxygen species [2]. While at high doses the diabetogenic potential of STZ is explained by its capacity to selectively promote insulin-producing b cell death by apoptosis or Correspondence: Miodrag L. Lukic, Department of Microbiology and Immunology, Faculty of Medicine and Health Sciences, UAE University, P O Box 17666, Al Ain, United Arab Emirates Fax: +971-3-7671966 e-mail:
[email protected] Abbreviations: CNS: central nervous system iNOS: inducible nitric oxide synthase MLD: multiple low doses PI: propidium iodide STZ: streptozotocin f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Received 2/8/05 Revised 19/9/05 Accepted 4/11/05 [DOI 10.1002/eji.200535325]
Key words: Diabetes IFN-c IL-17 IL-23
necrosis, at low doses, STZ generates H2O2 [3] and induces expression of glutamic acid decarboxylase autoantigens [4]. Glutamic acid decarboxylase is a strong trigger of b cell-specific autoimmunity, both in humans and experimental models of diabetes [5–7]. It has also been demonstrated that multiple low dose (MLD)-STZ-induced diabetes in susceptible strains of mice requires a Th-1 cell-dependent inflammatory reaction [6, 7]. We [8–10] and others [11–13] have used this model to study the role of proinflammatory cytokines in the development of type-1 diabetes. The model offers an opportunity to study the effects of cytokines on synchronized group of diabetic animals. The IL-12 family of cytokines, which includes IL-12, IL-23 and IL-27 (reviewed in [14]), are required for Th-1 cell differentiation. This family of cytokines is produced www.eji.de
Eur. J. Immunol. 2006. 36: 216–223
by antigen-presenting cells such as macrophages, dendritic cells (reviewed in [15]) and microglia in the central nervous system (CNS) [16]. While both macrophages and a small number of dendritic cells are present in intact adult pancreatic islets [17], blood-borne macrophages are the first cells to infiltrate the islets after MLD-STZ diabetes induction [18, 19]. Dendritic cells are also known to either enhance [20] or prevent [21] autoimmunity leading to diabetes, depending on their activation status. IL-12 induces IFN-c production, a process which appears to down-regulate rather than stimulate T celldependent autoimmunity, at least in the CNS [22]. In contrast, IL-23 and IL-27 play pivotal roles in the establishment and maintenance of organ-specific autoimmunity as demonstrated in experimental allergic encephalomyelitis (EAE) [23] and collagen-induced arthritis [24]. Increased expression of IL-23 has also been demonstrated in skin lesions of patients with psoriasis vulgaris [25]. Whereas the pro-diabetic effect of IL-12 produced by macrophages and dendritic cells in situ, appears to be controversial, the role of the other two members of the IL-12 family, IL-23 and IL-27, has not been studied. In humans, polymorphism of the IL-12B gene, which encodes the p40 subunits of IL-23, is associated with late onset of type-1 diabetes [26]. Here we provide the first evidence that in vivo administration of IL-23 induces the onset of diabetes in mice administered subdiabetogenic doses of MLD-STZ. This effect of IL-23 was associated with expression of IFN- c and IL-17 in the pancreatic islets and an enhancement of TNF-a and IL-18 expression early after diabetes induction. These effects were accompanied also by enhanced mononuclear cellular infiltration and b cell loss in the islets.
Clinical immunology
IL-23 promotes development of MLD-STZ diabetes in male C57BL/6 mice In the first series of experiments we administered ten daily i.p. injections of 400 ng IL-23 starting on the first day of a 4-day regimen of MLD-STZ. The control animals likewise received 4 40 mg/kg STZ but only the carrier solution (without IL-23), i.e., for 10 days. IL-23 significantly enhanced diabetogenesis. At the end of the experiment, four out of seven control animals were mildly diabetic, while all animals treated with IL-23 had significant hyperglycemia (Fig. 1a); venous glucose levels were significantly higher in IL-23-treated mice than controls after day 16 of MLD-STZ treatment. This effect was associated with weight loss in IL-23-treated animals compared to controls (Fig. 1b). Similar results were obtained in the two additional experiments (data not shown).
Results The MLD-STZ model To study the role of IL-23 in diabetes induction, we used a subdiabetogenic regimen of MLD-STZ treatment. It has been established that five daily doses of 40 mg/kg/day are required for delayed onset, sustained and progressive hyperglycemia and insulitis in male C57BL/6 mice [7, 8]. As shown in Fig. 1, four injections of STZ did not induce significant hyperglycemia and only mild singlecell insulitis (Fig. 2a vs. b). We proceeded to determine whether IL-23 would enhance the diabetogenic process by repeated administration of IL-23 with control animals receiving the carrier solution only. In accordance with a previous report [3] and our preliminary data, female C57BL/6 mice were resistant to the MLD-STZ regimen (data not shown), and therefore the modulatory effects IL-23 were studied in male mice only. f 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Effect of IL-23 on plasma glucose (a) and percentage weight gained or lost (b) induced by MLD-STZ in C57BL/6 mice. All mice were injected with 40 mg/kg/day STZ for 4 consecutive days together with either 400 ng IL-23 or 0.1% BSA/saline (controls) from the beginning of the STZ regimen and continued for 10 days. The results are representative of three experiments; p
