Dual-Label Immunohistochemical Study of Interleukin-4-and Interferon-γ-Expressing Cells within the Pancreas of the NOD Mouse During Disease Acceleration with Cyclophosphamide

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Abstract
Beta cell destruction has been shown to occur when rodent or human islets are exposed in vitro to inflammatory cytokines, such as interleukin-1β″ (IL-1β), tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). Other cytokines such as interleukin-4 (IL-4) or inter-leukin-10 (IL-10), when given to NOD mice, prevent insulin-dependent diabetes mellitus (IDDM). In this study, we have employed immunofluorescence histochemistry to study the expression of IFN-γ and IL-4 in the pancreas of female NOD mice at various time-points (days 0, 4, 7, 11 and at onset of diabetes) following disease acceleration with cyclophosphamide (Cy). Dual-label confocal and light microscopy were employed to determine the precise cellular sources of the two cytokines. IL-4 immunolabelling was observed in a few immune cells at days 0, 4, and 7 within the pancreatic islets but in larger numbers at day 11 and at onset of diabetes. The cytokine was co-localized predominantly in CD4 cells, while only a small minority of CD8 cells and macrophages also expressed IL-4. At days 0, 4, 7 and 11, weak to moderate immunolabelling for IL-4 was also observed in beta cells. In contrast, immunolabelling for IFN-γ within the islets was not observed until day 11 and this labelling persisted at onset of diabetes. It was immunolocalized in macrophages and to a lesser extent in CD4 cells. Only a few CD8 cells were immunopositive for IFN-γ. At day 11, a proportion of beta cells showed weak immunolabelling for IFN-γ. During the study period, immunolabelling for IFN-γ was also observed in a proportion of endothelial cells located in the intra-islet and exocrine regions of Cy and diluent-treated mice. From day 11 onwards, both the cytokines were observed in some of the peri-vascular regions. Our results demonstrate that during Cy-induced diabetes, there is increasing expression of both IL-4 and IFN-γ in specific immune cells within the inflamed islets in the late prediabetic stage and at onset of diabetes. Further studies are required to correlate our protein immunohistochemical findings with in situ cytokine gene expression and to determine whether there is a clear Thl cytokine protein bias at clinical onset of diabetes and immediately preceding it