Cytokines activate caspase-3 in insulinoma cells of diabetes-prone NOD mice directly and via upregulation of Fas

In type 1 diabetes, autoimmune inflammation of pancreatic islets of Langerhans ('insulitis') results in destruction of insulin-producing cells. Cytokines released from islet-infiltrating mononuclear cells are known to be cytotoxic both directly and by upregulating Fas for FasL-induced apoptosis. To investigate the role of caspase-3, a major effector of apoptosis in beta-cell death, we asked whether cytokine- and/or FasL-induced apoptosis was associated with increased activity of caspase-3 in NIT-1 insulinoma cells and islets of autoimmune diabetes-prone NOD mice. Measurement of caspase-3 activity using a fluorogenic cleavage assay was validated in NOD mouse thymocytes undergoing dexamethasone (Dex)-induced apoptosis. For cytokine-induced apoptosis, NIT- 1 cells or islets were exposed to IL-1beta and IFN-gamma for 24 h. Caspase-3-like activity was increased 2.1 +/- 0.7 and 2.4 +/- 0.9-fold in lysates of cytokine-treated NIT-1 cells and NOD mouse islets, respectively. However, NIT-1 cells exhibited 2.1% (4.7 pg active caspase-3/ mug protein) and islets 0.8% (1.9 pg active caspase-3/mug protein) of the active caspase-3 content observed in Dex-treated thymocytes (225.1 pg active caspase-3/mug protein). After 24 h cytokine-exposure, the percentage of Fas-positive NIT-1 cells increased from 1.4 +/- 1.1 to 29.7 +/- 11.6%. Addition of FasL for a further 3 h increased caspase-3-like activity an additional 1.8-fold in cytokine treated NIT-1 cells. In summary, exposure of NOD mouse insulinoma cells or islets to IL-1beta and IFN-gamma for 24 h induced caspase3-like activity that, in the case of insulinoma cells at least, can be further enhanced by interaction of cytokine-induced Fas receptor with FasL. Compared to thymocytes, insulinoma cells and islets from NOD mice were characterised by low basal and cytokine induced caspase-3 activity. (C) 2004 Elsevier Ltd. All rights reserved.