Cytokines Induce Both Necrosis and Apoptosis via a Common Bcl-2-Inhibitable Pathway in Rat Insulin-Producing Cells*

The presence of activated macrophages within pancreatic islets in insulin-dependent diabetes mellitus suggests an involvement of b-cell death by necrosis. The aim of this study was to investigate the frequencies and mechanisms of cytokine-induced b-cell apoptosis and necrosis and the possible protection mediated by the antiapoptotic gene bcl-2. A combination of interleukin-1b, interferon-g, and tumor necrosis factor-a increased both necrosis (17% of cells) and apoptosis (5% of cells) in isolated whole rat islets, as determined by vital staining and fluorescence microscopy. Hyperexpression of Bcl-2, achieved by stable transfection using a multicopy viral vector containing a bcl-2 complementary DNA in rat insulin-producing RINm5F cells, counteracted both apoptosis and necrosis. Cytokine-induced cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (which, in other cell types, may occur downstream or independently of a Bcl-2preventable mitochondrial permeability transition) was observed in controlbut neither in bcl-2-transfected cells nor in the presence of the iNOS inhibitor N-methyl-L-arginine. Tumor necrosis factor-a alone did not clearly induce cell death or poly(ADP-ribose) polymerasecleavage. These findings suggest that cytokines induce both necrosis and apoptosis in insulin-producing cells via a common Bcl-2-preventable nitric oxide-dependent pathway, which may involve mitochondrial permeability transition. The necrosis:apoptosis ratio might be increased by a relative lack of caspase activity. (Endocrinology 141: 2003–2010, 2000) I diabetes mellitus (IDDM) is regarded as a chronic autoimmune disease resulting from progressive destruction of the insulin-producing b-cells in the pancreas (1). Studies in animal models of IDDM have shown that macrophages are the first cells to infiltrate the islets of Langerhans, followed by lymphocytes. An increased expression of cytokines in the insulitis has been reported, and different combinations of cytokines have been shown to decrease the function and viability of b-cells in vitro, partially through the production of nitric oxide and/or reactive oxygen species by the b-cells themselves or by cells in their vicinity, such as macrophages and endothelial cells (2). It has been hypothesized that initial damage to b-cells could result in the release of intracellular components, leading to inflammation and activation of a b-cell directed immune response, followed by destruction of all insulin-producing cells (2). It is generally believed that programmed cell death has evolved in multicellular organisms as a means to dispose of unwanted or damaged cells by a controlled manner (reviewed in Refs. 3 and 4). The implementation of the apoptotic program, which is dependent on the activation of caspases, results in the dismantling of the cell and recognition and ingestion by phagocytocing cells without eliciting an inflammatory response (3, 4). By contrast, cell death by necrosis is known to result in activation of macrophages and to provoke inflammation and immune responses (3, 4). It has recently become clear that many forms of both apoptosis and necrosis share a common effector phase of mitochondrial permeability transition (MPT) (reviewed in Ref. 5). MPT may result from the opening of a multiprotein complex pore located at the contact site between the inner and outer mitochondrial membrane, which leads to disruption of the mitochondrial membrane potential (‚Cm), uncoupling of the respiratory chain with cessation of ATP production, hyperproduction of reactive oxygen species (ROS), depletion of reduced glutathione, and release of Ca, some of which may provoke necrosis (5). However, MPT is also associated with the release of apoptosis inducing factor and cytochrome c from mitochondria, which may result in activation of caspases involved in the degradation phase of apoptosis, such as caspase-3, which cleaves poly(ADP-ribose) polymerase (PARP) and other substrates (reviewed in Refs. 4 and 6). The principal role of the antiapoptotic protein Bcl-2 in preventing cell death was recently shown to be the inhibition of the MPT-pore opening (5, 7, 8). Moreover, receptors such as the tumor necrosis factor (TNF)R1 and CD95/Fas, which share a death signaling pathway involving FADD/MORT1 (reviewed in Ref. 9), may activate the initiation phase of apoptosis and are both known to induce MPT (5, 10–12). However, in some (but not all) cell types, signaling via FADD/ MORT1, in addition to MPT, rapidly activates the degradation phase of apoptosis with cleavage of PARP through a pathway that seems independent of MPT (11). The latter pathway may depend on a direct activation of caspase-8 (FLICE) upon recruitment of FADD/MORT1, by Received August 5, 1999. Address all correspondence and requests for reprints to: Dr. J. Saldeen, Department of Medical Cell Biology, Biomedicum, Uppsala University, P.O. Box 571, S-751 23 Uppsala, Sweden. E-mail: johan. saldeen@medcellbiol.uu. se. * This work was supported, in part, by Swedish Medical Research Council Grants (72X-11564205 C, 72P-12995–02B), the Swedish Diabetes Association, the Nordic Insulin Fund, the Juvenile Diabetes Foundation International, the Wallenberg Fund, the Swedish Society of Medicine, and the Family Ernfors Fund. 0013-7227/00/$03.00/0 Vol. 141, No. 6 Endocrinology Printed in U.S.A. Copyright © 2000 by The Endocrine Society