Liu D, Darville M, Eizirik D L
Gene Expression Unit, Diabetes Research Center, Vrije Universiteit Brussel, B-1090 Brussels, Belgium.
Endocrinology. 2001 Jun;142(6):2593-9. doi: 10.1210/endo.142.6.8188.
Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by progressive destruction of insulin-producing pancreatic beta-cells. Both viral infections and the cytokines interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) have been suggested as potential mediators of beta-cell death in early T1DM. We presently investigated whether the viral replicative intermediate double stranded RNA [here used as synthetic polyinosinic-polycytidylic acid (PIC)] modifies the effects of IL-1beta and IFN-gamma on gene expression and viability of rat pancreatic beta-cells. For this purpose, fluorescence-activated cell sorting-purified rat beta-cells were exposed for 6-16 h (study of gene expression by RT-PCR) or 6-9 days (study of viability by nuclear dyes) to PIC and/or IL-1beta and IFN-gamma. PIC increased the expression of Fas and Mn superoxide dismutase messenger RNAs by 5- to 10-fold. IL-1beta and a combination of PIC and IFN-gamma (but not PIC or IFN-gamma alone) induced expression of inducible nitric oxide (NO) synthase (iNOS) and consequent NO production. Induction of iNOS expression by PIC and IFN-gamma requires nuclear factor-kappaB activation, as suggested by transfection experiments with iNOS promoter-luciferase reporter constructs into primary beta-cells. Combinations of IL-1beta plus IFN-gamma, PIC plus IFN-gamma, or PIC plus IL-1beta induced a 2- to 3-fold increase in the number of apoptotic beta-cells. Blocking of iNOS activity significantly decreased PIC- plus IL-1beta-induced, but not PIC- plus IFN-gamma-induced, apoptosis. In conclusion, PIC alone or in combination with cytokines modifies the expression of several genes in pancreatic beta-cells. Two of these genes, Fas and iNOS, may contribute to beta-cell death. The transcription factor nuclear factor-kappaB is required for PIC-induced iNOS expression. PIC has an additive effect on cytokine-induced beta-cell death by both NO-dependent (in the case of IL-1beta) and NO-independent (in the case of IFN-gamma) mechanisms. These findings suggest that viral intermediates in synergism with local cytokine production may play an important role in beta-cell apoptosis in early T1DM.
1型糖尿病（T1DM）是一种自身免疫性疾病，由产生胰岛素的胰腺β细胞进行性破坏所致。病毒感染以及细胞因子白细胞介素-1β（IL-1β）和干扰素-γ（IFN-γ）均被认为是早期T1DM中β细胞死亡的潜在介质。我们目前研究了病毒复制中间体双链RNA[此处用作合成聚肌苷酸-聚胞苷酸（PIC）]是否会改变IL-1β和IFN-γ对大鼠胰腺β细胞基因表达和活力的影响。为此，将荧光激活细胞分选纯化的大鼠β细胞暴露于PIC和/或IL-1β及IFN-γ中6至16小时（通过逆转录聚合酶链反应研究基因表达）或6至9天（通过核染料研究活力）。PIC使Fas和锰超氧化物歧化酶信使核糖核酸的表达增加了5至10倍。IL-以及PIC与IFN-γ的组合（但不是单独的PIC或IFN-γ）诱导诱导型一氧化氮（NO）合酶（iNOS）的表达及随后的NO产生。如将iNOS启动子-荧光素酶报告基因构建体转染到原代β细胞的实验所示，PIC和IFN-γ诱导iNOS表达需要核因子-κB激活。IL-1β加IFN-γ、PIC加IFN-γ或PIC加IL-1β的组合使凋亡β细胞数量增加了2至3倍。抑制iNOS活性显著降低了PIC加IL-1β诱导的凋亡，但未降低PIC加IFN-γ诱导的凋亡。总之，单独的PIC或与细胞因子联合使用会改变胰腺β细胞中几个基因的表达。其中两个基因，Fas和iNOS，可能导致β细胞死亡。转录因子核因子-κB是PIC诱导iNOS表达所必需的。PIC通过NO依赖性（在IL-1β的情况下）和NO非依赖性（在IFN-γ的情况下）机制对细胞因子诱导的β细胞死亡具有相加作用。这些发现表明，病毒中间体与局部细胞因子产生协同作用可能在早期T1DM的β细胞凋亡中起重要作用。
