SUMMARY<p><p>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. Nitric oxide (NO) is a highly diffusible, short-lived free radical gas, which plays a significant role in several physiological processes in a diversity of tissues and organisms. Prolonged exposure of rodent or human pancreatic beta-cells to combinations of cytokines induces the expression of the inducible form of nitric oxide synthase (iNOS) and Fas, NO production, and cell death. It also induces the expression of potential "defense" genes, such as manganese superoxide dismutase (MnSOD) and heat shock protein (hsp) 70. Recent studies have shown that NO, in addition to having cytotoxic actions, may also regulate gene transcription. It remains unclear whether NO mediates cytokine-induced gene expression and subsequent beta-cell death. Previous studies using NO synthase blockers yielded conflicting results, which may be due to non-specific effects of these agents. <p>In the first part of our work, we examined the role of NO in beta-cell dysfunction and death by using an iNOS knockout mice (iNOS-/-, background C57BL/6x129SvEv). We evaluated the effects of cytokines on gene expression, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR), and viability, as determined by nuclear dyes, of pancreatic islet cells or fluorescence-activated cell sorter (FACS)-purified beta-cells isolated from iNOS knockout mice or their respective controls (C57BL/6x129SvEv). The combination of cytokines used was interleukin-1beta (50 U/ml) + gamma-interferon (1000 U/ml) + tumor necrosis factor-alpha (1000 U/ml). The lack of cytokine-induced iNOS activity in the iNOS-/- islet cells was confirmed by RT-PCR and nitrite determination. Cytokines induced a > 3-fold increase in Fas and MnSOD mRNA expression in wild-type (wt) and iNOS-/- islets. On the other hand, hsp 70 was induced in wt but not in iNOS-/- islets. Prolonged (6-9 days) exposure of wt islets to cytokines lead to an 80-90% decrease in islet cell viability, whereas viability decreased by only 10-30% in iNOS-/- islet cells. To determine the mode of cytokine-induced cell death, FACS-purified beta-cells were exposed to the same cytokines. After 9 days, the apoptosis index was similarly increased in wt (39 +/- 3%) and iNOS-/- (33 +/- 4 %) beta-cells. On the other hand, cytokines increased necrosis in wt (20 +/- 4 %) but not in iNOS-/- (7 +/- 3 %) beta-cells. From these data, we conclude that: 1) NO is required for cytokine-induced hsp 70 mRNA expression, but not for Fas and MnSOD expression; 2) cytokines induce both apoptosis and necrosis in mouse beta-cells; 3) cytokine-induced apoptosis is mostly NO-independent, whereas necrosis requires NO formation.<p>In the second part of our work, we examined the role of the viral product double-stranded RNA (dsRNA) in beta-cell dysfunction and death. DsRNA is produced by many viruses during their replicative cycle. We investigated whether dsRNA (here utilized as synthetic poly IC (PIC)) modifies the effects of IL-1beta and IFN-gamma on gene expression and viability of rat pancreatic beta-cells and the role of NO in this process. FACS-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 or IFN-gamma. PIC increased the expression of Fas and Mn superoxide dismutase mRNAs by 5-10-fold. IL-1beta and a combination of PIC + IFN-gamma& / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished
| Identifer | oai:union.ndltd.org:ulb.ac.be/oai:dipot.ulb.ac.be:2013/211206 |
| Date | 05 March 2004 |
| Creators | Liu, Dongbo |
| Contributors | Eizirik, Decio L., Devière, Jacques, Welsh, Nils, Mathieu, Chantal, Le Moine, Olivier, Robberecht, Patrick, Galand, Paul |
| Publisher | Universite Libre de Bruxelles, Université libre de Bruxelles, Faculté de Médecine – Médecine, Bruxelles |
| Source Sets | Université libre de Bruxelles |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis, info:ulb-repo/semantics/doctoralThesis, info:ulb-repo/semantics/openurl/vlink-dissertation |
| Format | 1 v., No full-text files |
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