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JG Mabley, G Hasko, L Liaudet, F Soriano, GJ Southan, AL Salzman and C Szabo

Insulin-dependent diabetes mellitus (IDDM) is a disease characterized by the autoimmune destruction of the pancreatic beta-cells, which requires the expression of a number of immune-related genes including major histocompatibility complex proteins, cytokines, chemokines, and cytotoxic enzymes, many of which are regulated by the transcription factor, NFkappaB. Inhibition of the entire NFkappaB family of transcription factors may be harmful, as these factors are involved in many normal physiological processes. However, identifying and targeting specific NFkappaB subunits critical for the pathogenesis of disease may prove to be valuable in designing new therapeutic strategies. To assess the potential role of the NFkappaB subunit, p50, in the development of IDDM, mice with gene disruption for NFkappaB (p50) were investigated for susceptibility to IDDM. We found that p50-deficient mice were fully resistant against multiple low-dose streptozotocin-induced diabetes, a model of diabetes with a strong autoimmune component. The site of involvement of NFkappaB (p50) lies at an early, critical juncture of immune activation and proinflammatory mediator production, because: (1) isolated islets of Langerhans from NFkappaB (p50)-deficient mice were not protected from the islet dysfunction induced by in vitro application of proinflammatory cytokines; (2) p50-deficient mice were not resistant to diabetes induced by a single high dose of streptozotocin, a model with a large oxidant component and no autoimmune involvement; and (3) diabetes induced up-regulation of nitric oxide and interleukin-12 was blocked in the p50-deficient mice. Our data suggest that NFkappaB (p50) has an essential role in the development of autoimmune diabetes. Selective therapeutic blockade of this subunit may be beneficial in preventing IDDM.

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JG Mabley, JM Cunningham, N John, MA Di Matteo and IC Green

The aim of this study was to examine if the growth factor, transforming growth factor beta 1 (TGF beta 1), could prevent induction of nitric oxide synthase and cytokine-mediated inhibitory effects in the insulin-containing, clonal beta cell line RINm5F. Treatment of RINm5F cells for 24 h with interleukin-1 beta (IL-1 beta) (100 pM) induced expression of nitric oxide synthase and inhibited glyceraldehyde-stimulated insulin secretion. Combinations of IL-1 beta (100 pM), tumour necrosis factor-alpha (100 pM) and interferon-gamma (100 pM) reduced RINm5F cell viability (determined by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium (MTT) reduction assay) and de novo protein synthesis, as measured by incorporation of radiolabelled amino acids into perchloric acid-precipitable protein. Pretreatment of RINm5F cells with TGF beta 1 (10 pM) for 18 or 24 h, prior to the addition of either IL-1 beta or combined cytokines, prevented cytokine-induced inhibition of insulin secretion, protein synthesis and the loss of cell viability. TGF beta 1 pretreatment inhibited cytokine-induced expression and activity of nitric oxide synthase in RINm5F cells as determined by Western blotting and by cytosolic conversion of radiolabelled arginine into labelled citrulline and nitric oxide. Chemically generated superoxide also induced expression of nitric oxide synthase possibly due to direct activation of the nuclear transcription factor NF kappa B, an effect prevented by both an antioxidant and TGF beta 1 pretreatment. In conclusion, the mechanism of action of TGF beta 1 in blocking cytokine inhibitory effects was by preventing induction of nitric oxide synthase.