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The purpose of this study was to evaluate the effects of interferon-␥ (IFN-␥) alone and in combination with interleukin 1␤ (IL-1␤) on inducible nitric-oxide synthase (iNOS) mRNA and protein expression, nitrite production, and insulin secretion by islets of Langerhans. Treatment of rat islets with IL-1␤ results in a concentration-dependent increase in the production of nitrite that is maximal at 5 units/ml. Individually, 0.1 unit/ml IL-1␤ or 150 units/ml rat IFN-␥ do not stimulate iNOS expression or nitrite production by rat islets; however, in combination, these cytokines induce the expression of iNOS and the production of nitrite to levels similar in magnitude to the individual effects of 5 units/ml IL-1␤. The islet ␤-cell, selectively destroyed during insulin-dependent diabetes mellitus, appears to be one islet cellular source of iNOS as 150 units/ml rat IFN-␥ and 0.1 unit/ml IL-1␤ induced similar effects in primary ␤-cells purified by fluorescence-activated cell sorting and in the rat insulinoma cell line, RINm5F. iNOS expression and nitrite production by rat islets in response to 150 units/ml rat IFN-␥ and 0.1 unit/ml IL-1␤ are correlated with an inhibition of insulin secretion and islet degeneration that are prevented by the iNOS inhibitor aminoguanidine. The mechanism by which IFN-␥ increases the sensitivity of ␤-cells for IL-1-induced iNOS expression appears to be associated with an increase in the stability of iNOS mRNA. Last, cellular damage during physical dispersion of islets results in the release of sufficient amounts of IL-1␤ to induce iNOS expression and nitrite production in the presence of exogenously added rat IFN-␥. The cellular source of IL-1␤ under these conditions is believed to be resident islet macrophages as depletion of macrophages prior to dispersion prevents IFN-␥-induced iNOS expression and nitrite formation by dispersed islet cells. These studies show that the T-lymphocyte cytokine, IFN-␥, increases the sensitivity of rat islets to the effects of IL-1␤ on iNOS expression and nitrite production by 10-fold, in part, through the stabilization of iNOS mRNA. Our studies also support an effector role for IFN-␥, in concert with resident islet macrophage release of IL-1␤, in mediating ␤-cell destruction during the development of autoimmune diabetes.Insulin-dependent diabetes mellitus is an autoimmune disease characterized by the selective destruction of insulin secreting ␤-cells found in islets of Langerhans. Many lines of evidence support a role for the involvement of cytokines as effector molecules that participate in the development of diabetes. Mandrup-Poulsen et al.(1) first showed that treatment of isolated rat islets with conditioned media derived from activated mononuclear cells results in a potent inhibition of insulin secretion followed by islet destruction. The active component of this conditioned media was determined to be the cytokine IL-1 1 (2). IL-1-induced inhibition of insulin secretion is both timeand concentration-dependent and requires mRNA transcription and new protein ...

BackgroundThere are at least two phases of β-cell death during the development of autoimmune diabetes: an initiation event that results in the release of β-cell-specific antigens, and a second, antigen-driven event in which β-cell death is mediated by the actions of T lymphocytes. In this report, the mechanisms by which the macrophage-derived cytokine interleukin (IL)-1 induces β-cell death are examined. IL-1, known to inhibit glucose-induced insulin secretion by stimulating inducible nitric oxide synthase expression and increased production of nitric oxide by β-cells, also induces β-cell death.Methods and FindingsTo ascertain the mechanisms of cell death, the effects of IL-1 and known activators of apoptosis on β-cell viability were examined. While IL-1 stimulates β-cell DNA damage, this cytokine fails to activate caspase-3 or to induce phosphatidylserine (PS) externalization; however, apoptosis inducers activate caspase-3 and the externalization of PS on β-cells. In contrast, IL-1 stimulates the release of the immunological adjuvant high mobility group box 1 protein (HMGB1; a biochemical maker of necrosis) in a nitric oxide-dependent manner, while apoptosis inducers fail to stimulate HMGB1 release. The release of HMGB1 by β-cells treated with IL-1 is not sensitive to caspase-3 inhibition, while inhibition of this caspase attenuates β-cell death in response to known inducers of apoptosis.Patient SummaryBackgroundType 1 diabetes (also called autoimmune diabetes or juvenile diabetes) is an autoimmune disease. For unknown reasons, at some point in childhood or adolescence, the body's own immune system starts attacking and destroying the insulin-producing islet cells in the pancreas. Once the majority of islet cells are destroyed, patients can no longer produce insulin to regulate their blood sugar and must depend on strict diets and insulin injections. Scientists are trying to understand the early events during the development of the disease. There are two fundamentally different kinds of cell death in cells of higher animals and humans, called apoptosis and necrosis. Apoptosis (also called programmed cell death) is an organized, clean way in which cells die without spilling their contents and without causing an inflammatory immune response. They are simply gobbled up by other cells that serve as the body's garbage collectors. Necrosis, on the other hand, is a more messy process and one that does activate the immune system and cause local inflammation.Why Was This Study Done?The scientists who did this study are interested in the early stages of islet cell death. Specifically, they wanted to know whether islet cells during the early events of autoimmune diabetes die via apoptosis or necrosis. Earlier experiments to address this question had yielded no clear-cut results.What Did the Researchers Do and Find?All the experiments for this study were done in cultured cells in the laboratory. For the most part, the researchers used rodent islet cells, and then they confirmed the crucial finding in human islet cells. They ...

The effects of double-stranded RNA (synthetic polyinosinic-polycytidylic acid; poly(I-C)) on macrophage expression of inducible nitric-oxide synthase (iNOS), production of nitric oxide, and release of interleukin-1 (IL-1) were investigated. Individually, poly(I-C), interferon-␥ (IFN-␥), and lipopolysaccharide (LPS) stimulate nitrite production and iNOS expression by RAW 264.7 cells. In combination, the effects of poly(I-C) ؉ IFN-␥ are additive, while poly(I-C) does not further potentiate LPS-induced nitrite production. These results suggest that poly(I-C) and LPS may stimulate iNOS expression by similar signaling pathways, which may be independent of pathways activated by IFN-␥. LPS-induced iNOS expression is associated with the activation of NF-B. We show that inhibition of NF-B by pyrrolidinedithiocarbamate prevents poly(I-C) ؉ IFN-␥-, poly(I-C) ؉ LPS-, and LPS-induced iNOS expression, nitrite production and IB degradation by RAW 264.7 cells. The effects of poly(I-C) on iNOS ex-pression appear to be cell-type specific. Poly(I-C), alone or in combination with IFN-␥, does not stimulate, nor does poly(I-C) potentiate, IL-1-induced nitrite production by rat insulinoma RINm5F cells. In addition, we show that the combination of poly(I-C) ؉ IFN-␥ stimulates iNOS expression, nitrite production, IB degradation, and the release of IL-1 by primary mouse macrophages, and these effects are prevented by pyrrolidinedithiocarbamate. These findings indicate that double-stranded RNA, in the presence of IFN-␥, is a potent activator of macrophages, stimulating iNOS expression, nitrite production, and IL-1 release by a mechanism which requires the activation of NF-B.

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