Production and function of IL-12 in islets and beta cells

Abstract

Aims/hypothesis IL-12 is an important cytokine in early inflammatory responses and is implicated in the immunemediated pathogenesis of pancreatic islets in diabetes. However, little is known about the direct effects of IL-12 on islets and beta cells. Methods In this study, beta cell function, gene expression and protein production were assessed in primary human donor islets and murine beta cell lines in response to stimulation with IL-12 or a pro-inflammatory cytokine cocktail (TNF-α, IL-1β and IFN-γ). Results The pro-inflammatory cytokine cocktail induced islet dysfunction and potently increased the expression and production of IL-12 ligand and IL-12 receptor in human islets. In human islets, the receptor for IL-12 co-localised to the cell surface of insulin-producing cells. Both IL-12 ligand and IL-12 receptor are expressed in the homogeneous beta cell line INS-1. IL-12 induced changes in gene expression, including a dose-dependent upregulation of IFNγ (also known as IFNG), in INS-1 cells. A neutralising antibody to IL-12 directly inhibited IFNγ gene expression in human donor islets induced by either IL-12 or pro-inflammatory cytokine stimulation. Functionally, IL-12 impaired glucose-stimulated insulin secretion (GSIS) in INS-1 cells and human donor islets. A neutralising antibody to IL-12 reversed the beta cell dysfunction (uncoupling of GSIS or induction of caspase-3 activity) induced by pro-inflammatory cytokines. Conclusions/interpretation These data identify beta cells as a local source of IL-12 ligand and suggest a direct role of IL-12 in mediating beta cell pathology.

Selective inhibition of 12-lipoxygenase protects islets and beta cells from inflammatory cytokine-mediated beta cell dysfunction

“…Compounds 5 or 9 were added 30 min before stimuli and retained for the remainder of the experiment. cDNA was prepared from isolated RNA as previously reported [13,27,31]. Speciesspecific Taqman primers were used for ALOX12, IL-12p40 (also known as IL12B), IFNγ (IFNG), monocyte chemoattractant protein-1 (MCP1), actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Life Technologies).…”

“…After stimulation islets/cells were incubated in low-glucose KRB for 1 h at 37°C. For GSIS, insulin, following 30 min incubation in 3 mmol/l glucose-KRB (low-glucose) or 16.7 mmol/l glucose-KRB (high-glucose), was measured by ELISA (Mercodia, Winston Salem, NC, USA) as previously reported [27].…”

“…For fluorescence microscopy, treated islets (up to 200 per condition) were washed with cold PBS and incubated for 30 min at 4°C in PBS containing 0.1 μmol/l YO-PRO-1 and 1 μg/ml propidium iodide (Life Technologies). Fluorescence was quantified as previously reported [13,27]. Briefly, after islets were washed in cold PBS, green/red/bright field images were captured with an Axiophot microscope (Zeiss, Jena, Germany) and green fluorescent intensity was quantified using Axiovision image analysis software (Zeiss).…”

“…Products of 12-lipoxygenase lead to ER stress, oxidative stress and generation of proinflammatory cytokines [11,13,14,26]. Very recent evidence has shown that 12-lipoxygenase can generate IL-12 production in the islet [23,27]. Local IL-12 production may propagate an immune inflammatory state that further induces beta cell damage [27].…”

“…Very recent evidence has shown that 12-lipoxygenase can generate IL-12 production in the islet [23,27]. Local IL-12 production may propagate an immune inflammatory state that further induces beta cell damage [27]. In human islets obtained from donors with type 2 diabetes, 12-lipoxygenase is upregulated and is associated with early islet dysfunction and increased migration of CD45 + leucocytes [11].…”