Interleukin-1 Stimulates β-Cell Necrosis and Release of the Immunological Adjuvant HMGB1

BACKGROUND: There 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 FINDINGS: To 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. CONCLUSIONS: These findings indicate that IL-1 induces β-cell necrosis and support the hypothesis that macrophage-derived cytokines may participate in the initial stages of diabetes development by inducing β-cell death by a mechanism that promotes antigen release (necrosis) and islet inflammation (HMGB1 release)

Nitric Oxide Mediates IL-1-Induced β-Cell Death as Determined by the MTT Assay

<div><p>RINm5F cells (A and B) or rat islets (C and D) were treated with IL-1 (10 units/ml) and NMMA (2 mM) for either 24 or 48 h, as indicated. The cells were isolated and viability determined using the MTT assay (A and C). The culture supernatants were isolated and nitrite production was determined using the Griess assay (B and D).</p> <p>(E and F) The effects of exogenously produced nitric oxide supplied by the donor compound DEANO (500 μM) and the apoptosis inducer camptothecin (25 μM) on RINm5F cell viability (E) and the apoptosis inducer staurosporine on rat islet cell viability (F) were determined by MTT assay.</p> <p>Cell viability data is expressed as percent death. Results for cell viability and nitrite production are the average ± standard error of the mean (SEM) of three independent experiments. *<i>p</i> < 0.05, significantly different from untreated controls. **<i>p</i> < 0.05, significantly different from IL-1-treated condition.</p></div

Caspase-3 Inhibition Prevents β-Cell Apoptosis but Does Not Prevent IL-1-Induced Necrosis

<p>The caspase-3 inhibitor Z-DQMD-CHO (100 μM) attenuated RINm5F cell death induced by a 24-h incubation with camptothecin as determined by MTT assay (A) or annexin V staining (B). This caspase-3 inhibitor failed to prevent RINm5F cell death in response to a 48-h incubation with 10 units/ml IL-1 as determined by the MTT assay (C). Z-DQMD-CHO also did not attenuate HMGB1 release by RINm5F cells treated for 24 h with 500 μM DEANO (D) or for 48 h with 10 units/ml IL-1 (E). The levels of HMGB1 released into the supernatant (s) and contained in the cells (p) were determined by Western blot analysis. Results for MTT assay (A and B) and annexin V staining (C) are the average ± SEM of three independent experiments. *<i>p</i> < 0.05, significantly different from untreated control. **<i>p</i> < 0.05, significantly different from camptothecin-treated condition. Results for HMGB1 release are representative of three independent experiments.</p

IL-1 Stimulates RINm5F Cell DNA Damage in a Nitric Oxide-Dependent Manner

<p>Treatment of RINm5F cells for 24 h (B) or 48 h (C) with IL-1 (10 units/ml) resulted in DNA damage, as determined by TUNEL staining (green fluorescence). DNA damage was attenuated by NMMA (E and F). The apoptosis inducer camptothecin (25 μM) stimulated DNA damage (D), and this DNA damage is morphologically distinct from that induced by IL-1. Nuclei are stained with DAPI (blue). The morphological differences in TUNEL staining, the loss of nuclear membrane integrity in response to IL-1 (B and C) and the condensation of DNA in response to camptothecin (D) are highlighted using white arrows. The levels of DNA damage in control cells (A) were less than 5%. Results are representative of three independent experiments.</p

Nitric Oxide-Dependent Release of HMGB1 from Cytokine-Treated Human Islets

<div><p>Human islets (200 islets in 400 μl of complete CMRL-1066) were treated with a combination of IL-1 (50 units/ml), IFN-γ (500 units/ml), and TNF (50 nM) in the presence or absence of the NOS inhibitor AG (2 mM) or treated with staurosporine (1 μM) alone.</p> <p>(A) Following 48h -incubation the supernatant was removed and nitrite production was determined using the Griess assay. The levels of HMGB1 released into the supernatant (s) and contained in the islets (p) were determined by Western blot analysis.</p> <p>(B) Results indicate that cytokine-stimulated HMGB1 release correlates with nitric oxide production, and that inhibition of nitric oxide production using AG attenuates HMGB1 release from human islets. Similar results have been obtained in three of five independent preparations of human islets.</p></div

Effects of IL-1 on β-Cell Viability as Determined by Neutral Red Uptake

<p>RINm5F cells were treated for 24 h or 48 h with IL-1 (10 units/ml), NMMA (2 mM) (A), or camptothecin (25 μM) (B), and the cells were isolated and percentage of death determined using the neutral red uptake assay. Results are average ± SEM of three independent experiments. *<i>p</i> < 0.05, significantly different from untreated controls. **<i>p</i> < 0.05, significantly different from IL-1-treated condition.</p

IL-1 Stimulates Islet Cell DNA Damage in a Nitric Oxide-Dependent Manner

<p>Treatment of rat islet cells for 24 h with IL-1 resulted in DNA damage as determined by TUNEL staining, and ∼95% of this damage occurred in insulin-containing cells (red fluorescence) (B). Nitric oxide appears to mediate IL-1-induced DNA damage, as it was attenuated by the NOS inhibitor NMMA (2 mM) (D). The apoptosis inducer staurosporine (1 μM) also stimulated DNA damage in both insulin containing and non-insulin containing cells (C). Less than 10% of control, untreated islet cells were TUNEL-positive (A). Results are representative of three independent experiments.</p