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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2008-0396
Copyright © 2008 by The Endocrine Society Increased Interleukin (IL)-1β Messenger Ribonucleic Acid Expression in β-Cells of Individuals with Type 2 Diabetes and Regulation of IL-1β in Human Islets by Glucose and AutostimulationMarianne Böni-Schnetzler, Jeffrey Thorne, Géraldine Parnaud, Lorella Marselli, Jan A. Ehses, Julie Kerr-Conte, Francois Pattou, Philippe A. Halban, Gordon C. Weir and Marc Y. DonathClinic of Endocrinology and Diabetes (M.B.-S., J.A.E., M.Y.D.), University Hospital of Zurich, 8091 Zurich, Switzerland; Section of Islet Transplantation and Cell Biology (J.T., L.M., G.C.W.), Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts 02215; Department of Genetic Medicine and Development (G.P., P.A.H.), University of Geneva, 1211 Geneva 4, Switzerland; and Thérapie Cellulaire du Diabète (J.K.-C., F.P.), Institut National de la Santé et de la Recherche Médicale Unit M 859, Faculté de Médecine, 59045 Lille Cedex, France Address all correspondence and requests for reprints to: Marianne Böni-Schnetzler, Ph.D., Clinic of Endocrinology and Diabetes, Department of Medicine, University Hospital, CH-8091 Zurich, Switzerland. E-mail: marianne.boeni{at}usz.ch.
Context: Elevated glucose levels impair islet function and survival, and it has been proposed that intraislet expression of IL-1β contributes to glucotoxicity. Objective: The objective was to investigate IL-1β mRNA expression in near-pure β-cells of patients with type 2 diabetes (T2DM) and study the regulation of IL-1β by glucose in isolated human islets. Methods: Laser capture microdissection was performed to isolate β-cells from pancreas sections of 10 type 2 diabetic donors and nine controls, and IL-1β mRNA expression was analyzed using gene arrays and PCR. Cultured human islets and fluorescence-activated cell sorter-purified human β-cells were used to study the regulation of IL-1β expression by glucose and IL-1β.
Results: Gene array analysis of RNA from β-cells of individuals with T2DM revealed increased expression of IL-1β mRNA. Real-time PCR confirmed increased IL-1β expression in six of 10 T2DM samples, with minimal or no expression in nine control samples. In cultured human islets, IL-1β mRNA and protein expression was induced by high glucose and IL-1β autostimulation and decreased by the IL-1 receptor antagonist IL-1Ra. The glucose response was negatively correlated with basal IL-1β expression levels. Autostimulation was transient and nuclear factor- Conclusion: Evidence that IL-1β mRNA expression is up-regulated in β-cells of patients with T2DM is presented, and glucose-promoted IL-1β autostimulation may be a possible contributor.
Chronically elevated glucose levels impair islet function and proliferation and induce apoptosis, leading to the concept of islet glucotoxicity (1, 2). Whereas it has been well documented over the last 20 yr that cytokines are crucial in the etiopathology of type 1 diabetes (3, 4), only recently was it postulated that cytokines also play a role in islet dysfunction and death in type 2 diabetes (T2DM) (5, 6, 7). It has been proposed that intraislet expression of inflammatory cytokines, in particular of IL-1β, contributes to the pathogenesis of type 2 diabetes (6, 8). This hypothesis was based on observations of increased IL-1β expression in pancreas sections of patients with T2DM by immunofluorescence and by in situ hybridization as well as in hyperglycemic Psammomys obesus (8). Furthermore, in vitro, high-glucose concentrations induced IL-1β release from some but not all human islet cell preparations (8). Nevertheless, the role of IL-1β in the deleterious effects of high glucose or the type 2 diabetes milieu on human pancreatic islets has been challenged based on ex vivo studies (9, 10). However, a recent clinical study demonstrated that the blockade of IL-1 in type 2 diabetic patients with the IL-1 receptor antagonist (IL1-Ra) results in improved blood glucose levels and insulin secretion in the absence of changes in peripheral insulin resistance and body mass index (BMI) (11). IL-1β has some unique features not shared by other chemokines and cytokines. Most notably, the signal transduction pathway via the IL-1 receptor (type I) is unusually effective and less than 10 molecules of IL-1β bound per cell can induce biological responses (12, 13, 14). Because of its effective signaling and cytotoxic effects on cells, processing, release, and receptor binding of IL-1β to target cells is tightly controlled. Unlike other secreted cytokines, IL-1β does not have a leader sequence and has to be processed from pro-IL-1β to IL-1β by inflammasomes before secretion (15). Secreted IL-1β associates with binding proteins such as the soluble form of the nonsignaling type II IL-1 receptor, thereby inhibiting its signaling to target cells (12, 16). These IL-1β binding proteins are also the reason that conventional ELISA detection of IL-1β is problematic (16). Furthermore, IL-1β-producing cells also synthesize their own antagonist IL-1Ra, which binds to the IL-1 receptor without having agonistic properties and thereby modulates inflammatory responses (17, 18). The low level of expression and the above described control mechanisms render IL-1β protein difficult to detect. IL-1β is typically produced by activated immune cells, but it is also expressed at lower levels by many different cell types (19). In islets, IL-1β expression has been demonstrated in resident lymphoid cells (20, 21), ductal cells, and vascular endothelial cells (22) as well as insulin producing β-cells (8, 21, 23). To demonstrate increased IL-1β mRNA expression in pancreatic sections of type 2 diabetic patients, we used laser capture microdissection to obtain near pure samples of β-cells from 10 patients with T2DM and nine controls. To elucidate the regulation of IL-1β expression and to understand the observed variable effects of glucose on IL-1β induction, we examined IL-1β expression in vitro using 12 different human islet preparations. Furthermore, we studied the effects of IL-1β and glucose on islet derived inflammatory factors IL-8 and IL-6.
Laser capture microdissection (LCM) Nearly pure β-cells were obtained from human pancreatic frozen sections of 10 T2DM donors and nine controls by LCM. The technical details and β-cell purity were described previously (24). Briefly, LCM was performed under direct microscopic visualization of the autofluorescent signal positive areas. We performed 150–250 pulses per section on 20–30 sections per donor corresponding to a total of 5,000–15,000 cells. RNA was extracted (25), amplified, biotinylated, and hybridized to GeneChip human X3P array (Affymetrix, Santa Clara, CA) and analyzed as described (24).
RT-PCR of LCM-derived RNA was done using TaqMan reverse transcription reagents and universal PCR master mix (Applied Biosystems, Foster City, CA). Ribosomal L32 transcripts served as a reference and primers were designed using Primer Express software (Applied Biosystems). For triplicate PCR determinations, SYBR green (IL-1β, IL-8) or TaqMan technology (RPL32) was used. The relative amount of mRNA was calculated by the Human islet cultures and treatment with glucose, IL-1β, and IL-1Ra Islets were isolated from pancreata of organ donors aged between 35 and 70 yr with a BMI between 20.7 and 40.6 kg/m2. Islet purity ranged between 75 and 90% as judged by dithizone staining and glucose-stimulated insulin secretion was 3.3 ± 0.96-fold (mean ± SD, n = 8). Islets were plated on extracellular matrix-coated dishes (Novamed Ltd., Jerusalem, Israel) at a density of 150–200 islets per 35 mm Ø dish in CMRL 1066 medium containing 5.5 mM glucose and 10% fetal calf serum (8). After 3 d of preculture, experiments were started by adding medium with 5.5 or 33.3 mM glucose for a further 4 d without medium change. IL-1Ra (Amgen, Seattle, WA) was added at 1 µg/ml and rhIL-1β (R&D Systems Inc., Minneapolis, MN) at 0.2 ng/ml at the start of the experiments. Human β-cell purification The detailed protocol for purification of human β-cells is described elsewhere (26) and depends on the preferential labeling of β-cells with the fluorescent Zn2+-chelator Newport Green (27, 28) and subsequent fluorescence-activated cell sorter (FACS) sorting. The resulting cell population was comprised of greater than 90% β-cells based on immunostaining with either antiinsulin or antipancreatic duodenal homeobox-1. Then 105 β-cells/dish were cultured and treated as described for the whole islets. Quantitative PCR of cDNA from islet and β-cell cultures RNA extraction and cDNA synthesis was done as described (29). For quantitative PCR, the real-time PCR system 7500 (Applied Biosystems) and the following commercial TaqMan assays were used: IL-1β, Hs00174097_m1; IL-1Ra, Hs00277299_m1; hypoxanthine-guanine phosphoribosyl transferase HPRT-1, Hs99999909_m1; IL-6, Hs00174131_m1; IL-8, Hs00174103_m1; CD31, Hs00169777_m1; insulin, Hs00154355_m1; CD68, Hs02741908_m1; caspase-1, Hs00236158_m1; prohormone convertase (PC)-1, Hs01026108_m1; PC2, Hs00159922_m1; and eukaryotic 18s rRNA, Hs99999901_s1 (Applied Biosystems, Rotkreuz, Switzerland). Triplicate cycle threshold values were normalized to 18s and values above cycle 36 were not used. Detection of IL-8 and IL-6 with Luminex technology Culture media were collected and stored at –20 C. IL-6 and IL-8 concentrations were assayed using a 2-Plex LINCO kit containing beads coupled with antibodies specific for IL-6 and IL-8 (Millipore, Billerica, MA). Recording was done with a Bioplex analyzer from Bio-Rad (Hercules, CA).
Nuclear factor- Islets were precultured for 3 d, media were removed, and cells were treated for 2 h with 5 µM BAY 11–7083 (ANAWA, Wangen, Switzerland) or solvent control [0.01% dimethylsulfoxide (DMSO)]. Cells were washed with media and stimulated for 24 h with or without 0.2 ng/ml IL-1β before RNA extraction. Western blotting Western blotting of protein extracts from islets was done as described (29). Anti-IL-1β antibody was from Xoma (Berkeley, CA), horseradish peroxidase-labeled goat antihuman IgG (Pierce Biotechnology, Rockford, IL), antiactin (C-2), and a horseradish peroxidase-labeled goat antimouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA). Signal detection was assessed with a chemoluminescence imager (LAS-3000; Bucher Biotech, Basel, Switzerland). Statistics Data were analyzed with the GraphPad Prism program (GraphPad, San Diego, CA). Statistical significance was determined using the t test for normally distributed samples, the Mann Whitney for nonnormal distribution, and ANOVA with Bonferroni’s post hoc test for multiple comparisons. Significance was set at P < 0.05.
IL-1β mRNA expression is increased in β-cells from patients with T2DM IL-1β and IL-8 mRNA expression was analyzed from gene profiles of near-pure samples of β-cells obtained by LCM from frozen pancreatic tissue sections of 10 T2DM and nine nondiabetic cadaver donors. The control and diabetic groups had a matched BMI of 30.8 ± 6.0 kg/m2 in the diabetic and 30.9 ± 5.4 kg/m2 in the control group. The average duration for known diabetes was 5.3 ± 2.3 yr (n = 7), and the cause of death was in most cases a cardiovascular accident (supplemental Table 1, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org).
Gene array analysis (Table 1
Increased expression of IL-1β and IL-8 in samples from T2DM was confirmed by real-time quantitative PCR. We found strongly increased IL-1β mRNA levels (77.52 ± 36.84 fold) in six of 10 patients with T2DM, whereas minimal expression of IL-1β (1.39 ± 0.26 fold) was observed in three of nine controls (P = 0.021). IL-8 expression was also observed by real-time PCR in six of 10 T2DM samples and four of nine control samples (Table 1  ). The mean IL-8 mRNA levels of the diabetics of 15.12 ± 5.23-fold vs. 3.19 ± 1.9-fold in controls also showed a trend to increased expression (P = 0.086) in the diabetic group. The gene array data correlated with the PCR data for both IL-1β (r2 = 0.67, P = 0.007) and IL-8 (r2 = 0.73, P = 0.0016). There were no correlations for the IL-1β PCR data with the BMI or cold ischemia time. A weak correlation was found for blood glucose and the IL-8 gene array data (r2 = 0.358, P = 0.018) but no correlation with the IL-8 PCR results. However, the blood glucose levels were significantly correlated with both the IL-1β-positive PCR results (r2 = 0.784, P = 0.003) and the IL-1β gene array data (r2 = 0.565, P = 0.001). IL-1β mRNA levels are induced by glucose and the responsiveness to glucose correlates with basal IL-1β levels
IL-1β mRNA expression and the effect of high glucose concentrations were evaluated in vitro using cultured human islets from 12 different donors. Untreated islets from different donors expressed IL-1β mRNA at unusually variable levels when compared with other basal transcript levels such as PC1/3 and PC2, insulin, macrophage marker CD68, and endothelial cell marker CD31 (Fig. 1
Glucose reduces IL-1Ra mRNA levels
We next analyzed the effects of glucose on IL-1Ra mRNA levels in the 12 human islet cultures (Fig. 1C IL-1β expression is increased by IL-1β autostimulation
Induction of IL-1β expression by IL-1β itself (autostimulation) was reported for different cell types (30, 31, 32, 33) but not yet for islets. Treatment of human islets with 0.2 ng/ml exogenous IL-1β [a concentration in the range released by islets (8, 9)] significantly stimulated IL-1β mRNA expression by 4.6 ± 1.3-fold (Fig. 2A
Because primary islet cell cultures contain different cell types, we examined whether IL-1β autostimulation can be observed in FACS-purified human β-cells. IL-1β strongly induced autostimulation in purified β-cells (15.3 ± 4.11; Fig. 2B Glucose induces biologically active IL-1β
To test whether glucose treatment results in release of biologically active IL-1β, we used the antagonist IL-1Ra, which blocks ligand-induced IL-1 receptor activation without having agonistic properties (17, 34). We incubated control and glucose- and IL-1β-treated human islet cultures with IL-1Ra and measured IL-1β and IL-8 mRNA and protein expression. Figure 3
Autostimulation and glucose-induced IL-1β and IL-8 production was also demonstrated at the protein level by measuring released IL-8 and performing Western blot analysis of pro-IL-1β (32 kDa). The latter enabled us to distinguish islet cell-produced IL-1β from exogenously added IL-1β. A representative blot with islet extracts from six different donors is shown in Fig. 3C . In all islet preparations, IL-1β strongly induced and IL-1Ra inhibited pro-IL-1β expression. Glucose increased pro-IL-1β expression in three of six islet donors, a frequency expected based on the results of Fig. 1 . Furthermore, glucose increased the level of IL-8 in culture supernatants (Fig. 3D). IL-1Ra antagonized glucose-induced pro-IL-1β and IL-8 (Fig. 3, C and D).
Dose response, kinetics, and NF-
Next, we determined the IL-1β and glucose dose dependence of IL-1β mRNA induction (Fig. 4
Low picogram per milliliter concentrations of IL-1β regulate IL-6 and IL-8 expression in human islets
IL-1β is a master regulator of various inflammatory factors including IL-8 and IL-6 (19). To test that this holds true in islets, we antagonized IL-1β-induced IL-6 and IL-8 protein release with IL-1Ra (Fig. 5
The concentrations of IL-1β released by islets were in the low picogram per milliliter range (8, 9). A dose dependence with picogram per milliliter concentrations of exogenous IL-1β demonstrated that these low amounts stimulated the release of IL-8 and IL-6 protein in human islet cultures (Fig. 5 , F and G). Already 20 pg/ml of IL-1β significantly induced the release of nanogram per milliliter concentrations of IL-8, and maximal effects were observed at 200 pg/ml of IL-1β. Similarly low doses of IL-1β also induced nanogram per milliliter concentrations of rodent CXC-chemokine ligand 1 in INS-1E cells (data not shown).
In vivo expression of cytokines, in particular the tightly regulated and transiently expressed IL-1β, is difficult to assess. In the present study, the gene expression profiles of β-cells captured by LCM of pancreas sections from patients with T2DM were analyzed, and IL-1β expression was confirmed by real-time PCR. These samples contain β-cell selected tissue strongly depleted of glucagon, somatostatin, acinar, and duct cells when compared with isolated whole islets (24). We observed IL-1β mRNA expression in β-cells of six of 10 patients with T2DM and, at a much lower level, in three of nine controls. Because only low amounts of material can be obtained by LCM, analysis of IL-1β protein was not possible. In a recent study with whole islets isolated from patients with T2DM, no significant difference in IL-1β expression could be observed, compared with nondiabetic islets (9). These islets were cultured at 5.5 mM glucose for 3–4 d before analysis, and IL-1β expression may have returned to lower levels during that time. Furthermore, the isolation stress and the high number of non-β-cells are additional confounding factors, particularly in T2DM islets displaying reduced numbers of β-cells, compared with controls. The observed IL-1β expression pattern was very variable in individuals with T2DM. In the array screening, we observed no differences between control and T2DM samples for β-cell specific genes as well as markers for macrophage and endothelial cells. It is thus unlikely that the variability of IL-1β expression is due to a technical problem related to RNA isolation or variable contamination by non-β-cells. Furthermore, we found no correlation between IL-1β expression and age, BMI, cause of death, or cold ischemia time. We did, however, find significant correlation of IL-1β expression with glycemia, suggesting that variable blood glucose levels could contribute to the variable IL-1β expression levels. A further possible cause for the variable IL-1β levels could be its mode of expression. In other cell types, IL-1β expression oscillated and was amplified by autostimulation (30, 31, 32, 33). In keeping with this, we indeed observed transient IL-1β expression and autostimulation in islets in vitro. With such changing expression levels, constant measurements can hardly be expected when sampling is done at just one time point. Highly variable circulating IL-1β concentrations were recently also reported in patients with type 1 diabetes, whereas other cytokines did not show such fluctuations (36). Furthermore, it is possible that only some of the patients with T2DM presented with an inflammatory phenotype. We also analyzed the effects of elevated glucose on IL-1β mRNA expression in vitro using 12 different islet preparations. In contrast to the ex vivo samples obtained by LCM, normal untreated islets always expressed IL-1β. However, basal expression levels were unusually variable in the different islet preparations, compared with other genes that did not show such a high variability. The most constant expression was observed with the macrophage marker CD68. The variability was also not due to IL-1β induction during the 6- to 7-d culture period because IL-1β levels changed only by a factor of 2 within subjects, and this cannot account for the up to 20-fold between-subject difference observed among different preparations. Variable basal IL-1β expression could be the result of the stress of islet isolation. In previous studies various inflammatory cyto- and chemokines including IL-1β were induced upon islet isolation (24, 37, 38) and persisted 2–11 d after isolation. Also, differences in the attachment of the islets on the extracellular matrix, which has been shown to induce IL-1β (23), or differences in the genetic background of the donors may contribute to the variability. Independent of the underlying cause, variable basal IL-1β could be the reason that we did not observe glucose stimulated IL-1β in all islet preparations. This notion is supported by the finding of a negative correlation between basal IL-1β and glucose-stimulated IL-1β. Furthermore, in the presence of exogenously added IL-1β, glucose did not further stimulate IL-1β mRNA. Taken together, this suggests that elevated basal IL-1β levels blunt the effects of glucose on IL-1β expression in some but not all islet preparations. A glucose dose response revealed that a concentration as low as 11 mM glucose already significantly increased IL-1β mRNA expression. Elevated glucose concentrations also increased IL-1β at the protein level and up-regulated inflammatory factor IL-8 mRNA and protein expression. IL-1Ra blocked glucose-induced IL-1β and IL-8, indicating that the effect of glucose requires IL-1 receptor activation via increased secretion of biologically active IL-1β.
Islet IL-1β expression was partly mediated by NF- A variety of different cell types present within islets can produce IL-1β (8, 20, 21, 22, 23). Here we demonstrate autostimulation in FACS-purified β-cells. This finding together with IL-1β expression in samples obtained by LCM supports the idea that human β-cells themselves can produce IL-1β. IL-1β expression was recently also demonstrated in sorted rat β-cells (23). This does not rule out that additional islet cells (e.g. macrophages) also produce IL-1β in inflamed islets of patients with T2DM (39). The LCM data also revealed that IL-1β and IL-8 expression was correlated, and in vitro, we found that glucose induced IL-8 mRNA and protein release by an IL-1-dependent mechanism. IL-1β concentrations measured in islet culture supernatants are in the low picogram per milliliter range (8, 9), and we show that these low concentrations were sufficient to stimulate IL-8 and IL-6, which were released in nanogram per milliliter concentrations. IL-8 produced by cultured human islets is functional and mediates chemoattraction of macrophages (40). The induction of proinflammatory mediators together with IL-1β autostimulation, which may act in an auto- and/or paracrine way, implicates IL-1β in the mediation and amplification of intraislet inflammatory processes. We hypothesize that glucose-induced intraislet IL-1β may contribute to an inflammatory state in at least some patients with T2DM.
We thank G. Sigfried-Kellenberger for excellent technical assistance. Some batches of human islets of Langerhans were provided by the Cell Isolation and Transplantation Center at the University of Geneva School of Medicine, thanks to the ECIT Islets for Research distribution program sponsored by the Juvenile Diabetes Research Foundation. We thank P. Marchetti for providing us pancreas sections from T2D donors (supported by a European Foundation for the Study of Diabetes/Pfizer Resource Grant).
The work of M.B.-S., J.A.E., and M.Y.D. was supported by grants from the Swiss National Science Foundation, the Juvenile Diabetes Research Foundation, the University Research Priority Program Integrative Human Physiology at the University of Zürich, and a European Foundation for the Study of Diabetes/Merck Sharp & Dohme basic research grant. The work of G.C.W., L.M., and J.T. was supported by grants from the National Institutes of Health (NCRR ICR U4Z RR 16606 and U19DK6125), the American Diabetes Association, and the Diabetes Research and Wellness Foundation. P.A.H. was supported by Grant 7-2005-1158 from the Juvenile Diabetes Research Foundation. Disclosure Summary: M.B.-S., J.T., G.P., L.M., J.A.E., J.K.-C., F.P., P.A.H., G.C.W., and M.Y.D. have nothing to declare. First Published Online July 29, 2008
Abbreviations: BMI, Body mass index; DMSO, dimethylsulfoxide; FACS, fluorescence-activated cell sorter; IL1-Ra, IL-1 receptor antagonist; LCM, laser capture microdissection; NF- Received February 19, 2008. Accepted July 21, 2008.
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Abstract
Introduction
B dependent. Glucose-induced IL-1β was biologically active and stimulated IL-8 release. Low picogram per milliliter concentrations of IL-1β up-regulated inflammatory factors IL-8 and IL-6. 
1), and endothelial cell markers (VE-cadherin, CD31, CD54, von Willebrand factor, CD34, CD51). 
phosphorylation BAY 11–7082 (
