Irf-1 knockout (Irf-1
−/−) mice were obtained from T.W. Mak (Ontario Cancer Institute, University of Toronto, ON, Canada) and have been back-crossed to C57BL/6 mice six times . C57BL/6 mice were used as controls and were obtained from stocks purchased from Harlan (Horst, the Netherlands). NOD mice, inbred in our animal facility (Proefdierencentrum Leuven, Leuven, Belgium) since 1989, were used as diabetes-prone animals, with diabetes detected and defined as described . All mice were housed under semi-barrier conditions. The institutional review committee for animal experiments approved all the procedures for mouse care and animal killing.
Islet isolation, culture and treatment
To obtain pancreatic islets, pancreases from Irf-1
−/− or control C57BL/6 mice were removed and islets were isolated by collagenase digestion . Batches of 100 islets were collected and cultured overnight in RPMI 1640 medium (with GlutaMAX–I), containing 100 U/ml penicillin, 100 µg/ml streptomycin and 10% FCS [vol./vol.] (Invitrogen, Merelbeke, Belgium). Thereafter, islets were kept for 1 or 3 days in culture medium in the absence or presence of inflammatory cytokines as follows: recombinant human IL-1β (50 U/ml; kind gift of C.W. Reynolds, National Cancer Institute, Bethesda, MD, USA) in combination with recombinant mouse IFN-γ (1,000 U/ml; PeproTech, London, UK). In some experiments, islets were pre-incubated for 30 min with recombinant human IL-1 receptor antagonist (IL-1Ra; Kineret; Amgen, Thousand Oaks, CA, USA) at a concentration of 500 ng/ml .
Islet viability and function
Islet viability was evaluated using Hoechst 342 (20 µg/ml)/propidium iodide (10 µg/ml) (Molecular Probes, Invitrogen) as described [25, 26].
In vitro function of pancreatic islets was assessed by glucose-stimulated insulin release. Islets from Irf-1
−/− or control C57BL/6 mice were washed twice with KRB (115 mmol/l NaCl, 24 mmol/l NaHCO3, 5 mmol/l KCl, 1 mmol/l MgCl2, 2.5 mmol/l CaCl2 and 25 mmol/l HEPES, pH 7.4). After 30 min of sedimentation in KRB at 37°C, islets were incubated first at low (3 mmol/l) and then at high (20 mmol/l) concentrations of glucose in culture medium. At the end of incubation, supernatant fractions were assayed using an insulin ELISA kit (Mercodia, Uppsala, Sweden). Stimulation index (SI) was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation.
For glucose tolerance tests, mice were fasted overnight and received an intraperitoneal glucose load (2 g/kg body weight). Before and at 15, 30, 60, 90 and 120 min after glucose administration, glucose levels were measured in venous blood using a glucose meter (AccuChek Aviva; Roche Diagnostics Belgium, Vilvoorde, Belgium).
Monocyte chemoattractant protein-1 and nitrite measurement
Culture supernatant fractions from Irf-1
−/− or control C57BL/6 islets were collected at 1 and 3 days after treatment with or without cytokines. The concentration of monocyte chemoattractant protein (MCP)-1 in the supernatant fraction was measured using a commercial kit (mouse MCP-1 ELISA; eBioscience, Immunosource, Halle-Zoersel, Belgium). Nitrite production was determined by Griess assay (Sigma-Aldrich, Bornem, Belgium).
Cell migration was evaluated using a classical chemotaxis assay [27, 28]. Briefly, islets from Irf-1
−/− or control C57BL/6 mice were cultured for 3 days in serum-free synthetic medium, using BioWhittaker Ultraculture medium (Lonza, Verviers, Belgium), supplemented with GlutaMAX–I (Invitrogen), 100 U/ml penicillin and 100 µg/ml streptomycin, in absence or presence of recombinant human IL-1β (50 U/ml) plus recombinant mouse IFN-γ (1,000 U/ml). Chemotaxis assay was performed for 1 h at 37°C using Transwell filter membranes (5 μm pore size; Costar, Boston, MA, USA) containing 1 × 106 leucocytes, isolated from spleens of 10- to 12-week-old female NOD mice, in 100 µl assay buffer (Hanks’ buffered salt solution supplemented with 20 mmol/l HEPES and 0.2% bovine serum albumin [wt/vol.]) in the upper compartment and 600 µl of test solution in the lower compartment. The migrated cells were collected and counted in a flow cytometer (FACSort; BD Biosciences, Erembodegem, Belgium). The number of migrated cells represents the number of counts registered during a 2 min acquisition. The chemotactic index was calculated as the number of leucocytes attracted by test solution (supernatant fraction of islet preparations) divided by the number of leucocytes attracted by medium alone (negative control). Recombinant mouse MCP-1 (PeproTech) at concentrations of 10 and 50 ng/ml was used as reference chemoattractant.
Culture and transfection of INS-1E cells with siRNA against Irf-1
Rat insulin-producing INS-1E cells (a kind gift of C. Wollheim, Center Medical Universitaire, Geneva, Switzerland) were cultured as described . Two different siRNAs against rat Irf-1 were purchased from Invitrogen and designed using a commercial software (BLOCK-iT RNAi Express/Stealth Select; Invitrogen): si-IRF-1#1 (5′-CCCUGGCUAGAGAUGCAGAUUAAUU-3′) and si-IRF-1#2 (5′-GCCCUCCAUUCAGGCUAUUCCUUGU-3′). Allstars negative control siRNA (Qiagen Benelux, Venlo, The Netherlands) was used as a control for siRNA transfection. Transfection of siRNAs in INS-1E cells was done using the lipid carrier DharmaFECT (Dharmacon, Chicago, IL, USA) as described previously . Lipid–RNA complexes were formed in Optimem in a proportion of 0.7 µl of DharmaFECT to 150 nmol/l of siRNA at room temperature for 20 min. The complex was added to cells for overnight transfection in antibiotic-free medium at a final concentration of 30 nmol/l siRNA. The transfection efficiency was ≥90% as measured using an FITC-conjugated siRNA (siGLO; Dharmacon). Afterwards, cells were cultured for a 24 h recovery period and subsequently exposed to recombinant human IL-1β (10 U/ml) and recombinant rat IFN-γ (100 U/ml; R&D Systems, Abingdon, UK) for 24 h.
Western blot experiments
Western blot analysis of IRF-1 levels in cytokine-treated INS-1E cells was performed as described previously , using antibodies against IRF-1 (dilution 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or α-tubulin (dilution 1:5,000; Sigma-Aldrich) as primary antibodies and horseradish peroxidase-conjugated donkey anti-rabbit IgG as secondary antibodies (dilution 1:5,000; Lucron Bioproducts, De Pinte, Belgium). The protein-specific signals were detected using chemiluminescence Supersignal (Pierce, Rockford, IL, USA) and quantified using Aida1D analysis software (Fujifilm, London, UK).
Mouse islets or INS-1E cells cultured for 1 day were used for RNA extraction as described [29, 31]. cDNA was created using Superscript II RT (Invitrogen) and quantitative PCR analysis was performed with a single colour real-time PCR detection system (MyiQ; Bio-Rad Laboratories, Hercules, CA, USA). Primer and probe sequences for the determination of rodent cDNAs for housekeeping genes (Actb and Gapdh), for chemokine genes Mcp-1 (also known as Ccl2), Ip-10 (also known as Cxcl10) and Mip-3α (also known as Ccl20), and for Il-1β (also known as Il1b) and Inos (also known as Nos2) were as described previously [31–33]. The target cDNA present in each sample was corrected for the respective Actb values in whole islets and for the respective Gapdh values in INS-1E cells.
Islet transplantation and evaluation of graft function
Freshly isolated Irf-1
−/− or control C57BL/6 islets (n = 500) were transplanted under the kidney capsule of overtly diabetic NOD mice as described previously . Islet primary non-function was defined as blood glucose levels never reaching normoglycaemia within 48 h after islet transplantation, while graft rejection was defined as a return to hyperglycaemia (non-fasting glycaemic values ≥11.1 mmol/l in three consecutive readings after initial normoglycaemia). Recipient mice were killed the day of graft rejection or in separate experiments for histological examination on days 3 and 5 post-transplantation. In a separate experiment, mice were treated with IL-1Ra (100 mg kg−1 day−1) for 15 days, starting 1 day before islet transplantation as described .
Paraffin-embedded kidneys containing islet grafts were sectioned, stained with haematoxylin and eosin, and analysed by light microscopy to assess the overall infiltration grade of the islet allografts. In addition, sections obtained from graft specimens were stained for insulin using guinea pig anti-insulin (dilution 1:100, A0564; Dako, Glostrup, Denmark), for T cells using rabbit anti-CD3 (dilution 1:200, A0452; Dako) and for macrophages using goat anti-F4/80 (dilution 1:500, sc-26642; Santa Cruz Biotechnology, Heidelberg, Germany) as described previously [10, 34]. All sections were visualised with a fluorescence microscope (AxioImager Z1; Carl Zeiss Micro Imaging, Oberkochen, Germany) using an EC Plan-Neofluar 20×/0.5 objective lens. Acquisition was done with AxioVision 4.6 software (Carl Zeiss Micro Imaging) and finally processed by ImageJ (US National Institutes of Health, Bethesda, MD, USA).
Tail skins (2 cm2) from Irf-1
−/− and control C57BL/6 mice were placed in graft beds on the dorsum of allogeneic NOD mice. Grafts were scored by an observer blinded for source of skin graft and were considered rejected when less than 50% viable tissue was present.
Data analysis and statistical methods
NCSS 2000 (Kaysville, UT, USA) software was used for statistical analysis. Data are expressed as mean ± SEM. Peto's log-rank test was performed to compare two or more survival curves. χ
2 test was used to compare incidence of primary non-function. Student’s t test and ANOVA were used for multiple comparisons, whenever appropriate. Significance was defined at the 0.05 level.