Reagents, cells and cell cultures
All reagents were from Sigma (St Louis, MO, USA) and all dishes for culturing cells from Sarstedt (Numbrecht, Germany), unless stated differently. To make the stock solution for further dilution in RPMI 1640, palmitic acid (PA) was mixed overnight at 37°C in Krebs Ringer HEPES buffer containing 20% BSA (fraction V, Roche, Basel, Switzerland). The following recombinant cytokines were used in the experiments: rat IFN-γ (R&D Systems, MI, USA, 10 ng/ml), mouse IL-17 (R&D Systems 50 ng/ml), mouse IL-1β (10 ng/ml), rat TNF-α (10 ng/ml), human IL-2 (100 ng/ml), rat IL-6 (R&D Systems, 10 ng/ml). SB202190 was used as a selective inhibitor of p38 MAPK activation and was applied to cell cultures at least 30 minutes prior to additional treatments. Hemoglobin (Hb, 20 mg/ml) was used as the NO quencher, and was applied to cultures at least 1 hour before additional treatments. Rat insulinoma cells RINm5F (RIN) were grown under standard conditions (37°C, 5% CO2) in RPMI 1640 medium supplemented with 5% fetal calf serum (FCS, PAA Chemicals, Pasching, Austria), L-glutamine, 2-mercaptoethanol and antibiotics (culture medium) in tissue culture flasks until reaching approximately 80% confluence. Then, they were detached with trypsin solution (0.25%) and ethylenediaminetetraacetic acid (EDTA, 0.02%) in PBS. Cells were washed and seeded into 96-well flat-bottom plates (1 × 104/well) for the MTT test, cell-based ELISA and Griess reaction, into 24-well plates (1 × 105/well) for co-cultivations, into 6-well plates (2.5 × 105/well) for cytofluorimetric analysis, or tissue culture flasks (25 cm3, 1 × 106) for western blot, ca. 16 hours before treatment. Subsequently, fresh culture medium with appropriate reagents and/or cells was added to RIN cell cultures. Pancreatic islets were isolated from male Dark Agouti (DA) rats using the collagenase digestion method. The pancreata were minced and subsequently incubated with collagenase type V solution (1 mg/ml) in PBS at 37°C for 10 min with vigorous shaking. After incubation, HBSS was added to stop the digestion. The islets were handpicked and seeded for the experiments into 96-well flat-bottom plates (4 × 101/well) in culture medium (10% FCS). The islets were used in experiments after an overnight rest. Lymph node cells (LNC) were isolated from cervical lymph nodes from DA rats and spleen cells from spleens of Albino Oxford (AO) rats, DA rats, CBA mice, Balb/C mice and C57BL/6 mice. For the purification of T cells from LNC of DA rats, anti-rat CD3-biotin conjugated antibody (BD Biosciences), MACS streptavidin microbeads and MACS separation columns were used according to the instructions of the manufacturer (Miltenyi Biotec, Aubum, CA). The obtained cells were more than 98% positive for CD4 or CD8 as deduced by cytofluorometry (FACS Calibur, BD Biosciences), and were stimulated with plate bound anti-CD3 (1 μg/ml) and anti CD28 (1 μg/ml) antibodies (eBioscience, San Diego, CA). The population of CD3- cells, obtained by the same procedure as cells not bound to CD3-biotin conjugated antibody were more than 98% negative for CD3 (as deduced by cytofluorimetry) and were stimulated with LPS (1 μg/ml, Sigma). Cell free supernatants of CD3+ and CD3- cultures were collected after 48 hours of cultivation. RIN cells and LNC (1 × 106/well) were co-cultivated in the absence or presence of tissue culture inserts (Nunc, Denmark). Spleen cell cultures (5 × 106/ml) were stimulated with concanavalin A (ConA, 2.5 μg/ml) for 48 hours and subsequently cell-free supernatants (Sn) were collected. Except in experiments where 10% Sn was combined with 32 μM PA, 40% Sn was employed. α-Methyl-D-mannoside (10 mg/ml) was used to neutralize the biological activity of ConA. The animals were obtained from the breeding facility of the Institute for Biological Research "Siniša Stanković" and were kept under standardized conditions. All experiments were conducted in accordance with local and international legislations regarding the wellbeing of laboratory animals.
Cell viability assay
In order to asses the viability of RIN cells, pancreatic islets or LNC we used the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) to formazan. At the end of appropriate treatments, cell culture supernatants were removed from the plates and MTT solution (1 mg/ml) was applied. Alternatively, pancreatic islets were collected in tubes, spun down, supernatants removed and the cell pellet dissolved in the MTT solution. Finally, in co-cultivation experiments, LNC were removed from cell cultures, spun down, supernatants removed and the residue dissolved in MTT solution. MTT solution was also added to the RIN cells remaining in the plates. Incubation with MTT lasted for 30 minutes at 37°C. Dimethyl sulfoxide (DMSO) was added to the pellet of pancreatic islets, to LNC and to plated RIN cells to dissolve the formazan crystals. The absorbance was measured at 570 nm, with a correction at 690 nm, using an automated microplate reader (LKB 5060-006, LKB, Vienna, Austria).
Measurement of NO generation
Cytofluorimetric assay was used for direct measurement of NO release with 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM, 1 μM), which was added to cultures 1 hour prior to the end of the treatment period. After washing in PBS, the cells were detached, resuspended in PBS and analyzed (excitation at 488 nm, emission at 510 nm) on a FACS Calibur flow cytometer (BD Biosciences).
Apoptosis detection and caspase-3 assay
Apoptotic cells were detected using an annexinV-FITC/EtD-III staining kit (Biotium, Hayward, CA), according to the manufacturer's protocol. Briefly, after treatment, RIN cells were detached, resuspended in Annexin Binding Buffer containing AnnexinV and EtDIII, and incubated in the dark at room temperature for 15 minutes. Subsequently, samples were diluted with four volumes of Annexin Binding Buffer and analysed with FACS Calibur flow cytometer (BD Biosciences) using CellQuest Pro software (BD Biosciences). The activity of caspase-3 was determined in cultures using the Caspase-3 DEVD-R110 Fluorimetric and Colorimetric Assay Kit (Biotium, CA), according to the manufacturer's protocol. The ability of cell lysates to cleave the specific caspase-3 substrate was quantified fluorometrically using an excitation wavelength of 485 nm and an emission wavelength of 535 nm with a microplate reader (Chameleon, Hidex, Turku, Finland). The results are expressed as amount of substrate conversion (μM), deduced from a standard curve generated from known concentrations of the dye R110.
Western blot
Whole-cell lysates were prepared in a solution containing 62.5 mM Tris-HCl (pH 6.8, 2% w/v sodium dodecyl sulfate (SDS), 10% glycerol, 50 mM dithiothreitol (DTT), 0.01% w/v bromophenol blue, 1 mM phenylmethylsulphonyl fluoride (PMSF), 1 μg/ml aprotinin, 2 mM EDTA and were electrophoresed on a 12% SDS-polyacrylamide gel. The samples were electro-transferred to polyvinylidene difluoride membranes at 5 mA/cm2, using semi-dry blotting system (Fastblot B43, Biorad, Munich, Germany). The blots were blocked with 5% w/v nonfat dry milk in PBS with 0.1% Tween-20 and probed with specific antibodies to p38 and phosphorylated-p38 at 1:1000 dilution (both from Cell Signaling Technology, Boston, MA), followed by incubation with secondary antibody at 1:10000 dilution (ECL donkey anti-rabbit horseradish peroxidase (HRP)-linked, GE Healthcare, Buckinghamshire, England, UK). Detection was by chemiluminescence (ECL, GE Healthcare).
Cell-based ELISA
The expression of inducible nitric oxide synthase (iNOS) was determined in triplicate by cell-based ELISA using specific antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), according to a previously described protocol [19]. Briefly, after adequate treatment RIN cells were fixed in 4% paraformaldehyde (PFA), endogenous peroxidase activity was quenched with hydrogen-peroxide and the cells exposed to primary anti-iNOS antibody 1:200 dilution and secondary HRP-conjugated detection antibody (GE Healthcare) at 1:2000 dilution. The substrate for HRP was 3,3',5,5'-tetra-methylbenzidine and the reaction was stopped with 1 M HCl. The absorbance was measured at 450 nm and the cells were stained with crystal violet in order to correct for differences in cell numbers. The final results were obtained by division of the absorbance at 450 nm after adding the stop solution and the absorbance at 570 nm after the additional crystal violet staining (A450/A570).
Statistical Analysis
Data are presented as the mean +/- SD of values obtained in independent experiments. Student's t-test was performed for analysis of the differences between means observed in the experiments.