Patients and controls
American College of Rheumatology criteria for SLE and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores were adopted for diagnosis and disease activity assessment of SLE patients, respectively [18
]. Patients whose SLEDAI score did not decrease after treatment for 1 month with prednisolone (1 mg/kg/day) were defined as steroid resistant (SR), while patients whose SLEDAI score decreased with treatment were defined as steroid sensitive (SS) [20
]. A total of 22 patients (18 women and four men) were recruited into the SR group. A total of 40 patients (33 women and seven men) matched for age and gender were selected into the SS group. All 62 patients were seen by the Department of Rheumatology at Renji Hospital. Twenty-seven healthy volunteers, with no signs of acute or chronic disease, served as age and gender controls.
Serum samples and peripheral blood mononuclear cells (PBMCs) were collected at the onset of treatment. The following demographic and clinical data were collected: gender, age, number of years since diagnosis, SLEDAI score before and after steroid treatment, and cumulative dose of prednisolone. Anticoagulated blood samples were collected from patients and controls.
All patients and controls were informed about the purpose of our study and consented to participate in the study. This study was approved by the institutional review board of Shanghai Jiaotong University.
Isolation and incubation of peripheral blood mononuclear cells
PBMCs were isolated from anticoagulated blood using Ficoll-HyPaque gradient centrifugation (Sigma-Aldrich, St Louis, MO, USA), which was performed at 400 × g for 30 minutes at 25°C. The PBMC-enriched interphase was collected and washed with PBS. Trypan blue staining detected the viability of freshly isolated cells. For in vitro experiments, PBMCs were resuspended in RPMI 1640 medium (Gibco BRL, Gaithersburg, MD, USA) supplemented with 2 mM L,L-glutamine, 100 μg/ml penicillin G and 100 μg/ml streptomycin, and 100 mM Hepes, 50 μM 2-mercaptoethanol, at a concentration of 5 × 106 cells/ml. Either recombinant MIF or GC was added to the cells and incubated at 37°C in a 5% carbon dioxide atmosphere.
ELISA for macrophage migration inhibitory factor
The DuoSet ELISA kit for human MIF (R&D Systems, Minneapolis, MN, USA) was used to measure MIF in serum and cell culture supernates. A 96-well microplate was coated with capture antibody (mouse anti-human MIF) and incubated overnight at room temperature. After a total of three washes with wash buffer, the plate was blocked with reagent diluent and then incubated at room temperature for 2 hours for sample addition. Diluted samples and standards (recombinant human MIF) were added in duplicate to each well for 2 hours at room temperature, followed by the addition of detection antibody (goat anti-human MIF) for another 2 hours. Streptavidin-horseradish peroxidase was then added to each well for 20 minutes at room temperature; the plate was not placed in direct light. Color was developed by incubating with substrate solution for 20 minutes at room temperature. The reaction was terminated with a stop solution, and a microplate reader set to 450 nm (Bio-Rad Laboratories, Hercules, CA, USA) was used to immediately determine the optical density of each well immediately.
Preparation of cytosolic and nuclear extracts
PBMCs were collected in a 1.5 ml centrifugal tube and washed with 1 ml ice-cold PBS. The suspensions were centrifuged at 750 × g for 8 minutes, and pellets were resuspended in 100 μl ice-cold buffer A (10 mM HEPES, pH 7.9; 10 mM KCl; 1.5 mM MgCl2; 0.5 mM dithiothreitol) with the addition of phenylmethansulfonyl fluoride. The mixture was then incubated on ice for 10 minutes followed by centrifugation at 18,000 × g for 10 minutes. The supernatants (cytosolic extracts. were collected and stored at -80°C. The remaining pellets were resuspended in 20 μl ice-cold buffer B (20 mM HEPES, pH 7.9; 20% v/v glycerol; 420 mM NaCl; 0.5 mM ethylenediaminetetraacetic acid; 1.5 mM MgCl2; 0.5 mM dithiothreitol; 0.5 mM phenylmethansulfonyl fluoride and incubated for 30 minutes on ice. After 10 minutes of centrifugation at 18,000 × g, the supernatants (nuclear extracts) were collected and stored at -80°C for further study.
Protein samples (20 μg) were subjected to 15% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The blots were then probed with mouse anti-human MIF (R&D Systems), rabbit anti-human IκB (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or β-actin antibodies, followed by reaction with a horseradish peroxidase-conjugated secondary antibody. Signals were detected using an enhanced chemiluminescence detection kit and were quantified with installed density-analysis software.
Electrophoretic mobility shift assays
The nuclear extracts (2 μg protein) were incubated with Dig-labeled NF-κB oligonucleotide (Roche, Mannheim, Germany). The assay was performed in a 20 μl total volume containing 2 μg nuclear extract, 4 μl gel shift binding buffer (20 mM Tris-HCl, pH 7.9; 5 mM MgCl2; 0.5 mM dithiothreitol; 0.5 mM ethylenediaminetetraacetic acid; 20% glycerol), 1 μg poly(dI-dC), 1 μg poly-L-lysine and 2 μl probe. The reaction was incubated at room temperature for 15 minutes, loaded on a 4% native polyacrylamide gel, and run in a 0.25 × Tris-Borate-EDTA (TBE) buffer. The gel was dried and subjected to autoradiography. NF-κB-specific bands were confirmed by competition with a 50-fold excess of an unlabeled NF-κB probe, which resulted in no shifted band, or by preparing the reaction with excess labeled nonspecific probe, which did not reduce the intensity of the NF-κB band. Quantity One 4.4 software (Bio-Rad Laboratories, Hercules, CA, USA) was used to analyze the results.
siRNA and plasmid transfection
Three different siRNAs (s8780, s194615, s194614) with putative high silencing efficiency designed for the MIF gene were used (Applied Biosystems, Foster City, CA, USA). We chose to use electroporation, which is the most effective way to introduce siRNA into primary cells. Cells transfected with nontarget double-stranded siRNA served as negative controls. Expression of MIF mRNA was determined by real-time RT-PCR.
Total RNA from PBMCs was isolated with the TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to product recommendations. RNA was then reverse transcribed using the Reverse Transcription Kit (Takara Bio Inc., Otsu, Japan). Expression levels of MIF genes were determined by real-time PCR with the SYBR Green PCR Master Mix (Takara Bio Inc.). PCR reactions were carried out in triplicate with the 7900 Real-time PCR System (Applied Biosystems). Forward primer: 5'-GAACCGCTCCTACAGCAAGCT-3'; Reverse primer: 5'-GCGAAGGTGGAGTTGTTCCA-3'. The thermal cycling conditions included 10 min at 95°C and then 40 cycles of amplification for 3 second at 95°C and 20 second at 60°C. The quantity of mRNA was calculated by normalizing the Cycle Threshold (CT) value of MIF to the CT of the housekeeping gene GAPDH in the same sample, according to the following formula: The average GAPDH CT was subtracted from the average MIF CT; the result represents the ΔCT. This ΔCT is specific and can be compared with the ΔCT of a calibration sample. The subtraction of control ΔCT from the ΔCT of interfered group is referred as ΔΔCT. The relative quantification of expression of MIF was determined by using 2-ΔΔCT.
Cell proliferation assay
The Cell Counting Kit-8 (Dojindol Laboratories, Kumamoto, Japan) was employed to measure the concentration of dexamethasone that was 50% lethal to PBMCs from healthy donors, rather than from SS or SR patients with SLE in vitro. Cells were seeded in 96-well plates and cultured with various concentrations of dexamethasone. Cells in each group were plated in triplicate. The optical density at 450 nm wavelength, which correlates to the number of viable cells, was measured and cell growth curves were then drawn.
Fisher's exact test or the chi-squared test was used to compare clinical parameters between the SS group and the SR group. P < 0.05 was considered significant. The levels of serum MIF, intracellular MIF and intracellular IκB did not conform to a normal distribution, so we conducted Kruskal-Wallis H tests to compare the three groups. If P < 0.05, Wilcoxon signed-ranked tests were used to analyze any two groups. Given the multiple groups (SS group, SR group, controls), Bonferroni correction was conducted and P < 0.166 was considered significant. Data are expressed as the median (25% to 75% percentiles). SPSS version 10.0 was used for the analysis (SPSS Inc., Chicago, IL, USA).