Negative Relationship Between Expression of Glucocorticoid Receptor α and Disease Activity: Glucocorticoid Treatment of Patients with Systemic Lupus Erythematosus

MATERIALS AND METHODS

Subjects

Fifty-five female patients with SLE and 20 healthy female controls were included in the trial (No. 2006-094). The average age of the 55 patients was 30 ± 8 years, and the average age of the 20 controls was 27 ± 6 years. All SLE patients met the diagnostic criteria for SLE established by the American Rheumatism Association in 1997¹⁷, and their scores on the SLE Disease Activity Index (SLEDAI) were > 9¹⁸ (Table 1). All SLE patients were steroid-naive. Patients who had been treated with prednisone or cytotoxic drugs were excluded from the study.

The expression of GRα mRNA and protein in PBMC of SLE patients was measured before and after GC administration. All the patients received a GC (methylprednisolone) as part of their routine therapy (without cytotoxic drugs). Patients who at the start of the study had fever, arthritis, arthralgia, myalgia, serositis, skin erythematosus, but not those with organ lesions, received methylprednisolone injection 0.5–1 mg/kg/day in 2 successive weeks; patients with organ lesions, such as renal involvement and obvious vasculitis, were given methylprednisolone injection 1–2 mg/kg/day for 2 weeks. We classified the patients into 2 groups according to their response to methylprednisolone: steroid-sensitive and steroid-insensitive. The criteria for classification in the steroid-sensitive group were remission or partial remission of clinical symptoms, and a decrease in the SLEDAI score after administration of methylprednisolone. The criteria for the steroid-insensitive group were that patients showed no response to GC in the clinic and an unchanging or increasing SLEDAI score.

Preparation of PBMC

Heparinized venous peripheral blood was obtained from all SLE patients and controls. The blood was diluted by addition of an equal volume of 0.9% NaCl. Six milliliters of diluted blood were carefully layered over 3 ml of Lymphoprep (Amersham Pharmacia Biotech, Uppsala, Sweden) and centrifuged at 800 xg for 20 min at room temperature in a swing-out rotor. After centrifugation the mononuclear cells formed a distinct band at the sample interface. The harvested fraction was diluted with buffered RPMI-1640 medium (Gibco BRL, Gaithersburg, MD, USA) to reduce the density of the solution, and the cells were pelleted by centrifugation for 10 min at 250 xg. Platelets were removed by layering the cells suspended in buffered RPMI-1640 medium and centrifugation for 15 min at 350 xg. The pellets were used as mononuclear cells.

Reverse transcriptase-polymerase chain reaction (RT-PCR)

Total RNA was extracted from PBMC of SLE patients and controls with TRIzol reagent (Invitrogen, Eugene, OR, USA) according to the instructions of the manufacturer and quantified by measuring absorbance at 260 nm. Reverse transcription and cDNAs were amplified in the same test tube using the Access RT-PCR system (Promega, Madison, WI, USA). These cDNAs were amplified with the following primer sets: GRα, 5’-CCT AAG GAC GGT CTG AAG AGC-3’ and 5’-GCC AAG TCT TGG CCC TCT AT-3’; ß-actin, 5’-CAA CTC CAT CAT GAA GTG TAA C-3’ and 5’-CCA CAC GGA GTA CTT GCG CTG-3’, as described³. The reaction mixture (50 μl) contained 2 μl RNA, 1 μl AMV reverse transcriptase (5 U/μl), 10 μl AMV/Tfl 5x reaction buffer, 1 μl dNTP mix (10 mM of each dNTP), 2 μl of primers (50 pmol each of upstream and downstream primers), 2 μl of MgSO4 (25 mM), 1 μl of Tfl DNA polymerase (5 U/μl), and 31 μl of nuclease-free water. First, cDNA synthesis was carried out at 45°C for 45 min and denaturation at 94°C for 2 min. Next, the PCR program used for GRα was incubated in a thermal cycler at 94°C for 30 s, 50°C for 1 min, and 68°C for 1 min, for 35 cycles; and for ß-actin at 94°C for 30 s, 56°C for 30 s, and 68°C for 30 s for 27 cycles. After final extension for 10 min at 68°C, PCR products were separated on 2% agarose gels containing ethidium bromide. A DL2000 DNA size standard (TaKaRa, Shiga, Japan) was electrophoresed on the same gel to determine product size. The gel was photographed and the amount of GRα and ß-actin from each sample was analyzed by scanning densitometry. To normalize the results, the amount of GRα was divided by the amount of ß-actin.

Western blot analysis

After 2 washes with phosphate buffered saline (PBS), the PBMC were lysed in cold lysis buffer (PBS containing 1% non-idet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate supplemented with 100 μg/ml phenylmethylsulfonyl fluoride and 1 μg/ml aprotinin). After centrifugation at 13,000 xg for 20 min, the supernatants were collected and the total protein concentrations were determined by spectrophotometer, using bovine serum albumin as standard. Proteins (20 μg) were resolved by electrophoresis through a 7.5% polyacrylamide gel and electrophoretically transferred to nitrocellulose membrane. After blocking nonspecific binding sites by immersing the membrane in 5% blocking reagent dissolved in Tris-buffered saline-Tween for 1 h at 37°C, the membrane was incubated overnight at 4°C with polyclonal anti-hGRα at 1:400 dilution (Santa Cruz Biochemical, Santa Cruz, CA, USA). The membrane was washed and then incubated with horseradish peroxidase-conjugated whole antibody at 1:1000 dilution (Amersham Biosciences) for 40 min at 37°C. After washing, the membrane was covered with ECL detection reagents (Amersham Biosciences) for 1 min at room temperature. The membrane wrapped with Pewrap was exposed to X-ray film for 2–5 min, developed, and exposed. The film was photographed and the amount of GRα and ß-actin from each sample was analyzed by scanning densitometry. To normalize the results, the amount of GRα was divided by the amount of ß-actin.

Statistical analysis

Statistical analyses were performed using SPSS v. 15.0 (SPSS, Chicago, IL, USA). All data were expressed as mean ± SD. Differences between groups were evaluated by one-way ANOVA and paired-samples t test; p < 0.05 was considered significant. Correlation between 2 variables was analyzed by Spearman rank correlation analysis.