Recombinant human SAA was purchased from Peprotech (Rocky Hills, NJ, USA). According to the manufacturer, the endotoxin level of the product is 0.1 ng/mg protein. MSU crystals were purchased from Alexis (Lausen, Switzerland). Polyclonal anti-IL-1β, pro-IL-1β and anti-cleaved caspase-1 (D57A2) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-caspase-1 polyclonal antibodies (sc-622) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-NLRP3 antibodies were obtained from Abcam (Cambridge, UK). Anti-cathepsin B antibodies and caspase-1 inhibitor (z-YVAD-FMK) were obtained from Calbiochem (San Diego, CA, USA)
Preparation of synovial fibroblasts
Synovial tissues were obtained from patients with rheumatoid arthritis at the time of total joint replacement. Synovial fibroblasts were isolated from the synovial tissues by enzymatic digestion. The study was approved by the Ethics Committees Nagasaki Medical Center and informed consent was obtained from each of the individuals. Synovial fibroblasts were used from passages 4 through 6 during which time they are a homogeneous population of cells (<1% CD 45 positive).
Measurement of cytokine secretion and immunoblot analysis
Synovial fibrablasts (5 × 104) were seeded in 24-well plates containing RPMI1640 supplemented with 10% heat-inactivated FBS and stimulated with MSU for 24 hours. In some experiments, synovial fibrablasts were pre-treated with SAA for 12 hours before stimulation. Cell-free supernatants were collected by centrifugation at 400 g for five minutes and assayed for IL-1β or IL-1α with enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) without the steps for concentrations or precipitations. The same supernatants were also subjected to 12% SDS-PAGE, followed by immunoblotting with Abs for human IL-1β (dilution 1:400), caspase-1 (dilution 1:500), and cathepsin B (dilution 1:500) with an ECL Western blotting kit (Amersham, Little Chalfont, UK). Endotoxin was measured by chromogenic limulus test (Toxicolor LS-50M Kit, SEIKAGAKU CORPORATION, Tokyo, Japan).
Small interfering RNA experiments
Synovial fibroblasts were transfected with 100 nM non-targeting control small interfering RNA (siRNA; AllStars Negative Control siRNA; Qiagen, Hilden, Germany) or with 50 nM two NLRP3 siRNAs (CIAS1_6 and CIAS1_9; Qiagen), combined with the HiPerFect Transfection Reagent (Qiagen) under serum-free condition, as instructed by the manufacturer. The medium was subsequently replaced, pretreated with SAA for 12 h and stimulated with another 24 h with MSU with medium containing 10% FBS. The cell-culture medium was collected for IL-1β ELISA analysis. In some experiments cells were harvested for total RNA purification after SAA pretreatment and analyzed by semi-quantitative RT-PCR (NLRP3), as described below.
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from synovial fibroblasts using the RNeasy total RNA isolation protocol (Qiagen, Crauley, UK) according to the manufacturer's protocol. First-strand cDNA was synthesized from 1 μg of total cellular RNA using an RNA PCR kit (Takara Bio Inc., Otsu, Japan) with random primers. Thereafter, cDNA was amplified using specific primers respectively. The specific primers used were as follows:
NLRP3: forward primer 5'- AAAGAGATGAGCCGAAGTGGG -3' reverse primer 5'- TCAATGCTGTCTTCCTGGCA -3' β-actin; forward primer 5'-GTGGGGCGCCCCAGGCACCA-3' reverse primer 5'-CTCCTTAATGTCACGCACGATTTC-3'.
The product sizes were 79 bp for NLRP3 and 234 bp for β-actin. The thermocycling conditions (35 cycles) 94°C for 60 s and 62°C for 60 s, and 72°C for 60 s.
The amplification of the IL-1-β transcripts was also accomplished on a Light Cycler (Roche Diagnostics, Mannheim, Germany) using specific primers. The housekeeping gene fragment of glyceraldehydes-3-phosphates dehydrogenase (GAPDH) was used for verification of equal loading.
Cell lysis and immunoblot
Synovial fibroblasts were stimulated with SAA with the indicated concentrations of SAA for 24 h. Cells were washed by ice-cold PBS and lysed with a lysis buffer (1% Nonidet P 40, 50 mM Tris, pH 7.5, 100 mM NaCl, 50 mM NaF, 5 mM EDTA , 20 mM β-glycerophosphate, 1.0 mM sodium orthovanadate, 10 μg/mL aprotinin and 10 μg/mL leupeptin) for 20 minutes at 4°C. Insoluble material was removed by centrifugation at 15,000 × g for 15 minutes at 4°C. The supernatant was saved and the protein concentration was determined using the Bio-Rad protein assay kit (Bio Rad, Hercules, CA, USA). An identical amount of protein (50 μg) for each lysate was subjected to 10% SDS-polyacrylamide gel electrophoresis, and then transferred to a nitrocellulose membrane. Immunoblot analysis using anti-NLRP3, pro-IL-β and β-acitin antibodies was performed with an ECL Western blotting kit (GE Healthcare, BUCKS, UK). In brief, the membrane was probed with primary antibodies and washed and incubated with donkey anti-rabbit secondary antibody conjugated with horseradish peroxidase (1:10,000 diluation; GE Healthcare). After being washed, the membrane was reacted with an ECL advance Western blot detection kit (GE Healthcare). Protein bands were visualized using a lumino-image analyzer (LAS3000; Fujifilm, Toyo, Japan).
Differences between groups were examined for statistical significance using Wilcoxon-Mann-Whitney U test. P-values less than 0.05 were considered statistically significance.