Synovial membrane samples were obtained following informed consent from patients with RA at arthroscopic biopsy (n = 7) or joint replacement surgery (n = 1). RA was diagnosed according to the criteria of the American College of Rheumatology . Histologically normal synovium (n = 1) was obtained from the knee joint of a patient undergoing lower limb amputation.
Isolation and culture of synovial cells
Synovial cells were obtained by enzymatic digestion of synovial membrane with 1mg/ml collagenase type I (Worthington Biochemical, Freehold, NJ, USA) in RPMI (GibcoBRL, Paisley, UK) for 4 h at 37°C in 5% carbon dioxide. Dissociated cells were plated in RPMI supplemented with 10% foetal calf serum (GibcoBRL), 10 ml of 1 mmol/l HEPES (GibcoBRL), penicillin (100 units/ml), streptomycin (100 units/ml) and fungizone (0.25 μg/ml). The cells were grown to confluency (approximately 10days) at 37°C in a 5% carbon dioxide atmosphere, then harvested with trypsin and passaged. Synoviocytes were found to be morphologically homogenous fibroblast-like cells and were used between the third and seventh passage. To confirm synoviocyte cultures were not contaminated by monocytes, staining for the monocyte marker CD14 was carried out. Cells were placed in serum-free medium 24 h before total RNA extraction.
Reverse transcription-polymerase chain reaction
Total RNA, isolated from freshly obtained synovial biopsies or cultured primary synoviocytes, was converted by RT into cDNA. For each total RNA sample, the following materials were used for RT at 42°C for 50 min: 1 μg total RNA and 200units SuperscriptII (GibcoBRL); RT buffer as supplied; 100 mmol/l dithiothreitol, 40units RNasin Ribonucleic Inhibitor (Promega, Madison, WI, USA); 1.25 mmol/l each of dATP, dCTP, dGTP and dTTP; and 500ng of oligo dTs. PCR was performed with the following materials: 2.0 μl cDNA; 1.25 mmol/l of each of dATP, dCTP, dGTP and dTTP; 2.5 units AmpliTaq (Perkin Elmer, Brachburg, NJ,USA); 2.5 mmol/l MgCl2 (GAPDH) and 2.0 mmol/l MgCl2(A-SAA); 2.5 μl 10 ×PCRII buffer (Perkin Elmer) and 20 ng of each specific PCR primer pair in a 25 μl total volume. Specific primers for human A-SAA were used to amplify a 335 base-pair (bp) A-SAA product: sense primer (5' -AAG CTT CTT TCC GTT CCT TGG-3') and antisense primer(5' -GAG AGC AGA GTG AAG AGG AAG C-3'). The A-SAA primers used span an intron. Thus, the PCR generates an unequivocally RNA-derived band, based on its size. GAPDH primers were designed to generate a 635-bp product: sense primer (5' -CCA CCC ATG GCA AAT TCC ATG GCA-3') and antisense primer (5' -TCT AGA CGG CAG GTC AGG TCC ACC-3'). After preincubation (94°C, 10 min) each PCR sample underwent a 35-cycle amplification regimen of denaturation (94°C, 1 min), primer annealing (60-56°C, 1 min) and extension (72°C, 1 min), with a final extension (72°C, 10 min) in a thermal cycler (MJ Research, Inc, Cambridge, MA, USA).
Northern blot analysis
Total RNA was isolated (RNeasy, Qiagen, Crawley, UK) from cultured primary synoviocytes and quantified by ultraviolet absorption. Of total RNA, 10 μg was electrophoresed on a standard northern gel and transferred onto a nylon membrane (BioRad, Richmond, CA, USA). The human A-SAA cDNA  was radiolabelled to a high specific activity using [α-32P] dCTP and a random primer labelling system (Promega). All membranes were probed under high stringency conditions. Blots were exposed to film at -80°C using intensifying screens and autoradiographic intensity was quantified using an imaging densitometer.
Southern blot analysis
PCR products generated were run on a 2% agarose gel and transferred onto a nylon membrane (BioRad) using standard procedures. Human A-SAA and GAPDH cDNA probes were radiolabelled to a high specific activity using [α-32P] dCTP and a random labelling system (Promega). All membranes were probed under high stringency conditions. Blots were exposed to film at -80°C using intensifying screens.
Measurement of acute-phase serum amyloid A by ELISA
A-SAA protein levels were measured using a sandwich enzyme immunoassay (Biotrin International, Dublin, Ireland). Supernatants derived from primary RA synoviocyte cultures were harvested. Samples were added to a microtitre plate, which was precoated with IgG (anti-A-SAA) and incubated for 1 h. The microtitre plate was washed and IgG (anti-A-SAA)-horseradish peroxidase conjugate added. After 1 h incubation and plate washing, substrate was added. The absorbance was measured at 450nm, using 650nm as a reference. Colour intensity is directly proportional to the amount of A-SAA in the sample. The detection limit of the assay was determined as 2.25 μg/l .
Synovial tissue was placed in the cryopreservative embedding media OCT compound (Tissue Tek, Sakura, Finetek, Europe BV, Zoeterwoude, The Netherlands) and immediately frozen in liquid nitrogen. Sections (7 μ m) were cut on a microtome (Microm HM 505N, GmbH 69190 Walldorf, Germany), placed on glass slides coated with 2% 3-amino-propyl-triethoxy-silane (Sigma-Aldrich Ireland Ltd, Dublin, Ireland) in acetone and dried overnight at room temperature. Isolated RA synoviocytes were trypsinized and placed into a six-well plate with apyrogenic cell culture coverslips. Once grown to confluency, the medium was removed and the cells were treated with methanol for 15 min. Synoviocytes were stained essentially as described for the tissue sections. Tissue sections were allowed to reach room temperature, fixed in acetone, air-dried and incubated for 1 h at room temperature with blocking serum (Vectastain Rabbit Elite Kit, Vector Laboratories Ltd, Peterborough, UK). The slides were incubated with avidin for 15 min, rinsed and then incubated with biotin for 15 min. The primary polyclonal antibody for A-SAA (1:1200-1:1600; rabbit antihuman) was incubated for 1 h at room temperature. Secondary antibodies (antirabbit and antimouse; Vectastain) were prepared and added to the relevant sections and incubated for 30 min. The secondary antibody was washed off and the slides were incubated with Avidin:Biotinylated enzyme complex solution for 30 min and incubated for 6 min with 3,3' -diaminobenzidine and counterstained in haemotoxylin stain for 1 min.