Targeting the P 2 X 7 receptor in rheumatoid arthritis : biological rationale for P 2 X 7 antagonism

Supplementary methods In vitro culture methodology BzATP-induced release of IL-1β and IL-18 from human isolated peripheral blood monocytes: Human peripheral blood monocytes were prepared from the blood of healthy human volunteers collected in EDTA blood tubes (product code 021066001; Sarstedt Monovette®, Germany). Dextran T500 (product code 17-0320-01; Amersham Pharmacia Biotech, Sweden) solution (1 mL of a 6% [w/v] solution in 0.9% [w/v] NaCl) was added to each 9 mL tube. The tube was mixed by inversion and left to stand for 45 minutes at room temperature, allowing the red blood cells to sediment. The upper, straw-coloured, white blood cell (WBC)-containing layer was layered (typically 5 mL) onto 2.5 mL Nycoprep 1.068 (product code 1002351; Nycomed Pharma, Switzerland) in 15 mL centrifuge tubes (Costar 430791). Tubes were centrifuged at 600 g for 15 minutes (brake off). The upper plasma layer (typically 2–3 mL) and the remaining buffy coat layer (typically 4–5 mL) were removed and collected separately into 50 mL centrifuge tubes (Costar 62-547-254). Tubes were centrifuged at 700 g for 10 minutes. To reduce platelet contamination, the buffy coat pellet was re-suspended in 15 mL autologous plasma (5% [v/v] in phosphate-buffered saline [PBS]), made up from the supernatant of the centrifuged plasma. Tubes were centrifuged at 78 g for 10 minutes (brake off). This was repeated three more times. After the final spin, the cells were re-suspended in RPMI priming buffer at a concentration of 0.5 × 106 cells/mL. Lipopolysaccharide (LPS) (10 ng/mL, Sigma L4391, Lot 89H4001) was added to the cell suspension and 200 μL was then transferred to the wells of a U-bottomed tissue culture-treated 96-well plate (product code 3799; Costar) and the plate was incubated for 4 hours at 37°C (5% CO2, 95% humidity). The supernatants were removed by aspiration, the plate blot-dried and RPMI assay buffer (160 μL) added to each well. Agonist benzoylbenzoyl adenosine triphosphate (BzATP) (20 μL, pre-diluted in RPMI assay buffer from a 100 mM stock) and either vehicle (1% [v/v] DMSO in RPMI assay buffer, 20 μL) or the appropriate concentration of AZD9056 (20 μL in 1% [v/v] DMSO in RPMI assay buffer) were added simultaneously in a total volume of 40 μL (from premixed combinations of the appropriate solutions). Plates were incubated in a water tray for 30 minutes at 37°C (5% CO2, 95% humidity). After this time, supernatants were transferred to a 96well Sero-Wel plate and frozen for subsequent cytokine measurements using anti-human interleukin-1β (hIL-1β) capture antibody, lyophilised recombinant hIL-1β standard and biotinylated anti-hIL-1β (all in product code OptEIA set 2687KI) or IL-18 enzymelinked immunosorbent assay (ELISA) (product code MBL7620; R&D systems, UK). ATP-induced release of IL-1β from human blood: Blood was collected into lithium heparin-coated (final concentration 10–30 IU/mL) tubes (product code 02.1065.001; Sarstedt, Leicester, UK) by venepuncture from each of 11 blood donors from the AstraZeneca R&D Charnwood healthy volunteer panel. Heparinised blood was diluted 1:1 with HEPES-buffered RPMI. Following addition of LPS (1 ng/mL, Sigma L4391), aliquots (160 μL/well) of diluted blood were added to 96-well plates (product code 611F96; Bibby Sterilin, Stone, UK) and incubated for 160 minutes at 37°C. Vehicle (1% [v/v] DMSO in HEPESbuffered RPMI) or the appropriate solution of AZD9056 in 1% DMSO in HEPES-buffered RPMI was added in a volume of 20 μL to the appropriate wells and the incubation continued for a further 20 minutes. ATP (3 mM) was then added and cells were incubated for 30 minutes at 37°C. The final concentration of DMSO in all wells was 0.1%. All incubations were performed in duplicate. At the end of the incubation period, the plates were centrifuged at 700 g for 10 minutes. The supernatant was removed from the wells and stored at –20°C until measurement of IL-1β levels by ELISA using anti-hIL1β (MAB601), biotinylated anti-hIL1β (BAF201) and hIL-1β standard (201LB) from R&D Systems, Minneapolis, USA. BzATP-induced release of IL-1β from human rheumatoid arthritis synovial cells: Synovial tissue (rheumatoid joint synovium from either hip, knee or elbow replacement surgery) was transported in assay buffer (DMEM containing L-glutamine [2 mM], penicillin [200 units/mL] and streptomycin [0.2 mg/mL]) either at room temperature (King’s Mill Hospital) or on ice (Western General Hospital, Edinburgh, UK) and, upon receipt, was stored in a refrigerator (4°C) awaiting isolation of cells. Cells were isolated between 12 and 48 hours after surgery.