Methods Cytokines, culture of human RA synovial fibroblasts, and

Methods Cytokines, culture of human RA synovial fibroblasts, and chemical inhibitors TNF was purchased from R D Systems. Fibroblasts were isolated from RA synovium obtained from RA patients undergoing arthroplasty or synovectomy as described previously. For all hu man specimens used in this study, we obtained written informed consent and all aspects of the study were approved by the University of Michigan Institutional Review Board. Allergies were not reported and no skin tests were performed on these RA patients. MAPK inhibitors, an NF ��B inhibitor or a JAK2 inhibitor were used at 10 uM of each inhibitor, e cept PDTC at 200 uM. All inhibitors were purchased from Calbiochem. All e periments were performed in serum free media e cept e periments for IL 18 detection.

Cell lysis and western blotting To study the effect of TNF on caspase 1 e pression, RA synovial fibroblasts were incubated with TNF in RPMI 1640 and processed, as previously described. We used a rabbit anti human caspase 1 antibody over night at 4 C and then horseradish pero idase conjugated antibody for 1 hour at room tem perature. Blots were scanned and analyzed for band intensities, as previously described. Caspase 1 activity assay RA synovial fibroblasts were pre incubated with the chemical inhibitors for 2 hours and then treated with TNF for 24 hours in serum free RPMI 1640. Cells were washed and then lysed with the lysis buffer from the caspase 1 activity assay kit. Cell lysates were centrifuged, and the supernatant was assessed. Caspase 1 activity in the supernatant was deter mined using a colorimetric caspase 1 activity assay kit.

IL 18 detection in conditioned media RA synovial fibroblasts were stimulated with TNF in RPMI 1640 with 10% fetal bovine serum supplementation for 72 hours. Conditioned me dium was collected and concentrated 10 fold using Amicon Ultra 3,000 MW concentrators from Millipore. Equal volumes of conditioned media were loaded and processed for western blotting as previously described above e cept that primary poly clonal rabbit anti human IL 18 antibody was used. ELISA for IL 18 and IL 18BP RA synovial fibroblasts were stimulated with TNF for 8 to 48 hours in RPMI with 10% FBS and conditioned medium was collected and concentrated as described above. IL 18 was assessed in conditioned media and cell lysates using an ELISA kit from Bender MedSystems.

IL 18BP was assessed Carfilzomib in condi tioned media using an ELISA kit from R D Systems. RNA e traction and quantitative real time polymerase chain reaction Following the manufacturers protocol, RNA was isolated from RA synovial fibroblasts and processed as described previously. IL 18 bioactivity The biologic activity of IL 18 was measured using human myelomonocytic KG 1 cells, as previously described. KG 1 cells, with or without mouse monoclonal anti IL 18 antibody at 1 ug ml, were dispensed into the wells of 96 well Falcon microtiter plates.

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