BioProject

TNFalpha and IL1beta play a pathogenic role in rheumatoid arthritis. Both cytokines are known to activate cytokine and metalloproteinase secretion by synovial fibroblasts. More...

TNFalpha and IL1beta play a pathogenic role in rheumatoid arthritis. Both cytokines are known to activate cytokine and metalloproteinase secretion by synovial fibroblasts. In the present study, we wanted to investigate whether TNFalpha and IL1beta displayed differential effects on cultured Fibroblast-like Synovial Cells derived from RA patients. Global gene expression analyses indicated that both cytokines induced similar genes in these cells. Overall design: Fibroblast-like cells (FLS) were purified from synovial biopsies from RA patients. Briefly, minced synovial fragments were digested in 1 mg/ml hyaluronidase solution (Sigma Aldrich, St Louis, MO) for 15 minutes at 37°C and 6 mg/ml collagenase type IV (Invitrogen, Paisley, UK) for 2 hours at 37°C. Next, cells were washed, resuspended in high-glucose Dulbecco’s Modified Eagle Medium (Invitrogen) supplemented with 1% antibiotics-antimycotics (Invitrogen) and 1% MEM sodium pyruvate (Invitrogen), and seeded at 10,000 cells/cm2 in 6-well plates. When the cells reached confluence, adherent cells were detached using sterile 0.5% trypsin-EDTA (Invitrogen) and used as FLS between passages 3 and 9. Cells were seeded in 24-well plated at 25.000/well and incubated overnight with or without the following cytokines : TNF-α (R&D Systems, Minneapolis, MN) 10 ng/ml, IL-1β (R&D Systems) 10 ng/ml. After overnight incubation with the indicated cytokines, cells were harvested and total RNA was extracted using the Nucleospin® RNA II extraction kit (Macherey-Nagel, Düren, Germany), in order to be labeled according to a standard Affymetrix procedure and hybridized on HGU133 Plus 2.0 Human Genome slides. Less...