Figure 5

Effect of neutralisation of tumour necrosis factor (TNF)-α and/or interleukin (IL)-1 on the expression of matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs), and link protein in osteoarthritis synovial cell cultures. Two million cells per well were plated into 4 wells on a 24-well plate and left untreated, incubated with the p75 TNF-soluble receptor-immunoglobulin fusion protein etanercept (Enbrel), incubated with a neutralising anti-IL-1β antibody, or incubated with a combination of etanercept and anti-IL-1β, as described in Materials and methods. After incubation for 48 hours, the cells were washed with phosphate-buffered saline and the RNA extracted using Tri-reagent for reverse transcription-polymerase chain reaction analysis with oligonucleotide primers specific for MMP-1, MMP-3, MMP-13, TIMP-1, TIMP-2, TIMP-3, and link protein. In all panels, analysis of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used for comparison of gene expression.