Figure 2

Effect of tumor necrosis factor alpha (TNFα) on the expression of interleukin-34 (IL-34) in rheumatoid arthritis (RA) and osteoarthritis (OA) fibroblast-like synovial cells (FLS). (A) Paired samples of non-inflamed (n = 7) and inflamed (n = 7) synovial fluid (SF) from individual RA patients were collected and analyzed for IL-34 by an enzyme-linked immunosorbent assay (ELISA). Closed triangles indicate individual data points; horizontal bars show group means (P < 0.01, inflamed versus non-inflamed RA SF samples). (B) Fibroblast-like synovial cells (FLS) were prepared and cultured as described in Materials and Methods. RA FLS (n = 9) and OA FLS (n = 7) were cultured with different cytokines (10 ng/ml of TNFα, IL-1β, or IL-17) for 24 hours, and the concentration of IL-34 in the media was measured by ELISA. Closed squares or circles indicate individual RA or OA data points, respectively; horizontal bars show group means (P < 0.01, IL-34 concentration in RA versus OA after TNFα treatment). (C) OA and RA FLS were treated with different cytokines (10 ng/ml of TNFα, IL-1β, or IL-17) for 24 hours. IL-34 mRNA levels in cell lysates were determined by quantitative reverse transcriptase-PCR (qRT-PCR). The relative expression of IL-34 mRNA was obtained using the cycle threshold (Ct) method after normalization to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are presented as fold-change of gene expression compared to untreated controls. Bars indicate the mean and SD of IL-34 mRNA expression relative to that of GAPDH (*P < 0.05, N.S., not significant, compared to none treated).