Figure 4

Production of receptor activator of NF-κB ligand (RANKL) and tumour necrosis factor-α (TNF-α) in vitro and expression of macrophage colony-stimulating factor (M-CSF) and RANKL in vivo. (a,b) RANKL and TNF-α concentrations measured in supernatant of anti-CD3 antibody-stimulated splenocytes. Splenocytes of naive and immunised mice (day 21 after immunisation) were cultured in the presence of M-CSF and stimulated with 1 μg/ml anti-CD3 antibody. RANKL (a) and TNF-α (b) detected in supernatants by enzyme-linked immunosorbent assay 6 days after stimulation. *P < 0.05 compared with wild-type mice (Mann–Whitney U-test). (c) Reverse transcriptase PCR performed on RNA of isolated inflamed synovia of two interferon-γ receptor knock-out (IFN-γR KO) mice (KO1, KO2) and two wild-type mice (WT1, WT2) showing transcription of RANKL and M-CSF within the inflamed synovium. The housekeeping gene β-actin was used to normalise the levels of cDNA. (d) Analysis of calcitonin receptor expression level by real-time quantitative PCR on synovium and spleen from three wild-type and three IFN-γR KO mice. Values are the numbers of calcitonin receptor mRNA copies per 1000 copies of hypoxanthine transferase. CIA, collagen-induced arthritis.