Figure 3

Localization of IL-18 on rheumatoid arthritis (RA) synovial fibroblasts with or without stimulation with TNF (20 ng/ml for 2 hours) was examined by immunofluorescence. RA synovial fibroblasts were pre-incubated for 2 hours with PD98059 or AG490 (ERK1/2 and JAK pathway chemical inhibitors at 10 μM, respectively) and then stimulated with TNF (20 ng/ml) for 48 hours. Control IgG (A) and IL-18 (B) detection without stimulation showed no staining. After 48 hours of TNF stimulation, we observed staining in the cytoplasm (C). Upon TNF stimulation, after blocking the ERK1/2 pathway, no detection was observed in the cytoplasm (D). Upon TNF stimulation, after blocking the JAK pathway, IL-18 was detected in the cytoplasm with some granularity (E). Images shown are representative of three independent experiments. Schematic representation of TNF induction of mature IL-18 by induction of functional caspase-1 through the JAK pathway (F).