ll The plates were incubated, and culture supernatants were har

ll. The plates were incubated, and culture supernatants were harvested 24 hours later. The IFN�� concentration in this media was determined by ELISA. IL 18 bioactivity was determined based on the difference in IFN�� levels bet ween cultures with and those without mouse anti IL 18 monoclonal antibody. Immunofluorescence staining RA synovial fibroblasts were plated in 8 well Labtek chamber slides and processed as described previously. Briefly, cells were untreated or stimulated with TNF for 48 hours with or without preincubation with PD98059 or AG490 for 2 hours. After 48 hours, cells were washed, fi ed, permeabilized, and blocked. IL 18 primary antibody, which reacts with both immature and mature IL 18 forms, was used after washing in combination with Ale a Fluor conjugated goat anti rabbit antibody.

After washing, nuclei were stained with 4,6 diamidino 2 phenylindole. Slides were dehydrated, mounted, and coverslipped. Immuno fluorescence staining was detected using an Olympus FV 500 microscope. Statistical analysis Statistically significant differences between groups were calculated using Students t test. P values less than 0. 05 were considered significant. All AV-951 statistical data are e pressed as the mean standard error of the mean. Results TNF induced functional caspase 1 in RA synovial fibroblasts To determine whether pro IL 18 was potentially cleaved by active caspase 1 to the IL 18 active form, we e a mined caspase 1 e pression in cell lysates and IL 18 e pression in cell lysates and conditioned media at the protein level, without or with TNF stimulation.

TNF induced caspase 1 at the protein level in cell lysates in a time dependent manner and the mature IL 18 secretion in the conditioned media assessed by western blot and ELISA. The pro IL 18 level in cell lysates did not change over time, suggesting that pro IL 18 is cleaved to IL 18 and then secreted. These data indicate that TNF induced functional caspase 1 to cleave pro IL 18. Role of the JAK pathway in TNF induced caspase 1 To identify signaling events that are critical for TNF induced caspase 1, RA synovial fibroblasts were in cubated with chemical signaling inhibitors for 2 hours, followed by TNF stimulation. Only JAK pathway inhibition significantly decreased TNF induced caspase 1 at the transcriptional level in RA synovial fibroblasts.

TNF induced caspase 1 protein e pression was markedly re duced when the JAK pathway was blocked in RA synovial fibroblasts. According to our blot, this reduction is due mainly to a reduction of pro caspase 1 e pression. At the end, we assessed the functional activity of capsase 1. Blocking the JAK pathway strongly reduced TNF induced caspase 1 activity. Furthermore, blocking the JNK pathway already slightly decreased the TNF induced caspase 1 activity. These data indicate that the JAK pathway is a critical pathway for TNF induced caspase 1 and IL 18 bioactivity. Blocking JAK results in reduction of TNF induced IL 18 bioactivity in RA synovial fibroblasts

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