Figure 2.

Suppressor of cytokine signalling 1 can increase interleukin-1β production by regulating the inhibitory effects of interferon-γ. (A) Mouse macrophages were pretreated with IFNγ for 1–24 h before stimulation with LPS for 4 h. Cells were then lysed and pro-IL-1β analysed by immunoblot. Pretreatment of macrophages with IFNγ alone did not induce IL-1β production. Tubulin is shown as a loading control. (B) Western blotting was performed as in (A) using IFNγ^−/− (control) and IFNγ^−/−SOCS1^−/− macrophages. The IL-1β level returns to normal after 16 h IFNγ pretreatment in control mice, but is decreased greatly at this time point in SOCS1-deficient cells. (C) The expression levels of IL-1β relative to tubulin from three independent experiments. ^**P<0.01. (D) IFNγ^−/− and IFNγ^−/−SOCS1^−/− macrophages were primed with IFNγ or GM-CSF for different times, followed by stimulation with LPS for 4 h. IL-1β release after inflammasome activation using MDP–TiO₂ was decreased in SOCS1-deficient cells at the 16 h time point when quantified by ELISA. ^*P<0.05. (E) IFNγ^−/− and IFNγ^−/−SOCS1^−/− macrophages were stimulated with IFNγ for 1–16 h and western blot performed for pSTAT1. STAT1 and tubulin are shown as loading controls. ELISA, enzyme-linked immunosorbent assay; GM-CSF; granulocyte–macrophage colony-stimulating factor; IFNγ, interferon-γ; IL-1β; interleukin-1β; LPS, lipopolysaccharide; MDP; muramyl dipeptide; SOCS1, suppressor of cytokine signalling 1; STAT1, signal transducer and activator of transcription 1.