IL-32-induced TSLP production and mRNA expression via activation of caspase-1 and NF-κB. (A) Cells (3 × 106) were treated with IL-32 (0.01, 0.1, and 1 μg/ml) or LPS (10 ng/ml) for 2 h. Caspase-1 and nuclear NF-κB were determined by Western blot analysis. (B) Cells (3 × 106) were treated with caspase-1 inhibitor (50 nM) for 1 h and then stimulated with IL-32 (0.1 μg/ml) or LPS (10 ng/ml) for 2 h. Caspase-1 activity was determined by a colorimetric kit. (C) Cells (3 × 105) were treated with caspase-1 inhibitor (50 and 500 nM) for 1 h and then stimulated with IL-32 or LPS for 24 h. TSLP and IL-1β production was determined by ELISA method. (D) Cells were treated with PDTC (10 μM) for 1 h and then stimulated with IL-32 or LPS for 5 h. The mRNA expressions TSLP were measured by the RT-PCR method (upper). The NF-κB-luciferase activity was assayed by luciferase assay (lower). Blank, unstimulated cells; Cas inhi, caspase-1 inhibitor 50 nM. *P < 0.05; significantly different from the unstimulated cells' value, **P < 0.05; significantly different from the IL-32 or LPS-stimulated cells' value. ELISA, enzyme-linked immunosorbent assay; IL, interleukin; LPS, lipopolysaccharide; NF-κB, nuclear factor-κB; PBMC, peripheral blood mononuclear cells; PDTC, pyrrolidine dithiocarbamate; RT-PCR, reverse transcriptase polymerase chain reaction; TSLP, thymic stromal lymphopoietin.