Cleavage of pyrin by caspase-1. (A) 35S-labeled pyrin was incubated with 50 units recombinant caspase-1 at 37°C for various periods as indicated, subjected to SDS-PAGE, and visualized by autoradiography. (B) Pyrin was cotransfected into Cos-7 cells with caspase-1 and pro–IL-1β. Cells were treated with increasing amounts of z-WEHD-fmk as indicated. After 24 hours, cell lysates underwent SDS-PAGE and Western blotting with a polyclonal Ab developed against the N-terminal 374 aa of pyrin (top). Cell culture supernatants were also collected and assayed for IL-1β by ELISA (bottom). (C) PT67 cells were cotransfected with pNBC-myc (construct expressing B30.2 domain–deleted pyrin) and with caspase-1. After 24 hours, cell lysates were immunoprecipitated using antimyc Ab, and subjected to SDS-PAGE followed by Western blotting with antimyc Ab (top left panel) or Coomassie blue staining (top right panel). The result of N-terminal Edman sequencing of the eluted, Coomassie-stained band corresponding to the C-terminal cleaved fragment is shown under the schematic diagram of pyrin. (D) WT or D330A pyrin was cotransfected with caspase-1 into PT67 cells. Lysates were subjected to SDS-PAGE and Western blotting with the same antipyrin Ab as in panel B. (E) PBMCs from healthy controls were cultured in RPMI supplemented with 10% fetal bovine serum in 6-well culture plates, treated with LPS (1 μg/mL) from E coli 0127:B7 (Sigma-Aldrich), IFN-γ (100 ng/mL), IL-1β (3.75 ng/mL), IL-2 (1.6 ng/mL), IL-4 (12.5 ng/mL), IL-6 (8.5 ng/mL), IL-10 (100 ng/mL) or IL-12 (10 ng/mL; BD Biosciences, San Jose, CA). After 24 hours, cells were lysed and 12 μg total protein was subjected to SDS-PAGE followed by Western blot with the same antipyrin Ab as in panel B (top). PBMCs were also stimulated with LPS or various amounts (from 10 pg/mL to 100 ng/mL) of IL-10 (middle) or IFN-γ (bottom) for 24 hours. Cells were lysed and analyzed as in top panel.
