Caspase-1 activation and mature interleukin-1β release are uncoupled events in monocytes

Figure 1 Human α-defensin 5 blocks the release from but not the processing of interleukin-1β in fresh and overnight cultured human monocytes. Freshly isolated or overnight cultured human monocytes were primed with lipopolysaccharide (LPS) (20 ng/mL) for 2 h before being labeled with 35S-methionine/cysteine for 1 h and then washed and treated with Adenosine triphosphate (ATP, 1 mmol/L) and/or human α-defensin 5 (HD-5) (50 μg/mL) for another 1.5 h. Media and cell-associated fractions were harvested separately. Interleukin-1β (IL-1β) was recovered from each by immunoprecipitation with anti-hIL-1β antibodies (1:1000) that recognize both the pro-IL-1β (31 kDa) and mature IL-1β (17 kDa) proteins. The resulting immunoprecipitates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Images are representative from three independent experiments.

Figure 2 Human α-defensin 5 blocks interleukin-1β release from monocytes stimulated with Adenosine triphosphate or nigericin. Overnight-cultured, lipopolysaccharide (LPS)-activated, 35S-Met/cys-labeled human monocytes were treated with Adenosine triphosphate (1 mmol/L) or nigericin (Nig) (30 μmol/L) in the presence or absence of human α-defensin 5 (HD-5) (50 μg/mL) for 1.5 h. Media and cell-associated fractions were harvested separately. Interleukin-1β (IL-1β) was recovered from each by immunoprecipitation and resulting immunoprecipitates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Images are representative from three independent experiments.

Figure 3 Adenosine triphosphate/nigericin-induced caspase-1 activation and externalization in human monocytes are not blocked by human α-defensin 5. Lipopolysaccharide (LPS)-activated monocytes were stimulated with Adenosine triphosphate (ATP, 1 mmol/L) or nigericin (Nig) (30 μmol/L) in the presence or absence of human α-defensin 5 (HD-5) (50 μg/mL) for 1.5 h. Media were harvested, precipitated, and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis with anti-caspase-1 p10 antibodies (1:1000) that recognize both the pro-caspase-1 (45 kDa) and cleaved caspase-1 p10 subunit proteins.