Abstract
Mononuclear phagocytes utilize caspase-1 activation as a means to respond to danger signals. Although caspase-1 was discovered using highly concentrated cell extracts that spontaneously activate caspase-1, it is now clear that in live cell models caspase-1 activation occurs in the process of its cellular release and is not an intracellular event. Therefore, we compared the characteristics of caspase-1 activation in the cell lysate model to that of caspase-1 that is released in response to exogenous inflammasome activation. Whereas both models generated active caspase-1, the cell-lysate induced caspase-1 required highly concentrated cell lysates and had a short half-life (~15 min) whereas, the activation induced released caspase-1 required 2-3 log fold fewer cells and was stable for greater than 12 h. Both forms were able to cleave proIL-1beta but unexpectedly, the released activity was unable to be immunodepleted by caspase-1 antibodies. Size exclusion chromatography identified two antigenic forms of p20 caspase-1 in the activation induced released caspase-1: one at the predicted size of tetrameric, p20/p10 caspase-1 and the other at >200 kDa. However, only the high molecular weight form had stable functional activity. These results suggest that released caspase-1 exists in a unique complex that is functionally stable and protected from immunodepletion whereas cell-extract generated active caspase-1 is rapidly inhibited in the cytosolic milieu.