CONSTITUTIVE APOPTOSIS IN HUMAN NEUTROPHILS REQUIRES SYNERGY BETWEEN CALPAINS AND THE PROTEASOME DOWNSTREAM OF CASPASES

Programmed cell death invariably requires the activation of proteolyti c cascades that are not yet well defined but are initiated after apica l caspase activation. We provide evidence that calpains and the protea some function synergistically downstream of caspases to assist the con stitutive apoptotic program of aging neutrophils, which plays an impor tant role in resolution of inflammatory responses. Inhibitor studies i ndicated that ''tetherrng'' of preapoptotic senescent neutrophils to h uman macrophages required caspase activity. However, the development o f morphological features characteristic of apoptosis, including nuclea r morphology, PS exposure, surface protein shedding, and the capacity to be ingested:by macrophages, required the downstream action of eithe r calpains or the proteasome. Calpain activities were constitutively a ctive in freshly isolated neutrophils and responsible for rearrangemen ts in the protein composition and structure of the plasmalemmal cytosk eleton as they aged in culture and underwent apoptosis. This included a dissociation of protein(s) from F-actin, a candidate mechanism for i ncreased susceptibility to cleavage, and a loss in immunodetectable al pha-actinin and ezrin, two actin-binding, membrane-anchoring proteins. These results clarify roles for different classes of proteases in a p hysiologically important form of constitutive apoptosis.