NEW PARADIGM FOR LYMPHOCYTE GRANULE-MEDIATED CYTOTOXICITY - TARGET-CELLS BIND AND INTERNALIZE GRANZYME-B, BUT AN ENDOSOMOLYTIC AGENT IS NECESSARY FOR CYTOSOLIC DELIVERY AND SUBSEQUENT APOPTOSIS

Lymphocyte granule-mediated apoptosis is postulated to entail the form ation of membrane pores by perforin. Then soluble granzyme reaches the cytosol either through these pores or by reparative pinocytosis. We d emonstrate here that Jurkat cells bind and internalize granzyme B via high affinity binding sites without toxic consequence. Apoptosis occur s, however, if sublytic perforin is added to targets washed free of so luble granzyme B, We suggest that granule-mediated apoptosis mimics vi ral strategies for cellular entry. Accordingly, co-internalization of granzyme B with adenovirus, a virus that escapes endosomes to reach th e cytosol, also induced apoptosis. Poly(ADP-ribose) polymerase cleavag e and processing of CPP32, ICE-LAP3, and Mch2 were detected at 30 min, while cytosolic acidification and DNA fragmentation occurred at 60 mi n. Annexin V binding and membrane permeabilization arose at 4 h. The c oncurrent activation of the Ced-3 proteases differed from the rate at which each cysteine protease is cleaved in vitro by granzyme B. Thus, granzyme B may not directly process these proteases in whole cells but rather may function by activating a more proximal enzyme. These resul ts indicate that adenovirus-mediated delivery of granzyme B is suitabl e for elucidating biochemical events that accompany granule-mediated a poptosis.