New Paradigm for Lymphocyte Granule-mediated Cytotoxicity

Abstract

Lymphocyte granule-mediated apoptosis is postulated to entail the formation of membrane pores by perforin. Then soluble granzyme reaches the cytosol either through these pores or by reparative pinocytosis. We demonstrate here that Jurkat cells bind and internalize granzyme B via high affinity binding sites without toxic consequence. Apoptosis occurs, however, if sublytic perforin is added to targets washed free of soluble granzyme B. We suggest that granule-mediated apoptosis mimics viral strategies for cellular entry. Accordingly, co-internalization of granzyme B with adenovirus, a virus that escapes endosomes to reach the cytosol, also induced apoptosis. Poly(ADP-ribose) polymerase cleavage and processing of CPP32, ICE-LAP3, and Mch2 were detected at 30 min, while cytosolic acidification and DNA fragmentation occurred at 60 min. Annexin V binding and membrane permeabilization arose at 4 h. The concurrent 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 results indicate that adenovirus-mediated delivery of granzyme B is suitable for elucidating biochemical events that accompany granule-mediated apoptosis.

Pit cells (hepatic natural killer cells) of the rat induce apoptosis in colon carcinoma cells by the perforin/granzyme pathway

“…Cells that show granzyme B relocalization undergo apoptotic death. 28,29 In this study, EGTA and the granzyme inhibitor DCI blocked pit cell-induced DNA fragmentation in the CC531s cells (Fig. 5).…”

“…8 Both perforin and granzyme(s) are necessary to induce apoptosis in target cells. 28 Recently, it has been shown that granzyme B is endocytosed by target cells independently of perforin, possibly through receptor-mediated endocytosis. In the absence of added perforin, granzyme B shows a cytoplasmic localization.…”

Blebbing confers resistance against cell lysis

“…34,35 During perforin-induced cell death, the integrity of the plasma membrane is restored by repair responses that protect the cells from necrosis and permit granzymes to trigger apoptosis. 5 We assume that blebbing is intimately involved in protecting perforin-permeabilized cells from necrosis.…”

Granzyme B-mediated apoptosis – the elephant and the blind men?

“…The adventitious proteolysis in the effected subset cannot be estimated by controls incubated with either PFN or granzyme alone. The adenoviral delivery system offers rapid loading of the granzyme without untoward necrosis 13 and has allowed us to appreciate the liabilities of PFN delivery by failing to verify that GrB directly cleaves PARP in target cells as we had previously reported. Finally, whenever possible, analysis of proteolytic digests (i.e.…”

“…Complicating matters, microinjection of rat GrB into target cells failed to induce cell death. 12 Although the cytosolic delivery of GrB was inferred by studies where the granzyme was delivered intracellularly with adenoviral particles, 13 this critical issue was eventually clarified by microinjection of the more active human form by the Bleackley and Froelich labs.…”