New Paradigm for Lymphocyte Granule-mediated Cytotoxicity

doi:10.1074/jbc.271.46.29073

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*

From the ^‡ Department of Medicine, Evanston Hospital, Northwestern University, Evanston, Illinois 60201, the
^¶ Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, the
** Medicine Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892, the
^‡‡ Division of Biochemistry, Scripps Research Institute, San Diego, California 92037, the
^§§ Poly(ADP-Ribose) Metabolism Group, Unit of Health & Environment, Hospital and Research Center of Laval University, Québec, Québec GIV 4G2, Canada, and the
^¶¶ Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

^§ To whom correspondence should be addressed:
Evanston Hospital, Research Department, WH Bldg., Room B624, 2650 Ridge Ave, Evanston, IL 60201
. Tel.: 847-570-2348; Fax: 847-560-1253, E-mail: granzyme{at}merle.acns.nwu.edu.

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.

Footnotes

↵^∥ Recipient of National Institutes of Health Postdoctoral Fellowship CA68769.
↵* This work was supported by grants from the Rice Foundation (to C. J. F.), the Stein Fund (to R. G.), the Cancer Research Society of Canada (Strategic Grant on Nutrition and Cancer) (to G. M. S.), the National Cancer Institute and Medical Research Council of Canada (to G. M. S and R. C. B.) and Grant NIH AG-13501 (to B. B. and R. G.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PFP

perforin

pfu

plaque forming units

AD

replication-deficient adenovirus type 2

GrB

granzyme B

PARP

poly(ADP-ribose) polymerase

PI

propidium iodide

FITC

fluorescein isothiocyanate

FITC-TUNEL

terminal deoxyribonucleotidyl transferase labeling of DNA strand breaks with FITC-dUTP

DEVD-FMK

carbobenzoxy-DEVD-fluoromethylketone.
↵² K. Orth, unpublished data.
↵³ G. Hommel-Berrey, M. R. Bochan, A. H. Montel, W. Goebel, C. J. Froelich, and Z. Brahmi, submitted for publication.
↵⁴ Froelich, C. J., Hanna, W. L., Poirier, G. G., Duriez, P., D'Amour, D., Salvesen, G. S., Alnemri, E. S., Earnshaw, W. C., and Shah, G. M. (1996) Biochem. Biophys. Res. Commun., in press.
↵⁵ K. Orth and C. Froelich, unpublished data.
- Received July 10, 1996.
- Revision received August 26, 1996.