HUMAN GRANZYME-B IS ESSENTIAL FOR DNA FRAGMENTATION OF SUSCEPTIBLE TARGET-CELLS

被引:47
作者
SU, B
BOCHAN, MR
HANNA, WL
FROELICH, CJ
BRAHMI, Z
机构
[1] INDIANA UNIV,SCH MED,DEPT MED,INDIANAPOLIS,IN
[2] INDIANA UNIV,SCH MED,DEPT MICROBIOL IMMUNOL,INDIANAPOLIS,IN 46202
[3] NORTHWESTERN UNIV,EVANSTON HOSP,SCH MED,DEPT MED,RHEUMATOL RES SECT,EVANSTON,IL 60201
关键词
GRANZYME B; PERFORIN; DNA FRAGMENTATION; GRANULES;
D O I
10.1002/eji.1830240921
中图分类号
R392 [医学免疫学]; Q939.91 [免疫学];
学科分类号
100102 ;
摘要
We have partially characterized the granules of the human NK cell line, YT-INDY, and assessed granule-mediated lysis and DNA fragmentation of assorted targets. Biochemical studies demonstrated significant quantities of granzyme B (asp-ase) and a heretofore undescribed chymase but no tryptase (i. e., granzyme A or 3) or distinct met-ase. YT-INDY expressed mRNA for granzyme B, perforin and CCPX. The existence of perforin was confirmed by immunoblot. The granules lysed both human and murine NK-sensitive and NK-resistant targets. YT-INDY and NK3.3, two human cytotoxic cells, were also lysed. EGTA reduced lysis by only 50%, suggesting that a perforin-independent lytic pathway is associated with the granules. In addition, 4-(2-aminoethyl) benzenesulfonylfluoride hydrochloride (AEBSF), an inhibitor that selectively blocked the chymase and 3,4-dichloroisocoumarin (DCI), an inhibitor that inactivated both chymase and asp-ase activities, marginally affected lysis. By gel electrophoresis and I-125-labeled deoxyuridine release assay, only murine cells (SP2/0 and YAC-1) underwent DNA fragmentation, and cleavage was completely inhibited by DCI, whereas EGTA, AEBSF and aurintricarboxylic acid (ATA) had no effect. The results, therefore, underscore the central role of granzyme B in granule-mediated DNA fragmentation, emphasize that the protease acts via an ATA-resistant endonuclease pathway and stress that nucleolysis does not invariably accompany granule-mediated cytolysis. Finally, ATA inhibited the asp-ase activity of isolated but not granule-associated granzyme B. ATA, therefore, is not a specific endonuclease inhibitor and results obtained with ATA should be viewed cautiously.
引用
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页码:2073 / 2080
页数:8
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