Surface cathepsin B protects cytotoxic lymphocytes from self-destruction after degranulation.
The granule exocytosis cytotoxicity pathway is the major molecular mechanism for cytotoxic T lymphocyte (CTL) and natural killer (NK) cytotoxicity, but the question of how these cytotoxic lymphocytes avoid self-destruction after secreting perforin has remained unresolved. We show that CTL and NK cells die within a few hours if they are triggered to degranulate in the presence of nontoxic thiol cathepsin protease inhibitors. The potent activity of the impermeant, highly cathepsin B-specific membrane inhibitors CA074 and NS-196 strongly implicates extracellular cathepsin B. CTL suicide in the presence of cathepsin inhibitors requires the granule exocytosis cytotoxicity pathway, as it is normal with CTLs from gld mice, but does not occur in CTLs from perforin knockout mice. Flow cytometry shows that CTLs express low to undetectable levels of cathepsin B on their surface before degranulation, with a substantial rapid increase after T cell receptor triggering. Surface cathepsin B eluted from live CTL after degranulation by calcium chelation is the single chain processed form of active cathepsin B. Degranulated CTLs are surface biotinylated by the cathepsin B-specific affinity reagent NS-196, which exclusively labels immunoreactive cathepsin B. These experiments support a model in which granule-derived surface cathepsin B provides self-protection for degranulating cytotoxic lymphocytes.
Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Building 10, Bethesda, MD 20892, USA.
Kithiganahalli N
KN
Balaji
Norbert
N
Schaschke
Werner
W
Machleidt
Marta
M
Catalfamo
Pierre A
PA
Henkart
D000818:Animals; D017252:CD3 Complex / Q000276:immunology; D002401:Cathepsin B / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D015550:Cell Degranulation / Q000502:physiology*; D002462:Cell Membrane / Q000201:enzymology; D055316:Cystatin C; D015891:Cystatins / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003601:Cytotoxicity Tests, Immunologic; D003978:Diazomethane / Q000031:analogs & derivatives* / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D005089:Exocytosis; D006801:Humans; D007659:Ketones / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009842:Oligopeptides / Q000494:pharmacology; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology / Q000502:physiology
C400541:CA 074 methyl ester; D017252:CD3 Complex; C525030:CST3 protein, human; C525031:Cst3 protein, mouse; D055316:Cystatin C; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D007659:Ketones; D009842:Oligopeptides; C090218:benzyloxycarbonylleucyl-leucyl-tyrosine diazomethyl ketone; C057383:MDL 201053; D003978:Diazomethane; D002401:Cathepsin B