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Surface cathepsin B protects cytotoxic lymphocytes from self-destruction after degranulation.

Balaji KN, Schaschke N, Machleidt W, Catalfamo M, Henkart PA - J. Exp. Med. (2002)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Building 10, Bethesda, MD 20892, USA.

ABSTRACT
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.

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Perforin cleavage by cathepsin B. (A) Perforin from CTL extracts was treated with 258 ng/ml purified cathepsin B for various times and analyzed by blotting with anti-perforin antibody. (B) Mean densitometric analysis of perforin degradation by cathepsin B. Three experiments are shown.
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fig7: Perforin cleavage by cathepsin B. (A) Perforin from CTL extracts was treated with 258 ng/ml purified cathepsin B for various times and analyzed by blotting with anti-perforin antibody. (B) Mean densitometric analysis of perforin degradation by cathepsin B. Three experiments are shown.

Mentions: The above data clearly implicate surface cathepsin B in cytotoxic lymphocyte self-protection, but do not directly address the molecular target of cathepsin B. Given previous data showing that membrane-associated perforin is highly susceptible to proteolysis (26), and the proximity of degranulated surface cathepsin B to perforin associated with effector cell membranes, perforin is an obvious candidate. To see if this protease is capable of degrading perforin, we incubated purified cathepsin B with CTL extracts containing perforin and monitored digestion with Western blots. The results show that perforin is efficiently digested by cathepsin B, a process that is inhibited by CA074 (Fig. 7) .


Surface cathepsin B protects cytotoxic lymphocytes from self-destruction after degranulation.

Balaji KN, Schaschke N, Machleidt W, Catalfamo M, Henkart PA - J. Exp. Med. (2002)

Perforin cleavage by cathepsin B. (A) Perforin from CTL extracts was treated with 258 ng/ml purified cathepsin B for various times and analyzed by blotting with anti-perforin antibody. (B) Mean densitometric analysis of perforin degradation by cathepsin B. Three experiments are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196055&req=5

fig7: Perforin cleavage by cathepsin B. (A) Perforin from CTL extracts was treated with 258 ng/ml purified cathepsin B for various times and analyzed by blotting with anti-perforin antibody. (B) Mean densitometric analysis of perforin degradation by cathepsin B. Three experiments are shown.
Mentions: The above data clearly implicate surface cathepsin B in cytotoxic lymphocyte self-protection, but do not directly address the molecular target of cathepsin B. Given previous data showing that membrane-associated perforin is highly susceptible to proteolysis (26), and the proximity of degranulated surface cathepsin B to perforin associated with effector cell membranes, perforin is an obvious candidate. To see if this protease is capable of degrading perforin, we incubated purified cathepsin B with CTL extracts containing perforin and monitored digestion with Western blots. The results show that perforin is efficiently digested by cathepsin B, a process that is inhibited by CA074 (Fig. 7) .

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Building 10, Bethesda, MD 20892, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus