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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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Surface cathepsin B on TCR-activated T cells is active and the target of NS-196. (A) Detection of biotinylated NS-196 on the CTL surface after degranulation. Flow cytometry of CD8+ cloned human CTL RS-56 after culture on wells coated with anti-CD3 or isotope control for 2 h, followed by treatment with or without 1 μM NS-196, which was detected by FITC-streptavidin. (B) Identification of cathepsin B as the molecular target of NS-196. CTL clone RS-56 was incubated for 2 h on anti-CD3– or IgG-coated wells, followed by incubation with or without 0.1 μM NS-196. Cells were lysed with Triton X-100 and immunodepleted with beads containing anticathepsin B antibody or control rabbit IgG. The remaining lysate was run on a 12% nonreduced SDS gel, blotted onto nitrocellulose, probed with Streptavidin-HRP, and developed using ECL. The right lane shows the biotinylation pattern when the whole CTL lysate was labeled with 0.1 μM NS-196 and run directly (1/10 of the cell-equivalent input of other lanes).
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fig6: Surface cathepsin B on TCR-activated T cells is active and the target of NS-196. (A) Detection of biotinylated NS-196 on the CTL surface after degranulation. Flow cytometry of CD8+ cloned human CTL RS-56 after culture on wells coated with anti-CD3 or isotope control for 2 h, followed by treatment with or without 1 μM NS-196, which was detected by FITC-streptavidin. (B) Identification of cathepsin B as the molecular target of NS-196. CTL clone RS-56 was incubated for 2 h on anti-CD3– or IgG-coated wells, followed by incubation with or without 0.1 μM NS-196. Cells were lysed with Triton X-100 and immunodepleted with beads containing anticathepsin B antibody or control rabbit IgG. The remaining lysate was run on a 12% nonreduced SDS gel, blotted onto nitrocellulose, probed with Streptavidin-HRP, and developed using ECL. The right lane shows the biotinylation pattern when the whole CTL lysate was labeled with 0.1 μM NS-196 and run directly (1/10 of the cell-equivalent input of other lanes).

Mentions: Because lysosomal cathepsin B might be unstable and inactive at neutral pH, the hypothesis that it provides self-protection for cytotoxic lymphocytes by cleaving perforin is open to question. On the other hand, it has been shown that cathepsin B on the surface of tumor cells is enzymatically active (23). We probed for the activity of CTL surface cathepsin B by testing whether it is reactive with the biotinylated cathepsin B–specific affinity reagent NS-196, which sensitizes CTL to activation-induced suicide (Fig. 2 D). This reagent undergoes covalent reaction with the active site cysteine in a step that mimics proteolysis. As shown by flow cytometry in Fig. 6 A, the NS-196 reagent biotinylates resting CTL surfaces to a small extent (variable in different experiments), but after degranulation a greatly increased biotinylation is observed, as predicted if the immunoreactive cathepsin B (Fig. 4) is enzymatically active. Fig. 6 B shows streptavidin blots of NS-196–treated CTL with and without degranulating stimuli. NS-196 biotinylates a single 32-kD protein in untreated CTL (compatible with the slight biotinylation seen in Fig. 6 A), and this band is greatly enhanced by the degranulation stimulus. Immunodepletion of the lysate with anticathepsin B specifically removes the biotinylated 32-kD band, demonstrating that the only protein significantly labeled by NS-196 during its inactivation of self-protection is cathepsin B.


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)

Surface cathepsin B on TCR-activated T cells is active and the target of NS-196. (A) Detection of biotinylated NS-196 on the CTL surface after degranulation. Flow cytometry of CD8+ cloned human CTL RS-56 after culture on wells coated with anti-CD3 or isotope control for 2 h, followed by treatment with or without 1 μM NS-196, which was detected by FITC-streptavidin. (B) Identification of cathepsin B as the molecular target of NS-196. CTL clone RS-56 was incubated for 2 h on anti-CD3– or IgG-coated wells, followed by incubation with or without 0.1 μM NS-196. Cells were lysed with Triton X-100 and immunodepleted with beads containing anticathepsin B antibody or control rabbit IgG. The remaining lysate was run on a 12% nonreduced SDS gel, blotted onto nitrocellulose, probed with Streptavidin-HRP, and developed using ECL. The right lane shows the biotinylation pattern when the whole CTL lysate was labeled with 0.1 μM NS-196 and run directly (1/10 of the cell-equivalent input of other lanes).
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Related In: Results  -  Collection

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fig6: Surface cathepsin B on TCR-activated T cells is active and the target of NS-196. (A) Detection of biotinylated NS-196 on the CTL surface after degranulation. Flow cytometry of CD8+ cloned human CTL RS-56 after culture on wells coated with anti-CD3 or isotope control for 2 h, followed by treatment with or without 1 μM NS-196, which was detected by FITC-streptavidin. (B) Identification of cathepsin B as the molecular target of NS-196. CTL clone RS-56 was incubated for 2 h on anti-CD3– or IgG-coated wells, followed by incubation with or without 0.1 μM NS-196. Cells were lysed with Triton X-100 and immunodepleted with beads containing anticathepsin B antibody or control rabbit IgG. The remaining lysate was run on a 12% nonreduced SDS gel, blotted onto nitrocellulose, probed with Streptavidin-HRP, and developed using ECL. The right lane shows the biotinylation pattern when the whole CTL lysate was labeled with 0.1 μM NS-196 and run directly (1/10 of the cell-equivalent input of other lanes).
Mentions: Because lysosomal cathepsin B might be unstable and inactive at neutral pH, the hypothesis that it provides self-protection for cytotoxic lymphocytes by cleaving perforin is open to question. On the other hand, it has been shown that cathepsin B on the surface of tumor cells is enzymatically active (23). We probed for the activity of CTL surface cathepsin B by testing whether it is reactive with the biotinylated cathepsin B–specific affinity reagent NS-196, which sensitizes CTL to activation-induced suicide (Fig. 2 D). This reagent undergoes covalent reaction with the active site cysteine in a step that mimics proteolysis. As shown by flow cytometry in Fig. 6 A, the NS-196 reagent biotinylates resting CTL surfaces to a small extent (variable in different experiments), but after degranulation a greatly increased biotinylation is observed, as predicted if the immunoreactive cathepsin B (Fig. 4) is enzymatically active. Fig. 6 B shows streptavidin blots of NS-196–treated CTL with and without degranulating stimuli. NS-196 biotinylates a single 32-kD protein in untreated CTL (compatible with the slight biotinylation seen in Fig. 6 A), and this band is greatly enhanced by the degranulation stimulus. Immunodepletion of the lysate with anticathepsin B specifically removes the biotinylated 32-kD band, demonstrating that the only protein significantly labeled by NS-196 during its inactivation of self-protection is cathepsin B.

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