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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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Activation-induced suicide of cytotoxic effectors in the presence of membrane-impermeant cathepsin B inhibitors. (A) Human CD8+ T cell blasts, resting blood CD8+ cells, CD4+ T cell blasts, and NK cells were incubated for 4 h with and without 50 μM cathepsin inhibitors ZLLY-DMK or ZFA-FMK. Cell death was assessed by propidium iodide from wells coated with control IgG (open inset bars) or the indicated degranulating stimuli (open bars). Striped bars, no inhibitor; solid bars, ZLLY-DMK; gray bars, ZFA-FMK.(B) Alloactivated mouse CD8+ T cell blasts were incubated for 4 h on anti-CD3–coated wells (solid bars) or control IgG (open inset bars) with or without 10 μM cystatin C or 50 μM ZLLY-DMK, and cell death was assessed by propidium iodide. (C) Alloactivated CD8+ BALB/c T cells were incubated on wells coated with anti-CD3 (solid bars) or control IgG (open inset bars) for 4 h in presence or absence of the indicated cathepsin inhibitors and stained with propidium iodide. (D) Same conditions as in C, comparing NS-196 and ZLLY-DMK.
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fig2: Activation-induced suicide of cytotoxic effectors in the presence of membrane-impermeant cathepsin B inhibitors. (A) Human CD8+ T cell blasts, resting blood CD8+ cells, CD4+ T cell blasts, and NK cells were incubated for 4 h with and without 50 μM cathepsin inhibitors ZLLY-DMK or ZFA-FMK. Cell death was assessed by propidium iodide from wells coated with control IgG (open inset bars) or the indicated degranulating stimuli (open bars). Striped bars, no inhibitor; solid bars, ZLLY-DMK; gray bars, ZFA-FMK.(B) Alloactivated mouse CD8+ T cell blasts were incubated for 4 h on anti-CD3–coated wells (solid bars) or control IgG (open inset bars) with or without 10 μM cystatin C or 50 μM ZLLY-DMK, and cell death was assessed by propidium iodide. (C) Alloactivated CD8+ BALB/c T cells were incubated on wells coated with anti-CD3 (solid bars) or control IgG (open inset bars) for 4 h in presence or absence of the indicated cathepsin inhibitors and stained with propidium iodide. (D) Same conditions as in C, comparing NS-196 and ZLLY-DMK.

Mentions: The experiments described above indicate that activated mouse and human CD8+ T cells die when induced to degranulate in the presence of cathepsin inhibitors. To define the cell types that are capable of undergoing this death, purified subpopulations of human blood lymphocytes were cultured under activating conditions to induce degranulation. As shown in Fig. 2 A, human CD8+ T cell blasts, highly active as cytotoxic effector cells, died within 4 h when incubated on anti-CD3–coated wells in the presence of cathepsin inhibitors. On the other hand, resting human CD8+ T cells and CD4+ blasts did not die when incubated on wells coated with both anti-CD3 and anti-CD28. Highly cytolytic CD56+ cultured human NK cells showed a pronounced death when triggered to degranulate with immobilized anti-CD16 (34) in the presence of cathepsin inhibitors. As with CTLs, these inhibitors showed no evidence of toxicity in the absence of the degranulating stimulus. Thus, the lymphocyte death response after a degranulation stimulus in the presence of cathepsin inhibitors reflects their cytotoxic potential via the granule exocytosis pathway.


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)

Activation-induced suicide of cytotoxic effectors in the presence of membrane-impermeant cathepsin B inhibitors. (A) Human CD8+ T cell blasts, resting blood CD8+ cells, CD4+ T cell blasts, and NK cells were incubated for 4 h with and without 50 μM cathepsin inhibitors ZLLY-DMK or ZFA-FMK. Cell death was assessed by propidium iodide from wells coated with control IgG (open inset bars) or the indicated degranulating stimuli (open bars). Striped bars, no inhibitor; solid bars, ZLLY-DMK; gray bars, ZFA-FMK.(B) Alloactivated mouse CD8+ T cell blasts were incubated for 4 h on anti-CD3–coated wells (solid bars) or control IgG (open inset bars) with or without 10 μM cystatin C or 50 μM ZLLY-DMK, and cell death was assessed by propidium iodide. (C) Alloactivated CD8+ BALB/c T cells were incubated on wells coated with anti-CD3 (solid bars) or control IgG (open inset bars) for 4 h in presence or absence of the indicated cathepsin inhibitors and stained with propidium iodide. (D) Same conditions as in C, comparing NS-196 and ZLLY-DMK.
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Related In: Results  -  Collection

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fig2: Activation-induced suicide of cytotoxic effectors in the presence of membrane-impermeant cathepsin B inhibitors. (A) Human CD8+ T cell blasts, resting blood CD8+ cells, CD4+ T cell blasts, and NK cells were incubated for 4 h with and without 50 μM cathepsin inhibitors ZLLY-DMK or ZFA-FMK. Cell death was assessed by propidium iodide from wells coated with control IgG (open inset bars) or the indicated degranulating stimuli (open bars). Striped bars, no inhibitor; solid bars, ZLLY-DMK; gray bars, ZFA-FMK.(B) Alloactivated mouse CD8+ T cell blasts were incubated for 4 h on anti-CD3–coated wells (solid bars) or control IgG (open inset bars) with or without 10 μM cystatin C or 50 μM ZLLY-DMK, and cell death was assessed by propidium iodide. (C) Alloactivated CD8+ BALB/c T cells were incubated on wells coated with anti-CD3 (solid bars) or control IgG (open inset bars) for 4 h in presence or absence of the indicated cathepsin inhibitors and stained with propidium iodide. (D) Same conditions as in C, comparing NS-196 and ZLLY-DMK.
Mentions: The experiments described above indicate that activated mouse and human CD8+ T cells die when induced to degranulate in the presence of cathepsin inhibitors. To define the cell types that are capable of undergoing this death, purified subpopulations of human blood lymphocytes were cultured under activating conditions to induce degranulation. As shown in Fig. 2 A, human CD8+ T cell blasts, highly active as cytotoxic effector cells, died within 4 h when incubated on anti-CD3–coated wells in the presence of cathepsin inhibitors. On the other hand, resting human CD8+ T cells and CD4+ blasts did not die when incubated on wells coated with both anti-CD3 and anti-CD28. Highly cytolytic CD56+ cultured human NK cells showed a pronounced death when triggered to degranulate with immobilized anti-CD16 (34) in the presence of cathepsin inhibitors. As with CTLs, these inhibitors showed no evidence of toxicity in the absence of the degranulating stimulus. Thus, the lymphocyte death response after a degranulation stimulus in the presence of cathepsin inhibitors reflects their cytotoxic potential via the granule exocytosis pathway.

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