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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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CTL suicide during target cell lysis. (A) Lysis of H-2d–bearing L1210 tumor cells by H-2d–reactive CTL in a 4-h assay. (B) Lysis of 51Cr-labeled CTL (104/well) used in A when incubated for 4 h with L1210 target cells in the presence of 10 μM CA074. ▴, CA074; ▵, no inhibitor. (C–D) H-2b–reactive CTL were mixed with 51Cr-labeled EL4 cells in the presence or absence of 10 μM CA074. After 4 h, 125 μl supernatant were harvested for counting (C) and replaced with 125 μl of medium for ∼12 h of additional incubation. D shows the second harvest. As a toxicity control for CA074 in D, the CTLs in C were incubated with 10 μM CA074 for 16 h before the cytotoxicity assay with 51Cr-labeled EL-4 cells. □, no inhibitor; ▿, 10 μM CA074. (E) Mean inhibition of CTL lytic activity from multiple experiments like those shown in C and D, calculated by lytic units (horizontal shift between cytotoxicity dose response curves).
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fig3: CTL suicide during target cell lysis. (A) Lysis of H-2d–bearing L1210 tumor cells by H-2d–reactive CTL in a 4-h assay. (B) Lysis of 51Cr-labeled CTL (104/well) used in A when incubated for 4 h with L1210 target cells in the presence of 10 μM CA074. ▴, CA074; ▵, no inhibitor. (C–D) H-2b–reactive CTL were mixed with 51Cr-labeled EL4 cells in the presence or absence of 10 μM CA074. After 4 h, 125 μl supernatant were harvested for counting (C) and replaced with 125 μl of medium for ∼12 h of additional incubation. D shows the second harvest. As a toxicity control for CA074 in D, the CTLs in C were incubated with 10 μM CA074 for 16 h before the cytotoxicity assay with 51Cr-labeled EL-4 cells. □, no inhibitor; ▿, 10 μM CA074. (E) Mean inhibition of CTL lytic activity from multiple experiments like those shown in C and D, calculated by lytic units (horizontal shift between cytotoxicity dose response curves).

Mentions: 51Cr-labeled B6 anti-BALB/c MLR cells were mixed with varying numbers of L1210 cells in the presence or absence of 10 μM CA074 for 4 h at 37°C in 5% CO2 in 96-well plates (see Fig. 3, A and B) . After 4 h, 100 μl supernatant were harvested for counting. EL-4 target cells were labeled with Na251Cr2O7 (200 μCi in 0.5 ml HBSS plus 10% FCS) for 45 min at 37°C (see Fig. 3, C–E). After washing twice with complete medium, 104 target cells were incubated with varying numbers of BALB/c anti-B6 MLR cells in the presence or absence of 10 μM CA074 for 4 h at 37°C in 5% CO2 in 96-well plates. After 4 h, 125 μl supernatant were harvested for counting and replaced with 125 μl medium for ∼12 h of additional incubation. As a toxicity control for CA074, CTLs were mixed with 10 μM CA074 for 16 h before carrying out cytotoxicity assays with 51Cr-labeled EL-4 cells.


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)

CTL suicide during target cell lysis. (A) Lysis of H-2d–bearing L1210 tumor cells by H-2d–reactive CTL in a 4-h assay. (B) Lysis of 51Cr-labeled CTL (104/well) used in A when incubated for 4 h with L1210 target cells in the presence of 10 μM CA074. ▴, CA074; ▵, no inhibitor. (C–D) H-2b–reactive CTL were mixed with 51Cr-labeled EL4 cells in the presence or absence of 10 μM CA074. After 4 h, 125 μl supernatant were harvested for counting (C) and replaced with 125 μl of medium for ∼12 h of additional incubation. D shows the second harvest. As a toxicity control for CA074 in D, the CTLs in C were incubated with 10 μM CA074 for 16 h before the cytotoxicity assay with 51Cr-labeled EL-4 cells. □, no inhibitor; ▿, 10 μM CA074. (E) Mean inhibition of CTL lytic activity from multiple experiments like those shown in C and D, calculated by lytic units (horizontal shift between cytotoxicity dose response curves).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196055&req=5

fig3: CTL suicide during target cell lysis. (A) Lysis of H-2d–bearing L1210 tumor cells by H-2d–reactive CTL in a 4-h assay. (B) Lysis of 51Cr-labeled CTL (104/well) used in A when incubated for 4 h with L1210 target cells in the presence of 10 μM CA074. ▴, CA074; ▵, no inhibitor. (C–D) H-2b–reactive CTL were mixed with 51Cr-labeled EL4 cells in the presence or absence of 10 μM CA074. After 4 h, 125 μl supernatant were harvested for counting (C) and replaced with 125 μl of medium for ∼12 h of additional incubation. D shows the second harvest. As a toxicity control for CA074 in D, the CTLs in C were incubated with 10 μM CA074 for 16 h before the cytotoxicity assay with 51Cr-labeled EL-4 cells. □, no inhibitor; ▿, 10 μM CA074. (E) Mean inhibition of CTL lytic activity from multiple experiments like those shown in C and D, calculated by lytic units (horizontal shift between cytotoxicity dose response curves).
Mentions: 51Cr-labeled B6 anti-BALB/c MLR cells were mixed with varying numbers of L1210 cells in the presence or absence of 10 μM CA074 for 4 h at 37°C in 5% CO2 in 96-well plates (see Fig. 3, A and B) . After 4 h, 100 μl supernatant were harvested for counting. EL-4 target cells were labeled with Na251Cr2O7 (200 μCi in 0.5 ml HBSS plus 10% FCS) for 45 min at 37°C (see Fig. 3, C–E). After washing twice with complete medium, 104 target cells were incubated with varying numbers of BALB/c anti-B6 MLR cells in the presence or absence of 10 μM CA074 for 4 h at 37°C in 5% CO2 in 96-well plates. After 4 h, 125 μl supernatant were harvested for counting and replaced with 125 μl medium for ∼12 h of additional incubation. As a toxicity control for CA074, CTLs were mixed with 10 μM CA074 for 16 h before carrying out cytotoxicity assays with 51Cr-labeled EL-4 cells.

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