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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 CTL is released by EGTA and is the single chain form. CD8+ cloned human CTL were stimulated with plate-bound anti-CD3 for 2 h at 37°C, washed with PBS, and (A) incubated with PBS or (B) 0.53 mM EDTA in PBS for 10 min at 37°C. After washing, cells were incubated with anti-cathepsin B (heavy line) followed by anti–mouse Ig-FITC (dashed line shows control with no anticathepsin B). (C) Anti-CD3–treated CTL as in A and B were incubated with 2 mM Mg-EGTA or Ca-EDTA in HBSS for 10 min at 37°C. Supernatants were analyzed by blotting with anti-cathepsin B. The last lane shows whole CTL dissolved in SDS sample buffer and run directly (1/20 of the cell-equivalent input of other lanes).
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fig5: Surface cathepsin B on TCR-activated CTL is released by EGTA and is the single chain form. CD8+ cloned human CTL were stimulated with plate-bound anti-CD3 for 2 h at 37°C, washed with PBS, and (A) incubated with PBS or (B) 0.53 mM EDTA in PBS for 10 min at 37°C. After washing, cells were incubated with anti-cathepsin B (heavy line) followed by anti–mouse Ig-FITC (dashed line shows control with no anticathepsin B). (C) Anti-CD3–treated CTL as in A and B were incubated with 2 mM Mg-EGTA or Ca-EDTA in HBSS for 10 min at 37°C. Supernatants were analyzed by blotting with anti-cathepsin B. The last lane shows whole CTL dissolved in SDS sample buffer and run directly (1/20 of the cell-equivalent input of other lanes).

Mentions: Because the association of active cathepsin B with tumor cell surfaces has been shown to be dependent on divalent cations (22), we tested whether this was also the case with CTL. Fig. 5, A and B , show that the EDTA treatment that removes surface cathepsin B from tumor cells also removes it from recently degranulated CTL. Fig. 5 C shows an experiment in which the released cathepsin B was immunoprecipitated from supernatants after treating such CTL under selective conditions for Ca+2 and Mg+2 chelation. Western blots of such supernatants (Fig. 5 C) showed that the 32-kD “one-chain” form of cathepsin B was only detectable when CTL had been precultured on anti-CD3–coated wells to trigger degranulation, and when they were subsequently incubated in Mg-EGTA to chelate free calcium.


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 CTL is released by EGTA and is the single chain form. CD8+ cloned human CTL were stimulated with plate-bound anti-CD3 for 2 h at 37°C, washed with PBS, and (A) incubated with PBS or (B) 0.53 mM EDTA in PBS for 10 min at 37°C. After washing, cells were incubated with anti-cathepsin B (heavy line) followed by anti–mouse Ig-FITC (dashed line shows control with no anticathepsin B). (C) Anti-CD3–treated CTL as in A and B were incubated with 2 mM Mg-EGTA or Ca-EDTA in HBSS for 10 min at 37°C. Supernatants were analyzed by blotting with anti-cathepsin B. The last lane shows whole CTL dissolved in SDS sample buffer and run directly (1/20 of the cell-equivalent input of other lanes).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196055&req=5

fig5: Surface cathepsin B on TCR-activated CTL is released by EGTA and is the single chain form. CD8+ cloned human CTL were stimulated with plate-bound anti-CD3 for 2 h at 37°C, washed with PBS, and (A) incubated with PBS or (B) 0.53 mM EDTA in PBS for 10 min at 37°C. After washing, cells were incubated with anti-cathepsin B (heavy line) followed by anti–mouse Ig-FITC (dashed line shows control with no anticathepsin B). (C) Anti-CD3–treated CTL as in A and B were incubated with 2 mM Mg-EGTA or Ca-EDTA in HBSS for 10 min at 37°C. Supernatants were analyzed by blotting with anti-cathepsin B. The last lane shows whole CTL dissolved in SDS sample buffer and run directly (1/20 of the cell-equivalent input of other lanes).
Mentions: Because the association of active cathepsin B with tumor cell surfaces has been shown to be dependent on divalent cations (22), we tested whether this was also the case with CTL. Fig. 5, A and B , show that the EDTA treatment that removes surface cathepsin B from tumor cells also removes it from recently degranulated CTL. Fig. 5 C shows an experiment in which the released cathepsin B was immunoprecipitated from supernatants after treating such CTL under selective conditions for Ca+2 and Mg+2 chelation. Western blots of such supernatants (Fig. 5 C) showed that the 32-kD “one-chain” form of cathepsin B was only detectable when CTL had been precultured on anti-CD3–coated wells to trigger degranulation, and when they were subsequently incubated in Mg-EGTA to chelate free calcium.

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