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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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Alloactivated mouse CD8+ T cell death induced by anti-CD3 in the presence of cathepsin inhibitors. (A) CD8+ T cells from B6 mice activated by a primary MLR culture were incubated for 4 h with and without the indicated cathepsin inhibitors on wells coated with anti-CD3 or anti-CD45 (filled bars) or control hamster or mouse IgG (inset open bars). Cell death was measured microscopically by nuclear propidium iodide staining (measuring membrane integrity, solid bars) or apoptotic nuclear morphology using Hoechst 33342 (gray bars). The cathepsin inhibitors ZLLY-DMK, ZFA-FMK, and controls GF-DMK and ZFβA-FMK, were used at final concentrations of 50 μM. Error bars show SEM of triplicate samples. (B) Murine-alloactivated CD8+ MLR blasts were incubated with 50 μM cathepsin inhibitor ZLLY-DMK in the presence of 10 μg/ml IgG anti-Fas antibody, 1μM concanamycin A (Ccm A), or 2.5 mM EGTA. Cell death was assessed after 4 h by propidium iodide staining in anti-CD3– (solid bars) or control hamster IgG–coated wells (inset open bars). (C) Alloactivated CD8+ MLR blasts from perforin knockout or gld mice were incubated 4 h with or without 50 μM cathepsin inhibitor ZLLY-DMK. Cell death by propidium iodide was measured in wells coated with anti-CD3 (solid bars) or control IgG (open inset bars). (D) Kinetics of death of human CTL clone RS-56 induced by plate-bound anti-CD3 in the presence of 50 μM cathepsin inhibitor ZFA-FMK. □, no anti-CD3 or ZFA-FMK. (E) Density dependence of CTL death as in D, measured at 4 h. ▿, CTL incubated with 50 μM ZFA-FMK on plate-bound anti-CD3; ⋄, same as ▿ with 2 × 106 kD dextran added to a final concentration of 5% to increase medium viscosity to block fratricidal killing; ▪, anti-CD3 with no ZFA-FMK; •, ZFA-FMK with no anti-CD3.
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fig1: Alloactivated mouse CD8+ T cell death induced by anti-CD3 in the presence of cathepsin inhibitors. (A) CD8+ T cells from B6 mice activated by a primary MLR culture were incubated for 4 h with and without the indicated cathepsin inhibitors on wells coated with anti-CD3 or anti-CD45 (filled bars) or control hamster or mouse IgG (inset open bars). Cell death was measured microscopically by nuclear propidium iodide staining (measuring membrane integrity, solid bars) or apoptotic nuclear morphology using Hoechst 33342 (gray bars). The cathepsin inhibitors ZLLY-DMK, ZFA-FMK, and controls GF-DMK and ZFβA-FMK, were used at final concentrations of 50 μM. Error bars show SEM of triplicate samples. (B) Murine-alloactivated CD8+ MLR blasts were incubated with 50 μM cathepsin inhibitor ZLLY-DMK in the presence of 10 μg/ml IgG anti-Fas antibody, 1μM concanamycin A (Ccm A), or 2.5 mM EGTA. Cell death was assessed after 4 h by propidium iodide staining in anti-CD3– (solid bars) or control hamster IgG–coated wells (inset open bars). (C) Alloactivated CD8+ MLR blasts from perforin knockout or gld mice were incubated 4 h with or without 50 μM cathepsin inhibitor ZLLY-DMK. Cell death by propidium iodide was measured in wells coated with anti-CD3 (solid bars) or control IgG (open inset bars). (D) Kinetics of death of human CTL clone RS-56 induced by plate-bound anti-CD3 in the presence of 50 μM cathepsin inhibitor ZFA-FMK. □, no anti-CD3 or ZFA-FMK. (E) Density dependence of CTL death as in D, measured at 4 h. ▿, CTL incubated with 50 μM ZFA-FMK on plate-bound anti-CD3; ⋄, same as ▿ with 2 × 106 kD dextran added to a final concentration of 5% to increase medium viscosity to block fratricidal killing; ▪, anti-CD3 with no ZFA-FMK; •, ZFA-FMK with no anti-CD3.

Mentions: If thiol cathepsin endoproteases participate in cytotoxic lymphocyte self-protection, inhibiting them during degranulation would be expected to induce effector cell suicide. The experiment in Fig. 1 A shows that surface-bound anti-CD3 induces death within 4 h of ∼10–15% of murine CD8+ T cells from a primary MLR culture. However, in the presence of two irreversible inhibitors of thiol cathepsin endoproteases, ZFA-FMK (28) and ZLLY-DMK (29), 35–55% of these cells died, with similar numbers obtained by measuring apoptotic nuclear morphology or lytic membrane damage. These peptide-based cathepsin inhibitors displayed no toxicity in the absence of CD3 cross-linking (inset bars). As specificity controls for the reactivity of these inhibitors, we used the β amino acid–containing homologue ZF(β)A-FMK and the cathepsin C inhibitor GF-DMK, which contain identical reactive groups but do not inactivate cathepsin endoproteases. These control compounds failed to promote CTL death when CD3 was cross-linked (Fig. 1 A). As additional controls, CTLs were incubated on wells coated with anti-CD45, which induces attachment without degranulation. Cathepsin inhibitors did not promote death under these conditions (Fig. 1 A).


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)

Alloactivated mouse CD8+ T cell death induced by anti-CD3 in the presence of cathepsin inhibitors. (A) CD8+ T cells from B6 mice activated by a primary MLR culture were incubated for 4 h with and without the indicated cathepsin inhibitors on wells coated with anti-CD3 or anti-CD45 (filled bars) or control hamster or mouse IgG (inset open bars). Cell death was measured microscopically by nuclear propidium iodide staining (measuring membrane integrity, solid bars) or apoptotic nuclear morphology using Hoechst 33342 (gray bars). The cathepsin inhibitors ZLLY-DMK, ZFA-FMK, and controls GF-DMK and ZFβA-FMK, were used at final concentrations of 50 μM. Error bars show SEM of triplicate samples. (B) Murine-alloactivated CD8+ MLR blasts were incubated with 50 μM cathepsin inhibitor ZLLY-DMK in the presence of 10 μg/ml IgG anti-Fas antibody, 1μM concanamycin A (Ccm A), or 2.5 mM EGTA. Cell death was assessed after 4 h by propidium iodide staining in anti-CD3– (solid bars) or control hamster IgG–coated wells (inset open bars). (C) Alloactivated CD8+ MLR blasts from perforin knockout or gld mice were incubated 4 h with or without 50 μM cathepsin inhibitor ZLLY-DMK. Cell death by propidium iodide was measured in wells coated with anti-CD3 (solid bars) or control IgG (open inset bars). (D) Kinetics of death of human CTL clone RS-56 induced by plate-bound anti-CD3 in the presence of 50 μM cathepsin inhibitor ZFA-FMK. □, no anti-CD3 or ZFA-FMK. (E) Density dependence of CTL death as in D, measured at 4 h. ▿, CTL incubated with 50 μM ZFA-FMK on plate-bound anti-CD3; ⋄, same as ▿ with 2 × 106 kD dextran added to a final concentration of 5% to increase medium viscosity to block fratricidal killing; ▪, anti-CD3 with no ZFA-FMK; •, ZFA-FMK with no anti-CD3.
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Related In: Results  -  Collection

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fig1: Alloactivated mouse CD8+ T cell death induced by anti-CD3 in the presence of cathepsin inhibitors. (A) CD8+ T cells from B6 mice activated by a primary MLR culture were incubated for 4 h with and without the indicated cathepsin inhibitors on wells coated with anti-CD3 or anti-CD45 (filled bars) or control hamster or mouse IgG (inset open bars). Cell death was measured microscopically by nuclear propidium iodide staining (measuring membrane integrity, solid bars) or apoptotic nuclear morphology using Hoechst 33342 (gray bars). The cathepsin inhibitors ZLLY-DMK, ZFA-FMK, and controls GF-DMK and ZFβA-FMK, were used at final concentrations of 50 μM. Error bars show SEM of triplicate samples. (B) Murine-alloactivated CD8+ MLR blasts were incubated with 50 μM cathepsin inhibitor ZLLY-DMK in the presence of 10 μg/ml IgG anti-Fas antibody, 1μM concanamycin A (Ccm A), or 2.5 mM EGTA. Cell death was assessed after 4 h by propidium iodide staining in anti-CD3– (solid bars) or control hamster IgG–coated wells (inset open bars). (C) Alloactivated CD8+ MLR blasts from perforin knockout or gld mice were incubated 4 h with or without 50 μM cathepsin inhibitor ZLLY-DMK. Cell death by propidium iodide was measured in wells coated with anti-CD3 (solid bars) or control IgG (open inset bars). (D) Kinetics of death of human CTL clone RS-56 induced by plate-bound anti-CD3 in the presence of 50 μM cathepsin inhibitor ZFA-FMK. □, no anti-CD3 or ZFA-FMK. (E) Density dependence of CTL death as in D, measured at 4 h. ▿, CTL incubated with 50 μM ZFA-FMK on plate-bound anti-CD3; ⋄, same as ▿ with 2 × 106 kD dextran added to a final concentration of 5% to increase medium viscosity to block fratricidal killing; ▪, anti-CD3 with no ZFA-FMK; •, ZFA-FMK with no anti-CD3.
Mentions: If thiol cathepsin endoproteases participate in cytotoxic lymphocyte self-protection, inhibiting them during degranulation would be expected to induce effector cell suicide. The experiment in Fig. 1 A shows that surface-bound anti-CD3 induces death within 4 h of ∼10–15% of murine CD8+ T cells from a primary MLR culture. However, in the presence of two irreversible inhibitors of thiol cathepsin endoproteases, ZFA-FMK (28) and ZLLY-DMK (29), 35–55% of these cells died, with similar numbers obtained by measuring apoptotic nuclear morphology or lytic membrane damage. These peptide-based cathepsin inhibitors displayed no toxicity in the absence of CD3 cross-linking (inset bars). As specificity controls for the reactivity of these inhibitors, we used the β amino acid–containing homologue ZF(β)A-FMK and the cathepsin C inhibitor GF-DMK, which contain identical reactive groups but do not inactivate cathepsin endoproteases. These control compounds failed to promote CTL death when CD3 was cross-linked (Fig. 1 A). As additional controls, CTLs were incubated on wells coated with anti-CD45, which induces attachment without degranulation. Cathepsin inhibitors did not promote death under these conditions (Fig. 1 A).

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