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Apoptotic pathways are selectively activated by granzyme A and/or granzyme B in CTL-mediated target cell lysis.

Pardo J, Bosque A, Brehm R, Wallich R, Naval J, Müllbacher A, Anel A, Simon MM - J. Cell Biol. (2004)

Bottom Line: Their physiological relevance in CTL-mediated target cell apoptosis is elusive.Thus, perf is the principal regulator in CTL-mediated and gzm-facilitated intracellular processes.The ability of gzmA and gzmB to induce multiple independent cell death pathways may be the hosts response to circumvent evasion strategies of pathogens and tumors.

View Article: PubMed Central - PubMed

Affiliation: Departmento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, E-50009 Zaragoza, Spain.

ABSTRACT
Purified cytolytic T lymphocyte (CTL) proteases granzyme (gzm)A and gzmB with sublytic dose of perforin (perf) initiate distinct proapoptotic pathways. Their physiological relevance in CTL-mediated target cell apoptosis is elusive. Using ex vivo virus-immune CD8(+) T cells from mice deficient in perf, gzmA and/or gzmB, and the Fas-resistant EL4.F15 tumor target cell, we show that (a) CTL from gzmA(-/-) or gzmB(-/-) mice similarly induced early proapoptotic features, such as phosphatidyl serine (PS) exposure on plasma membrane, Delta Psi(m) loss, and reactive oxygen radical generation, though with distinct kinetics; (b) CTL from gzmA(-/-) but not from gzmB(-/-) mice activate caspase 3 and 9; (c) PS exposure induced by CTL from gzmA(-/-) or gzmB(-/-) mice is prevented, respectively, by caspase inhibitors or by reactive oxygen scavengers without interfering with target cell death; and (d) all gzm-induced apoptotic features analyzed depend critically on perf. Thus, perf is the principal regulator in CTL-mediated and gzm-facilitated intracellular processes. The ability of gzmA and gzmB to induce multiple independent cell death pathways may be the hosts response to circumvent evasion strategies of pathogens and tumors.

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Caspase inhibitors completely block PS exposure induced by gzmA-deficient CTL. (A–F) EL4.F15 cells were incubated with ex vivo virus-specific CD8+ cells (MACS selected, ≥95% CD8+ cells) from B6, gzmA−/−, or gzmB−/− mice for 2 h at a 10:1 effector/target ratio in the presence or absence of the LCMV peptide gp33. (A–D) CML cultures were incubated in the presence or absence of 100 μM of the pan caspase inhibitor Z-VAD-fmk or the caspase 3 inhibitor Z-DEVD-fmk. Subsequently, PS exposure on plasma membrane (annexin-V-FITC; A) and PI uptake (B) and, in parallel, ΔΨm loss (DiOC6(3) staining; C) and ROS generation (2-HE; D) were analyzed by three-color flow cytometry in the cell population negative for CD8 expression (target cells). Results are the mean of three independent experiments and are given as the difference of percentages in the presence or absence of gp33 ± SD. (E and F) Inhibition of caspase 3 or 9 activity reduces, respectively, caspase 9 or 3 activation induced by B6 and gzmA−/− CTL. Caspase 3 activity was analyzed using an FITC-labeled mAb against the active form of the enzyme (E), whereas caspase 9 activity was analyzed by using the specific fluorescent substrate FAM-LEHD-fmk (F) by two-color flow cytometry in the cell population negative for CD8 expression (target cells). CMLs were also developed in the presence of the caspase 3 (100 μM Z-DEVD-fmk; F, bottom, gray) or caspase 9 (100 μM Z-LEHD-fmk; E, bottom, orange; and F, middle, orange) inhibitors. Numbers correspond to the percentage of cells positive for the labeling in each case, indicated by the horizontal bars shown in the top panels.
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fig3: Caspase inhibitors completely block PS exposure induced by gzmA-deficient CTL. (A–F) EL4.F15 cells were incubated with ex vivo virus-specific CD8+ cells (MACS selected, ≥95% CD8+ cells) from B6, gzmA−/−, or gzmB−/− mice for 2 h at a 10:1 effector/target ratio in the presence or absence of the LCMV peptide gp33. (A–D) CML cultures were incubated in the presence or absence of 100 μM of the pan caspase inhibitor Z-VAD-fmk or the caspase 3 inhibitor Z-DEVD-fmk. Subsequently, PS exposure on plasma membrane (annexin-V-FITC; A) and PI uptake (B) and, in parallel, ΔΨm loss (DiOC6(3) staining; C) and ROS generation (2-HE; D) were analyzed by three-color flow cytometry in the cell population negative for CD8 expression (target cells). Results are the mean of three independent experiments and are given as the difference of percentages in the presence or absence of gp33 ± SD. (E and F) Inhibition of caspase 3 or 9 activity reduces, respectively, caspase 9 or 3 activation induced by B6 and gzmA−/− CTL. Caspase 3 activity was analyzed using an FITC-labeled mAb against the active form of the enzyme (E), whereas caspase 9 activity was analyzed by using the specific fluorescent substrate FAM-LEHD-fmk (F) by two-color flow cytometry in the cell population negative for CD8 expression (target cells). CMLs were also developed in the presence of the caspase 3 (100 μM Z-DEVD-fmk; F, bottom, gray) or caspase 9 (100 μM Z-LEHD-fmk; E, bottom, orange; and F, middle, orange) inhibitors. Numbers correspond to the percentage of cells positive for the labeling in each case, indicated by the horizontal bars shown in the top panels.

Mentions: We tested if in the course of CTL-mediated cytolysis, activation of caspases is essential for gzm-induced PS exposure on plasma membrane, cell death (PI incorporation), ΔΨm loss, and ROS generation. For this purpose, LCMV-immune CD8+ T cells from B6, gzmA−/−, or gzmB−/− mice were incubated with EL4.F15 cells in the presence of the universal caspase inhibitor Z-VAD-fmk or with the caspase 3 inhibitor Z-DEVD-fmk. As shown in Fig. 3 (A and B; mean of three independent experiments), the number of annexinV+ and PI+ cells was drastically reduced by either Z-DEVD-fmk or Z-VAD-fmk when gzmA−/−-derived CTL were used, only partially in the presence of B6 CTL, and not at all with gzmB−/− CTL. Furthermore, ΔΨm loss and ROS generation induced by B6- and gzmA−/−- but not gzmB−/−-derived CTL were also reduced, though only partially, in the presence of Z-DEVD-fmk or Z-VAD-fmk (Fig. 3, C and D). These data suggest that CTL-mediated induction of plasma membrane perturbation is critically dependent on functional active caspases when elicited by gzmB but not by gzmA and reveals caspase-dependent and -independent mechanisms of gzmB-induced mitochondrial depolarization. In both cases, a DEVD-sensitive caspase, in particular caspase 3, may be the main executor of these caspase-dependent processes.


Apoptotic pathways are selectively activated by granzyme A and/or granzyme B in CTL-mediated target cell lysis.

Pardo J, Bosque A, Brehm R, Wallich R, Naval J, Müllbacher A, Anel A, Simon MM - J. Cell Biol. (2004)

Caspase inhibitors completely block PS exposure induced by gzmA-deficient CTL. (A–F) EL4.F15 cells were incubated with ex vivo virus-specific CD8+ cells (MACS selected, ≥95% CD8+ cells) from B6, gzmA−/−, or gzmB−/− mice for 2 h at a 10:1 effector/target ratio in the presence or absence of the LCMV peptide gp33. (A–D) CML cultures were incubated in the presence or absence of 100 μM of the pan caspase inhibitor Z-VAD-fmk or the caspase 3 inhibitor Z-DEVD-fmk. Subsequently, PS exposure on plasma membrane (annexin-V-FITC; A) and PI uptake (B) and, in parallel, ΔΨm loss (DiOC6(3) staining; C) and ROS generation (2-HE; D) were analyzed by three-color flow cytometry in the cell population negative for CD8 expression (target cells). Results are the mean of three independent experiments and are given as the difference of percentages in the presence or absence of gp33 ± SD. (E and F) Inhibition of caspase 3 or 9 activity reduces, respectively, caspase 9 or 3 activation induced by B6 and gzmA−/− CTL. Caspase 3 activity was analyzed using an FITC-labeled mAb against the active form of the enzyme (E), whereas caspase 9 activity was analyzed by using the specific fluorescent substrate FAM-LEHD-fmk (F) by two-color flow cytometry in the cell population negative for CD8 expression (target cells). CMLs were also developed in the presence of the caspase 3 (100 μM Z-DEVD-fmk; F, bottom, gray) or caspase 9 (100 μM Z-LEHD-fmk; E, bottom, orange; and F, middle, orange) inhibitors. Numbers correspond to the percentage of cells positive for the labeling in each case, indicated by the horizontal bars shown in the top panels.
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Related In: Results  -  Collection

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fig3: Caspase inhibitors completely block PS exposure induced by gzmA-deficient CTL. (A–F) EL4.F15 cells were incubated with ex vivo virus-specific CD8+ cells (MACS selected, ≥95% CD8+ cells) from B6, gzmA−/−, or gzmB−/− mice for 2 h at a 10:1 effector/target ratio in the presence or absence of the LCMV peptide gp33. (A–D) CML cultures were incubated in the presence or absence of 100 μM of the pan caspase inhibitor Z-VAD-fmk or the caspase 3 inhibitor Z-DEVD-fmk. Subsequently, PS exposure on plasma membrane (annexin-V-FITC; A) and PI uptake (B) and, in parallel, ΔΨm loss (DiOC6(3) staining; C) and ROS generation (2-HE; D) were analyzed by three-color flow cytometry in the cell population negative for CD8 expression (target cells). Results are the mean of three independent experiments and are given as the difference of percentages in the presence or absence of gp33 ± SD. (E and F) Inhibition of caspase 3 or 9 activity reduces, respectively, caspase 9 or 3 activation induced by B6 and gzmA−/− CTL. Caspase 3 activity was analyzed using an FITC-labeled mAb against the active form of the enzyme (E), whereas caspase 9 activity was analyzed by using the specific fluorescent substrate FAM-LEHD-fmk (F) by two-color flow cytometry in the cell population negative for CD8 expression (target cells). CMLs were also developed in the presence of the caspase 3 (100 μM Z-DEVD-fmk; F, bottom, gray) or caspase 9 (100 μM Z-LEHD-fmk; E, bottom, orange; and F, middle, orange) inhibitors. Numbers correspond to the percentage of cells positive for the labeling in each case, indicated by the horizontal bars shown in the top panels.
Mentions: We tested if in the course of CTL-mediated cytolysis, activation of caspases is essential for gzm-induced PS exposure on plasma membrane, cell death (PI incorporation), ΔΨm loss, and ROS generation. For this purpose, LCMV-immune CD8+ T cells from B6, gzmA−/−, or gzmB−/− mice were incubated with EL4.F15 cells in the presence of the universal caspase inhibitor Z-VAD-fmk or with the caspase 3 inhibitor Z-DEVD-fmk. As shown in Fig. 3 (A and B; mean of three independent experiments), the number of annexinV+ and PI+ cells was drastically reduced by either Z-DEVD-fmk or Z-VAD-fmk when gzmA−/−-derived CTL were used, only partially in the presence of B6 CTL, and not at all with gzmB−/− CTL. Furthermore, ΔΨm loss and ROS generation induced by B6- and gzmA−/−- but not gzmB−/−-derived CTL were also reduced, though only partially, in the presence of Z-DEVD-fmk or Z-VAD-fmk (Fig. 3, C and D). These data suggest that CTL-mediated induction of plasma membrane perturbation is critically dependent on functional active caspases when elicited by gzmB but not by gzmA and reveals caspase-dependent and -independent mechanisms of gzmB-induced mitochondrial depolarization. In both cases, a DEVD-sensitive caspase, in particular caspase 3, may be the main executor of these caspase-dependent processes.

Bottom Line: Their physiological relevance in CTL-mediated target cell apoptosis is elusive.Thus, perf is the principal regulator in CTL-mediated and gzm-facilitated intracellular processes.The ability of gzmA and gzmB to induce multiple independent cell death pathways may be the hosts response to circumvent evasion strategies of pathogens and tumors.

View Article: PubMed Central - PubMed

Affiliation: Departmento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, E-50009 Zaragoza, Spain.

ABSTRACT
Purified cytolytic T lymphocyte (CTL) proteases granzyme (gzm)A and gzmB with sublytic dose of perforin (perf) initiate distinct proapoptotic pathways. Their physiological relevance in CTL-mediated target cell apoptosis is elusive. Using ex vivo virus-immune CD8(+) T cells from mice deficient in perf, gzmA and/or gzmB, and the Fas-resistant EL4.F15 tumor target cell, we show that (a) CTL from gzmA(-/-) or gzmB(-/-) mice similarly induced early proapoptotic features, such as phosphatidyl serine (PS) exposure on plasma membrane, Delta Psi(m) loss, and reactive oxygen radical generation, though with distinct kinetics; (b) CTL from gzmA(-/-) but not from gzmB(-/-) mice activate caspase 3 and 9; (c) PS exposure induced by CTL from gzmA(-/-) or gzmB(-/-) mice is prevented, respectively, by caspase inhibitors or by reactive oxygen scavengers without interfering with target cell death; and (d) all gzm-induced apoptotic features analyzed depend critically on perf. Thus, perf is the principal regulator in CTL-mediated and gzm-facilitated intracellular processes. The ability of gzmA and gzmB to induce multiple independent cell death pathways may be the hosts response to circumvent evasion strategies of pathogens and tumors.

Show MeSH
Related in: MedlinePlus