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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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Related in: MedlinePlus

PS exposure, mitochondrial depolarization, and ROS generation is induced by gzmA−/− and gzmB−/− but not by perf−/− and gzmA×B−/− CTL. EL4.F15 cells were incubated with ex vivo virus-immune CD8+ cells (MACS selected, ≥95% CD8+ cells) from either B6, gzmA−/−, or gzmB−/− (A) or from B6, perf −/−, or gzmA×B−/− (B) mice, in the presence or absence of the LCMV peptide gp33. (Aa) percentage of virus-specific CD8+ cells, determined by staining with gp33-labeled tetramers (H-2Db; tet-PE). (Ab) EL4.F15 cells were incubated with effector cells for 4 h (open symbols, −gp33; closed symbols, +gp33) at different effector/target ratios, and DNA fragmentation was analyzed by 3H-thymidine release. (Ac and Ad) EL4.F15 cells were incubated with effector cells for 2 h at a 10:1 effector/target ratio. Subsequently, PS exposure on plasma membrane (annexin-V-FITC) and PI uptake (c) and, in parallel, ΔΨm loss (3,3-dihexyloxacarbocyanin [DiOC6(3)] staining) and ROS generation (2-hydroxiethidine [2-HE]; d) were analyzed by three-color flow cytometry in the cell population negative for CD8 expression (target cells). EL4.F15 cells were also incubated with 200 μM glyotoxin (c) or 200 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP; d) as positive controls for PS exposure and ΔΨm loss, respectively. Numbers indicate percentage of cells in each quadrant. (Ae) EL4.F15 cells were incubated with effector cells for 30, 60, or 120 min at a 10:1 effector/target ratio, and PS exposure and PI uptake or ΔΨm loss and ROS generation were analyzed as described in Ac and Ad. Data presented are the mean of at least three independent experiments and are given as the difference of percentages in the presence versus the absence of gp33 ± SD. (B) a, same as Aa; b, same as Ab; c, target cell death (open symbols, −gp33; closed symbols, +gp33) was monitored after incubation of CML for 2 h at an effector/target cell ratio of 10:1 by the cell survival assay. Bd, same as Ac; Be, same as Ad.
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fig1: PS exposure, mitochondrial depolarization, and ROS generation is induced by gzmA−/− and gzmB−/− but not by perf−/− and gzmA×B−/− CTL. EL4.F15 cells were incubated with ex vivo virus-immune CD8+ cells (MACS selected, ≥95% CD8+ cells) from either B6, gzmA−/−, or gzmB−/− (A) or from B6, perf −/−, or gzmA×B−/− (B) mice, in the presence or absence of the LCMV peptide gp33. (Aa) percentage of virus-specific CD8+ cells, determined by staining with gp33-labeled tetramers (H-2Db; tet-PE). (Ab) EL4.F15 cells were incubated with effector cells for 4 h (open symbols, −gp33; closed symbols, +gp33) at different effector/target ratios, and DNA fragmentation was analyzed by 3H-thymidine release. (Ac and Ad) EL4.F15 cells were incubated with effector cells for 2 h at a 10:1 effector/target ratio. Subsequently, PS exposure on plasma membrane (annexin-V-FITC) and PI uptake (c) and, in parallel, ΔΨm loss (3,3-dihexyloxacarbocyanin [DiOC6(3)] staining) and ROS generation (2-hydroxiethidine [2-HE]; d) were analyzed by three-color flow cytometry in the cell population negative for CD8 expression (target cells). EL4.F15 cells were also incubated with 200 μM glyotoxin (c) or 200 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP; d) as positive controls for PS exposure and ΔΨm loss, respectively. Numbers indicate percentage of cells in each quadrant. (Ae) EL4.F15 cells were incubated with effector cells for 30, 60, or 120 min at a 10:1 effector/target ratio, and PS exposure and PI uptake or ΔΨm loss and ROS generation were analyzed as described in Ac and Ad. Data presented are the mean of at least three independent experiments and are given as the difference of percentages in the presence versus the absence of gp33 ± SD. (B) a, same as Aa; b, same as Ab; c, target cell death (open symbols, −gp33; closed symbols, +gp33) was monitored after incubation of CML for 2 h at an effector/target cell ratio of 10:1 by the cell survival assay. Bd, same as Ac; Be, same as Ad.

Mentions: To study a physiologically relevant model for granule exocytosis–executed apoptotic cell death, in particular mediated by gzms and independent of Fas, we used an in vitro CTL-target cytotoxic assay. Ex vivo–derived virus-immune CD8+ T cells (day eight after infection) from B6 mice or those deficient in gzm(s) (gzmA−/−, gzmB−/−, and gzmA×B−/−) or perf (perf−/−), infected with LCMV 8 d before, were used as effector cells. As target cell, the tumor cell line EL4.F15 was chosen because it is Fas resistant and at the same time sensitive to both gzmA- and gzmB-induced apoptosis (Pardo et al., 2002). Confirming previous studies, splenocytes from 8-d LCMV-immune B6, gzmA−/−, and gzmB−/− mice contained similar numbers (11, 6, and 9%, respectively) of gp33/D btetramer+/CD8+ T cells (Fig. 1 Aa). As shown previously (Simon et al., 1997; Pardo et al., 2002), enriched CD8+ T cells from all three mouse strains readily induced specific nucleolysis in gp33-pulsed EL4.F15 target cells (4-h assay), which was similar in magnitude for B6 and gzmA−/− and somewhat lower for gzmB−/− T cells (Fig. 1 Ab).


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)

PS exposure, mitochondrial depolarization, and ROS generation is induced by gzmA−/− and gzmB−/− but not by perf−/− and gzmA×B−/− CTL. EL4.F15 cells were incubated with ex vivo virus-immune CD8+ cells (MACS selected, ≥95% CD8+ cells) from either B6, gzmA−/−, or gzmB−/− (A) or from B6, perf −/−, or gzmA×B−/− (B) mice, in the presence or absence of the LCMV peptide gp33. (Aa) percentage of virus-specific CD8+ cells, determined by staining with gp33-labeled tetramers (H-2Db; tet-PE). (Ab) EL4.F15 cells were incubated with effector cells for 4 h (open symbols, −gp33; closed symbols, +gp33) at different effector/target ratios, and DNA fragmentation was analyzed by 3H-thymidine release. (Ac and Ad) EL4.F15 cells were incubated with effector cells for 2 h at a 10:1 effector/target ratio. Subsequently, PS exposure on plasma membrane (annexin-V-FITC) and PI uptake (c) and, in parallel, ΔΨm loss (3,3-dihexyloxacarbocyanin [DiOC6(3)] staining) and ROS generation (2-hydroxiethidine [2-HE]; d) were analyzed by three-color flow cytometry in the cell population negative for CD8 expression (target cells). EL4.F15 cells were also incubated with 200 μM glyotoxin (c) or 200 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP; d) as positive controls for PS exposure and ΔΨm loss, respectively. Numbers indicate percentage of cells in each quadrant. (Ae) EL4.F15 cells were incubated with effector cells for 30, 60, or 120 min at a 10:1 effector/target ratio, and PS exposure and PI uptake or ΔΨm loss and ROS generation were analyzed as described in Ac and Ad. Data presented are the mean of at least three independent experiments and are given as the difference of percentages in the presence versus the absence of gp33 ± SD. (B) a, same as Aa; b, same as Ab; c, target cell death (open symbols, −gp33; closed symbols, +gp33) was monitored after incubation of CML for 2 h at an effector/target cell ratio of 10:1 by the cell survival assay. Bd, same as Ac; Be, same as Ad.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172484&req=5

fig1: PS exposure, mitochondrial depolarization, and ROS generation is induced by gzmA−/− and gzmB−/− but not by perf−/− and gzmA×B−/− CTL. EL4.F15 cells were incubated with ex vivo virus-immune CD8+ cells (MACS selected, ≥95% CD8+ cells) from either B6, gzmA−/−, or gzmB−/− (A) or from B6, perf −/−, or gzmA×B−/− (B) mice, in the presence or absence of the LCMV peptide gp33. (Aa) percentage of virus-specific CD8+ cells, determined by staining with gp33-labeled tetramers (H-2Db; tet-PE). (Ab) EL4.F15 cells were incubated with effector cells for 4 h (open symbols, −gp33; closed symbols, +gp33) at different effector/target ratios, and DNA fragmentation was analyzed by 3H-thymidine release. (Ac and Ad) EL4.F15 cells were incubated with effector cells for 2 h at a 10:1 effector/target ratio. Subsequently, PS exposure on plasma membrane (annexin-V-FITC) and PI uptake (c) and, in parallel, ΔΨm loss (3,3-dihexyloxacarbocyanin [DiOC6(3)] staining) and ROS generation (2-hydroxiethidine [2-HE]; d) were analyzed by three-color flow cytometry in the cell population negative for CD8 expression (target cells). EL4.F15 cells were also incubated with 200 μM glyotoxin (c) or 200 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP; d) as positive controls for PS exposure and ΔΨm loss, respectively. Numbers indicate percentage of cells in each quadrant. (Ae) EL4.F15 cells were incubated with effector cells for 30, 60, or 120 min at a 10:1 effector/target ratio, and PS exposure and PI uptake or ΔΨm loss and ROS generation were analyzed as described in Ac and Ad. Data presented are the mean of at least three independent experiments and are given as the difference of percentages in the presence versus the absence of gp33 ± SD. (B) a, same as Aa; b, same as Ab; c, target cell death (open symbols, −gp33; closed symbols, +gp33) was monitored after incubation of CML for 2 h at an effector/target cell ratio of 10:1 by the cell survival assay. Bd, same as Ac; Be, same as Ad.
Mentions: To study a physiologically relevant model for granule exocytosis–executed apoptotic cell death, in particular mediated by gzms and independent of Fas, we used an in vitro CTL-target cytotoxic assay. Ex vivo–derived virus-immune CD8+ T cells (day eight after infection) from B6 mice or those deficient in gzm(s) (gzmA−/−, gzmB−/−, and gzmA×B−/−) or perf (perf−/−), infected with LCMV 8 d before, were used as effector cells. As target cell, the tumor cell line EL4.F15 was chosen because it is Fas resistant and at the same time sensitive to both gzmA- and gzmB-induced apoptosis (Pardo et al., 2002). Confirming previous studies, splenocytes from 8-d LCMV-immune B6, gzmA−/−, and gzmB−/− mice contained similar numbers (11, 6, and 9%, respectively) of gp33/D btetramer+/CD8+ T cells (Fig. 1 Aa). As shown previously (Simon et al., 1997; Pardo et al., 2002), enriched CD8+ T cells from all three mouse strains readily induced specific nucleolysis in gp33-pulsed EL4.F15 target cells (4-h assay), which was similar in magnitude for B6 and gzmA−/− and somewhat lower for gzmB−/− T cells (Fig. 1 Ab).

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