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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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Ape-1 degradation is induced by B6, gzmA−/−, gzmB−/−, and gzmA×B−/− but not perf−/− CTL. EL4.F15 cells were incubated with ex vivo virus-specific CD8+ cells (MACS selected, ≥95% CD8+ cells) from wild-type B6, gzmA−/−, or gzmB−/− mice (A) or from perf−/− or gzmA×B−/− mice (B) for 4 h at a 10:1 effector/target ratio in the presence (green) or absence (blue) of gp33 peptide. Subsequently, Ape-1 expression was analyzed in the cell population negative for CD8 expression by two-color flow cytometry. Red histograms indicate the cells labeled with the control isotype antibody. Numbers correspond to the percentage of cells positive for the labeling in each case, indicated by the horizontal bars.
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fig5: Ape-1 degradation is induced by B6, gzmA−/−, gzmB−/−, and gzmA×B−/− but not perf−/− CTL. EL4.F15 cells were incubated with ex vivo virus-specific CD8+ cells (MACS selected, ≥95% CD8+ cells) from wild-type B6, gzmA−/−, or gzmB−/− mice (A) or from perf−/− or gzmA×B−/− mice (B) for 4 h at a 10:1 effector/target ratio in the presence (green) or absence (blue) of gp33 peptide. Subsequently, Ape-1 expression was analyzed in the cell population negative for CD8 expression by two-color flow cytometry. Red histograms indicate the cells labeled with the control isotype antibody. Numbers correspond to the percentage of cells positive for the labeling in each case, indicated by the horizontal bars.

Mentions: To test if the apurinic endonuclease-1, Ape-1, a known substrate of gzmA (Fan et al., 2003b), is cleaved during CTL-mediated lysis of targets, LCMV-immune CD8+ T cells from either B6, gzmA−/−, or gzmB−/− mice were incubated with gp33-pulsed EL4.F15 cells (4 h), and changes in Ape-1 expression were tested by monitoring intracellular staining of CD8-negative target cells with FITC-labeled Ape-1–reactive polyclonal antibody H-300. As shown in Fig. 5 A, 95% of mock-treated EL4.F15 cells stained with the antibody preparation. When gp33-pulsed target cells were incubated with either B6-, gzmA−/−-, or gzmB−/−-derived CTL, the numbers of Ape-1–positive cells were significantly reduced, compared with control mock-treated targets (from 85 to 65%, 74 to 48%, and 76 to 54%, respectively; Fig. 5 A). When, in a further experiment (Fig. 5 B), B6 CTL were compared with perf−/− and gzmA×B−/− CTL using similar conditions, the number of target cells staining for Ape-1 were also significantly reduced by gzmA×B−/− CTL (from 77 to 66%), though to a lesser extend compared with B6 CTL (from 79 to 54%). No reduction occurred with perf−/−-derived CTL. These results indicate that cleavage of Ape-1 in targets, following CTL attack, is strictly dependent on perf and also occurs, at least partially, in the absence of both gzms.


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)

Ape-1 degradation is induced by B6, gzmA−/−, gzmB−/−, and gzmA×B−/− but not perf−/− CTL. EL4.F15 cells were incubated with ex vivo virus-specific CD8+ cells (MACS selected, ≥95% CD8+ cells) from wild-type B6, gzmA−/−, or gzmB−/− mice (A) or from perf−/− or gzmA×B−/− mice (B) for 4 h at a 10:1 effector/target ratio in the presence (green) or absence (blue) of gp33 peptide. Subsequently, Ape-1 expression was analyzed in the cell population negative for CD8 expression by two-color flow cytometry. Red histograms indicate the cells labeled with the control isotype antibody. Numbers correspond to the percentage of cells positive for the labeling in each case, indicated by the horizontal bars.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172484&req=5

fig5: Ape-1 degradation is induced by B6, gzmA−/−, gzmB−/−, and gzmA×B−/− but not perf−/− CTL. EL4.F15 cells were incubated with ex vivo virus-specific CD8+ cells (MACS selected, ≥95% CD8+ cells) from wild-type B6, gzmA−/−, or gzmB−/− mice (A) or from perf−/− or gzmA×B−/− mice (B) for 4 h at a 10:1 effector/target ratio in the presence (green) or absence (blue) of gp33 peptide. Subsequently, Ape-1 expression was analyzed in the cell population negative for CD8 expression by two-color flow cytometry. Red histograms indicate the cells labeled with the control isotype antibody. Numbers correspond to the percentage of cells positive for the labeling in each case, indicated by the horizontal bars.
Mentions: To test if the apurinic endonuclease-1, Ape-1, a known substrate of gzmA (Fan et al., 2003b), is cleaved during CTL-mediated lysis of targets, LCMV-immune CD8+ T cells from either B6, gzmA−/−, or gzmB−/− mice were incubated with gp33-pulsed EL4.F15 cells (4 h), and changes in Ape-1 expression were tested by monitoring intracellular staining of CD8-negative target cells with FITC-labeled Ape-1–reactive polyclonal antibody H-300. As shown in Fig. 5 A, 95% of mock-treated EL4.F15 cells stained with the antibody preparation. When gp33-pulsed target cells were incubated with either B6-, gzmA−/−-, or gzmB−/−-derived CTL, the numbers of Ape-1–positive cells were significantly reduced, compared with control mock-treated targets (from 85 to 65%, 74 to 48%, and 76 to 54%, respectively; Fig. 5 A). When, in a further experiment (Fig. 5 B), B6 CTL were compared with perf−/− and gzmA×B−/− CTL using similar conditions, the number of target cells staining for Ape-1 were also significantly reduced by gzmA×B−/− CTL (from 77 to 66%), though to a lesser extend compared with B6 CTL (from 79 to 54%). No reduction occurred with perf−/−-derived CTL. These results indicate that cleavage of Ape-1 in targets, following CTL attack, is strictly dependent on perf and also occurs, at least partially, in the absence of both gzms.

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