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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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PS exposure induced by gzmB−/− CTL is blocked in the absence of ROS, but mitochondrial depolarization is still observed. Gp33-pulsed EL4.F15 cells were incubated with ex vivo virus-specific CD8+ cells (MACS selected, ≥95% CD8+ cells) from wild-type B6, gzmA−/−, and/or gzmB−/− mice for 2 h at a 10:1 effector/target ratio, in the presence or absence of the ROS scavenger NAC at 15 mM. 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. Results are the mean of two independent experiments and are given as the difference of percentages in the presence or absence of gp33 (SD was always lower that 5% of the mean). (E) Alternatively, CML cultures were incubated in the absence (red or green) or presence (blue) of NAC (15 mM). Caspase 3 activation was determined using an FITC-labeled mAb against the active form of the enzyme (left), whereas caspase 9 activity was determined by using the specific fluorescent substrate FAM-LEHD-fmk (right) by two-color flow cytometry in the cell population negative for CD8 expression (target cells). Numbers correspond to the percentage of cells positive for the labeling in each case, indicated by the horizontal bars.
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fig4: PS exposure induced by gzmB−/− CTL is blocked in the absence of ROS, but mitochondrial depolarization is still observed. Gp33-pulsed EL4.F15 cells were incubated with ex vivo virus-specific CD8+ cells (MACS selected, ≥95% CD8+ cells) from wild-type B6, gzmA−/−, and/or gzmB−/− mice for 2 h at a 10:1 effector/target ratio, in the presence or absence of the ROS scavenger NAC at 15 mM. 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. Results are the mean of two independent experiments and are given as the difference of percentages in the presence or absence of gp33 (SD was always lower that 5% of the mean). (E) Alternatively, CML cultures were incubated in the absence (red or green) or presence (blue) of NAC (15 mM). Caspase 3 activation was determined using an FITC-labeled mAb against the active form of the enzyme (left), whereas caspase 9 activity was determined by using the specific fluorescent substrate FAM-LEHD-fmk (right) by two-color flow cytometry in the cell population negative for CD8 expression (target cells). Numbers correspond to the percentage of cells positive for the labeling in each case, indicated by the horizontal bars.

Mentions: As shown in the first paragraph of Results, incubation of EL4.F15 cells with either B6, gzmA−/−, or gzmB−/− LCMV-immune CD8+ T cells leads to ROS generation. To evaluate the requirement for ROS generation in gzmA- and gzmB-mediated apoptotic processes, the respective CML cultures were incubated with targets (2 h) previously treated with the antioxidant N-acetylcysteine (NAC) and tested for the annexinV±/PI± phenotype, ΔΨm loss, and presence of active caspases 3 and 9. As expected, ROS formation is similarly suppressed after pretreatment of EL4.F15 cells with NAC, irrespective of the source of CTL (Fig. 4 D). The number of cells with the annexinV+/PI+ phenotype was significantly reduced in NAC-treated as compared with mock-treated EL4.F15 cells after culture with B6- and gzmA−/−-derived CTL and reduced to near background level when cultured with gzmB−/−-derived CTL (Fig. 4, A and B). NAC protected EL4.F15 cells only partially from ΔΨm loss, independent of the effector cell population (Fig. 4 C). Pretreatment of EL4.F15 cells with NAC had no effect on the activation of caspase 3, as monitored by mAb C92-605 fluorescence (Fig. 4 E, left), but resulted in considerably reduced activation of caspase 9 after incubation with either B6- or gzmA−/−-derived CTL, as determined by staining with FAM-LEHD-fmk (Fig. 4 E, right). Upon addition of both NAC and LEHD-fmk to cultures of B6 CTL and EL4.F15 target cells, inhibition of caspase 3 activation did not exceed that obtained with LEHD-fmk alone (Fig. 3 E and not depicted). The fact that suppression of plasma membrane perturbation (annexin+/PI± phenotype) by NAC is more pronounced when gzmB−/−- compared with gzmA−/−-derived CTL are used suggests that the generation of ROS is critical in gzmA-induced cell surface membrane perturbation, but less so in gzmB-mediated but 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)

PS exposure induced by gzmB−/− CTL is blocked in the absence of ROS, but mitochondrial depolarization is still observed. Gp33-pulsed EL4.F15 cells were incubated with ex vivo virus-specific CD8+ cells (MACS selected, ≥95% CD8+ cells) from wild-type B6, gzmA−/−, and/or gzmB−/− mice for 2 h at a 10:1 effector/target ratio, in the presence or absence of the ROS scavenger NAC at 15 mM. 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. Results are the mean of two independent experiments and are given as the difference of percentages in the presence or absence of gp33 (SD was always lower that 5% of the mean). (E) Alternatively, CML cultures were incubated in the absence (red or green) or presence (blue) of NAC (15 mM). Caspase 3 activation was determined using an FITC-labeled mAb against the active form of the enzyme (left), whereas caspase 9 activity was determined by using the specific fluorescent substrate FAM-LEHD-fmk (right) by two-color flow cytometry in the cell population negative for CD8 expression (target cells). Numbers correspond to the percentage of cells positive for the labeling in each case, indicated by the horizontal bars.
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Related In: Results  -  Collection

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fig4: PS exposure induced by gzmB−/− CTL is blocked in the absence of ROS, but mitochondrial depolarization is still observed. Gp33-pulsed EL4.F15 cells were incubated with ex vivo virus-specific CD8+ cells (MACS selected, ≥95% CD8+ cells) from wild-type B6, gzmA−/−, and/or gzmB−/− mice for 2 h at a 10:1 effector/target ratio, in the presence or absence of the ROS scavenger NAC at 15 mM. 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. Results are the mean of two independent experiments and are given as the difference of percentages in the presence or absence of gp33 (SD was always lower that 5% of the mean). (E) Alternatively, CML cultures were incubated in the absence (red or green) or presence (blue) of NAC (15 mM). Caspase 3 activation was determined using an FITC-labeled mAb against the active form of the enzyme (left), whereas caspase 9 activity was determined by using the specific fluorescent substrate FAM-LEHD-fmk (right) by two-color flow cytometry in the cell population negative for CD8 expression (target cells). Numbers correspond to the percentage of cells positive for the labeling in each case, indicated by the horizontal bars.
Mentions: As shown in the first paragraph of Results, incubation of EL4.F15 cells with either B6, gzmA−/−, or gzmB−/− LCMV-immune CD8+ T cells leads to ROS generation. To evaluate the requirement for ROS generation in gzmA- and gzmB-mediated apoptotic processes, the respective CML cultures were incubated with targets (2 h) previously treated with the antioxidant N-acetylcysteine (NAC) and tested for the annexinV±/PI± phenotype, ΔΨm loss, and presence of active caspases 3 and 9. As expected, ROS formation is similarly suppressed after pretreatment of EL4.F15 cells with NAC, irrespective of the source of CTL (Fig. 4 D). The number of cells with the annexinV+/PI+ phenotype was significantly reduced in NAC-treated as compared with mock-treated EL4.F15 cells after culture with B6- and gzmA−/−-derived CTL and reduced to near background level when cultured with gzmB−/−-derived CTL (Fig. 4, A and B). NAC protected EL4.F15 cells only partially from ΔΨm loss, independent of the effector cell population (Fig. 4 C). Pretreatment of EL4.F15 cells with NAC had no effect on the activation of caspase 3, as monitored by mAb C92-605 fluorescence (Fig. 4 E, left), but resulted in considerably reduced activation of caspase 9 after incubation with either B6- or gzmA−/−-derived CTL, as determined by staining with FAM-LEHD-fmk (Fig. 4 E, right). Upon addition of both NAC and LEHD-fmk to cultures of B6 CTL and EL4.F15 target cells, inhibition of caspase 3 activation did not exceed that obtained with LEHD-fmk alone (Fig. 3 E and not depicted). The fact that suppression of plasma membrane perturbation (annexin+/PI± phenotype) by NAC is more pronounced when gzmB−/−- compared with gzmA−/−-derived CTL are used suggests that the generation of ROS is critical in gzmA-induced cell surface membrane perturbation, but less so in gzmB-mediated but 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