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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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Cell death induced by B6, gzmA−/−, and gzmB−/− CTL is not affected by caspase inhibitors or ROS scavengers. (A) EL4.F15 cells were incubated with ex vivo LCMV-immune CD8+ T cells (MACS selected, ≥95% CD8+ cells) from B6, gzmA−/−, or gzmB−/− mice for 4 h at a 10:1 effector/target ratio, in the presence (opened symbols) or absence (closed symbols) of the viral protein gp33 (top panels). Alternatively, gp33-pulsed EL4.F15 cells were incubated with the respective effector cell populations in the presence of either 100 μM Z-VAD-fmk (square), 100 μM Z-DEVD-fmk (middle, triangle), or 15 mM NAC (bottom, circle). Subsequently, target cell death was monitored by propagating CML cultures at conditions unsuitable for CTL. (Ba) L1210.Fas cells were cultured in the presence or absence of αFas antibody (Jo-2; 1 μg/ml) for 20 h; cell cultures supplemented with Jo-2 were in addition treated with either Z-VAD-fmk or Z-DEVD-fmk (100 μM) or with 24-h SN of effector cell-target cell cultures (A; B6 CD8+ T cells +EL4.F15 + gp33). Cell death was analyzed by Trypan blue exclusion and results are expressed as the mean ± SD of two independent experiments. (Bb) EL4.F15 cells were cultured in the presence or absence of gliotoxin (100 or 200 μM) for 3 h; cell cultures supplemented with gliotoxin were in addition treated with either 15 mM NAC or with 24 h SN of effector cell-target cell cultures (A; B6 CD8+ T cells +EL4.F15 + gp33). Generation of ROS was analyzed by 2-HE staining. Data shown are representative of three independent experiments.
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fig6: Cell death induced by B6, gzmA−/−, and gzmB−/− CTL is not affected by caspase inhibitors or ROS scavengers. (A) EL4.F15 cells were incubated with ex vivo LCMV-immune CD8+ T cells (MACS selected, ≥95% CD8+ cells) from B6, gzmA−/−, or gzmB−/− mice for 4 h at a 10:1 effector/target ratio, in the presence (opened symbols) or absence (closed symbols) of the viral protein gp33 (top panels). Alternatively, gp33-pulsed EL4.F15 cells were incubated with the respective effector cell populations in the presence of either 100 μM Z-VAD-fmk (square), 100 μM Z-DEVD-fmk (middle, triangle), or 15 mM NAC (bottom, circle). Subsequently, target cell death was monitored by propagating CML cultures at conditions unsuitable for CTL. (Ba) L1210.Fas cells were cultured in the presence or absence of αFas antibody (Jo-2; 1 μg/ml) for 20 h; cell cultures supplemented with Jo-2 were in addition treated with either Z-VAD-fmk or Z-DEVD-fmk (100 μM) or with 24-h SN of effector cell-target cell cultures (A; B6 CD8+ T cells +EL4.F15 + gp33). Cell death was analyzed by Trypan blue exclusion and results are expressed as the mean ± SD of two independent experiments. (Bb) EL4.F15 cells were cultured in the presence or absence of gliotoxin (100 or 200 μM) for 3 h; cell cultures supplemented with gliotoxin were in addition treated with either 15 mM NAC or with 24 h SN of effector cell-target cell cultures (A; B6 CD8+ T cells +EL4.F15 + gp33). Generation of ROS was analyzed by 2-HE staining. Data shown are representative of three independent experiments.

Mentions: To further evaluate the requirement of caspase activation or ROS in gzm-mediated target cell death, LCMV-immune CD8+ T cells from either B6 gzmA−/− or gzmB−/− mice were incubated with gp33 peptide or mock-treated EL4.F15 cells (Fig. 6 A, top) or in the presence of either Z-VAD-fmk or Z-DEVD-fmk (Fig. 6 A, middle) or after pretreatment of target cells with NAC (Fig. 6 A, bottom). Target cell survival was estimated by extended culture under growth conditions unsuitable for CTL and by adding fresh inhibitors every 24 h. Neither treatment of target cells with Z-VAD-fmk nor Z-DEVD-fmk was able to rescue target cells from death, irrespective of the CTL population used. Furthermore, pretreatment of EL4.F15 cells with NAC did not prevent their death by either B6, gzmA−/−, or gzmB−/− CD8+ T cells. The lack of inhibition of target cell death was most likely not due to loss of inhibitor activity of any of the three agents. This is indicated by the finding that supernatant of cell cultures still retained most of the inhibitor activity after 24 h of culture, as tested by its effect on either anti-Fas mAb-mediated killing of L1210.Fas cells (Fig. 6 Ba, for Z-VAD-fmk and Z-DEVD-fmk activities) or on generation of gliotoxin-induced ROS in EL4.F15 cells (Fig. 6 Bb, for NAC activity). However, even with this control, the possibility that during CTL-mediated apoptosis caspases were not completely inhibited and contributed to cell death cannot be formally excluded.


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)

Cell death induced by B6, gzmA−/−, and gzmB−/− CTL is not affected by caspase inhibitors or ROS scavengers. (A) EL4.F15 cells were incubated with ex vivo LCMV-immune CD8+ T cells (MACS selected, ≥95% CD8+ cells) from B6, gzmA−/−, or gzmB−/− mice for 4 h at a 10:1 effector/target ratio, in the presence (opened symbols) or absence (closed symbols) of the viral protein gp33 (top panels). Alternatively, gp33-pulsed EL4.F15 cells were incubated with the respective effector cell populations in the presence of either 100 μM Z-VAD-fmk (square), 100 μM Z-DEVD-fmk (middle, triangle), or 15 mM NAC (bottom, circle). Subsequently, target cell death was monitored by propagating CML cultures at conditions unsuitable for CTL. (Ba) L1210.Fas cells were cultured in the presence or absence of αFas antibody (Jo-2; 1 μg/ml) for 20 h; cell cultures supplemented with Jo-2 were in addition treated with either Z-VAD-fmk or Z-DEVD-fmk (100 μM) or with 24-h SN of effector cell-target cell cultures (A; B6 CD8+ T cells +EL4.F15 + gp33). Cell death was analyzed by Trypan blue exclusion and results are expressed as the mean ± SD of two independent experiments. (Bb) EL4.F15 cells were cultured in the presence or absence of gliotoxin (100 or 200 μM) for 3 h; cell cultures supplemented with gliotoxin were in addition treated with either 15 mM NAC or with 24 h SN of effector cell-target cell cultures (A; B6 CD8+ T cells +EL4.F15 + gp33). Generation of ROS was analyzed by 2-HE staining. Data shown are representative of three independent experiments.
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fig6: Cell death induced by B6, gzmA−/−, and gzmB−/− CTL is not affected by caspase inhibitors or ROS scavengers. (A) EL4.F15 cells were incubated with ex vivo LCMV-immune CD8+ T cells (MACS selected, ≥95% CD8+ cells) from B6, gzmA−/−, or gzmB−/− mice for 4 h at a 10:1 effector/target ratio, in the presence (opened symbols) or absence (closed symbols) of the viral protein gp33 (top panels). Alternatively, gp33-pulsed EL4.F15 cells were incubated with the respective effector cell populations in the presence of either 100 μM Z-VAD-fmk (square), 100 μM Z-DEVD-fmk (middle, triangle), or 15 mM NAC (bottom, circle). Subsequently, target cell death was monitored by propagating CML cultures at conditions unsuitable for CTL. (Ba) L1210.Fas cells were cultured in the presence or absence of αFas antibody (Jo-2; 1 μg/ml) for 20 h; cell cultures supplemented with Jo-2 were in addition treated with either Z-VAD-fmk or Z-DEVD-fmk (100 μM) or with 24-h SN of effector cell-target cell cultures (A; B6 CD8+ T cells +EL4.F15 + gp33). Cell death was analyzed by Trypan blue exclusion and results are expressed as the mean ± SD of two independent experiments. (Bb) EL4.F15 cells were cultured in the presence or absence of gliotoxin (100 or 200 μM) for 3 h; cell cultures supplemented with gliotoxin were in addition treated with either 15 mM NAC or with 24 h SN of effector cell-target cell cultures (A; B6 CD8+ T cells +EL4.F15 + gp33). Generation of ROS was analyzed by 2-HE staining. Data shown are representative of three independent experiments.
Mentions: To further evaluate the requirement of caspase activation or ROS in gzm-mediated target cell death, LCMV-immune CD8+ T cells from either B6 gzmA−/− or gzmB−/− mice were incubated with gp33 peptide or mock-treated EL4.F15 cells (Fig. 6 A, top) or in the presence of either Z-VAD-fmk or Z-DEVD-fmk (Fig. 6 A, middle) or after pretreatment of target cells with NAC (Fig. 6 A, bottom). Target cell survival was estimated by extended culture under growth conditions unsuitable for CTL and by adding fresh inhibitors every 24 h. Neither treatment of target cells with Z-VAD-fmk nor Z-DEVD-fmk was able to rescue target cells from death, irrespective of the CTL population used. Furthermore, pretreatment of EL4.F15 cells with NAC did not prevent their death by either B6, gzmA−/−, or gzmB−/− CD8+ T cells. The lack of inhibition of target cell death was most likely not due to loss of inhibitor activity of any of the three agents. This is indicated by the finding that supernatant of cell cultures still retained most of the inhibitor activity after 24 h of culture, as tested by its effect on either anti-Fas mAb-mediated killing of L1210.Fas cells (Fig. 6 Ba, for Z-VAD-fmk and Z-DEVD-fmk activities) or on generation of gliotoxin-induced ROS in EL4.F15 cells (Fig. 6 Bb, for NAC activity). However, even with this control, the possibility that during CTL-mediated apoptosis caspases were not completely inhibited and contributed to cell death cannot be formally excluded.

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