Limits...
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.

Show MeSH

Related in: MedlinePlus

B6 and gzmA−/− but neither gzmB−/−, perf−/−, nor gzmA×B−/− CTL are able to induce caspase 3 and 9 activity. EL4.F15 cells were incubated with ex vivo virus-specific CD8+ cells (MACS selected, ≥95% CD8+ cells) from either B6, gzmA−/−, or gzmB−/− (A) or from B6, perf−/−, or gzmA×B−/− (B) mice (2 h, 10:1 effector/target ratio), in the presence (red and green) or absence (black) of the LCMV peptide gp33, as indicated. Activation of caspase 3 was monitored with either an FITC-labeled mAb against the active form of the enzyme (Aa and Ba) or the specific fluorescent substrates SRH-DEVD-fmk (Ab and Bb), by two-color flow cytometry in the cell population negative for CD8 expression (target cells) as described in Materials and methods. Similarly, activation of caspase 9 was monitored with the specific fluorescent substrates FAM-LEHD-fmk (Ac and Bc). CMLs were also developed in the presence of a blocking mAb anti-FasL, where indicated (Aa, bottom, dark blue; and Ab and Ac, middle, dark blue), and in the presence of the caspase 3 (100 μM Z-DEVD-fmk; b) or caspase 9 (100 μM Z-LEHD-fmk; c) inhibitors (Ab, Ac, Bb, and Bc, bottom, orange). Numbers correspond to the percentage of cells positive for the labeling in each case, indicated by the horizontal bars shown in the top panels.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172484&req=5

fig2: B6 and gzmA−/− but neither gzmB−/−, perf−/−, nor gzmA×B−/− CTL are able to induce caspase 3 and 9 activity. EL4.F15 cells were incubated with ex vivo virus-specific CD8+ cells (MACS selected, ≥95% CD8+ cells) from either B6, gzmA−/−, or gzmB−/− (A) or from B6, perf−/−, or gzmA×B−/− (B) mice (2 h, 10:1 effector/target ratio), in the presence (red and green) or absence (black) of the LCMV peptide gp33, as indicated. Activation of caspase 3 was monitored with either an FITC-labeled mAb against the active form of the enzyme (Aa and Ba) or the specific fluorescent substrates SRH-DEVD-fmk (Ab and Bb), by two-color flow cytometry in the cell population negative for CD8 expression (target cells) as described in Materials and methods. Similarly, activation of caspase 9 was monitored with the specific fluorescent substrates FAM-LEHD-fmk (Ac and Bc). CMLs were also developed in the presence of a blocking mAb anti-FasL, where indicated (Aa, bottom, dark blue; and Ab and Ac, middle, dark blue), and in the presence of the caspase 3 (100 μM Z-DEVD-fmk; b) or caspase 9 (100 μM Z-LEHD-fmk; c) inhibitors (Ab, Ac, Bb, and Bc, bottom, orange). 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: LCMV-immune CD8+ T cells from B6, gzmA−/−, and gzmB−/− mice were incubated with gp33-pulsed or mock-treated EL4.F15 target cells (2 h), and activation of intracellular caspase 3 was assessed using the mAb C92-605. Fig. 2 A shows that effector cells from B6 and gzmA−/− mice specifically and comparably induced activation of caspase 3 in ∼30% of target cells, whereas those from gzmB−/− mice did not (Fig. 2 Aa, top). The presence of blocking anti-FasL mAb did not alter activation of caspase 3 by the two CD8+ T cell populations (Fig. 2 Aa, bottom). This excludes a contribution of the Fas pathway and suggests that the CTL-mediated effect is solely elicited via the exocytosis pathway (Pardo et al., 2002). When caspase activation in EL4.F15 cells was tested under similar conditions by applying intracellular staining with the caspase 3–specific agent SRH-DEVD-fmk, comparable results were obtained (Fig. 2 Ab, top). Gp33-pulsed but not mock-treated target cells incubated with either B6- or gzmA−/−-derived LCMV-immune CD8+ T cells stained positive for active caspase 3 (44 vs. 6% and 60 vs. 15%, respectively). Again, the presence of blocking anti-FasL mAb did not alter activation of caspase 3 by B6 and gzmA−/− CTL (Fig. 2 Ab, middle, + gp33 peptide). In both cases, pretreatment of targets with the specific inhibitor Z-DEVD-fmk (Fig. 2 Ab, orange) leads to a drastic reduction in positively staining cells (Fig. 2 Ab, bottom). Surprisingly and in contrast to results obtained using the specific mAb for active caspase 3, a small fraction of gp33-pulsed EL4.F15 cells (30 vs. 17% for mock-treated cells) also stained positive with SRH-DEVD-fmk after incubation with gzmB−/− LCMV-immune CD8+ T cells (Fig. 2 Ab, top). However, the number of EL4.F15 cells staining for active caspase 3 were only marginally reduced by the specific inhibitor Z-DEVD-fmk (Fig. 2 Ab, bottom). This finding indicates that the apparent caspase 3 activation observed by using the fluorescent substrate (but not the mAb) is due to unspecific binding of the substrate to a molecule distinct from caspase 3 (Amstad et al., 2001).


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)

B6 and gzmA−/− but neither gzmB−/−, perf−/−, nor gzmA×B−/− CTL are able to induce caspase 3 and 9 activity. EL4.F15 cells were incubated with ex vivo virus-specific CD8+ cells (MACS selected, ≥95% CD8+ cells) from either B6, gzmA−/−, or gzmB−/− (A) or from B6, perf−/−, or gzmA×B−/− (B) mice (2 h, 10:1 effector/target ratio), in the presence (red and green) or absence (black) of the LCMV peptide gp33, as indicated. Activation of caspase 3 was monitored with either an FITC-labeled mAb against the active form of the enzyme (Aa and Ba) or the specific fluorescent substrates SRH-DEVD-fmk (Ab and Bb), by two-color flow cytometry in the cell population negative for CD8 expression (target cells) as described in Materials and methods. Similarly, activation of caspase 9 was monitored with the specific fluorescent substrates FAM-LEHD-fmk (Ac and Bc). CMLs were also developed in the presence of a blocking mAb anti-FasL, where indicated (Aa, bottom, dark blue; and Ab and Ac, middle, dark blue), and in the presence of the caspase 3 (100 μM Z-DEVD-fmk; b) or caspase 9 (100 μM Z-LEHD-fmk; c) inhibitors (Ab, Ac, Bb, and Bc, bottom, orange). Numbers correspond to the percentage of cells positive for the labeling in each case, indicated by the horizontal bars shown in the top panels.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: B6 and gzmA−/− but neither gzmB−/−, perf−/−, nor gzmA×B−/− CTL are able to induce caspase 3 and 9 activity. EL4.F15 cells were incubated with ex vivo virus-specific CD8+ cells (MACS selected, ≥95% CD8+ cells) from either B6, gzmA−/−, or gzmB−/− (A) or from B6, perf−/−, or gzmA×B−/− (B) mice (2 h, 10:1 effector/target ratio), in the presence (red and green) or absence (black) of the LCMV peptide gp33, as indicated. Activation of caspase 3 was monitored with either an FITC-labeled mAb against the active form of the enzyme (Aa and Ba) or the specific fluorescent substrates SRH-DEVD-fmk (Ab and Bb), by two-color flow cytometry in the cell population negative for CD8 expression (target cells) as described in Materials and methods. Similarly, activation of caspase 9 was monitored with the specific fluorescent substrates FAM-LEHD-fmk (Ac and Bc). CMLs were also developed in the presence of a blocking mAb anti-FasL, where indicated (Aa, bottom, dark blue; and Ab and Ac, middle, dark blue), and in the presence of the caspase 3 (100 μM Z-DEVD-fmk; b) or caspase 9 (100 μM Z-LEHD-fmk; c) inhibitors (Ab, Ac, Bb, and Bc, bottom, orange). 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: LCMV-immune CD8+ T cells from B6, gzmA−/−, and gzmB−/− mice were incubated with gp33-pulsed or mock-treated EL4.F15 target cells (2 h), and activation of intracellular caspase 3 was assessed using the mAb C92-605. Fig. 2 A shows that effector cells from B6 and gzmA−/− mice specifically and comparably induced activation of caspase 3 in ∼30% of target cells, whereas those from gzmB−/− mice did not (Fig. 2 Aa, top). The presence of blocking anti-FasL mAb did not alter activation of caspase 3 by the two CD8+ T cell populations (Fig. 2 Aa, bottom). This excludes a contribution of the Fas pathway and suggests that the CTL-mediated effect is solely elicited via the exocytosis pathway (Pardo et al., 2002). When caspase activation in EL4.F15 cells was tested under similar conditions by applying intracellular staining with the caspase 3–specific agent SRH-DEVD-fmk, comparable results were obtained (Fig. 2 Ab, top). Gp33-pulsed but not mock-treated target cells incubated with either B6- or gzmA−/−-derived LCMV-immune CD8+ T cells stained positive for active caspase 3 (44 vs. 6% and 60 vs. 15%, respectively). Again, the presence of blocking anti-FasL mAb did not alter activation of caspase 3 by B6 and gzmA−/− CTL (Fig. 2 Ab, middle, + gp33 peptide). In both cases, pretreatment of targets with the specific inhibitor Z-DEVD-fmk (Fig. 2 Ab, orange) leads to a drastic reduction in positively staining cells (Fig. 2 Ab, bottom). Surprisingly and in contrast to results obtained using the specific mAb for active caspase 3, a small fraction of gp33-pulsed EL4.F15 cells (30 vs. 17% for mock-treated cells) also stained positive with SRH-DEVD-fmk after incubation with gzmB−/− LCMV-immune CD8+ T cells (Fig. 2 Ab, top). However, the number of EL4.F15 cells staining for active caspase 3 were only marginally reduced by the specific inhibitor Z-DEVD-fmk (Fig. 2 Ab, bottom). This finding indicates that the apparent caspase 3 activation observed by using the fluorescent substrate (but not the mAb) is due to unspecific binding of the substrate to a molecule distinct from caspase 3 (Amstad et al., 2001).

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