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Early activation of caspases during T lymphocyte stimulation results in selective substrate cleavage in nonapoptotic cells.

Alam A, Cohen LY, Aouad S, Sékaly RP - J. Exp. Med. (1999)

Bottom Line: Moreover, T cell activation triggers the selective processing and activation of downstream caspases (caspase-3, -6, and -7), but not caspase-1, -2, or -4, as demonstrated even in intact cells using a cell-permeable fluorescent substrate.The pathway leading to caspase activation involves death receptors and caspase-8, which is also processed after TCR triggering, but not caspase-9, which remains as a proenzyme.Most importantly, caspase activity results in a selective substrate specificity, since poly(ADP-ribose) polymerase (PARP), lamin B, and Wee1 kinase, but not DNA fragmentation factor (DFF45) or replication factor C (RFC140), are processed.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire d'Immunologie, Institut de Recherches Cliniques de Montréal, Montréal, Québec H2W 1R7, Canada.

ABSTRACT
Apoptosis induced by T cell receptor (TCR) triggering in T lymphocytes involves activation of cysteine proteases of the caspase family through their proteolytic processing. Caspase-3 cleavage was also reported during T cell stimulation in the absence of apoptosis, although the physiological relevance of this response remains unclear. We show here that the caspase inhibitor benzyloxycarbonyl (Cbz)-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) blocks proliferation, major histocompatibility complex class II expression, and blastic transformation during stimulation of peripheral blood lymphocytes. Moreover, T cell activation triggers the selective processing and activation of downstream caspases (caspase-3, -6, and -7), but not caspase-1, -2, or -4, as demonstrated even in intact cells using a cell-permeable fluorescent substrate. Caspase-3 processing occurs in different T cell subsets (CD4(+), CD8(+), CD45RA(+), and CD45RO(+)), and in activated B lymphocytes. The pathway leading to caspase activation involves death receptors and caspase-8, which is also processed after TCR triggering, but not caspase-9, which remains as a proenzyme. Most importantly, caspase activity results in a selective substrate specificity, since poly(ADP-ribose) polymerase (PARP), lamin B, and Wee1 kinase, but not DNA fragmentation factor (DFF45) or replication factor C (RFC140), are processed. Caspase and substrate processing occur in nonapoptotic lymphocytes. Thus, caspase activation is an early and physiological response in viable, stimulated lymphocytes, and appears to be involved in early steps of lymphocyte activation.

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Caspase-3 activation occurs in various lymphocyte subsets. (A and B) Caspase cleavage is detected in CD4+, CD8+, CD45RO+, or CD45RA+ lymphocytes. PBMCs were stimulated (+) or not (−) with anti-CD3 and IL-2 for 4 d at 37°C, and stained for CD4, CD8, CD45RO, or CD45RA expression. Each subset was sorted by flow cytometry after gating on living cells on the basis of forward/side scatter. The purity of sorted cells was >99%. Total population (Tot) or sorted cells were then lysed as in the legend to Fig. 3, and subjected to Western blot analysis for caspase-3 processing. (C) Caspase-3 is cleaved after B cell receptor triggering. Fresh PBMCs (To) were cultured with medium alone (NS), anti-CD3 and IL-2 (CD3), or SAC for 4 d, and whole PBMCs (lane 1–3) or sorted CD19+ B lymphocytes (lane 4) were lysed in Laemmli buffer and subjected to Western blot analysis using the anti–caspase-3 antibody. After cell sorting, viability was >95% as assessed by trypan blue exclusion. These results are representative of four (A and B) and two (C) independent experiments with PBMCs from different individuals.
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Figure 3: Caspase-3 activation occurs in various lymphocyte subsets. (A and B) Caspase cleavage is detected in CD4+, CD8+, CD45RO+, or CD45RA+ lymphocytes. PBMCs were stimulated (+) or not (−) with anti-CD3 and IL-2 for 4 d at 37°C, and stained for CD4, CD8, CD45RO, or CD45RA expression. Each subset was sorted by flow cytometry after gating on living cells on the basis of forward/side scatter. The purity of sorted cells was >99%. Total population (Tot) or sorted cells were then lysed as in the legend to Fig. 3, and subjected to Western blot analysis for caspase-3 processing. (C) Caspase-3 is cleaved after B cell receptor triggering. Fresh PBMCs (To) were cultured with medium alone (NS), anti-CD3 and IL-2 (CD3), or SAC for 4 d, and whole PBMCs (lane 1–3) or sorted CD19+ B lymphocytes (lane 4) were lysed in Laemmli buffer and subjected to Western blot analysis using the anti–caspase-3 antibody. After cell sorting, viability was >95% as assessed by trypan blue exclusion. These results are representative of four (A and B) and two (C) independent experiments with PBMCs from different individuals.

Mentions: The complex pattern of caspase-3 subunits suggested that they could originate from several proenzyme species observed in nonstimulated cells (Fig. 2 B), or from different cell subsets. To distinguish between these two possibilities, sorting of CD4+, CD8+, CD45RO+, and CD45RA+ cells was performed on viable, blastic cells. Processing of caspase-3 was similar in both CD4 and CD8 T cell subsets and occurred not only in CD45RO+, but also in the remaining CD45RA+/CD25+ cells (Fig. 3a and Fig. b), further confirming that caspase activation was an early event after TCR triggering. When PBMCs were stimulated for 4 d with the B cell mitogen SAC, and B lymphocytes were purified by CD19+ cell sorting, caspase-3 cleavage was observed in sorted, viable cells and resulted in the production of the 20-kD doublet and the 17-kD large subunit as in activated T lymphocytes (Fig. 3 C). These results indicate that caspase-3 processing occurs in several T cell subsets and in activated B cells.


Early activation of caspases during T lymphocyte stimulation results in selective substrate cleavage in nonapoptotic cells.

Alam A, Cohen LY, Aouad S, Sékaly RP - J. Exp. Med. (1999)

Caspase-3 activation occurs in various lymphocyte subsets. (A and B) Caspase cleavage is detected in CD4+, CD8+, CD45RO+, or CD45RA+ lymphocytes. PBMCs were stimulated (+) or not (−) with anti-CD3 and IL-2 for 4 d at 37°C, and stained for CD4, CD8, CD45RO, or CD45RA expression. Each subset was sorted by flow cytometry after gating on living cells on the basis of forward/side scatter. The purity of sorted cells was >99%. Total population (Tot) or sorted cells were then lysed as in the legend to Fig. 3, and subjected to Western blot analysis for caspase-3 processing. (C) Caspase-3 is cleaved after B cell receptor triggering. Fresh PBMCs (To) were cultured with medium alone (NS), anti-CD3 and IL-2 (CD3), or SAC for 4 d, and whole PBMCs (lane 1–3) or sorted CD19+ B lymphocytes (lane 4) were lysed in Laemmli buffer and subjected to Western blot analysis using the anti–caspase-3 antibody. After cell sorting, viability was >95% as assessed by trypan blue exclusion. These results are representative of four (A and B) and two (C) independent experiments with PBMCs from different individuals.
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Related In: Results  -  Collection

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Figure 3: Caspase-3 activation occurs in various lymphocyte subsets. (A and B) Caspase cleavage is detected in CD4+, CD8+, CD45RO+, or CD45RA+ lymphocytes. PBMCs were stimulated (+) or not (−) with anti-CD3 and IL-2 for 4 d at 37°C, and stained for CD4, CD8, CD45RO, or CD45RA expression. Each subset was sorted by flow cytometry after gating on living cells on the basis of forward/side scatter. The purity of sorted cells was >99%. Total population (Tot) or sorted cells were then lysed as in the legend to Fig. 3, and subjected to Western blot analysis for caspase-3 processing. (C) Caspase-3 is cleaved after B cell receptor triggering. Fresh PBMCs (To) were cultured with medium alone (NS), anti-CD3 and IL-2 (CD3), or SAC for 4 d, and whole PBMCs (lane 1–3) or sorted CD19+ B lymphocytes (lane 4) were lysed in Laemmli buffer and subjected to Western blot analysis using the anti–caspase-3 antibody. After cell sorting, viability was >95% as assessed by trypan blue exclusion. These results are representative of four (A and B) and two (C) independent experiments with PBMCs from different individuals.
Mentions: The complex pattern of caspase-3 subunits suggested that they could originate from several proenzyme species observed in nonstimulated cells (Fig. 2 B), or from different cell subsets. To distinguish between these two possibilities, sorting of CD4+, CD8+, CD45RO+, and CD45RA+ cells was performed on viable, blastic cells. Processing of caspase-3 was similar in both CD4 and CD8 T cell subsets and occurred not only in CD45RO+, but also in the remaining CD45RA+/CD25+ cells (Fig. 3a and Fig. b), further confirming that caspase activation was an early event after TCR triggering. When PBMCs were stimulated for 4 d with the B cell mitogen SAC, and B lymphocytes were purified by CD19+ cell sorting, caspase-3 cleavage was observed in sorted, viable cells and resulted in the production of the 20-kD doublet and the 17-kD large subunit as in activated T lymphocytes (Fig. 3 C). These results indicate that caspase-3 processing occurs in several T cell subsets and in activated B cells.

Bottom Line: Moreover, T cell activation triggers the selective processing and activation of downstream caspases (caspase-3, -6, and -7), but not caspase-1, -2, or -4, as demonstrated even in intact cells using a cell-permeable fluorescent substrate.The pathway leading to caspase activation involves death receptors and caspase-8, which is also processed after TCR triggering, but not caspase-9, which remains as a proenzyme.Most importantly, caspase activity results in a selective substrate specificity, since poly(ADP-ribose) polymerase (PARP), lamin B, and Wee1 kinase, but not DNA fragmentation factor (DFF45) or replication factor C (RFC140), are processed.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire d'Immunologie, Institut de Recherches Cliniques de Montréal, Montréal, Québec H2W 1R7, Canada.

ABSTRACT
Apoptosis induced by T cell receptor (TCR) triggering in T lymphocytes involves activation of cysteine proteases of the caspase family through their proteolytic processing. Caspase-3 cleavage was also reported during T cell stimulation in the absence of apoptosis, although the physiological relevance of this response remains unclear. We show here that the caspase inhibitor benzyloxycarbonyl (Cbz)-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) blocks proliferation, major histocompatibility complex class II expression, and blastic transformation during stimulation of peripheral blood lymphocytes. Moreover, T cell activation triggers the selective processing and activation of downstream caspases (caspase-3, -6, and -7), but not caspase-1, -2, or -4, as demonstrated even in intact cells using a cell-permeable fluorescent substrate. Caspase-3 processing occurs in different T cell subsets (CD4(+), CD8(+), CD45RA(+), and CD45RO(+)), and in activated B lymphocytes. The pathway leading to caspase activation involves death receptors and caspase-8, which is also processed after TCR triggering, but not caspase-9, which remains as a proenzyme. Most importantly, caspase activity results in a selective substrate specificity, since poly(ADP-ribose) polymerase (PARP), lamin B, and Wee1 kinase, but not DNA fragmentation factor (DFF45) or replication factor C (RFC140), are processed. Caspase and substrate processing occur in nonapoptotic lymphocytes. Thus, caspase activation is an early and physiological response in viable, stimulated lymphocytes, and appears to be involved in early steps of lymphocyte activation.

Show MeSH
Related in: MedlinePlus