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Specific activation of the cysteine protease CPP32 during the negative selection of T cells in the thymus.

Alam A, Braun MY, Hartgers F, Lesage S, Cohen L, Hugo P, Denis F, Sékaly RP - J. Exp. Med. (1997)

Bottom Line: This was observed both in anti-CD3- and dexamethasone-induced apoptosis.Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD.While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F.

View Article: PubMed Central - PubMed

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

ABSTRACT
Cysteine proteases of the CED-3 and ICE family have been recently proposed as the ultimate executioners in several mammalian cell death pathways. Among them, the cysteine protease CPP32 has been shown to participate in programmed cell death (PCD), or apoptosis, affecting lymphoid cells in vitro. In the thymus, negative selection is a mechanism through which developing thymocytes expressing a TcR with high affinity for self peptide-MHC complexes are eliminated by PCD. In order to investigate the role of CPP32 in thymic apoptosis, isolated thymocytes were submitted to cell surface CD3 crosslinking by immobilized anti-CD3 mAb or to dexamethasone treatment. Although apoptosis occurred in the absence or after crosslinking with anti-CD3 mAb, specific activation of CPP32, as assessed by the extent of proteolytic cleavage of the p32 zymogen, was only detected in thymocytes cultured in the presence of the immobilized antibody or dexamethasone. This activation was a very early event during apoptosis as it occurred before the exposure of phosphatidyl serine to the upper side of the cell membrane. This was observed both in anti-CD3- and dexamethasone-induced apoptosis. Moreover, using mice transgenic for pigeon cytochrome C (PCC)-specific TcR, we were able to show that, after injection of PCC, the activation of CPP32 and cleavage of its substrate occurred in thymocytes obtained from mice expressing a permissive MHC haplotype for PCC presentation (H-2k). Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD. While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F. Taken together, these results support the concept that CPP32 is among the earliest effectors of the pathway leading to negative selection of autoreactive thymocytes. Our results also suggest the involvement of a distinct CPP32-like cysteine protease in spontaneous apoptosis of thymocytes.

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The activation of CPP32 is a very early event of thymocyte  apoptosis. (A and B) Thymocytes from C56BL/6 mice were isolated and  incubated with dexamethasone. Samples were taken at 30-min intervals  and analyzed for levels of apoptosis with annexin V-FITC and PI (A).  Cell lysates were submitted to Western blotting analysis for the processing  of CPP32 (B). (C) Thymocytes were cultured for 20 h in the presence of  immobilized anti-CD3 mAb. Cells were then labeled with annexin  V-FITC, and annexin V− and annexin V+ cells were sorted at 4°C with  a FACStar® Plus. Sorted cells were lysed and extract contents were analyzed for processing of CPP32 by Western blotting.
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Figure 5: The activation of CPP32 is a very early event of thymocyte apoptosis. (A and B) Thymocytes from C56BL/6 mice were isolated and incubated with dexamethasone. Samples were taken at 30-min intervals and analyzed for levels of apoptosis with annexin V-FITC and PI (A). Cell lysates were submitted to Western blotting analysis for the processing of CPP32 (B). (C) Thymocytes were cultured for 20 h in the presence of immobilized anti-CD3 mAb. Cells were then labeled with annexin V-FITC, and annexin V− and annexin V+ cells were sorted at 4°C with a FACStar® Plus. Sorted cells were lysed and extract contents were analyzed for processing of CPP32 by Western blotting.

Mentions: As mentioned above, after 6 h of treatment with dexamethasone, usually 50–60% of thymocytes are in early apoptosis whereas 5–20% of cells are in late apoptosis. The massive activation of CPP32 at this time point (especially in the experiment presented in Fig. 1 C where only 6% of thymocytes are in late apoptosis), suggests that CPP32 activation occurs as soon as or before the exposure of the phosphatidylserine at the upper side of the cell membrane. The ability of zVAD-CH2F to inhibit annexin V staining and CPP32 activation in thymocytes (Fig. 4) further suggests that activation of cysteine proteases occurs before the loss of membrane asymmetry. To determine whether CPP32 activation is a very early event of thymocyte apoptosis, a time course of CPP32 activation was carried out on thymocytes stimulated with dexamethasone. As illustrated in Fig. 5 a, treatment with dexamethasone did not induce significant apoptosis, as evidenced by the absence of annexin V+ PI−, during the first 150 min. After this initial lag an abrupt increase in the relative number of cells in early apoptosis was noted. A plateau in percentage of annexin V+ PI− cells was reached at 4 h. Western blot analysis showed that activation of CPP32 (as indicated by the appearance of the p17 subunit) occurred 90 min after the addition of dexamethasone (Fig. 5 b). Moreover, this experiment confirmed that CPP32 activation is a very early event during dexamethasone-induced apoptosis since it takes place before the exposure of phosphatidylserine at the cell surface.


Specific activation of the cysteine protease CPP32 during the negative selection of T cells in the thymus.

Alam A, Braun MY, Hartgers F, Lesage S, Cohen L, Hugo P, Denis F, Sékaly RP - J. Exp. Med. (1997)

The activation of CPP32 is a very early event of thymocyte  apoptosis. (A and B) Thymocytes from C56BL/6 mice were isolated and  incubated with dexamethasone. Samples were taken at 30-min intervals  and analyzed for levels of apoptosis with annexin V-FITC and PI (A).  Cell lysates were submitted to Western blotting analysis for the processing  of CPP32 (B). (C) Thymocytes were cultured for 20 h in the presence of  immobilized anti-CD3 mAb. Cells were then labeled with annexin  V-FITC, and annexin V− and annexin V+ cells were sorted at 4°C with  a FACStar® Plus. Sorted cells were lysed and extract contents were analyzed for processing of CPP32 by Western blotting.
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Related In: Results  -  Collection

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Figure 5: The activation of CPP32 is a very early event of thymocyte apoptosis. (A and B) Thymocytes from C56BL/6 mice were isolated and incubated with dexamethasone. Samples were taken at 30-min intervals and analyzed for levels of apoptosis with annexin V-FITC and PI (A). Cell lysates were submitted to Western blotting analysis for the processing of CPP32 (B). (C) Thymocytes were cultured for 20 h in the presence of immobilized anti-CD3 mAb. Cells were then labeled with annexin V-FITC, and annexin V− and annexin V+ cells were sorted at 4°C with a FACStar® Plus. Sorted cells were lysed and extract contents were analyzed for processing of CPP32 by Western blotting.
Mentions: As mentioned above, after 6 h of treatment with dexamethasone, usually 50–60% of thymocytes are in early apoptosis whereas 5–20% of cells are in late apoptosis. The massive activation of CPP32 at this time point (especially in the experiment presented in Fig. 1 C where only 6% of thymocytes are in late apoptosis), suggests that CPP32 activation occurs as soon as or before the exposure of the phosphatidylserine at the upper side of the cell membrane. The ability of zVAD-CH2F to inhibit annexin V staining and CPP32 activation in thymocytes (Fig. 4) further suggests that activation of cysteine proteases occurs before the loss of membrane asymmetry. To determine whether CPP32 activation is a very early event of thymocyte apoptosis, a time course of CPP32 activation was carried out on thymocytes stimulated with dexamethasone. As illustrated in Fig. 5 a, treatment with dexamethasone did not induce significant apoptosis, as evidenced by the absence of annexin V+ PI−, during the first 150 min. After this initial lag an abrupt increase in the relative number of cells in early apoptosis was noted. A plateau in percentage of annexin V+ PI− cells was reached at 4 h. Western blot analysis showed that activation of CPP32 (as indicated by the appearance of the p17 subunit) occurred 90 min after the addition of dexamethasone (Fig. 5 b). Moreover, this experiment confirmed that CPP32 activation is a very early event during dexamethasone-induced apoptosis since it takes place before the exposure of phosphatidylserine at the cell surface.

Bottom Line: This was observed both in anti-CD3- and dexamethasone-induced apoptosis.Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD.While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F.

View Article: PubMed Central - PubMed

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

ABSTRACT
Cysteine proteases of the CED-3 and ICE family have been recently proposed as the ultimate executioners in several mammalian cell death pathways. Among them, the cysteine protease CPP32 has been shown to participate in programmed cell death (PCD), or apoptosis, affecting lymphoid cells in vitro. In the thymus, negative selection is a mechanism through which developing thymocytes expressing a TcR with high affinity for self peptide-MHC complexes are eliminated by PCD. In order to investigate the role of CPP32 in thymic apoptosis, isolated thymocytes were submitted to cell surface CD3 crosslinking by immobilized anti-CD3 mAb or to dexamethasone treatment. Although apoptosis occurred in the absence or after crosslinking with anti-CD3 mAb, specific activation of CPP32, as assessed by the extent of proteolytic cleavage of the p32 zymogen, was only detected in thymocytes cultured in the presence of the immobilized antibody or dexamethasone. This activation was a very early event during apoptosis as it occurred before the exposure of phosphatidyl serine to the upper side of the cell membrane. This was observed both in anti-CD3- and dexamethasone-induced apoptosis. Moreover, using mice transgenic for pigeon cytochrome C (PCC)-specific TcR, we were able to show that, after injection of PCC, the activation of CPP32 and cleavage of its substrate occurred in thymocytes obtained from mice expressing a permissive MHC haplotype for PCC presentation (H-2k). Moreover, PCC induced apoptosis was blocked by the caspase inhibitor zVAD. While spontaneous apoptosis was not accompanied by detectable levels of CPP32 processing, it was characterized by the proteolysis of poly(ADP-ribose) polymerase (PARP) and was blocked by the cysteine protease inhibitor, zVAD-CH2F. Taken together, these results support the concept that CPP32 is among the earliest effectors of the pathway leading to negative selection of autoreactive thymocytes. Our results also suggest the involvement of a distinct CPP32-like cysteine protease in spontaneous apoptosis of thymocytes.

Show MeSH
Related in: MedlinePlus