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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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zVAD-CH2F blocks early apoptosis and CPP32 activity in  thymocytes undergoing negative selection. 3 wk-old H-2k transgenic  mice were injected with PCC. 16 h later thymocytes were isolated and  cultured with or without zVAD-CH2F. After 6 h, cells were compared  for levels of apoptosis with annexin V-FITC and PI. Cell extracts were  analyzed by Western blotting for processing of CPP32 and cleavage of  PARP.
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Figure 7: zVAD-CH2F blocks early apoptosis and CPP32 activity in thymocytes undergoing negative selection. 3 wk-old H-2k transgenic mice were injected with PCC. 16 h later thymocytes were isolated and cultured with or without zVAD-CH2F. After 6 h, cells were compared for levels of apoptosis with annexin V-FITC and PI. Cell extracts were analyzed by Western blotting for processing of CPP32 and cleavage of PARP.

Mentions: To confirm the involvement of CPP32 in negative selection we obtained apoptotic cells ex vivo after injection of PCC. Parameters were modified including the duration of PCC treatment (16 vs. 24 h) and the age of injected mice (3 vs. 5–6 wk). Under these conditions, we were able to detect substantial levels of apoptosis ex vivo in thymocytes of transgenic H-2k mice (13.0 ± 0.5% annexin V+ PI−, 12.0 ± 3.3% annexin V+ PI+ for H-2k mice) (Fig. 7 A). Control H-2b mice were injected and studied for levels of apoptotic cells using the same protocol (1.6 ± 0.9% annexin V+ PI−, 1.3 ± 0.5% annexin V+ PI+ for H-2b mice). In view of these results and together with the observation that CPP32 cleavage is a very early event of anti-CD3–induced apoptosis, experiments were carried out to determine if inhibition of cysteine protease activity could block death of cells already committed to apoptosis. Thymocytes isolated from PCC-injected H-2k transgenic mice were then incubated at 37°C in the presence or absence of zVAD-CH2F. As depicted in Fig. 7 A, zVAD was able to inhibit completely viable cells (annexin V− PI−) from undergoing apoptosis. Indeed the percentage of annexin V− PI− remained constant throughout the experiment (75.0%; Fig. 7 A) while the relative number of annexin V− PI− cells decreased in untreated cells (61.3%; Fig. 7 A). In contrast this inhibitor did not prevent the passage of cells from early apoptosis (annexin V+ PI−) to late apoptosis (annexin V+ PI+) (Fig. 7 A). This suggests that once thymocytes express phosphatidylserine at the upper face of the cell membrane, these are condemned to die. Moreover these results confirm that cysteine proteases play a major role during apoptosis before the loss of membrane asymmetry.


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)

zVAD-CH2F blocks early apoptosis and CPP32 activity in  thymocytes undergoing negative selection. 3 wk-old H-2k transgenic  mice were injected with PCC. 16 h later thymocytes were isolated and  cultured with or without zVAD-CH2F. After 6 h, cells were compared  for levels of apoptosis with annexin V-FITC and PI. Cell extracts were  analyzed by Western blotting for processing of CPP32 and cleavage of  PARP.
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Related In: Results  -  Collection

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Figure 7: zVAD-CH2F blocks early apoptosis and CPP32 activity in thymocytes undergoing negative selection. 3 wk-old H-2k transgenic mice were injected with PCC. 16 h later thymocytes were isolated and cultured with or without zVAD-CH2F. After 6 h, cells were compared for levels of apoptosis with annexin V-FITC and PI. Cell extracts were analyzed by Western blotting for processing of CPP32 and cleavage of PARP.
Mentions: To confirm the involvement of CPP32 in negative selection we obtained apoptotic cells ex vivo after injection of PCC. Parameters were modified including the duration of PCC treatment (16 vs. 24 h) and the age of injected mice (3 vs. 5–6 wk). Under these conditions, we were able to detect substantial levels of apoptosis ex vivo in thymocytes of transgenic H-2k mice (13.0 ± 0.5% annexin V+ PI−, 12.0 ± 3.3% annexin V+ PI+ for H-2k mice) (Fig. 7 A). Control H-2b mice were injected and studied for levels of apoptotic cells using the same protocol (1.6 ± 0.9% annexin V+ PI−, 1.3 ± 0.5% annexin V+ PI+ for H-2b mice). In view of these results and together with the observation that CPP32 cleavage is a very early event of anti-CD3–induced apoptosis, experiments were carried out to determine if inhibition of cysteine protease activity could block death of cells already committed to apoptosis. Thymocytes isolated from PCC-injected H-2k transgenic mice were then incubated at 37°C in the presence or absence of zVAD-CH2F. As depicted in Fig. 7 A, zVAD was able to inhibit completely viable cells (annexin V− PI−) from undergoing apoptosis. Indeed the percentage of annexin V− PI− remained constant throughout the experiment (75.0%; Fig. 7 A) while the relative number of annexin V− PI− cells decreased in untreated cells (61.3%; Fig. 7 A). In contrast this inhibitor did not prevent the passage of cells from early apoptosis (annexin V+ PI−) to late apoptosis (annexin V+ PI+) (Fig. 7 A). This suggests that once thymocytes express phosphatidylserine at the upper face of the cell membrane, these are condemned to die. Moreover these results confirm that cysteine proteases play a major role during apoptosis before the loss of membrane asymmetry.

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