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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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Time course of apoptosis in isolated thymocytes cultured alone or in the presence of  immobilized anti-CD3 mAbs or  dexamethasone. The upper panel  shows the flow cytometry measurement of annexin V binding  (x axis) vs. propidium iodide uptake (y axis) on thymocytes cultured for 12 h in the presence of  immobilized anti-CD3. Viable  cells and cells in early apoptosis  are annexin V− PI− (A) and annexin V+ PI− (B), respectively.  Dead cells are annexin V+ PI+  (C). The lower panels display the  evolution of cell viability (A),  the percentage of cells in early  apoptosis (B) and the mortality  (C) in thymocyte populations incubated at 4°C (open square) or  cultured at 37°C alone (closed  squares), in the presence of immobilized anti-CD3 mAb (open circles)  or with dexamethasone (closed circles). This experiment produced  identical results in a total of four  independent occasions.
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Figure 1: Time course of apoptosis in isolated thymocytes cultured alone or in the presence of immobilized anti-CD3 mAbs or dexamethasone. The upper panel shows the flow cytometry measurement of annexin V binding (x axis) vs. propidium iodide uptake (y axis) on thymocytes cultured for 12 h in the presence of immobilized anti-CD3. Viable cells and cells in early apoptosis are annexin V− PI− (A) and annexin V+ PI− (B), respectively. Dead cells are annexin V+ PI+ (C). The lower panels display the evolution of cell viability (A), the percentage of cells in early apoptosis (B) and the mortality (C) in thymocyte populations incubated at 4°C (open square) or cultured at 37°C alone (closed squares), in the presence of immobilized anti-CD3 mAb (open circles) or with dexamethasone (closed circles). This experiment produced identical results in a total of four independent occasions.

Mentions: In four independent experiments, freshly isolated thymocytes were cultured in medium alone at 4 or 37°C in the presence of immobilized anti-CD3 mAb or dexamethasone. Cell viability was assessed by flow cytometry using PI and FITC-conjugated annexin V staining. Early apoptosis has been characterized by the loss of plasma membrane asymmetry and the subsequent exposure of phosphatidylserine at the cell surface (annexin V+) while cellular integrity is maintained as evidenced by exclusion of PI (22). The combined use of PI and FITC-annexin V therefore allows the simultaneous analysis of viable cells (PI− annexin V−), cells in early apoptosis (PI− annexin V+), and dead cells (PI+ annexin V+) (Fig. 1, top).


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)

Time course of apoptosis in isolated thymocytes cultured alone or in the presence of  immobilized anti-CD3 mAbs or  dexamethasone. The upper panel  shows the flow cytometry measurement of annexin V binding  (x axis) vs. propidium iodide uptake (y axis) on thymocytes cultured for 12 h in the presence of  immobilized anti-CD3. Viable  cells and cells in early apoptosis  are annexin V− PI− (A) and annexin V+ PI− (B), respectively.  Dead cells are annexin V+ PI+  (C). The lower panels display the  evolution of cell viability (A),  the percentage of cells in early  apoptosis (B) and the mortality  (C) in thymocyte populations incubated at 4°C (open square) or  cultured at 37°C alone (closed  squares), in the presence of immobilized anti-CD3 mAb (open circles)  or with dexamethasone (closed circles). This experiment produced  identical results in a total of four  independent occasions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199117&req=5

Figure 1: Time course of apoptosis in isolated thymocytes cultured alone or in the presence of immobilized anti-CD3 mAbs or dexamethasone. The upper panel shows the flow cytometry measurement of annexin V binding (x axis) vs. propidium iodide uptake (y axis) on thymocytes cultured for 12 h in the presence of immobilized anti-CD3. Viable cells and cells in early apoptosis are annexin V− PI− (A) and annexin V+ PI− (B), respectively. Dead cells are annexin V+ PI+ (C). The lower panels display the evolution of cell viability (A), the percentage of cells in early apoptosis (B) and the mortality (C) in thymocyte populations incubated at 4°C (open square) or cultured at 37°C alone (closed squares), in the presence of immobilized anti-CD3 mAb (open circles) or with dexamethasone (closed circles). This experiment produced identical results in a total of four independent occasions.
Mentions: In four independent experiments, freshly isolated thymocytes were cultured in medium alone at 4 or 37°C in the presence of immobilized anti-CD3 mAb or dexamethasone. Cell viability was assessed by flow cytometry using PI and FITC-conjugated annexin V staining. Early apoptosis has been characterized by the loss of plasma membrane asymmetry and the subsequent exposure of phosphatidylserine at the cell surface (annexin V+) while cellular integrity is maintained as evidenced by exclusion of PI (22). The combined use of PI and FITC-annexin V therefore allows the simultaneous analysis of viable cells (PI− annexin V−), cells in early apoptosis (PI− annexin V+), and dead cells (PI+ annexin V+) (Fig. 1, top).

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