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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3 November 1997
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November 03 1997
Specific Activation of the Cysteine Protease CPP32 during the Negative Selection of T Cells in the Thymus
Antoine Alam,
Antoine Alam
From the *Laboratoire d'Immunologie and the ‡Laboratoire de Différentiation des Lymphocytes, Institut de Recherches Cliniques de Montréal, Montréal H2W 1R7; the §Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal H3C 3J7; and the ‖Department of Experimental Medicine, McGill University, Montréal H3A 2B4, Canada
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Michel Y. Braun,
Michel Y. Braun
From the *Laboratoire d'Immunologie and the ‡Laboratoire de Différentiation des Lymphocytes, Institut de Recherches Cliniques de Montréal, Montréal H2W 1R7; the §Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal H3C 3J7; and the ‖Department of Experimental Medicine, McGill University, Montréal H3A 2B4, Canada
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Franca Hartgers,
Franca Hartgers
From the *Laboratoire d'Immunologie and the ‡Laboratoire de Différentiation des Lymphocytes, Institut de Recherches Cliniques de Montréal, Montréal H2W 1R7; the §Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal H3C 3J7; and the ‖Department of Experimental Medicine, McGill University, Montréal H3A 2B4, Canada
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Sylvie Lesage,
Sylvie Lesage
From the *Laboratoire d'Immunologie and the ‡Laboratoire de Différentiation des Lymphocytes, Institut de Recherches Cliniques de Montréal, Montréal H2W 1R7; the §Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal H3C 3J7; and the ‖Department of Experimental Medicine, McGill University, Montréal H3A 2B4, Canada
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Luchino Cohen,
Luchino Cohen
From the *Laboratoire d'Immunologie and the ‡Laboratoire de Différentiation des Lymphocytes, Institut de Recherches Cliniques de Montréal, Montréal H2W 1R7; the §Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal H3C 3J7; and the ‖Department of Experimental Medicine, McGill University, Montréal H3A 2B4, Canada
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Patrice Hugo,
Patrice Hugo
From the *Laboratoire d'Immunologie and the ‡Laboratoire de Différentiation des Lymphocytes, Institut de Recherches Cliniques de Montréal, Montréal H2W 1R7; the §Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal H3C 3J7; and the ‖Department of Experimental Medicine, McGill University, Montréal H3A 2B4, Canada
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François Denis,
François Denis
From the *Laboratoire d'Immunologie and the ‡Laboratoire de Différentiation des Lymphocytes, Institut de Recherches Cliniques de Montréal, Montréal H2W 1R7; the §Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal H3C 3J7; and the ‖Department of Experimental Medicine, McGill University, Montréal H3A 2B4, Canada
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Rafick-Pierre Sékaly
Rafick-Pierre Sékaly
From the *Laboratoire d'Immunologie and the ‡Laboratoire de Différentiation des Lymphocytes, Institut de Recherches Cliniques de Montréal, Montréal H2W 1R7; the §Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal H3C 3J7; and the ‖Department of Experimental Medicine, McGill University, Montréal H3A 2B4, Canada
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Antoine Alam
From the *Laboratoire d'Immunologie and the ‡Laboratoire de Différentiation des Lymphocytes, Institut de Recherches Cliniques de Montréal, Montréal H2W 1R7; the §Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal H3C 3J7; and the ‖Department of Experimental Medicine, McGill University, Montréal H3A 2B4, Canada
Michel Y. Braun
From the *Laboratoire d'Immunologie and the ‡Laboratoire de Différentiation des Lymphocytes, Institut de Recherches Cliniques de Montréal, Montréal H2W 1R7; the §Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal H3C 3J7; and the ‖Department of Experimental Medicine, McGill University, Montréal H3A 2B4, Canada
Franca Hartgers
From the *Laboratoire d'Immunologie and the ‡Laboratoire de Différentiation des Lymphocytes, Institut de Recherches Cliniques de Montréal, Montréal H2W 1R7; the §Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal H3C 3J7; and the ‖Department of Experimental Medicine, McGill University, Montréal H3A 2B4, Canada
Sylvie Lesage
From the *Laboratoire d'Immunologie and the ‡Laboratoire de Différentiation des Lymphocytes, Institut de Recherches Cliniques de Montréal, Montréal H2W 1R7; the §Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal H3C 3J7; and the ‖Department of Experimental Medicine, McGill University, Montréal H3A 2B4, Canada
Luchino Cohen
From the *Laboratoire d'Immunologie and the ‡Laboratoire de Différentiation des Lymphocytes, Institut de Recherches Cliniques de Montréal, Montréal H2W 1R7; the §Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal H3C 3J7; and the ‖Department of Experimental Medicine, McGill University, Montréal H3A 2B4, Canada
Patrice Hugo
From the *Laboratoire d'Immunologie and the ‡Laboratoire de Différentiation des Lymphocytes, Institut de Recherches Cliniques de Montréal, Montréal H2W 1R7; the §Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal H3C 3J7; and the ‖Department of Experimental Medicine, McGill University, Montréal H3A 2B4, Canada
François Denis
From the *Laboratoire d'Immunologie and the ‡Laboratoire de Différentiation des Lymphocytes, Institut de Recherches Cliniques de Montréal, Montréal H2W 1R7; the §Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal H3C 3J7; and the ‖Department of Experimental Medicine, McGill University, Montréal H3A 2B4, Canada
Rafick-Pierre Sékaly
From the *Laboratoire d'Immunologie and the ‡Laboratoire de Différentiation des Lymphocytes, Institut de Recherches Cliniques de Montréal, Montréal H2W 1R7; the §Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal H3C 3J7; and the ‖Department of Experimental Medicine, McGill University, Montréal H3A 2B4, Canada
Address correspondence to R.-P. Sékaly, Laboratoire d'Immunologie, Institut de Recherches Cliniques de Montréal, 110 avenue des Pins Ouest, Montréal H2W 1R7, Canada. Tel.: 514-987-5550; FAX: 514-987-5711; E-mail: [email protected]
The first two authors contributed equally to this work.
Received:
June 09 1997
Revision Received:
August 18 1997
Online ISSN: 1540-9538
Print ISSN: 0022-1007
1997
J Exp Med (1997) 186 (9): 1503–1512.
Article history
Received:
June 09 1997
Revision Received:
August 18 1997
Citation
Antoine Alam, Michel Y. Braun, Franca Hartgers, Sylvie Lesage, Luchino Cohen, Patrice Hugo, François Denis, Rafick-Pierre Sékaly; Specific Activation of the Cysteine Protease CPP32 during the Negative Selection of T Cells in the Thymus . J Exp Med 3 November 1997; 186 (9): 1503–1512. doi: https://doi.org/10.1084/jem.186.9.1503
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