A. Alam et al., SPECIFIC ACTIVATION OF THE CYSTEINE PROTEASE CPP32 DURING THE NEGATIVE SELECTION OF T-CELLS IN THE THYMUS, The Journal of experimental medicine, 186(9), 1997, pp. 1503-1512
Cysteine proteases of the CED-3 and ICE family have been recently prop
osed as the ultimate executioners in several mammalian cell death path
ways. Among them, the cysteine protease CPP32 has been shown to partic
ipate in programmed cell death (PCD), or apoptosis, affecting lymphoid
cells in vitro. In the thymus, negative selection is a mechanism thro
ugh which developing thymocytes expressing a TcR with high affinity fo
r self peptide-MHC complexes are eliminated by PCD. In order to invest
igate 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 abs
ence or alter crosslinking with anti-CD3 mAb, specific activation of C
PP32, as assessed by the extent of proteolytic cleavage of the p32 zym
ogen, was only detected in thymocytes cultured in the presence of the
immobilized antibody or dexamethasone. This activation was a very earl
y event during apoptosis as it occurred before the exposure of phospha
tidyl serine to the upper side of the cell membrane. This was observed
both in anti-CD3- and dexamethasone-induced apoptosis. Moreover, usin
g 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 e
xpressing a permissive MHC haplotype for PCC presentation (H-2k). More
over, 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(A
DP-ribose) polymerase (PARP) and was blocked by the cysteine protease
inhibitor, zVAD-CH2F. Taken together, these results support the concep
t that CPP32 is among the earliest effectors of the pathway leading to
negative selection of autoreactive thymocytes. Our results also sugge
st the involvement of a distinct oo CPP32-like cysteine protease in sp
ontaneous apoptosis of thymocytes.