Cornea graft endothelial cells undergo apoptosis by way of an alternate (caspase-independent) pathway.
OBJECTIVE
To look for apoptosis pathways involved in corneal endothelial cell death during acute graft rejection and to evaluate the potential role of nitric oxide in this process.


METHODS
Corneal buttons from Brown-Norway rats were transplanted into Lewis rat corneas. At different time intervals after transplantation, apoptosis was assessed by diamino-2-phenylindol staining and annexin-V binding on flat-mount corneas, and by terminal transferase dUTP nick end labeling (TUNEL), caspase-3 dependent and leukocyte elastase inhibitor (LEI)/LDNase II caspase-independent pathways on sections. Inducible nitric oxide synthase (NOS-II) expression and the presence of nitrotyrosine were assayed by immunohistochemistry.


RESULTS
Graft endothelial cells demonstrated nuclear fragmentation and LEI nuclear translocation, annexin-V binding, and membranes bleb formation. Apoptosis associated with caspase-3 activity or TUNEL-positive reaction was not observed at any time either in the graft or in the recipient corneal endothelial cells. During 14 days posttransplantation, the recipient corneal endothelial cells remained unaltered and their number unchanged in all studied corneas. NOS-II was expressed in infiltrating cells present within the graft. This expression was closely associated with the presence of nitrotyrosine in endothelial and infiltrating cells.


CONCLUSIONS
During the time course of corneal graft rejection, graft endothelial cells undergo apoptosis. Apoptosis is caspase 3 independent and TUNEL negative and is, probably, carried out by an alternative pathway driven by an LEI/L-Dnase II. Peroxynitrite formation may be an additional mechanism for cell toxicity and programmed cell death of the graft endothelial cells during the rejection process in this model.
Hôtel-Dieu Hospital, Service d'Ophtalmologie, Paris, France.
Jean-Louis
JL
Bourges
Fatemeh
F
Valamanesh
Alicia
A
Torriglia
Jean-Claude
JC
Jeanny
Michèle
M
Savoldelli
Gilles
G
Renard
David
D
BenEzra
Yvonne
Y
de Kozak
Francine
F
Behar-Cohen
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D020169:Caspases / Q000378:metabolism; D016039:Corneal Transplantation / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology* / Q000637:transplantation*; D051381:Rats; D011914:Rats, Inbred BN; D011917:Rats, Inbred Lew; D014184:Transplantation, Homologous
D020169:Caspases