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Blood, 1 June 2001, Vol. 97, No. 11, pp. 3521-3530
IMMUNOBIOLOGY
In vivo and in vitro apoptosis of human thymocytes are associated
with nitrotyrosine formation
Nathalie Moulian,
Frédérique Truffault,
Yvette Morot Gaudry-Talarmain,
Alain Serraf, and
Sonia Berrih-Aknin
From the Laboratoire de Physiologie Thymique, CNRS
ESA-8078 (an affiliation of Institut Paris-Sud sur les Cytokines),
Hôpital Marie Lannelongue, Le Plessis Robinson; and Laboratoire
de Neurobiologie Cellulaire et Moléculaire, CNRS UPR 9040, Gif
sur Yvette, France.
Most thymocytes are deleted by thymic selection. The mechanisms of
cell death are far from being clear. Peroxynitrite is a powerful
oxidant produced in vivo by the reaction of superoxide (O2  ) with nitric oxide
(NO) and is able to mediate apoptosis. The aim of this
study was to analyze whether NO and peroxynitrite could play a role in
human thymocyte apoptosis. The results indicate that
3-(4-morpholinyl)-sydnonimine (SIN-1, an
O2 and NO donor) and
chemically synthesized peroxynitrite, but not
S-nitroso-N-acetyl-D,L-penicillamine (SNAP, an
NO donor), have a strong apoptotic effect on human
thymocytes (annexin V staining and TUNEL reaction). This effect was
inhibited by exogenous superoxide dismutase (SOD), which interacts with
O2 and inhibits the formation of
peroxynitrite. Because peroxynitrite formation requires
NO, thymic stromal cells were investigated to determine
if they produced NO. Inducible NOS was synthesized in
cultured thymic epithelial cells in certain conditions of cytokine
stimulation, as shown by messenger RNA levels, protein analysis, and
nitrite production in the supernatants. SIN-1-treated thymocytes had
high levels of tyrosine nitration, abolished by the addition of
exogenous SOD. Tyrosine nitration was also detected in thymus extracts
and sections, suggesting the presence of peroxynitrite in situ.
In thymus sections, clusters of nitrotyrosine-positive cells were found in the cortex and corticomedullary areas colocalized with cells
positive in the TUNEL reaction. These data indicate an association between human thymocyte apoptosis and nitrotyrosine formation. Thus,
the results support the notion of a physiologic role for peroxynitrite
in human thymocyte apoptosis.
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