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IMMUNOBIOLOGY
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 The thymus plays a central role in T-cell
differentiation and T-cell repertoire selection. Clonal deletion of
immature thymocytes is an important mechanism ensuring self-tolerance.
Few of the thymocytes generated in the thymus survive to become mature
T cells, the remainder being deleted through thymic
selection.1 The cellular and molecular mechanisms
underlying the deletion of immature thymocytes are unclear, but
these processes are known to be driven by interactions between
developing thymocytes and thymic stromal cells.2 The
thymus stroma is a complex structure. One of the main cell types is the
thymic epithelial cell (TEC), which has a key role in T-cell
development and in the acquisition of self-tolerance.3-5
Many intracellular reactions, including respiration, reduce oxygen to
superoxide (O2
The possible role of oxidative stress in the human thymus has not yet
been studied. We therefore examined the role of ROS in human thymocyte
apoptosis. We show that peroxynitrite, but not NO Thymic tissues
Thymocyte isolation and culture
To analyze their potential production of iNOS, thymocytes were activated either by phorbol myristate acetate (PMA) (2.5 ng/mL) and ionomycin (0.25 mg/mL) for 24 hours or with concanavalin A (10 µg/mL) for 3 days. Total RNA was then extracted, and iNOS was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Measurement of apoptosis Apoptosis was analyzed by quantifying phosphatidylserine residues exposed on the external cell membrane.19 One microliter of human recombinant fluorescein isothiocyanate (FITC)-conjugated annexin V (Boehringer Mannheim, Meylan, France) and 2 µg/mL propidium iodide (PI) were added to 100 µL cell suspension in binding buffer (10 mM HEPES/NaOH, pH 7.4; 140 mM NaCl; 5 mM CaCl2). After 15 minutes of incubation in the dark, dual-color flow cytometry was performed.Apoptosis was also quantified by measuring DNA fragmentation and by using the TdT-mediated dUTP nick end labeling (TUNEL) method with the Boehringer Mannheim kit following the manufacturer's instructions. Briefly, 106 cells were fixed in phosphate-buffered saline (PBS) containing 2% paraformaldehyde. After 2 washes with PBS, cells were permeabilized in hypotonic solution containing 0.1% Triton X-100 and 0.1% sodium citrate for 2 minutes on ice. Cells were washed twice in PBS and resuspended in TUNEL reaction mixture containing terminal deoxynucleotidyl transferase and FITC-coupled dUTP. After 2 washes, the label incorporated at damaged DNA sites was visualized on a FACScan flow cytometer (Becton Dickinson, Grenoble, France) using Cell Quest software. Setup of conditions of flow cytometry analysis Trypan blue staining was used to determine cell viability before each experiment. When 106 thymocytes were cultured in control conditions, 0.86 × 106 ± 0.06 × 106 viable cells were recovered after 18 hours. We then measured thymocyte binding of FITC-conjugated annexin V by flow cytometry (Figure 2). According to forward- and side-scatter criteria, 2 regions (R1 and R2) were identified. As shown in Figure 2, R1 cells were mainly apoptotic (annexin V+ PI ) and necrotic (annexin V+ PI+)
thymocytes; R2 mainly contained living cells. After 18 hours of
culture, about 30% of all thymocytes were apoptotic (FITC-annexin V+ and PI) and were essentially found in R1.
Of note, freshly isolated human thymocytes contained only a few
apoptotic cells, found in R1 (data not shown). Except when indicated,
the flow cytometry analysis was performed in the whole population of
thymocytes (R1 and R2).
In some experiments, thymocytes were labeled with anti-CD4-Cy5 (diluted 1:3) and anti-CD8-phycoerythrin (diluted 1:3) monoclonal antibodies (Dako, Trappes, France) before using the annexin V staining protocol. Immunofluorescence on frozen human thymic sections Thymic sections were from 4 different newborn individuals (1 week to 2 years). Cryostat thymic sections (5 µm) were fixed with acetone for 10 minutes. To detect nitrotyrosine, rabbit polyclonal antinitrotyrosine antibody (Upstate Biotechnology, Lake Placid, NY) was applied in a dilution of 1:100 for 60 minutes. The sections were then washed 3 times and revealed using goat antirabbit bound to tetramethylrhodamine isothiocyanate (GAR-TRITC; Immunotech, Marseille, France) at a 1:100 dilution for 30 minutes. Double labeling with monoclonal antikeratin antibodies at a 1:50 dilution (mix of LP34 and MNF116 monoclonal antibodies) (Dako) revealed by goat antimouse immunoglobulins coupled to fluorescein at a 1:100 dilution (GAM-FITC; Silenius, Eurobio, France) was performed to visualize the epithelial network in the thymus. Two types of controls were performed: (1) The antinitrotyrosine antibody (10 µL) was mixed in 1 mL PBS with free nitrotyrosine (10 M) for 1 hour at room temperature before being applied on thymic sections, and (2) the fluorescent second-layer antibodies were directly tested on thymic sections.Three-color immunofluorescence was performed as follows: After fixation and washings, the slides were incubated with antinitrotyrosine antibody (1:100 dilution for 1 hour) revealed by GAR-TRITC (1:100 dilution for 30 minutes), then with antikeratin-mix antibodies (1:50 dilution for 30 minutes) revealed by RAM-AMCA (rabbit antimouse aminoethylcoumarin acetic acid, Dako) diluted 1:10 and, lastly, with monoclonal anti-iNOS antibody coupled to FITC (diluted 1:25) for 2 hours (Transduction Laboratories, Lexington, KY). To assess the in situ cell death, we used a kit for detection of apoptosis based on labeling of DNA strand breaks (Boehringer Mannheim). Briefly, the slides were fixed with paraformaldehyde 2% for 30 minutes followed by 3 washes. They were then permeabilized in 0.1%Triton X-100 in PBS for 2 minutes on ice. The slides were then washed 3 times, and the TUNEL reaction mixture was added on the samples for 60 minutes at 37°C in the dark. The slides were then washed 3 times before applying the antinitrotyrosine antibody as described above. A negative control was included. It consists of incubating the sections with the label solution without terminal transferase instead of TUNEL reaction mixture. Fluorescence was then visualized using the Leica DMRB microscope (Wetzlar, Germany) equipped with a Sony digital charge-coupled device camera for capturing images (Koehn, Germany). Culture of thymic epithelium Primary cultures of human TECs were established as previously described.20 Briefly, small fragments of thymic tissue were washed in RPMI and transferred to culture dishes. The culture medium consisted of RPMI 1640 Glutamax I (Life Technologies) supplemented with 20% horse serum, 0.2% Ultroser (Life Technologies), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2.5 µg/mL fungizone and was replaced twice-weekly. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2. After 8 to 12 days of primary culture, the confluent monolayers were treated with 0.075% trypsin (Life Technologies) and 0.16% ethylenediaminetetraacetic acid (EDTA) for 5 minutes at 37°C. To study NOS regulation, TECs were subcultured in 24-well plates (5 × 105 cells/well) and incubated overnight to allow cells to adhere. After 2 washes, 1 ng/mL recombinant human interleukin (IL)-1 (Sigma), 10 ng/mL recombinant tumor necrosis factor
(TNF)- , and 500 U/mL recombinant human interferon (IFN)-
(Genzyme, Cergy Saint Christophe, France) were added, separately or
together, in RPMI containing 5% horse serum. When indicated, 0.5 mM
N -nitro-L-arginine methyl ester (L-NAME, Sigma) was also
added. After various incubation times, TECs were treated with
trypsin-EDTA solution and the cell suspension thus obtained was washed,
centrifuged, and used for flow cytometry, protein extraction, or total
RNA preparation. The epithelial nature of the cells was shown by means
of immunofluorescence with an antikeratin antibody (clone CK1, Dako);
the percentage of epithelial cells was consistently higher than 95%.
Fibroblasts represented around 5% of the cultured cells, as shown by
staining with an anticollagen III antibody (ICN, Orsay, France).
RNA preparation and RT-PCR Total RNA was prepared from TEC pellets by using the acid guanidinium thiocyanate-phenol-chloroform extraction method as described by Chomczynski et al.21 The cell pellet was homogenized in 0.1 mL denaturing solution (4 M guanidinium thiocyanate; 25 mM sodium citrate, pH 7; 0.5 N lauryl sarcosyl; and 0.1 M 2-mercaptoethanol). Extracted total RNA was purified with 0.5 vol 7.5 M ammonium acetate and 2.5 vol 100% ethanol and then centrifuged at 15 000 rpm for 30 minutes at 4°C. The pellet was washed in 75% ethanol, dried under a vacuum, and stored at 80°C after dissolution
in water. The total RNA concentration was determined from absorbance at 260 nm in a Gene Quant spectrophotometer (Amersham Pharmacia Biotech, Buckinghamshire, England). Purity was checked by measuring the 260 nm:280 nm optical density (OD) ratio.
The oligonucleotide primers used for RT-PCR (Genset, Paris, France) have the following sequences: iNOS primers, forward 5'-ATGTCTGGCAGGACGAGAAG-3' and reverse 5'-CTGAATGTGCTGTTTGCCTC-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers, forward 5'-ATCACCATCTTCCAGGAG-3' and reverse 5'-CCTGCTTCACC- ACCTTCTTG-3'. A 50 µL RT reaction mixture containing 2 µg total RNA, 5 µL 10 × RT buffer (Eurobio, Les Ulis, France), 1.5 mM each deoxynucleoside triphosphate (dNTP) (Eurobio), 40 U RNasin (Promega, Charbonnières, France), 1 µM reverse primer, and 4 U avian myeloblastosis virus (AMV) reverse transcriptase (Eurobio) was incubated at 42°C for 60 minutes. PCR was carried out in a total volume of 100 µL containing 10 µL RT reaction mix, 10 µL PCR buffer (Eurobio), 1.5 mM MgCl2, 0.5 µM each primer, 0.2 mM each dNTP, and 2.5 U EurobioTaqII polymerase (Eurobio). The mixture was overlaid with mineral oil and amplified in a PHC3 thermal cycler (Techne, Cambridge, United Kingdom) as follows: denaturing step, 94°C for 1 minute; annealing step, 55°C (iNOS) or 60°C (GAPDH) for 1 minute; extension step, 72°C for 2 minutes. The final elongation step lasted 10 minutes at 72°C. PCR products were analyzed on 1.5% agarose gel containing ethidium bromide. Western blotting Primary cultures of human TECs were solubilized in 1% Triton X-100; 150 mM NaCl; 50 mM Tris, pH 8.0; 5 mM EDTA, pH 8.0; 0.02% NaN3; 1 mM phenylmethylsulfonyl fluoride; and 0.15 U/mL aprotinin for 20 minutes at 4°C. Insoluble material was removed by centrifugation at 4°C for 10 minutes. The protein concentration was measured with the bicinchoninic acid (BCA) protein assay reagent (Pierce, Rockford, IL).Samples were suspended by boiling in sample buffer and were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 8% to 18% linear acrylamide gradients gels overlaid with 5% acrylamide stacking gels. Protein bands were electroblotted onto nitrocellulose filters. Blots were saturated in buffer containing 20 mM Tris, pH 7.4; 500 mM NaCl; 0.1% Tween-20; and 1% bovine serum albumin. Anti-iNOS (Transduction Laboratories) or antinitrotyrosine (Euromedex, Souffelweyersheim, France) antibodies diluted in this buffer were added and allowed to bind overnight. Bound immunoglobulins were detected indirectly by using peroxidase-conjugated antimouse immunoglobulin antibodies. Immunoreactivity was determined using the enhanced chemiluminescence reaction (Amersham). To check the specificity of the antinitrotyrosine reactivity, the antibody (10 µL) was absorbed with 90 µL free competing nitrotyrosine (10 mM) for 1 hour before being applied in the Western blot experiments as described above. Immunohistochemical analysis of cytocentrifuged cells Cultured TECs (5 × 104 per slide) were cytocentrifuged (Cytospin 2, Shandon, United Kingdom), air-dried, fixed in acetone, and tested with the labeled streptavidin biotin (LSAB) labeling kit (Dako). Briefly, the cells were incubated for 5 minutes with the blocking antibody (normal goat serum), then with 5 µg/mL anti-iNOS monoclonal antibody (Transduction Laboratories) for 30 minutes, and washed in PBS. Biotinylated antimouse immunoglobulins were then added for 30 minutes. After 3 washes in PBS, horseradish peroxidase-conjugated streptavidin was added for 10 minutes. After 3 washes the amino-3-ethyl-9 carbazole (AEC) substrate was added for 10 minutes, and the slides were rinsed with distilled water and mounted in glycerol/PBS.Nitrite analysis Nitrite production by TECs was measured as an index of NOS activity. Confluent cells in 24-well plates were washed twice with PBS and incubated with Dulbecco modified Eagle medium without phenol red, with or without cytokines, for the indicated times. The medium was then removed, and nitrite was measured by mixing 200 µL supernatant with 50 µL Griess reagent as previously described.22 Absorbance was read at 550 nm, and the nitrite concentration was determined from a calibration curve constructed with sodium nitrite standards.
ONOO Comparative effects of NO
Effect of synthesized ONOO
SOD inhibits the apoptotic effect of SIN-1.
SOD competes with NO
Double-positive immature thymocytes are the T-cell subpopulation most sensitive to SIN-1-induced apoptosis As shown in Figure 6, short incubation in the presence of SIN-1 depleted CD4+CD8+ cells (from 82% at the beginning of the culture to 54% after 4 hours of incubation with 0.5 mM SIN-1), and the percentages of CD4+ and CD8+ single-positive cells increased accordingly. The fall in CD4+CD8+ cell numbers observed in the R2 gate was compensated for by an increase in cell numbers in the R1 gate (Figure 2). After 4 hours of incubation with 0.5 mM SIN-1, the percentage of annexin V+ PI cells was higher
in the CD4+CD8+ population than in the single
mature cells (Table 1). This analysis was
performed in the R2 gate. Taken together, these data indicated that
CD4+CD8+ cells are more sensitive to
SIN-1-induced apoptosis than mature cells. At a later stage (18 hours), more than 80% of the cells were apoptotic, and all the
subpopulations were concerned (data not shown).
Tyrosine nitration levels increase during thymocyte apoptosis Because peroxynitrite has the functional property of inducing tyrosine nitration, we examined whether SIN-1-treated cells contain proteins with nitrated tyrosine residues. The level of tyrosine nitration was clearly increased after 4 hours of treatment with 1 mM SIN-1 and affected a few thymic proteins as shown in Western blot experiments (Figure 7A). This effect was strongly inhibited by 1000 U/mL SOD, which also reversed SIN-1-induced apoptosis of human thymocytes (Figure 5). In normal thymic extracts from children undergoing cardiac surgery, 2 major proteins with nitrated tyrosines were detected around 15 and 60 kd (Figure 7B). When the antinitrotyrosine antibody was incubated with free nitrotyrosine before its application in the Western blot experiment, the signal was strikingly reduced (data not shown).
The antinitrotyrosine reactivity was also evidenced by flow cytometry
after membrane permeabilization of human thymocytes. A kinetics study
shown in Figure 8A indicates that
nitrotyrosine-positive cell percentage increases in the first hours of
the culture of thymocytes, in parallel with the increase of
apoptotic cells determined by annexin V staining. Of note, the
percentage of apoptotic cells is higher before culture, probably due to
dying cells provoked by the mechanical dissociation. Preincubation of
the antinitrotyrosine antibody with free competing nitrotyrosine
reverses the immunoreactivity of the antinitrotyrosine antibody (Figure
8B).
Tyrosine nitration is inhibited by NOS inhibitors in coculture experiments In coculture experiments, thymocytes were incubated with TECs or with MITC (a thymic myoid cell line) used as thymic cell line control for 4 hours, and the percentage of nitrotyrosine-positive cells was determined (Figure 8C). When the coculture was performed in presence of L-NAME (0.01 mM), the percentage of nitrotyrosine-positive cells was decreased from 60.9% to 49.2% (19.2% decrease); in coculture with MITC, L-NAME (0.01 mM) had no effect because the percentage of nitrotyrosine-positive cells varies from 55% to 56%.Detection of nitrotyrosine on thymic sections The presence of nitrated proteins in tissues is regarded as a specific footprint of peroxynitrite,8,9 although nitrating activity could also be generated by myeloperoxidase in specific conditions.23 To investigate the relevance of peroxynitrite in vivo, we analyzed the presence of tyrosine nitrations on human thymic sections by immunofluorescence using a specific polyclonal antibody. The thymic samples were from 4 newborn individuals. Small spots of positive cells were clearly detectable (Figure 9Aii). Double staining with antikeratin antibodies shows that nitrotyrosine-positive spots are located in the thymic cortex and at the corticomedullary junction, depicted as a white dotted line on the microphotographies (Figure 9Ai-iii). At a higher magnification, we could visualize TECs included in nitrotyrosine-positive spots (Figure 9Aiv). The controls performed by absorbing the antinitrotyrosine activity with free competing nitrotyrosine (Figure 9Av) or by omitting the first layers were negative (Figure 9Avi).
We then wondered whether the spots of nitrotyrosine could be associated with an in vivo apoptosis. To this end, we performed double-staining studies on the thymic frozen sections using labeling of nitrotyrosine with the specific polyclonal antibody and of DNA strand breaks by the TUNEL reaction. TUNEL-positive cells were located in the thymic cortex and the corticomedullary junction as previously reported.24 Most spots of nitrotyrosine (Figure 9Bii) were associated with a strong TUNEL reaction (Figure 9Bi-v). The TUNEL control consisting of the same reaction mixture but without terminal transferase enzyme was negative (Figure 9Bvi). Expression of iNOS in the human thymus Because NO regulates the formation of
peroxynitrite, we wondered whether iNOS is produced by thymic cells.
Thymic sections. Incubation of thymic sections with anti-iNOS antibody revealed a significant staining of several cell types. A double staining with antikeratin antibody revealed that some of them are epithelial, as shown by the arrowheads (Figure 9Ci,ii), but others are located around the vessels (arrow) and are probably endothelial cells (Figure 9Ci,ii). Triple immunofluorescence on the thymic sections with antinitrotyrosine, anti-iNOS, and antikeratin antibodies revealed that nitrotyrosine-positive spots enclosed frequently iNOS+ cells (Figure 9Ciii-viii). In some spots of nitropositive cells, it was possible to identify iNOS epithelial-positive cells (Figure 9Ciii-viii). Cultured cells.
Thymocytes activated by PMA and ionomycin for 24 hours or concanavalin
A for 3 days did not synthesize iNOS, as assessed by RT-PCR (data not
shown). Figure 10A shows iNOS messenger
RNA expression in cultured TECs after stimulation with cytokines in
RT-PCR experiments. The expression was undetectable when TNF-
After 24 hours of stimulation, TECs were analyzed for their iNOS protein content by Western blotting and immunohistochemistry. The iNOS was undetectable in unstimulated cells, but it was clearly detected after stimulation with the combination of IFN- , TNF-, and
IL-1. The signal was of the expected size (130 kd) (Figure 10B). In immunohistochemical studies, cultured TECs were
cytocentrifuged and examined for iNOS expression with an anti-iNOS
monoclonal antibody; iNOS was not detected in unstimulated cultures,
but many cells expressed iNOS after cytokine stimulation (Figure 10C).
Finally, NOS activity was measured in TEC cultures using the amount of
nitrites in supernatants as an index. NO
The main results of this study are as follows: (1) Peroxynitrite
is a powerful inducer of human thymocyte apoptosis; NO Peroxynitrite induces human thymocyte apoptosis Peroxynitrite is a potent inducer of human thymocyte apoptosis in vitro. Interestingly, this mechanism does not occur in all thymic types of cells, because SIN-1 treatment of TECs does not induce apoptosis of TECs (data not shown). Apoptosis was defined by 3 criteria: annexin V staining (an early event), the TUNEL reaction (a late event), and the absolute number of viable cells (a very late event). As regards the role of peroxynitrite in thymic apoptosis in vivo, our results show that nitrotyrosine, a marker for peroxynitrite, is found in the thymic extracts and on thymic sections colocalized with apoptotic cells assessed by TUNEL reaction. Thus peroxynitrite induces apoptosis of human thymocytes in vitro and is associated with thymocyte apoptosis in vivo.As regards the activity of NO Because apoptosis of human thymocytes in vitro was induced by
peroxynitrite, one could hypothesize that in vivo NO has reacted with
the O2 Although tyrosine nitration and DNA fragmentation are associated ex
vivo, it is not clear which event is the cause. In our flow cytometry
analyses, nitrotyrosine appeared in thymocytes, as early as 1 hour and
before annexin V staining, indicating that tyrosine nitration was an
early event in the apoptosis process. However, we cannot
exclude that O2 Several reports point to a role of ROS in murine thymocyte apoptosis. A study documented the oxygen dependence of the initial steps of thymocyte death induced by glucocorticoids or gamma irradiation, and it showed that the antioxidant N-acetyl cysteine inhibited the induction of thymocyte apoptosis by these reagents.17 Thymocyte apoptosis induced by CD45 cross-linking was also inhibited by the addition of ROS scavengers.31 ROS can also regulate signals involved in caspase activation and apoptosis and contribute to peripheral activation-induced cell death.32,33 N-acetyl cysteine completely blocks activation-induced cell death in T-cell hybridomas and in myelin basic protein--specific T-cell lines.12,34 Recently, it was shown that peroxynitrite primes normal human T lymphocytes to undergo peroxynitrite-driven apoptotic death following activation in vitro.35 The mechanism involved is the inhibition of activation-induced protein tyrosine phosphorylation through nitration of tyrosine residues by peroxynitrite. Thus, peroxynitrite could play a physiologic role as a modulator of activation and death in the immune central and peripheral systems. Our coculture experiments indicate that tyrosine nitration of
thymocytes in contact with TECs is partially inhibited in the presence
of iNOS inhibitors. In addition, TECs were found to express iNOS on
thymic sections. These results bring additional arguments in favor of a
physiologic role of peroxynitrite in tyrosine nitration of thymocytes
and apoptosis. Insofar as spots of nitrotyrosine-positive cells are
mainly found in the cortex, that there is no need for a stimulation by
anti-CD3 to observe tyrosine nitration, and that CD4+CD8+ cells are more sensitive to
peroxynitrite-induced apoptosis than are single-positive cells, the
death analyzed in this study essentially concerns death during the
positive selection processes, ie, death by neglect. Whether the same
events could occur during negative selection of human thymocytes
deserves further investigation. In mice, it was shown that T-cell
receptor-stimulated murine thymocytes are much more sensitive to
NO Significance of tyrosine nitration in the thymus Peroxynitrite might lead to cell death by a mechanism involving nitration of critical tyrosine residues.9 Tyrosine nitration may interfere with intracellular signal transduction. Nitration of tyrosine residues is able to inhibit protein tyrosine phosphorylation in purified activated lymphocytes,35 and it inactivates protein tyrosine phosphatases in vitro.36 Nitration of surfactant protein A by peroxynitrite has been linked to decreased protein function.37 Similarly, tyrosine nitration inactivates prostacyclin synthase, which favors atherosclerotic processes.38 Nitration of structural proteins, including neurofilaments and actin, can disrupt filament assembly and have severe pathological consequences.9The presence of nitrotyrosine in the normal newborn human thymus suggests the presence of peroxynitrite ex vivo, although it was recently described that myeloperoxidase could also be a nitrating agent.23 The presence of nitrotyrosine was also obtained in mice, but its localization was different. In the human thymus, we found nitrotyrosine spots in the cortex and the corticomedullary junction; in mouse thymus nitrotyrosine was detected in the corticomedullary junction and medulla.39 In mice deficient in iNOS, expression of nitrotyrosine in the thymus was less than in normal mice,39 suggesting a role of iNOS in the expression of nitrated tyrosine residues. In the human thymus, the localization of nitrotyrosine spots in the cortex and the corticomedullary junction is compatible with an association with apoptotic mechanisms. Indeed, most human apoptotic thymocytes in situ were detected at the corticomedullary junction and scattered throughout the cortex.24 In our study, using TUNEL reaction we found that most nitrotyrosine-positive cells were colocalized with TUNEL-positive cells. Using antinitrotyrosine antibody in a Western blot analysis, we showed that 2 main proteins were detectable. Their molecular weights were around 60 kd and 15 kd. Tyrosine nitration is generally associated with protein dysfunction, suggesting that biochemical modifications of these proteins could be crucial for thymic survival. Because some spots of nitrotyrosine were negative in TUNEL reaction, we could speculate that nitrotyrosine formation is an event occurring earlier than DNA fragmentation assessed by the TUNEL reaction. Alternatively, nitrotyrosine-positive cells that are negative in TUNEL reaction could be associated with necrosis induced by the peroxynitrite. TECs frequently observed in nitrotyrosine spots are potentially peroxynitrite producer cells because they are reactive with an anti-iNOS antibody. However, more direct arguments are needed to characterize the nitrated proteins and to evaluate their role on thymic apoptosis. Protein nitration is increased in several pathological models. For example, nitrotyrosine residues have been detected with a specific monoclonal antibody in the brains of multiple sclerosis patients.40 Patients with adult respiratory distress syndrome have elevated nitrotyrosine levels.41 Nitrotyrosine has also been detected in vacuolated muscle fibers from patients with myositis inclusion body but not in normal muscles.42 Our data show that nitrotyrosine could also be present at a basal level in normal situations. Taken together, our results demonstrate that peroxynitrite is a powerful inducer of apoptosis of human thymocytes in vitro. The evidence of iNOS reactivity in TECs supports their role in NO synthesis. The presence of nitrotyrosine in the human thymus supports the presence of peroxynitrite in the thymus gland. Finally, the colocalization of thymic apoptotic cells with nitrated tyrosine residues suggests a significant role of peroxynitrite in human thymic apoptosis in vivo.
We are grateful to Claire Ducrocq (Institut de Chimie des Substances Naturelles, CNRS, Gif/Yvette, France) for chemical preparation of peroxynitrite and to C. Bruand for preparing the figures.
Submitted March 10, 2000; accepted February 1, 2001.
Supported by grants from Association Française contre les Myopathies, Centre National de la Recherche Scientifique, and Caisse Nationale d'Assurance Maladie des Travailleurs Salariés. N.M. is supported by a postdoctoral grant from Fondation Singer Polignac.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Sonia Berrih-Aknin, Laboratoire de Physiologie Thymique, UPS CNRS ESA-8078, Hôpital Marie Lannelongue, 133, avenue de la Résistance, 92350 Le Plessis Robinson, France; e-mail: sonia.berrih{at}ccml.u-psud.fr.
1. Sprent J, Webb SR. Intrathymic and extrathymic clonal deletion of T cells. Curr Opin Immunol. 1995;7:196-205[CrossRef][Medline] [Order article via Infotrieve]. 2. Anderson G, Moore NC, Owen JJT, Jenkinson EJ. Cellular interactions in thymocyte development. Ann Rev Immunol. 1996;14:73-99[CrossRef][Medline] [Order article via Infotrieve]. 3. Oosterwegel MA, Haks MC, Jeffry U, Murray R, Kruisbeek AM. Induction of TCR gene rearrangements in uncommitted stem cells by a subset of IL-7 producing, MHC class II-expressing thymic stromal cells. Immunity. 1997;6:351-360[CrossRef][Medline] [Order article via Infotrieve]. 4. Lo D, Reilly CR, Burkly LC, DeKoning J, Laufer TM, Glimcher LH. Thymic stromal cell specialization and the T-cell receptor repertoire. Immunol Res. 1997;16:3-14[Medline] [Order article via Infotrieve]. 5. Rubin RL, Kretz-Rommel A. Linkage of immune self-tolerance with the positive selection of T cells. Crit Rev Immunol. 1999;19:199-218[Medline] [Order article via Infotrieve]. 6. Schmidt HH, Walter U. NO at work. Cell. 1994;78:919-925[CrossRef][Medline] [Order article via Infotrieve].
7.
Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA.
Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide.
Proc Natl Acad Sci U S A.
1990;87:1620-1624
8.
Pryor WA, Squadrito GL.
The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide.
Am J Physiol.
1995;268:L699-L722
9.
Beckman JS, Koppenol WH.
Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly.
Am J Physiol.
1996;271:C1424-C1437 10. Groves JT. Peroxynitrite: reactive, invasive and enigmatic. Curr Opin Chem Biol. 1999;3:226-235[CrossRef][Medline] [Order article via Infotrieve]. 11. Albina JE, Cui S, Mateo RB, Reichner JS. Nitric oxide-mediated apoptosis in murine peritoneal macrophages. J Immunol. 1993;150:5080-5085[Abstract]. 12. Sandstrom PA, Mannie MD, Buttke TM. Inhibition of activation-induced death in T cell hybridomas by thiol antioxidants: oxidative stress as a mediator of apoptosis. J Leukoc Biol. 1994;55:221-226[Abstract]. 13. Fehsel K, Kröncke KD, Meyer KL, Huber H, Wahn V, Kolb-Bachofen V. Nitric oxide induces apoptosis in mouse thymocytes. J Immunol. 1995;155:2858-2865[Abstract]. 14. Tai XG, Toyo-oka K, Yamamoto N, et al. Expression of an inducible type of nitric oxide (NO) synthase in the thymus and involvement of NO in deletion of TCR-stimulated double-positive thymocytes. J Immunol. 1997;158:4696-4703[Abstract]. 15. Salgo MG, Squadrito GL, Pryor WA. Peroxynitrite causes apoptosis in rat thymocytes. Biochem Biophys Res Comnun. 1995;215:1111-1118[CrossRef][Medline] [Order article via Infotrieve]. 16. Ramakrishnan N, Catravas G. N-(2-mercaptoethyl)-1,3-propanediamine (WR-1065) protects thymocytes from programmed cell death. J Immunol. 1992;148:1817-1821[Abstract]. 17. McLaughlin KA, Osborne BA, Goldsby RA. The role of oxygen in thymocyte apoptosis. Eur J Immunol. 1996;26:1170-1174[Medline] [Order article via Infotrieve]. 18. Morot Gaudry-Talarmain Y, Moulian N, Meunier FA, et al. Nitric oxide and peroxynitrite affect differently acetylcholine release, choline acetyltransferase activity, synthesis, and compartmentation of newly formed acetylcholine in Torpedo marmorata synaptosomes. Nitric Oxide. 1997;1:330-345[CrossRef][Medline] [Order article via Infotrieve]. 19. Vermes I, Haanen C, Steffens-Nakken H, Reutlingsperger C. A novel assay for apoptosis: flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled annexin V. J Immunol Methods. 1995;184:39-51[CrossRef][Medline] [Order article via Infotrieve]. 20. Berrih S, Arenzana-Seisdedos F, Cohen S, Devos R, Charron D, Virelizier JL. Interferon-gamma modulates HLA class II antigen expression on cultured human thymic epithelial cells. J Immunol. 1995;135:1165-1171[Abstract]. 21. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159[Medline] [Order article via Infotrieve]. 22. Drapier JC, Hibbs JB Jr. Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in L-arginine-dependent inhibition of mitochondrial iron-sulfur enzymes in the macrophage effector cells. J Immunol. 1988;140:2829-2838[Abstract]. 23. Souza JM, Daikhin E, Yudkoff M, Raman CS, Ischiropoulos H. Factors determining the selectivity of protein tyrosine nitration. Arch Biochem Biophys. 1999;371:169-178[CrossRef][Medline] [Order article via Infotrieve]. 24. Le PT, Maecker HT, Cook JE. In situ detection and characterization of apoptotic thymocytes in human thymus: expression of Bcl2-in vivo does not prevent apoptosis. J Immunol. 1995;154:4371-4378[Abstract]. 25. Downing JEG. Multiple nitric oxide synthase systems in adult rat thymus revealed using NADPH diaphorase histochemistry. Immunology. 1994;82:659-664[Medline] [Order article via Infotrieve]. 26. Downing JE, Virag L, Jones IW. NAPDH diaphorase-positive dendritic profiles in rat thymus are discrete from autofluorescent cells, immunoreactive for inducible nitric oxide synthase, and shown strain-specific abundance differences. Immunology. 1998;95:148-155[CrossRef][Medline] [Order article via Infotrieve]. 27. Murphy M, Friend DS, Pike-Nobile L, Epstein LB. Tumor necrosis factor-alpha and IFN-gamma expression in human thymus: localization and overexpression in Down syndrome (trisomy 21). J Immunol. 1992;149:2506-2512[Abstract]. 28. Le PT, Singer KH. Human thymic epithelial cells: adhesion molecules and cytokine production. Int J Clin Lab Res. 1993;23:56-60[Medline] [Order article via Infotrieve].
29.
Throsby M, Herbelin A, Pleau JM, Dardenne M.
CD11c+ eosinophils in the murine thymus: developmental regulation and recruitment upon MHC class I-restricted thymocyte deletion.
J Immunol.
2000;165:1965-1975 30. Macho A, Hirsch T, Marzo I, et al. Glutathione depletion is an early and calcium elevation is a late event of thymocyte apoptosis. J Immunol. 1997;58:4612-4619. 31. Lesage S, Steff AM, Philippoussis F, et al. CD4+CD8+ thymocytes are preferentially induced to die following CD45 cross-linking, through a novel apoptotic pathway. J Immunol. 1997;159:4762-4771[Abstract]. 32. Kabelitz D, Pohl T, Pechhold K. Activation-induced cell death (apoptosis) of mature peripheral T lymphocytes. Immunol Today. 1993;14:338-339[CrossRef][Medline] [Order article via Infotrieve]. 33. Hildeman DA, Mitchell T, Teague TK, et al. Reactive oxygen species regulate activation-induced T cell apoptosis. Immunity. 1999;10:735-744[CrossRef][Medline] [Order article via Infotrieve]. 34. Williams MS, Henkart PA. Role of reactive oxygen intermediates in TCR-induced death of T cell blasts and hybridomas. J Immunol. 1996;157:2395-2402[Abstract].
35.
Brito C, Naviliat M, Tiscornia AC, et al.
Peroxynitrite inhibits T lymphocytes activation and proliferation by promoting impairment of tyrosine phosphorylation and peroxynitrite-driven apoptotic death.
J Immunol.
1999;162:3356-3366 36. Takakura K, Beckman JS, MacMillan-Crow LA, Crow JP. Rapid and irreversible inactivation of protein tyrosine phosphatases PTP1B, CD45, and LAR by peroxynitrite. Arch Biochem Biophys. 1999;15:197-207.
37.
Haddad IY, Ischiropoulos H, Holm BA, Beckman JS, Baker JR, Matalon S.
Mechanisms of peroxynitrite induced injury to pulmonary surfactant.
Am J Physiol.
1993;265:L555-L564
38.
Zou MH, Leist M, Ullrich V.
Selective nitration of prostacyclin synthase and defective vasorelaxation in atherosclerotic bovine coronary arteries.
Am J Pathol.
1999;154:1359-1365 39. Virag L, Scott GS, Cuzzocrea S, Marmer D, Salzman AL, Szabo C. Peroxinitrite-induced thymocyte apoptosis: the role of caspases and poly (ADP-ribose)synthetase (PARS) activation. Immunology. 1998;94:345-355[CrossRef][Medline] [Order article via Infotrieve].
40.
Bagasra O, Michaels FH, Zheng YM, et al.
Activation of the inducible form of nitric oxide synthase in the brains of patients with multiple sclerosis.
Proc Natl Acad Sci U S A.
1995;92:12041-12045 41. Haddad IY, Pataki G, Hu P, Galliani C, Beckman JS, Matalon S. Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury. J Clin Invest. 1994;94:2407-2413. 42. Yang CC, Alvarez RB, Engel WK, Askanas V. Increase of nitric oxide synthases and nitrotyrosine in inclusin-body myositis. Neuroreport. 1996;8:153-158[Medline] [Order article via Infotrieve].
© 2001 by The American Society of Hematology.
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  A. Gilboa-Geffen, P. P. Lacoste, L. Soreq, G. Cizeron-Clairac, R. Le Panse, F. Truffault, I. Shaked, H. Soreq, and S. Berrih-Aknin The thymic theme of acetylcholinesterase splice variants in myasthenia gravis Blood, May 15, 2007; 109(10): 4383 - 4391. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. R. Atkuri, L. A. Herzenberg, A.-K. Niemi, T. Cowan, and L. A. Herzenberg Importance of culturing primary lymphocytes at physiological oxygen levels PNAS, March 13, 2007; 104(11): 4547 - 4552. [Abstract] [Full Text] [PDF]
|
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 |
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 V. Mollace, C. Muscoli, E. Masini, S. Cuzzocrea, and D. Salvemini Modulation of Prostaglandin Biosynthesis by Nitric Oxide and Nitric Oxide Donors Pharmacol. Rev., June 1, 2005; 57(2): 217 - 252. [Abstract] [Full Text] [PDF]
|
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 |
|
 V. Bronte, T. Kasic, G. Gri, K. Gallana, G. Borsellino, I. Marigo, L. Battistini, M. Iafrate, T. Prayer-Galetti, F. Pagano, et al. Boosting antitumor responses of T lymphocytes infiltrating human prostate cancers J. Exp. Med., April 18, 2005; 201(8): 1257 - 1268. [Abstract] [Full Text] [PDF]
|
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|
 |
|
 C. Gras-Le Guen, A. Jarry, G. Vallette, C. Toquet, C. Colombeix, C. L. Laboisse, G. Potel, J-C. Roze, D. Bugnon, and T. Debillon Antibiotic therapy reduces nitrosative stress and programmed cell death in the rabbit foetal lung Eur. Respir. J., January 1, 2005; 25(1): 88 - 95. [Abstract] [Full Text] [PDF]
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|
 S. Kumar, L. Gupta, Y. S. Han, and C. Barillas-Mury Inducible Peroxidases Mediate Nitration of Anopheles Midgut Cells Undergoing Apoptosis in Response to Plasmodium Invasion J. Biol. Chem., December 17, 2004; 279(51): 53475 - 53482. [Abstract] [Full Text] [PDF]
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 E. L. Oleszak, J. R. Chang, H. Friedman, C. D. Katsetos, and C. D. Platsoucas Theiler's Virus Infection: a Model for Multiple Sclerosis Clin. Microbiol. Rev., January 1, 2004; 17(1): 174 - 207. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
) but not by SNAP (
) or DEA-NO (
). The results are expressed
as the mean numbers of viable cells in 4 different experiments. (B)
SIN-1 (0.5 mM) induced apoptosis (TUNEL reaction and annexin V
staining) of a large population after as little as 4 hours. Most cells
were apoptotic after 18 hours of treatment with 0.5 mM SIN-1. SNAP had
no effect after 18 hours of culture. The analysis was performed on the
whole population. The data are representative of 4 individual
experiments. The percentages in each panel indicate the percentage of
TUNEL-positive cells in the upper panel, and percentage of Annexin
V-positive cells in the lower panel.
) show
a moderate reduction of the nitrotyrosine-positive cells, but this is
not observed with the MITC line used as a control thymic cell line
(
