* Department of Surgery, University of Pittsburgh School of Medicine,
Department of Biostatistics, Graduate School of Public Health, and
Children’s Hospital of Pittsburgh, University of Pittsburgh, Pittsburgh, Pennsylvania; and
Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chunchon, Kangwondo, Korea
Correspondence: Dr. Henri R. Ford, Department of Surgery, Children’s Hospital of Pittsburgh, University of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213. E-mail: FordH{at}chplink.chp.edu
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Key Words: thymus • bax • heme oxygenase • catalase
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),
interleukin (IL)-1, IL-6, and tumor necrosis factor alpha (TNF-
),
alone or in combination with bacterial endotoxin [lipopolysaccharide
(LPS)], results in NO production in vitro [4
,
5
]. Furthermore, we have demonstrated previously
[6
, 7
], and others have confirmed
[8
], that NO is produced during cell-mediated immune
reactions and, in turn, can modulate the proliferative activity of the
lymphocytes that participate in the immune response. More recently, we
have shown that NO production is associated with increased tissue
destruction as well as decreased T-lymphocyte function in a nonlethal,
graft-versus-host disease (GvHD) model [9
]. In addition,
using a lethal GvHD model, we have shown that administration of a NO
synthase (NOS) inhibitor ameliorated lethality [10
].
Worrall et al. [11
] demonstrated that
inhibition of NO production prolongs cardiac-allograft survival,
improves graft function, and decreases the histologic grade of
rejection. Thus, the data demonstrate that NO is produced in alloimmune
reactions as well as in a variety of other cell-mediated, immune
responses such as the response to intracellular pathogens or to
autoimmune stimuli [12
]. In some reactions, NO synthesis
appears to attenuate lymphocyte responses and in other circumstances,
to augment them. NO has been shown to induce apoptosis in a variety of cell types including macrophages [13 ], dendritic cells [14 ], thymocytes [15 ], and neuronal cells [16 ]. Recently, NO has been implicated in the process of negative selection in the thymus [17 ]. The mechanisms by which immature thymocytes undergo apoptosis during negative selection are poorly defined. Cross-talk between T-cell receptor (TCR)-stimulated thymocytes and stromal cells is believed to contribute to thymocyte apoptosis [18 ]. Macrophages, dendritic cells, and endothelial cells in the thymic stroma may play a critical role in this process because these cells are able to produce NO upon stimulation [13 , 14 , 17 ]. Furthermore, previous studies have demonstrated that cross-linking TCR leads to stromal-cell activation, expression of inducible NOS (NOS 2), and, subsequently, to thymocyte apoptosis [19 , 20 ]. The mechanisms by which NO exerts its regulatory effect on lymphocyte or thymocyte function and viability have not been well-delineated. There are various potential mechanisms of NO-mediated cytotoxicity. NO is a free-radical that can diffuse through the plasma membrane readily [21 ]. It has a high affinity for metalloproteins such as hemoglobin and iron (Fe)-containing enzymes such as aconitase, mitochondrial complexes I and II, and ribonucleotide reductase. Therefore, NO can inhibit critical cellular functions such as mitochondrial oxidative phosphorylation and DNA synthesis [22 , 23 ]. Heme iron-dependent enzymes such as catalase, NADPH oxidase, and cytochrome p450 may also be inhibited. NO may also augment the toxic effect of reactive oxygen species (ROS) such as superoxide by forming peroxynitrite, which can damage cellular proteins directly through nitration reactions [24 ]. In addition, at physiologic pH, peroxynitrite undergoes protonation to form peroxynitrous acid, which can theoretically give rise to other toxic molecules such as nitrogen dioxide or the short-lived hydroxyl radical. Lastly, NO, through its oxidative product, peroxynitrite, can interact with macromolecules such as structural proteins (which result in nitration of tyrosine residues) and DNA (which leads to purine and pyrimidine deamination), thereby damaging the infrastructure as well as the genetic information within the cell. These NO-mediated injuries may be cytostatic, resulting in inhibition of mitosis and proliferation, or cytotoxic, resulting in cell death via necrosis or apoptosis, as previously shown for macrophages [13 ], thymocytes [15 ], and chondrocytes [25 ]. In this study, we examined the effect of exogenous NO on the biochemical and molecular pathways that regulate NO-mediated thymocyte apoptosis.
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Thymocyte preparation
Single-cell suspensions of thymocytes were prepared in RPMI
(BioWhittaker, Walkersville, MD) as previously described
[1
]. Briefly, the cells were washed three times and
cultured in duplicate at a final concentration of 5 x
106 cells/ml in a final volume of 2 ml. The culture medium,
which consisted of RPMI supplemented with 1 mM sodium pyruvate, 100
units/ml penicillin, 100 µg/ml streptomycin, 1 mM nonessential amino
acids, 2 mM L-glutamine, and 5 x 10-5 M
2-mercaptoethanol, was supplemented with 5% fetal bovine serum.
Reagents
The NO donor, S-nitroso-N-acetyl penicillamine (SNAP), was
synthesized from NaNO2 and N-acetyl-D, L-penicillamine
(Sigma Chemical Co., St. Louis, MO) as described previously
[26
]. SNAP was dissolved in dimethylsulfoxide (DMSO;
American Type Culture Collection, Manassas, VA), aliquoted, and kept
frozen at -20°C. The heme oxygenase 1 (HSP-32) inhibitor, tin
protoporphyrin IX (SNPP9), and the synthetic corticosteroid,
dexamethasone, were purchased from Sigma. The former was resuspended in
6 N HCl at a final concentration of 25 mM and the latter, in distilled
water at a final concentration of 100 mM.
DNA fragmentation assay
The protocol was adapted from McCarthy et al.
[27
] . Briefly, cells were washed three times in
phosphate-buffered saline (PBS), lysed with a buffer containing 5 mM
Trizma base, 1 mM EDTA, and 0.5% Triton X-100, and centrifuged to
obtain supernatant and pellet fractions. Both fractions were sonicated
(Sonics & Materials Inc., Danbury, CT) for 60 s on ice and plated
in 0.1 ml triplicate serial dilutions in Dynatech MicroFluor 96-well
plates (Dynatech Laboratories, Alexandria, VA). An equal volume (0.1
ml) of a 0.6 µg/ml solution of the fluorescent DNA tag DAPI dye
(Sigma), suspended in a buffer containing 10 mM Trizma base and 100 mM
NaCl, was then added to the samples. Relative pellet and supernatant
DNA concentrations were calculated from the emission at 465 nm, as
measured on a Dynatech MicroFluor plate reader. Following mathematical
conversion, percent DNA fragmentation was quantitated as: % DNA
fragmentation = (DNA in supernatant)/(DNA in supernatant+DNA in
pellet) x 100.
Coculture of thymocytes and red blood cells (RBCs)
Coculture of thymocytes and RBCs was performed as described by
Kim et al. [26
]. Briefly, approximately 1 ml
blood was collected per anesthetized mouse by cardiac puncture prior to
thymectomy. The blood was centrifuged at 2000 g at 4°C for
5 min. The pellet was washed twice and resuspended in 15 ml media.
Thymocytes were then cultured in media alone, media with RBCs, SNAP
alone, or SNAP with RBCs for 24 h. Following incubation, % DNA
fragmentation was measured.
Supernatant nitrite levels
Nitrite (NO2-) concentrations were
measured using the Griess reagent as described previously
[28
]. Aliquots (0.1 ml) of culture supernatants were
added to an equal volume of Griess reagent (1% sulfanilamide/0.1%
napthylethylene diamide dihydrochloride/2.5%
H2PO3) and incubated at room temperature for 10
min. The absorbance was measured in a microplate reader (Molecular
Dynamics Corp., Sunnyvale, CA) at 550 nm using
NaNO2 as a standard.
Characterization of thymocyte subpopulations by flow cytometry
Following a 12-h incubation with culture media or SNAP, cells
were incubated with phycoerythrin (PE)-labeled, anti-CD4
antibody (Pharmingen, San Diego, CA), and tri-color (TC)-labeled,
anti-CD8 antibody (Caltag Laboratories, Burlingame, CA) at 4°C for 30
min in the dark, then washed, and incubated with 100 µl of 4%
paraformaldehyde (PFA) for 1 h at room temperature followed by
TUNEL [terminal deoxynucleotidyl transferase (TdT)-mediated
deoxyuridine triphosphate (dUTP) nick end-labeling] assay. Apoptotic
thymocytes were detected by TUNEL reagents according to the
manufacturer’s protocol (Boehringer Mannheim, Mannheim, Germany).
Briefly, cells were permeabilized by incubation with 100 µl of 0.1%
Triton X-100/0.1% sodium citrate at 0°C for 2 min. After washing,
the cells were resuspended in TUNEL reaction mixture containing
fluorescein isothiocyanate (FITC)-dUTP and Tdt, incubated at 37°C for
1 h and washed twice. Cell fluorescence was analyzed by flow
cytometry (Becton Dickinson, San Jose, CA). For FasL expression,
anti-FasL antibody (Pharmingen) was used.
Enzyme activity assays
Thymocyte glutathione (GSH), reduced and oxidized, superoxide
dismutase (SOD), and catalase activity were measured by harvesting
2 x 107 thymocytes from 16 mm wells after a 24-h
incubation in media alone or in 0.5 mM SNAP. The cells were
resuspendend in PBS containing protease inhibitors [6 µg/ml
leupeptin, 2 µg/ml aprotinin, and 1 µg/ml phenylmethylsulfonyl
fluouride (PMSF); Sigma] and then sonicated with three 15-s bursts on
ice. The solution was centrifuged at 15,000 g for 15 min at
4°C, and then the supernatant was assayed for enzyme activity
[26
]. Catalase activity was determined
spectrophotometrically by measuring decreased absorbance at 240 nm
using hydrogen peroxide as a substrate. The activity was calculated
from molecular extinction coefficient of 43.6
M-1 for H2O2. GSH
peroxidase activity was determined in the presence of GSH reductase by
the decrease in NADPH concentration. SOD activity was measured
spectrophotometrically by monitoring the inhibition of the reduction of
ferricytochrome c at 550 nm in the xanthine oxidase and hypoxanthine
system.
Measurement of total Fe and heme Fe concentrations
Thymocyte total iron (Fe) and heme Fe were measured as described
by Kim et al. [26
].
Polyacrylamide gel electrophoresis (PAGE) and Western blot analysis
Whole-cell lysates were obtained at various intervals by
incubating the cells in a buffer containing 50 mM Tris, 30 mM NaCl, 10
mM sodium dodecyl sulfate (SDS), 100 mM Triton X, and protease
inhibitors (6 µg/ml leupeptin, 2 µg/ml aprotinin, and 1µg/ml
PMSF; Sigma). Protein concentration was determined by bicinchonic
acid method (Pierce Biochemicals, Madison, WI). The protein
samples (80 µg) were boiled in sample buffer for 60 s and loaded
on 12% (HSP-32, bax, bcl-2, p21) or 10% (p53)
SDS-PAGE. Proteins were transferred to nitrocellulose membranes, which
were prehybridized with 5% fat-free milk in PBS, pH 7.5, containing
0.01% Tween-20 (PBST) for 1 h at room temperature and then
hybridized with antibodies to bcl-2 (Pharmingen),
bax (Santa Cruz Biotechnology, Santa Cruz, CA),
p53 (1:500; Calbiochem, Cambridge, MA), HSP-32 (Stress-Gen
Biotechnologies Corp., Victoria, BC), or p21 (Calbiochem) in
1% milk-PBST for 1 h at room temperature. The membranes were
washed and incubated in the appropriate horseradish
peroxidase-conjugated, secondary antibody diluted in 1% milk PBST for
1 h at room temperature. Antibody binding was detected with
enhanced chemiluminescence (ECL) reagent (Amersham Life Science,
Arlington Heights, IL) and developed on Kodak X-Omat film for a period
of 30 s–10 min. The band-density volume was measured by using a
Personal Densitometer IS (Molecular Dynamics) and expressed in
arbitrary units.
Irradiation
Thymocytes were exposed to a total of 5 Gy gamma radiation using
a Cesium emitter irradiator (Gamma Cell 1000 Elite, Nordion
International Inc., Vancouver, BC).
Statistics
Data are presented as mean ± SD. Percent
apoptosis, Fe concentrations, and enzyme activity were compared among
groups using a two-tailed t-test with Bonferoni adjustment
for multiple comparisons (=0.01).
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 View larger version (17K): [in a new window] |
Figure 1. (A and B) The effect of SNAP on thymocyte apoptosis. BALB/c thymocytes
were cultured for 24 h in the presence of increasing
concentrations of SNAP. Percent DNA fragmentation (apoptosis; A) and
supernatant nitrite (NO2-) concentration (B)
were measured. The data represent the mean ± SD of
three separate experiments; *P < 0.001
versus media, penicillamine, or DMSO (see text); open areas or
SNAP (solid bars). (C) Thymocytes were cultured with SNAP or media for
24 h. The cells were labeled with TUNEL reaction mixture
containing FITC and dUTP and analyzed by flow cytometry. The majority
of the cells treated with media (open area) are viable (large peak on
left); the rate of spontaneous apoptosis (small peak on right) is 33%.
In contrast, cells treated with SNAP (dark area) show extensive
apoptosis (77%).
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Figure 2. Effect of RBCs on SNAP-induced thymocyte apoptosis. BALB/c thymocytes
were cultured in the presence of media, media with RBCs, SNAP, or SNAP
with RBCs for 24 h. Percent DNA fragmentation and supernatant
NO2- concentrations were measured. A
representative of three experiments is shown.
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Figure 3. Kinetics of SNAP-induced apoptosis. BALB/c thymocytes were incubated
with media, 0.5 mM SNAP, or DMSO for 6, 12, or 24 h. Irradiated
thymocytes (5 Gy) were incubated for identical periods. Percent
apoptosis was determined at each time point. The data represent the
mean ± SD of three separate experiments;
*P < 0.01 versus media or DMSO.
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Figure 4. The effect of length of exposure to SNAP on thymocyte apoptosis. BALB/c
thymocytes were cultured in 0.5 mM SNAP for 3, 6, 9, or 24 h. At
each time point, the cells were washed to remove SNAP and then
re-incubated in fresh tissue culture (SNAP-free) media. Percent DNA
fragmentation was determined at 24 h for each group. A
representative of three experiments is shown. (SD for
triplicate samples is shown.)
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Figure 5. Phenotypic analysis of TUNEL-positive and -negative thymocytes, which
were cultured in medium or SNAP for 12 h. Cells were labeled with
PE-anti-CD4 and TC-anti-CD8 antibodies followed by TUNEL assay, as
described in Materials and Methods. Cell fluorescence was analyzed by
flow cytometry. A representative of three experiments is shown.
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Table 1. Effect of SNAP on Thymocyte Catalase, Glutathione (GSH), and Superoxide
Dismutase (SOD) Activity
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Table 2. Effect of SNAP on Thymocyte Total Fe and Heme Fe Concentrations
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Figure 6. Thymocyte HSP-32 expression. Thymocytes were cultured with or without
0.5 mM SNAP. After 4, 8, or 24 h of culture, HSP-32 expression was
analyzed by Western blot. Up-regulation of HSP-32 was detected in
SNAP-treated thymocytes at 4 and 8 h. A representative blot of
three experiments is shown.
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Table 3. The Effect of Tin-Protoporphyrin IX (SNPP9) on NO-Induced Apoptosis
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Figure 7. p53 expression in SNAP-treated thymocytes, which were
cultured with or without 0.5 mM SNAP after 1 and 8 h of culture.
p53 expression was analyzed by Western blot, and the
band-density volume for each group was measured. The cell line C3L5
served as positive control. A representative of three experiments is
shown.
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Figure 8. Bax expression in SNAP-treated thymocytes, which were
cultured with or without 0.5 mM SNAP or irradiated. After 4 or 8 h, bax expression was analyzed by Western blot, and the
band-density volume for each group was measured. Jurkat cells served as
positive control. A representative of three experiments is shown.
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Table 4. The Effect of SNAP and Irradiation on Thymocytes from p53
Wild Type (WT) and p53 Null (KO) Mice
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It is well-documented that thymocyte apoptosis plays an important role in cellular physiology and pathophysiology in the immune system. A number of external signals have been shown to regulate thymocyte apoptosis in vivo. These signals include cytokines, hormones such as glucocorticoids, growth hormones, thyroid and sex hormones, and nutrients such as Zn2+ [33 ]. Fehsel et al. [15 ] showed that administration of LPS to mice resulted in an increased incidence of apoptosis in the thymic cortex, presumably because of the production of NO by adjacent dendritic and capillary endothelial cells, thus suggesting that NO may play a regulatory role in thymocyte apoptosis in vivo during periods of physiologic stress. Our data demonstrate that SNAP induces apoptosis in thymocytes in a dose-dependent manner. There was a direct correlation between the amount of SNAP added and the concentration of the stable end-product of NO metabolism—nitrite—detected in the supernatant, indicating that NO was released in the cultures. The fact that addition of RBCs to the cultures neutralized the effect of SNAP, combined with our observation that the parent compound, penicillamine, as well as the diluent DMSO had no effect on the cells, suggests that apoptosis was indeed the direct result of NO generation in culture. Fehsel et al. [15 ] and Tai et al. [17 ] also demonstrated that the CD4+8+ subset of thymocytes is the population that is most sensitive to NO, which is consistent with our observation that NO induces apoptosis in 50–60% of mostly CD4+8+ thymocytes and a relative increase in the percentage of CD4+ cells. High doses of SNAP did not lead to any further increase in apoptosis once the NO-sensitive population had already been affected.
Possible mechanisms by which NO may exert its immunoregulatory effects include: 1) inhibition of iron-dependent metabolic enzymes such as heme-dependent enzymes (i.e., catalase), cytochrome p450, Fe-sulfur proteins such as aconitase, NADH (ubiquinone reductase) and NADH (succinate oxireductase) [29 , 34 , 35 ], and ribonucleotide reductase, the rate-limiting enzyme in DNA synthesis [23 ]; 2) interaction with reactive oxygen species to alter redox reactions [36 ]; and 3) induction of apoptotic mediators including p53, as has been suggested in macrophages, thymocytes, and chondrocytes [13 , 15 , 25 ].
Thymocyte exposure to SNAP resulted in a significant decrease in the activity of the antioxidant enzyme catalase as well as a concomitant decrease in heme-Fe levels. These results indicate that despite normal SOD, GSH, and total Fe levels, exposure to NO may predispose thymocytes to oxidative injury by impairing their ability to convert H2O2 into water. These observations are consistent with the study by Forrest et al. [37 ], who showed that thymocytes subjected to oxidative stress by treating them with H2O2 in the presence of ferrous sulfate underwent apoptosis. However, they are in conflict with the data by Wang et al. [38 ], who demonstrated that decreased, spontaneous production of ROS precedes spontaneous and dexamethasone-induced thymocyte apoptosis in vitro. The significance of the decrease in catalase activity and heme-Fe levels remains speculative at best. Because the cellular membrane is impermeable to exogenous catalase, the enzyme cannot be added back successfully to thymocytes in culture to determine whether restoration of intracellular catalase levels would prevent NO-induced apoptosis. HSP-32, an inducible isoform of heme oxygenase, which may be responsible for the decrease in catalase activity, was up-regulated at 4 and 8 h but returned to baseline by 24 h. It suggests that oxidative stress may play a pivotal role in NO-induced thymocyte apoptosis. In addition, the fact that SNPP9, a specific inhibitor of HSP-32, blocked SNAP-induced apoptosis further suggests that oxidative injury may be part of the final, common pathway in NO-induced thymocyte apoptosis.
p53 is a pivotal cell-cycle checkpoint and mediator of apoptosis. Once the cell has sustained a significant degree of injury, p53 is activated, which, in turn, induces the cell-cycle inhibitor protein p21, which mediates G1 cell-cycle arrest. Interruption of the cell cycle at G1 allows the activation of DNA repair enzymes to decrease the possibility of chromosomal mutations, translocations, deletions, and inversions prior to replication. In situations where extensive damage to the DNA or cytoskeletal structures has occurred, p53 is able to induce apoptosis. Our findings are consistent with those of Fehsel et al. [15 ] and Messmer et al. [39 ], who showed that p53 is up-regulated after exposure to NO. However, we extend the observations by Fehsel et al. by demonstrating that NO-induced thymocyte apoptosis is a p53-dependent phenomenon, similar to radiation-induced thymocyte apoptosis. The role of p21 in this process, if any, is not entirely clear because its expression was unaffected. Miyashita, Reed, and co-workers [40 , 41 ] have performed extensive studies on the interactions between p53 and the bcl-2 family of apoptosis-modulating proteins. p53 can up-regulate bax gene expression by binding to the bax promoter [40 ]. In addition, p53 can down-regulate bcl-2 expression, reducing the ability of bcl-2 to heterodimerize with bax and thereby avert apoptosis. Thus, overexpression of bax, along with down-regulation or even constitutive expression of bcl-2, can lead to an increase in bax homodimers, a combination that has been shown to accelerate apoptotic cell death [42 ]. Our data show that p53 is up-regulated within 1 h and remains elevated up to 8 h following exposure to NO, and furthermore, it is necessary for NO-induced apoptosis. By 8 h, bax appears to be up-regulated, but bcl-2 expression is unchanged, even at 24 h. Thus, it is possible that p53 may transactivate bax in our system. FasL expression was unaffected by SNAP treatment.
Our data suggest that conditions that up-regulate NO production in the thymus may lead to accelerated thymocyte apoptosis. The source of NO production in the thymus is not entirely clear. Kirk and colleagues [43 ] demonstrated that NO synthesis occurs following stimulation of specific murine, T-cell clones, however NOS 2 activity was not demonstrated. Using activated T-cell clones specific for malaria, Taylor-Robinson et al. [44 ] demonstrated the expression of NOS 2 mRNA in T-helper cell (Th)1 but not Th2 cells. In vivo and in vitro data from our laboratory suggest that thymocytes do not possess the NOS 2 enzyme and, therefore, are incapable of producing NO, even when stimulated with LPS and cytokines. In fact, NOS 2 inhibitors do not prevent DNA fragmentation in thymocytes exposed to various apoptotic stimuli in vitro, including phorbol 12-myristate 13-acetate (PMA) and/or calcium ionophore (unpublished results). Lu et al. [14 ] demonstrated that in alloimmune reactions or in septic states, activated APCs such as macrophages or dendritic cells exhibit a marked increase in NOS 2 mRNA in vivo and in vitro. Despite the aforementioned, isolated studies suggesting that T lymphocytes make NO, APCs are probably the most potent and significant sources of NO within the thymus during physiologic stress.
The mechanism by which a decrease in catalase activity and up-regulation of p53, bax, and HSP-32 leads to thymocyte apoptosis is not clearly defined. Data from our laboratory suggest that the cysteine protease, caspase-1, may be the terminal effector of thymocyte apoptosis in this pathway [1 ]. Currently, we are investigating how p53 and bax can lead to cleavage of pro-caspase-1 in our system and ultimately affect thymocyte apoptosis.
Received July 11, 2000; revised December 28, 2000; accepted February 15, 2001.
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