Contrasting effects of NO and peroxynitrites on

Am J Physiol Cell Physiol
279: C452–C460, 2000.
Contrasting effects of NO and peroxynitrites on HSP70
expression and apoptosis in human monocytes
CHRISTOPHE ADRIE,1,2 CHRISTOPH RICHTER,3 MARIA BACHELET,1 NATHALIE BANZET,1
DOMINIQUE FRANÇOIS,1 A. TUAN DINH-XUAN,1 JEAN FRANÇOIS DHAINAUT,2
BARBARA S. POLLA,1,5 AND MARIE-JEANNE RICHARD1,4
1
Laboratory of Respiratory Physiology, Unité de Formation et de Recherche Cochin Port-Royal,
Paris V University, and 2Medical Intensive Care Unit, Cochin Port-Royal Hospital, Paris, France;
3
Institute of Biochemistry, Swiss Federal Institute of Technology, Zurich, Switzerland; and
4
Laboratory of Biology of Oxidative Stress, Biochemistry C, A. Michallon Hospital, Grenoble, France
Adrie, Christophe, Christoph Richter, Maria Bachelet, Nathalie Banzet, Dominique François, A. Tuan
Dinh-Xuan, Jean François Dhainaut, Barbara S. Polla,
and Marie-Jeanne Richard. Contrasting effects of NO and
peroxynitrites on HSP70 expression and apoptosis in human
monocytes. Am J Physiol Cell Physiol 279: C452–C460,
2000.—The free radicals nitric oxide (䡠NO) and superoxide
(O2⫺䡠) react to form peroxynitrite (ONOO⫺), a highly toxic
oxidant species. In this study we investigated the respective
effects of NO and ONOO⫺ in monocytes from healthy human
donors. Purified monocytes were incubated for 6 or 16 h with
a pure NO donor (S-nitroso-N-acetyl-DL-penicillamine, 0–2
mM), an 䡠NO/ONOO⫺ donor (3-morpholinosydnonimine chlorhydrate, 0–2 mM) with and without superoxide dismutase
(200 IU/ml), or pure ONOO⫺. We provide evidence that
3-morpholinosydnonimine chlorhydrate alone represents a
strong stress to human monocytes leading to a dose-dependent increase in heat shock protein-70 (HSP70) expression,
mitochondrial membrane depolarization, and cell death by
apoptosis and necrosis. These phenomena were abolished by
superoxide dismutase, suggesting that ONOO⫺, but not 䡠NO,
was responsible for the observed effects. This observation
was further strengthened by the absence of a stress response
in cells exposed to S-nitroso-N-acetyl-DL-penicillamine. Conversely, exposure of cells to ONOO⫺ alone also induced mitochondrial membrane depolarization and cell death by apoptosis and necrosis. Thus ONOO⫺ formation may well
explain the toxic effect generally attributed to 䡠NO.
(ROS) are generated from molecular oxygen and include the free radicals superoxide
(O2⫺䡠), hydroxyl (䡠OH), and nitric oxide (䡠NO), as well as
nonradical intermediates such as H2O2, peroxynitrite
(ONOO⫺), and singlet oxygen (1O2) (15, 41). During
normal cellular respiration in the mitochondria, ROS
are constantly produced at low rates. At these low
concentrations, ROS can act as second messengers and
mediators for cell activation. However, during infection
or inflammation or on exposure to various environmen-
tal toxic agents, ROS can accumulate to deleterious
levels, leading to cell damage and subsequent adaptive
responses (28).
NO is an endogenous mediator first characterized as
an endothelium-derived relaxing factor (21). It is now
recognized as a key mediator in many physiological
processes. Although 䡠NO generated by activated macrophages plays a key role in the defense against tumor
cells and pathogens, it has been reported to be protective or cytotoxic, according to its rate and amount of
production (19, 54). 䡠NO-mediated cytotoxicity might
underlie the pathogenesis of an organ in shock and
inflammation.
The stress response is a marker of cellular injury and
a conserved adaptive response to stressful conditions
such as oxidative stress. It includes induction of the
so-called heat shock (HS) proteins (HSP). The HSP
and, in particular, the cytosolic, inducible, 72-kDa
HSP70 protect human cells against the deleterious
effects of ROS, including 䡠NO. These protective effects
are exerted primarily at the mitochondrial level and
are associated with an inhibition of apoptosis when
HSP70 are induced before stresses (9, 39, 40, 52).
However, the protective effects of HSP70 cannot be
extrapolated to all cases of apoptosis (52, 53).
Several reports have suggested that 䡠NO is involved,
on the one hand, in HSP70 induction (30, 35, 36) and,
on the other hand, in mitochondrial dysfunction and
cell death. 䡠NO and O2⫺䡠, however, are weak oxidants.
Thus the question as to whether 䡠NO by itself or its
by-products, such as ONOO⫺, are responsible for these
various effects remains a matter of controversy. For
example, toxic effects such as DNA single-strand
breaks and activation of the enzyme poly(ADP-ribose)
polymerase, once attributed to 䡠NO, are now believed to
be mediated by ONOO⫺ (50, 51).
We addressed this issue in human monocytes, since
these cells play a key role in defense mechanisms
Address for reprint requests and other correspondence: M.-J. Richard, Laboratoire de Biochimie C, Hôpital A. Michallon, BP217X,
38043 Grenoble, France (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
nitric oxide; heat shock; cell stress; cell death
REACTIVE OXYGEN SPECIES
C452
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society
http://www.ajpcell.org
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Received 12 July 1999; accepted in final form 7 February 2000
NO AND STRESS RESPONSE IN HUMAN MONOCYTES
during infection and inflammation, as sources and as
targets of mediators such as ROS. Using 䡠NO/ONOO⫺
donors or exogenous ONOO⫺, we investigated the respective contribution of 䡠NO and ONOO⫺ in the monocyte stress response (as markers of oxidative stress)
and cytotoxicity. We analyzed HSP70 expression, mitochondrial membrane potential (⌬␺m), and cell death
by apoptosis or necrosis. Our results suggest that 䡠NO
is not by itself stressful to human monocytes, whereas
ONOO⫺ induces an oxidative stress response in these
cells.
MATERIALS AND METHODS
were stored at ⫺70°C and pH 12. Because of the potential
presence of H2O2 as contaminant in this solution, ONOO⫺
was applied to culture medium in the absence of cells at the
highest concentration used in our study, and the solution was
incubated for 30 min at 37°C to completely degrade ONOO⫺.
The medium on the cells was replaced with the blank reagent
obtained after complete degradation of ONOO⫺. This allowed
us to distinguish the effects of ONOO⫺ from those of its
by-products or contaminants (51).
Cells. Human peripheral mononuclear cells were isolated
by Ficoll gradient centrifugation, and monocytes were purified by adherence in 60-mm-diameter petri dishes for 45 min
and then washed with PBS. Cells were maintained in RPMI
1640 medium containing 10% FCS, 2 mM glutamine, and 25
mM HEPES in a humidified atmosphere containing 95%
air-5% CO2 at 37°C.
Cells were exposed to the pure 䡠NO donor SNAP (0.5, 0.75,
1, and 2 mM) or to Sin-1 (0.25, 0.5, 1, and 2 mM) for 6 or 16 h.
Sin-1 releases 䡠NO and O2⫺䡠 in an equimolar manner, thus
generating ONOO⫺, whereas SNAP releases only 䡠NO (20).
Cells were incubated with Sin-1 or SNAP in the presence or
absence of SOD (200 IU/ml, added 20 min before 䡠NO donors).
SOD, by dismutating O2⫺䡠, prevents the formation of ONOO⫺
during Sin-1 decomposition.
Nitrite/nitrate determination. 䡠NO is oxidized in cell medium to form several nitrogen oxides (NOx), in particular
nitrate (NO3⫺) and nitrite (NO2⫺). NOx can be measured in
medium by conversion to 䡠NO by use of a strong reducing
environment (3) consisting of vanadium and 1 N HCl at 90°C
as follows: NO3⫺ ⫹ 4H⫹ ⫹ 3e⫺3 䡠NO ⫹ 2H2O. The amount of
䡠NO produced was determined by chemiluminescence with
Fig. 1. Cytofluorometric analysis of heat shock (HS)
protein (HSP)-70 expression. Results are from 1 representative experiment in which HSP70 expression was
analyzed in control cells (A), heat-shocked (44°C, 30
min) cells (B), and cells exposed to 1 mM 3-morpholinosydnonimine chlorhydrate (Sin-1) for 6 h in the absence (C) or presence (D) of superoxide dismutase
(SOD, 200 IU/ml). There was a marked increase in
HSP70 expression after HS and after exposure to Sin-1.
Preincubation with SOD abolished the effects elicited
by Sin-1.
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Reagents and media. 3-Morpholinosydnonimine chlorhydrate (Sin-1) was kindly provided by Hoechst (Paris, France).
S-nitroso-N-acetyl-DL-penicillamine (SNAP), superoxide dismutase (SOD) from bovine erythrocytes (EC 1.15.1.1), and
saponin were purchased from Sigma Chemical (St. Louis,
MO), antibiotic-free RPMI 1640 medium, fetal calf serum
(FCS), HEPES, and glutamine from GIBCO (Paisley, Scotland), the monoclonal antibody SPA-810, specific for the
inducible HSP70, from StressGen Biotechnologies (Victoria,
Canada), the secondary anti-mouse antibody (IgG-FITC)
from Dako (Carpinteria, CA), 5,5⬘,6,6⬘-tetrachloro-1,1⬘,3,3⬘tetraethylbenzimidazolylcarbocyanine iodide (JC-1) from
Molecular Probes (Eugene, OR), and the annexin V-Fluos
staining kit from Boehringer Mannheim (Mannheim, Germany).
ONOO⫺ was synthesized from sodium nitrite and H2O2 by
use of a quenched flow reactor (8). Stock solutions (83 mM)
C453
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NO AND STRESS RESPONSE IN HUMAN MONOCYTES
parison of the mean. The criterion for statistical significance
was P ⬍ 0.05.
RESULTS
Effects of Sin-1 and SNAP on Hsp70 expression.
Inasmuch as ROS were previously reported to induce a
stress response in human monocytes, we tested
whether 䡠NO itself, or its by-product ONOO⫺, induced
HSP70 expression. Cells were exposed to Sin-1 with or
without SOD to distinguish the respective effects of
䡠NO and ONOO⫺. Sin-1 (1 mM, 6 h) induced HSP70
expression (Fig. 1C) to levels similar to those induced
by HS (Fig. 1B), although the distribution of HSP70positive and HSP70-negative cells was different with
HS (double peak) and with Sin-1 (single peak). In the
presence of SOD, HSP70 expression induced by Sin-1
was abolished (Fig. 1D). The expression of HSP70 was
detectable 3 h after exposure to Sin-1 and was no
longer detectable after 16 h (data not shown).
Fig. 2. Effects of Sin-1 and S-nitroso-N-acetyl-DL-penicillamine (SNAP)
on HSP70 expression. Cells were incubated with 0.25–2 mM Sin-1 for
6 h (A) or 0.5–2 mM SNAP for 16 h (B) with (SOD⫹) or without SOD
(SOD⫺). Values are means ⫾ SE; n ⫽ 4. *P ⬍ 0.05 vs. respective controls;
#
P ⬍ 0.05 vs. controls with SOD. Sin-1 induced an increase of HSP70
expression that is inhibited by preincubation with 200 mM SOD.
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use of a fast-responding analyzer (model NOA 280, Sievers
Instruments) (3).
Analysis of HSP70 expression. The level of HSP70 expression in monocytes was quantified by flow cytometry analysis,
as described previously (4). Briefly, a pellet of 106 cells was
resuspended in 100 ␮l of 3% paraformaldehyde in PBS, kept
for 10 min at room temperature, and then washed by addition of 1 ml of PBS with 1% BSA (PBS-BSA). For labeling,
cells were incubated in 50 ␮l of 0.6% saponin and the antibody against the cytosolic inducible HSP70 at a dilution of
1:100 for 10 min at room temperature. Unbound antibodies
were removed by washing twice in PBS-BSA. Bound antibodies were revealed with rabbit anti-mouse IgG-FITC conjugate
diluted at 1:30 for 10 min at room temperature. Analysis was
performed by flow cytometry (EPICS Elite flow cytometer;
Coulter, Miami, FL). HS (44°C for 30 min, with 4 h of
recovery) was used as positive control for HSP70 induction in
monocytes. Data are expressed as the ratio of median
fluorescence channels (data have been converted from
logarithmic to linear scale) of cells incubated with an irrelevant isotypic matched antibody (i.e., the negative control) to
that of cells incubated with the monoclonal anti-HSP70
antibody.
Determination of ⌬␺m. ⌬⌿m was measured by using the
lipophilic cation JC-1, which is able to selectively enter mitochondria. JC-1 exists in a monomeric form, emitting at 527
nm after excitation at 490 nm. Depending on ⌬⌿m, JC-1 is
able to form J-aggregates that are associated with a large
shift in emission (590 nm). Dye color changes reversibly from
green to greenish orange as mitochondrial membrane becomes more polarized. Cell staining was performed as follows: cell suspensions were adjusted to a density of 0.5 ⫻ 106
cells/ml and incubated in supplemented RPMI 1640 medium
with JC-1 (10 ␮g/ml) for 10 min at room temperature in the
dark. At the end of the incubation period, cells were washed
in PBS, resuspended in a total volume of 400 ␮l, and immediately analyzed by flow cytometry using a EPICS Elite flow
cytometer, as previously described (17, 47). H2O2 (4 mM, 4 h
at 37°C) was used as positive control for mitochondrial depolarization (40).
Flow cytometry analysis of cell death by apoptosis and
necrosis. Apoptosis was detected with annexin V, which has
high affinity for negatively charged phospholipids such as
phosphatidylserine. The simultaneous use of the DNA stain
propidium iodide, which is excluded from intact and apoptotic cells, allows for the adequate detection of necrotic cells
among the annexin V-positive cluster. Cells were washed,
stained with annexin V and propidium iodide in HEPES
buffer as described by the manufacturer, and analyzed by
flow cytometry with an EPICS Elite flow cytometer equipped
with a single 488-nm argon laser. In all cases, a total of 5,000
cells/sample were analyzed in list mode for green fluorescence through a 525-nm filter and for red fluorescence
through a 575-nm filter. All data were analyzed with Elite
software version 4.02. Cells exclusively positive for annexin
V were considered to be undergoing apoptosis, whereas cells
positive for propidium iodide and annexin V were considered
necrotic (18, 52).
Electron microscopy. After Sin-1 treatment, monocytes
were fixed in suspension (106 cells) with 2.5% glutaraldehyde
in 0.1 M PBS, pH 7.4, for 10 min at 4°C and as a pellet for 2 h.
After they were washed in PBS, ultrathin sections were cut,
counterstained, and then examined in a Philips EM-300
electron microscope operating at 60 kV.
Statistical analysis. Values are means ⫾ SE. The data
were analyzed using a one-way ANOVA for repeated measures followed by the Mann-Whitney test for post hoc com-
C455
NO AND STRESS RESPONSE IN HUMAN MONOCYTES
Table 1. NO2⫺/NO3⫺ production after exposure of human monocytes to Sin-1 or SNAP
Sin-1, mM
⫺
2
⫺
3
NO /NO , ␮M
SOD⫺
SOD⫹
0
0.25
0.5
1
2
37 ⫾ 27
56 ⫾ 59
268 ⫾ 96*
315 ⫾ 128
450 ⫾ 88*
600 ⫾ 127*
950 ⫾ 241†
735 ⫾ 198†
1,327 ⫾ 349†
1,643 ⫾ 538†
SNAP, mM
NO2⫺/NO3⫺, ␮M
SOD⫺
SOD⫹
0
0.5
0.75
1
2
19 ⫾ 23
35 ⫾ 43
175 ⫾ 89
187 ⫾ 69
229 ⫾ 40*
262 ⫾ 52*
484 ⫾ 47†
347 ⫾ 117†
678 ⫾ 311†
642 ⫾ 330†
We then tested the dose dependence of HSP70 induction by Sin-1, in the presence or absence of SOD, and
compared it with the pure NO donor SNAP. HSP70
expression increased gradually as a function of Sin-1
concentration up to 1 mM (Fig. 2A) but decreased at a
higher concentration (2 mM, 6 h), an observation probably related to the toxicity of Sin-1 at this concentration. SOD completely prevented the increased expression of HSP70 at all concentrations tested, indicating
that 䡠NO per se was unable to induce HSP70 expression in human monocytes, whereas ONOO⫺ generated
during Sin-1 decomposition appeared as an efficient
inducer of HSP70 expression. These conclusions were
further supported by the lack of induction of HSP70
expression obtained with the pure 䡠NO donor SNAP
(Fig. 2B) at any concentration tested (0.5–2 mM) after
6 or 16 h of exposure, despite dramatic increases in
NO2⫺/NO3⫺ concentrations in the supernatant (Table 1).
In addition, SOD had no effect in SNAP-treated cells
(Fig. 2B).
Effects of Sin-1 and SNAP on ⌬⌿m. A close link
between mitochondrial functions and apoptosis was
previously proposed (33). We thus investigated the
effects of 䡠NO/ONOO⫺ on ⌬⌿m (Figs. 3 and 4) and
compared them with those obtained in cells treated
with H2O2, an oxidant known to induce a marked
mitochondrial depolarization (Fig. 3B). Sin-1 (1 mM,
6 h) induced a decrease in ⌬⌿m (Fig. 3C) that was
prevented by SOD (Fig. 3D).
Exposure of human monocytes to Sin-1 induced dosedependent (0.25–2 mM) disruption of ⌬⌿m, which was
prevented by SOD (Fig. 4A). When cells were exposed
to increasing doses of SNAP (0–2 mM) for ⱖ6 h (16 h,
data not shown), no alterations in ⌬⌿m were observed
with or without addition of SOD (Fig. 4B). These results indicate that the expression of HSP70 in cells
exposed to Sin-1 is insufficient to afford protection from
alterations of ⌬⌿m induced by ONOO⫺ in human
monocytes. In addition, we observed that, parallel to
the lack of induction of HSP70 expression, 䡠NO per se
did not affect ⌬⌿m in human monocytes.
Effects of Sin-1 and SNAP on cell death. Inasmuch as
mitochondrial depolarization is generally recognized
as a prerequisite step in the pathways leading to ROSdependent apoptosis, the effects of 䡠NO/ONOO⫺ on
Fig. 3. Cytofluorometric analysis of mitochondrial membrane potential (⌬⌿m). Cytofluorometric analysis of ⌬⌿m
was assessed by staining with 5,5⬘,6,6⬘-tetrachloro1,1⬘,3,3⬘-tetraethylbenzimidazolylcarbocyanine iodide
(JC-1) staining in control cells (A), cells treated with 4
mM H2O2 for 4 h (B), and cells exposed to 1 mM Sin-1
for 6 h in the absence (C) and presence (D) of SOD (200
IU/ml). Results from 1 representative experiment are
shown. Percentage of cells with depolarized mitochondria is shown.
Downloaded from http://ajpcell.physiology.org/ by 10.220.32.246 on February 11, 2017
Values are means ⫾ SE; n ⫽ 3. Nitrite and nitrate (NO2⫺/NO3⫺) production after 6 h of incubation with 3-morpholinosydronimine
chlorhydrate (Sin-1) or S-nitroso-N-acetyl-DL-penicillamine (SNAP) without (SOD⫺) or with superoxide dismutase (SOD⫹). Dose-dependent
increase in NO2⫺/NO3⫺ monocyte exposure to SNAP or Sin-1 was not affected by SOD. * P ⬍ 0.05; † P ⬍ 0.01 vs. baseline.
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NO AND STRESS RESPONSE IN HUMAN MONOCYTES
monocyte death were analyzed. Monocytes exposed to
Sin-1 for 16 h underwent cell death by apoptosis or
necrosis depending on the concentration applied (Fig.
5A), with apoptosis reaching a maximum at 1 mM. At
a higher concentration (2 mM), apoptosis decreased
whereas necrosis increased up to 78 ⫾ 8%. As observed
for ⌬⌿m and for HSP70 expression, SOD prevented
Sin-1-induced cell death, indicating that ONOO⫺, but
not 䡠NO, was responsible for cell death by apoptosis or
by necrosis. High concentrations of SNAP for up to 16 h
did not elicit any effect on human monocyte viability,
further supporting the conclusions that ONOO⫺, but
not 䡠NO, was cytotoxic in these cells (Fig. 5B).
Effects of Sin-1 on ultrastructural morphology. Functional criteria for Sin-1-induced cell death paralleled
ultrastructural criteria of apoptosis or necrosis (Fig. 6).
Electron microscopy showed that a low concentration
of Sin-1 essentially led to an alteration of cytosolic
organelles with “cytosolic boiling,” swollen mitochondria, and perinuclear condensation. At higher concen-
trations, cells exhibiting features of apoptosis coexisted
with cells exhibiting necrosis features, such as plasma
membrane disruption and pycnotic nuclei. These morphological alterations were prevented by preincubation
with SOD.
Effects of ONOO⫺ on ⌬⌿m and cell death. Finally, to
further confirm our results, we investigated the effects
of exposure to increasing concentrations of ONOO⫺
(0–1,000 ␮M, 16 h) on human monocytes. ONOO⫺ induced ⌬␺m in a dose-dependent manner (Table 2) as well
as cell death by apoptosis or necrosis, depending on its
concentration (Table 2). The blank reagent obtained after
complete degradation of ONOO⫺ did not exert any cytotoxicity on human monocytes, excluding an effect due to
contaminants or decomposition products of ONOO⫺.
DISCUSSION
Here, we report that ONOO⫺ induced a stress response in human monocytes that was characterized by
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Fig. 4. Effects of Sin-1 and SNAP on ⌬⌿m. Cells were
incubated with 0.25–2 mM Sin-1 for 6 h (A) or 0.5–2
mM SNAP for 16 h (B) with or without SOD. Values are
means ⫾ SE; n ⫽ 3. * P ⬍ 0.01 vs. respective controls;
#
P ⬍ 0.01 vs. controls with SOD. Sin-1 induced a
dose-dependent mitochondrial membrane depolarization that was abolished by SOD. SNAP did not alter
⌬␺m.
NO AND STRESS RESPONSE IN HUMAN MONOCYTES
induction of HSP70 expression, mitochondrial membrane depolarization, and cell death by apoptosis or
necrosis, whereas 䡠NO per se, even at high concentrations, did not elicit such a stress response in these cells.
The HS/stress response is a conserved, physiological,
and transient response to cellular injuries, including
oxidative stress. The presence of abnormal, unfolded,
or misfolded proteins, alterations in membrane physical state, classical second messengers, specific mitochondrial alterations, or ROS could represent the cellular sensors for HSP induction (2, 13, 29, 38, 39).
Among ROS, we previously showed that H2O2 and 䡠OH,
but not the membrane-impermeant O2⫺䡠, are inducers
of an HS response (6). Here we studied the expression
of HSP70 in monocytes on exposure to 䡠NO or ONOO⫺.
Sin-1, which generates 䡠NO and O2⫺䡠, induced the intracellular overexpression of HSP70, which was abolished
by the addition of SOD, indicating that ONOO⫺, but
not 䡠NO, induced HSP70 accumulation. This conclusion
was supported by the lack of induction of HSP70 expression that we observed with SNAP, a slow genera-
tor of 䡠NO alone, even at high concentrations. Similarly, SNAP did not induce HSP in a human
keratinocyte cell line, even at toxic concentrations (unpublished observations).
Malyshev et al. (35, 36) identified 䡠NO as an inducer
of HSP70 during HS in rat tissues and human hepatoblastoma cells. They suggested that the ionized form of
䡠NO interacts with SH-groups of HS factor (HSF), leading to an S-nitrosothiol intermediate, catalyzing the
trimerization of HSF, accelerating the formation of
disulfides bonds between HSP molecules, which ultimately favors the DNA binding of HSF. Nevertheless,
䡠NO itself, as O2⫺䡠, is not a strong oxidant and cannot
nitrosate thiols (41), whereas the energetic form of
ONOO⫺ is a potent oxidant capable of oxidizing a
variety of biomolecules including thiols (31). The various studies that suggested that 䡠NO is involved in
HSP70 induction (30, 35, 36) have generally not considered the respective roles of 䡠NO and ONOO⫺. Our
results indicate that, at least in human monocytes, 䡠NO
is not involved in the HS response. Although tissue
specificity is another plausible explanation for the observed differences, we would favor the possibility that
ONOO⫺ is indeed the 䡠NO-related activator of HSP.
We demonstrated that HSF binding depends, at
least in part, on intracellular redox potential (27),
which is regulated by mitochondria, whereas HSP70
prevents mitochondrial membrane depolarization (40).
This spurred our interest in investigating the respective effects of 䡠NO/ONOO⫺ on ⌬⌿m. Here we report
that Sin-1 induced a dose-dependent mitochondrial
depolarization that was completely abolished on addition of SOD. The pure 䡠NO donor SNAP did not alter
⌬⌿m. Moreover, ONOO⫺ alone induced ⌬⌿m disruption similar to that induced by Sin-1. Various reports
showed that 䡠NO can reversibly inhibit mitochondrial
respiration by competing with oxygen at cytochrome
oxidase (11, 12, 16, 49), whereas the formation of
ONOO⫺ from 䡠NO and mitochondria-derived superoxide can cause the irreversible inhibition of complexes
I-III, leading to mitochondrial dysfunction (14, 34, 42).
Moreover, 䡠NO has been shown to induce apoptosis by
triggering mitochondrial permeability transition and
mitochondrial membrane depolarization (5, 26). Our
data, however, indicate that 䡠NO per se did not alter
⌬⌿m in human monocytes.
⌬␺m has been recently suggested as a prerequisite,
initial, and irreversible step toward cell death (33).
Indeed, functional integrity of mitochondria and maintenance of ATP levels appear to be the main determinants leading cells to undergo apoptosis or necrosis
(44). In addition, ATP-depleting agents induce HSP
synthesis (22), whereas HSP70 prevents protein aggregation under ATP deficiency. Thus mitochondria, as
the site for control of ATP levels and under the control
of HSP70, are central to the cellular “choice” between
cell survival and cell death (39).
Necrosis and apoptosis are two distinct mechanisms
of cell death that differ from each other by morphological, biochemical, and cytological characteristics. Cytotoxicity can lead to necrosis or apoptosis (programmed
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Fig. 5. Effects of Sin-1 on death of human monocytes. Incubation
with the 䡠NO/ONOO⫺ donor Sin-1 for 16 h induced apoptosis (A) and
necrosis (B) in human monocytes as assessed by annexin V and
propidium iodide. Preincubation with SOD (200 IU/ml) prevented
cell death induced by Sin-1. Values are means ⫾ SE; n ⫽ 5. * P ⬍ 0.01
vs. respective controls; # P ⬍ 0.01 vs. controls with SOD.
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NO AND STRESS RESPONSE IN HUMAN MONOCYTES
cell death). The latter represents an active process of
cell “suicide,” resulting in the demise and subsequent
removal of the affected cell by scavenger phagocytes
without liberation of inflammatory mediators, as observed during necrosis. ONOO⫺ is well established as a
very reactive species inducing cell lesions and cell
death by apoptosis or necrosis, the latter at higher
Table 2. Effects of ONOO⫺ on ⌬⌿m and cell death
ONOO⫺, ␮M
Cells With Depolarized
Mitochondria, %
Apoptosis, %
Necrosis, %
0
50
100
250
500
750
1,000
Blank reagent
11.5 ⫾ 1.05
15.8 ⫾ 3.5
13.5 ⫾ 2.8
20.7 ⫾ 6.9
23.3 ⫾ 5.3*
29.1 ⫾ 4.6*
33.8 ⫾ 10.8*
10.2 ⫾ 1.3
9.5 ⫾ 0.5
13 ⫾ 1.5
10.7 ⫾ 2.2
10.8 ⫾ 1.9
14.5 ⫾ 2.6*
20.9 ⫾ 2.5*
28.2 ⫾ 7.3
8.2 ⫾ 0.9
4.9 ⫾ 3.6
3.3 ⫾ 1.7
3.8 ⫾ 0.9
6 ⫾ 3.2
11.2 ⫾ 6
13.7 ⫾ 5.9
21 ⫾ 5.6*
4.4 ⫾ 2.6
Values are means ⫾ SE; n ⫽ 3. Exposure to peroxynitrite
(ONOO⫺) for 16 h induced mitochondrial membrane depolarization
and cell death by apoptosis or necrosis in human monocytes. Note
lack of effect of the blank reagent after complete degradation of
ONOO⫺. ⌬⌿m, mitochondrial membrane potential. * P ⬍ 0.05 vs.
baseline.
concentrations (10). Evidence for apoptosis induced by
䡠NO was provided by microscopic examination of chromatin condensation and by a specific pattern of internucleosomal fragmentation, i.e., DNA laddering detected by agarose gel electrophoresis (1, 46). In our
experimental conditions, Sin-1 induced apoptosis and
necrosis at low concentrations and necrosis only at
higher concentrations. Our present data are consistent
with other studies showing that the same biological,
chemical, or physical stresses may induce apoptosis or
necrosis depending on their concentration and/or the
cell type (9, 25, 52). Apoptosis and necrosis observed in
cells incubated with Sin-1 for 16 h were inhibited by
preincubation with SOD. SNAP did not induce cell
death even after a long incubation period, indicating
that Sin-1-mediated monocyte apoptosis was only dependent on ONOO⫺ formation.
Our present data suggest a close link between
HSP70 synthesis, ⌬⌿m disruption, and cell death, with
ONOO⫺ inducing all and 䡠NO none. These results are
in agreement with the hypothesis that mitochondria
play a key role as sensors of oxidative stress and
deliver intracellular signals for the induction of autoprotective mechanisms such as HSP70 and eventually
Downloaded from http://ajpcell.physiology.org/ by 10.220.32.246 on February 11, 2017
Fig. 6. Effects of Sin-1 on monocyte
ultrastructure. Morphological analysis
of the effects of Sin-1 (1 mM, 6 h) on
human monocytes is shown. A: control
monocyte displayed vacuoles, euchromatin, and heterochromatin. B: Sin-1
(0.5 mM, 16 h). Sin-1 essentially led to
an alteration of cytosolic organelles
with “cytosolic boiling,” swollen mitochondria, and perinuclear condensation. At higher concentrations, cells exhibiting features of apoptosis coexisted
with cells exhibiting features of necrosis, such as plasma membrane disruption and pycnotic nuclei (C). Apoptosis
and necrosis were completely prevented by preincubation with SOD (D).
NO AND STRESS RESPONSE IN HUMAN MONOCYTES
The authors thank Ewa Mariéthoz for technical assistance and
Sarah Kreps for reviewing the manuscript.
This study was supported by Institut National de la Santé et de la
Recherche Médicale.
Present address of B. S. Polla: INSERM U332, Institut Cochin de
Génétique Moléculaire, 75014 Paris, France.
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