1 Laboratory of Respiratory Physiology, Unité de Formation et de Recherche Cochin Port-Royal, Paris V University, and 2 Medical 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
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ABSTRACT |
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The free radicals nitric oxide
(·NO) and superoxide (O2·) 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 ·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 ·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 ·NO.
nitric oxide; heat shock; cell stress; cell death
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INTRODUCTION |
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REACTIVE
OXYGEN SPECIES (ROS) are generated from molecular oxygen and
include the free radicals superoxide (O2·),
hydroxyl (·OH), and nitric oxide (·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 environmental 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 ·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). ·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 ·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 ·NO is involved, on the
one hand, in HSP70 induction (30, 35,
36) and, on the other hand, in mitochondrial dysfunction
and cell death. ·NO and O2·, however, are weak
oxidants. Thus the question as to whether ·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 ·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 during infection and inflammation, as
sources and as targets of mediators such as ROS. Using
·NO/ONOO donors or exogenous ONOO
, we
investigated the respective contribution of ·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 ·NO is not by
itself stressful to human monocytes, whereas ONOO
induces
an oxidative stress response in these cells.
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MATERIALS AND METHODS |
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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).
ONOOCells. 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 ·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 ·NO and O2Nitrite/nitrate determination.
·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 ·NO by use of a strong reducing environment
(3) consisting of vanadium and 1 N HCl at 90°C as
follows: NO3
+ 4H+ + 3e
·NO + 2H2O. The
amount of ·NO produced was determined by chemiluminescence with 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 µl 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 µl 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 µg/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 µl, 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 comparison of the mean. The criterion for statistical significance was P < 0.05.
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RESULTS |
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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 ·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
·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 HSP70-positive 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).
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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 ·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).
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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 ROS-dependent apoptosis,
the effects of ·NO/ONOO on 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
·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 ·NO, was cytotoxic in these cells (Fig.
5B).
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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 concentrations, 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.
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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 µM, 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
.
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DISCUSSION |
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Here, we report that ONOO induced a stress response
in human monocytes that was characterized by induction of HSP70
expression, mitochondrial membrane depolarization, and cell death by
apoptosis or necrosis, whereas ·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 ·OH, but not the membrane-impermeant O2·, are inducers of an HS response
(6). Here we studied the expression of HSP70 in monocytes
on exposure to ·NO or ONOO
. Sin-1, which generates
·NO and O2
·, induced the intracellular
overexpression of HSP70, which was abolished by the addition of SOD,
indicating that ONOO
, but not ·NO, induced HSP70
accumulation. This conclusion was supported by the lack of induction of
HSP70 expression that we observed with SNAP, a slow generator of ·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 ·NO as
an inducer of HSP70 during HS in rat tissues and human hepatoblastoma
cells. They suggested that the ionized form of ·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, ·NO itself, as O2·, 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 ·NO is involved in HSP70
induction (30, 35, 36) have
generally not considered the respective roles of ·NO and
ONOO
. Our results indicate that, at least in human
monocytes, ·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 ·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 ·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 ·NO donor SNAP did not alter
m.
Moreover, ONOO
alone induced
m
disruption similar to that induced by Sin-1. Various reports showed
that ·NO can reversibly inhibit mitochondrial respiration by
competing with oxygen at cytochrome oxidase (11, 12, 16, 49), whereas the
formation of ONOO
from ·NO and mitochondria-derived
superoxide can cause the irreversible inhibition of complexes I-III,
leading to mitochondrial dysfunction (14, 34,
42). Moreover, ·NO has been shown to induce apoptosis by
triggering mitochondrial permeability transition and mitochondrial membrane depolarization (5, 26). Our data,
however, indicate that ·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 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
concentrations (10). Evidence for apoptosis induced by ·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 ·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 apoptosis. They
support the hypothesis that the oxidative injury associated with
simultaneous production of ·NO and other ROS is mediated through
ONOO
formation. DNA damage, once attributed to ·NO, is
now believed to be the consequence of ONOO
formation
(50, 51). Along these lines, inhibition of
tissue factor expression circulating in human monocytes after
stimulation by lipopolysaccharide (23) and rat pulmonary
epithelial type II cell cytotoxicity (24), once believed
to be induced by ·NO, appeared to be an indirect effect of ·NO by
subsequent ONOO
formation. However, in our study,
potential mitochondrial membrane disruption and cell death occurred
despite HSP70 expression.
HSP accumulation has long been used as a marker of cell and tissue
damage. This would seem to be in conflict with the previously described
antiapoptotic properties of HSP. However, there is evidence that HSP
could act to prevent and promote apoptosis. An overlap exists between
the signals (the protein denaturation, the oxidation of protein thiols,
or the increase in ceramide levels) that induce a protective stress
response and those that initiate apoptosis (for review see Ref. 45).
Although a number of signals are known to induce in parallel HSP
expression, mitochondrial depolarization, and initiation of the program
of cell destruction (6, 37, 53),
there is a paucity of evidence that the cells that accumulate HSP are
the cells that are destined to die. In this study, HSP70 expression was
regarded as a marker of stressful conditions induced by
ONOO in human monocytes.
In conclusion, our data suggest that, in contrast to
ONOO, ·NO per se is not stressful or cytotoxic for
human monocytes as assessed by HSP70 expression,
m,
and cell death. ONOO
formation may well explain the toxic
effect generally attributed to ·NO. Because of the widespread
production of O2
· by many cell types during
inflammation, all subsequent studies in which ·NO is used as the
donor should include selective scavengers such as SOD to distinguish
the effect of ·NO from those secondary to ONOO
formation.
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ACKNOWLEDGEMENTS |
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The authors thank Ewa Mariéthoz for technical assistance and Sarah Kreps for reviewing the manuscript.
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FOOTNOTES |
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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.
Address for reprint requests and other correspondence: M.-J. Richard, Laboratoire de Biochimie C, Hôpital A. Michallon, BP217X, 38043 Grenoble, France (E-mail: Marie-Jeanne.Richard{at}ujf-grenoble.fr).
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. §1734 solely to indicate this fact.
Received 12 July 1999; accepted in final form 7 February 2000.
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