Contrasting effects of NO and peroxynitrites on HSP70 expression and apoptosis in human monocytes

Christophe Adrie1,2, Christoph Richter3, Maria Bachelet1, Nathalie Banzet1, Dominique François1, A. Tuan Dinh-Xuan1, Jean François Dhainaut2, Barbara S. Polla1, and Marie-Jeanne Richard1,4

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (Delta psi 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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) 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 ·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 O2-· in an equimolar manner, thus generating ONOO-, whereas SNAP releases only ·NO (20). Cells were incubated with Sin-1 or SNAP in the presence or absence of SOD (200 IU/ml, added 20 min before ·NO donors). SOD, by dismutating O2-·, prevents the formation of ONOO- during Sin-1 decomposition.

Nitrite/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-right-arrow ·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 Delta psi m. Delta Psi 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 Delta Psi 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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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.

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 ·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 ·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).


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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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Table 1.   NO2-/NO3- production after exposure of human monocytes to Sin-1 or SNAP

Effects of Sin-1 and SNAP on Delta Psi m. A close link between mitochondrial functions and apoptosis was previously proposed (33). We thus investigated the effects of ·NO/ONOO- on Delta Psi 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 Delta Psi m (Fig. 3C) that was prevented by SOD (Fig. 3D).


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Fig. 3.   Cytofluorometric analysis of mitochondrial membrane potential (Delta Psi m). Cytofluorometric analysis of Delta Psi m was assessed by staining with 5,5',6,6'-tetrachloro-1,1',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.



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Fig. 4.   Effects of Sin-1 and SNAP on Delta Psi 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 Delta psi m.

Exposure of human monocytes to Sin-1 induced dose-dependent (0.25-2 mM) disruption of Delta Psi m, which was prevented by SOD (Fig. 4A). When cells were exposed to increasing doses of SNAP (0-2 mM) for >= 6 h (16 h, data not shown), no alterations in Delta Psi 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 Delta Psi m induced by ONOO- in human monocytes. In addition, we observed that, parallel to the lack of induction of HSP70 expression, ·NO per se did not affect Delta Psi 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 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 Delta Psi 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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Fig. 5.   Effects of Sin-1 on death of human monocytes. Incubation with the ·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.

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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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).

Effects of ONOO- on Delta Psi 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 Delta psi 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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Table 2.   Effects of ONOO- on Delta Psi m and cell death


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Delta Psi 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 Delta Psi m. Moreover, ONOO- alone induced Delta Psi 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 Delta Psi m in human monocytes.

Delta psi 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, Delta Psi 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, Delta psi 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.


    ACKNOWLEDGEMENTS

The authors thank Ewa Mariéthoz for technical assistance and Sarah Kreps for reviewing the manuscript.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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