Superoxide Formation and Macrophage Resistance to Nitric Oxide-mediated Apoptosis*

(Received for publication, December 13, 1995, and in revised form, December 2, 1996)

Bernhard Brüne Dagger , Christine Götz , Udo K. Meßmer , Katrin Sandau , Maija-Riitta Hirvonen § and Eduardo G. Lapetina

From the Faculty of Medicine, Department of Medicine IV, Experimental Division, University of Erlangen-Nürnberg, 91054 Erlangen, Federal Republic of Germany, the § Department of Toxicology, National Health Institute, 70211 Kuopio, Finland, and  Case Western Reserve University, Molecular Cardiovascular Research Center, School of Medicine, Cleveland, Ohio 44106-4958

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES


ABSTRACT

RAW 264.7 macrophages, when challenged with a combination of lipopolysaccharide (10 µg/ml) and interferon-gamma (100 units/ml), respond with endogenous NO· formation, which ultimately results in apoptotic cell death. Apoptosis is detected morphologically by chromatin condensation. Concomitantly we noticed the accumulation of the tumor suppressor protein p53. NO·-derived apoptosis was blocked by the NO·-synthase inhibitor NG-monomethyl-L-arginine. Repetitive treatment of RAW 264.7 macrophages with lipopolysaccharide/interferon-gamma , followed by subculturing viable cells, allowed us to select resistant macrophages which we called RES. RES cells still produced comparable amounts of nitrite/nitrate in response to agonist treatment but showed no apoptotic markers, i.e. chromatin condensation or p53 accumulation. However, RES macrophages undergo apoptosis in the presence of exogenously supplied NO·, released from the NO-donors S-nitrosoglutathione or spermine-NO. Assessment of cytochrome c reduction established that RES cells released twice the amount of superoxide compared to RAW 264.7 macrophages under both resting and stimulated conditions. We linked increased superoxide production to cellular macrophage resistance by demonstrating decreased apoptosis after simultaneous application of S-nitrosoglutathione or spermine-NO and the redox cycler 2,3-dimethoxy-1,4-naphthoquinone. Our results suggest that macrophage resistance toward NO·-mediated apoptosis is, at least in part, due to increased superoxide formation. Therefore, the balance between reactive nitrogen and reactive oxygen species regulates RAW 264.7 macrophage apoptosis.


INTRODUCTION

Nitric oxide (NO)1 is recognized for its participation in diverse biological processes in nearly all aspects of life (1-3). The formation of NO· occurs under both physiological and pathophysiological settings. The molecule is synthesized by a family of enzymes termed NO· synthases (NOS), which utilize arginine as their substrate in the generation of NO· and stoichiometric amounts of citrulline (4). For convenience, two types of NOS are recognized; constitutive isoforms, which are active for a relatively short time in response to intracellular Ca2+ fluctuations, and a cytokine-inducible isoform. For the latter to be active, mRNA translation and protein synthesis are required. The inducible NOS generates large amounts of NO· for an extended period. However, once NO· is produced by the action of NOS, it is extremely susceptible to both oxidation and reduction. This results in the concomitant formation of species with NO+-like activity (nitrosonium ion) or NO- (nitroxyl anion), respectively (5). In addition to reacting with oxygen, superoxide, and transition metals, NO· causes biological signaling via interactions with sulfhydryl groups. Because multiple NO surrogates are formed, transduction pathways are classified as either cyclic GMP-dependent or -independent. Cyclic GMP formation is initiated by NO· binding to the heme group of soluble guanylyl cyclase, thus causing enzyme activation. This leads to the conversion of guanosine triphosphate to guanosine cyclic 3':5'-monophosphate. Formation of cyclic GMP and downstream phosphorylation events are the basis of the transducing ability of NO, whereby a vascular message is carried from the endothelium to vascular smooth muscle; these events also describe the action of NO· on platelets (6). The cyclic GMP-independent mechanisms, however, are less well understood. Both cytostatic and cytotoxic actions have been observed. NO·-mediated toxicity affects pathogens, tumor cells, and susceptible host cells (1, 7, 8). In the search for an underlying mechanism, investigations are considering inhibition of iron-sulfur centers, protein modification due to S-nitrosylation or tyrosine nitration, DNA damage, and poly(ADP-ribosylation) activation with cellular energy deprivation (5-9).

Neurons (10, 11), pancreatic beta cells (12, 13), and macrophages (14, 15) among several other cells seem to be particularly sensitive to the toxic effects of NO·. In some systems NO· causes cell death by necrosis; in others, the progressive intra- or extracellular generation of NO· may cause apoptosis.

The most characteristic features of apoptosis are nuclear and cytoplasmic condensation, DNA fragmentation to high molecular weight fragments followed by DNA laddering, and apoptotic body formation (16, 17). Recently, we established p53 accumulation as an early event during NO·-mediated apoptosis in RAW 264.7 macrophages and the RINm5F beta cell line (18).

Recognizing NO· as an apogen in several experimental systems, we wanted to investigate signaling pathways and, more importantly, uncover cellular defense mechanisms against NO·-mediated apoptosis. To this end, we have used a RAW 264.7 derived cell line, termed RES. RES macrophages showed complete protection to apoptosis after endogenous NO·-generation in response to lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma ). Cellular resistance to NO·-mediated apoptosis was apparently conferred by increased superoxide formation.


EXPERIMENTAL PROCEDURES

Materials

Hoechst dye 33258, protein A-Sepharose, diphenylamine, bovine serum albumin, and cytochrome c were purchased from Sigma (Deisenhofen, Germany). Spermine-NO came from Biotrend (Cologne, Germany). Kaiser's glycerol gelatin was provided by Merck (Darmstadt, Germany). RPMI 1640 was ordered from Biochrom (Berlin, Germany). Cell culture supplements and fetal calf serum were from Life Technologies, Inc. (Berlin, Germany). DMNQ was kindly provided by Prof. Nicotera, University of Konstanz (Konstanz, Germany). All other chemicals were of the highest grade of purity commercially available.

Cell Culture

The mouse monocyte/macrophage cell line RAW 264.7 and derived RES-cells were maintained in RPMI 1640 supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal calf serum (complete RPMI), as described previously (15). All experiments were performed using complete RPMI. For nitrite/nitrate determinations cells were cultured in high glucose Dulbecco's modified Eagle's medium supplemented to make it comparable with RPMI 1640.

Nitrite/Nitrate Determination

Nitrite, a stable NO oxidation product, was determined using the Griess reaction. Cell-free culture supernatants were collected (200 µl), adjusted to 4 °C, mixed with 20 µl of sulfanilamide (dissolved in 1.2 M HCl), and 20 µl of N-naphthylethylenediamine dihydrochloride. After 5 min at room temperature the absorbance was measured at 560 nm with a reference wavelength at 690 nm. Nitrite concentrations were calculated using a NaNO2 standard. For details see Ref. 15. The total production of NO· was determined by analyzing the media for the presence of nitrite and nitrate using a described method (19). Briefly, the assay consists of two steps; the enzymatic reduction of nitrate to nitrite using purified nitrate reductase (EC 1.6.6.2.; Boehringer Mannheim), flavin adenine dinucleotide, and reduced beta -nicotinamide dinucleotide phosphate, followed by derivatization and spectrophotometric detection of nitrite after interfering NADPH has been enzymatically removed.

Morphological Investigations

4 × 105 cells were grown in 12-well plates. After overnight adhesion, cells were stimulated according to experimental protocols and scraped off the culture plates followed by fixation with 3% paraformaldehyde for 5 min onto glass slides. Samples were washed with phosphate-buffered saline, stained with 8 µg/ml Hoechst dye 33258 for 5 min, washed with distilled water, and mounted in Kaiser's glycerol gelatin. Nuclei were examined using an Axiovert 35 microscope (Carl Zeiss, Oberkochen, Germany) (15).

GSNO Synthesis

GSNO (S-nitroso derivative of glutathione) was synthesized as described previously (20). Briefly, glutathione was dissolved in HCl, at 4 °C, prior to the addition of NaNO2. The mixture was stirred at 4 °C for 40 min followed by the addition of 2.5 volumes of acetone. Precipitates were filtered, washed once with 80% acetone, two times with 100% acetone, three times with diethyl ether, and dried under vacuum. GSNO was characterized by high pressure liquid chromatography analysis and UV spectroscopy.

p53 Quantification

Detection of p53 was performed following previously published procedures (18). Briefly, 5 × 106 cells were lysed (50 mM Tris, 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, pH 8.0) for 30 min on ice and sonicated (Branson Sonifier, 10 s, duty cycle 100%, output control 1). After the addition of SDS sample buffer, samples (equalized to the same amount of protein, 100-150 of µg protein) were heated (95 °C, 5 min), separated on 10% SDS-polyacrylamide gels, and electrophoretically transferred onto nitrocellulose sheets. Nonspecific binding was blocked with TBS-T-M (140 mM NaCl, 50 mM Tris, pH 7.2, 0.06% Tween-20, 5% nonfat-dry milk), followed by the addition (1 h) of p53 antibody (1:20 in TBS-T-M, hybridoma supernatant against p53, clone Pab122, kindly provided by H. Stahl, Universität des Saarlandes, Homburg, Germany). Transblots were washed three times (TBS-T) before adding an anti-mouse peroxidase-labeled secondary polyclonal antibody (45 min, diluted 1:5000 in TBS-T-M from Promega). Blots were washed four times, before ECL development.

Cytochrome c Reduction

Superoxide-induced reduction of ferricytochrome c to ferrocytochrome c was monitored spectrophotometrically at 550 nm (21). 4 × 105 cells were stimulated for 16 h with 10 µg/ml LPS and 100 units/ml IFN-gamma . Medium was changed to Phenol Red-free medium and cells were further incubated for 3 h in the presence of 50 µM cytochrome c. Medium with cytochrome c served as a control.

Statistical Analyses

Each experiment was performed at least three times and statistical analysis were performed using the two tailed Student's t test. Significant differences (p > 0.05) are marked by asterisks. In case of photographs (apoptotic morphology or p53 Western blot analysis) representative data are shown.


RESULTS

Apoptotic Cell Death in RAW 264.7 and RES Macrophages in Response to Endogenously Formed NO·---A combination of 10 µg/ml LPS and 100 units/ml IFN-gamma caused apoptotic cell death in RAW 264.7 macrophages (Fig. 1, A and B). Based on characteristic apoptotic morphology, that is chromatin condensation visualized by Hoechst dye 33258 staining, the apoptotic response to LPS/IFN-gamma was completely suppressed by the addition of the NOS inhibitor NMMA. Thus, endogenous NO formation fully accounted for apoptotic cell death in RAW 264.7 macrophages. Fig. 1A gives statistical analysis, whereas Fig. 1B demonstrates the response of RAW 264.7 and RES macrophages to LPS/IFN-gamma or to the combination of LPS/IFN-gamma /NMMA.


Fig. 1. Apoptotic cell death in RAW 264.7 and RES macrophages in response to endogenously generated NO·. RAW 264.7 and RES macrophages (4 × 105 cells/assay) were cultured for 24 h without any additions (control), with 10 µg/ml LPS and 100 units/ml IFN-gamma (LPS,IFN-gamma ), or with both agonists in the presence of 1 mM NMMA (LPS,IFN-gamma ,NMMA). Cells were fixed and stained using Hoechst dye 33258 as outlined under "Experimental Procedures." A, results are expressed as % apoptotic positive cells, i.e. macrophages positive for chromatin condensation. The figure gives mean values ± S.D. of at least three separate experiments. B, representative morphological evaluation showing chromatin condensation of unstimulated RAW 264.7 and RES macrophages after LPS/IFN-gamma treatment or after LPS/IFN-gamma /NMMA addition. Arrowheads mark condensed chromatin.
[View Larger Version of this Image (33K GIF file)]


Although we observed extensive apoptotic cell death in RAW 264.7 macrophages after endogenous NO· production, some cells survived the damaging insult. These viable cells were subcultured until confluence was reached, then these cells were treated with LPS/IFN-gamma again. Repeated addition of LPS/IFN-gamma and subculturing of surviving cells was performed 10 times. Finally, when cells were challenged with the agonist combination, macrophages were totally resistant, no longer showing an apoptotic response (Fig. 1). Because of their altered cellular behavior toward endogenously generated NO·, these cells were termed "resistant macrophages," abbreviated as RES.

It is known that RAW 264.7 macrophages accumulate the tumor suppressor gene product p53 after endogenous generation of NO· (18). The data in Fig. 2 confirm p53 accumulation in RAW 264.7 macrophages after LPS (10 µg/ml)/IFN-gamma (100 units/ml) treatment. Unstimulated controls and NMMA-treated samples showed a very faint p53 protein band on the Western blot, which significantly increased in response to LPS/IFN-gamma addition. Agonists in the presence of the NOS inhibitor NMMA were ineffective in causing p53 accumulation in RAW 264.7 macrophages, thereby establishing a causative relationship between an active, inducible NOS and tumor suppressor protein accumulation. A time-response analysis revealed maximal p53 accumulation 14 h after the initial challenge.


Fig. 2. p53 accumulation in RAW 264.7 and RES macrophages in response to endogenously generated NO·. RAW 264.7 and RES macrophages (2 × 107 cells/assay) were cultured for 14 h without any additions (control), 1 mM NMMA (NMMA), with 10 µg/ml LPS and 100 units/ml IFN-gamma (LPS/IFN-gamma ), or with both agonists in the presence of 1 mM NMMA (LPS/IFN-gamma ,NMMA). p53 was detected by Western blot analysis using the monoclonal antibody Pab122, as described under "Experimental Procedures." The figure is representative of three similar experiments.
[View Larger Version of this Image (14K GIF file)]


We did not observe a p53 response in RES macrophages, and 14 h after LPS/IFN-gamma addition to RES cells p53 was still undetectable (Fig. 2). Therefore, based upon morphological criteria and biochemical analysis, RES macrophages showed no apoptotic markers after LPS/IFN-gamma stimulation. This is in contrast to the parental RAW 264.7 macrophages.

To confirm that comparable effective signaling mechanisms were present in both RAW 264.7 and RES macrophages, we determined nitrite and nitrate accumulation in the cell supernatant. Increased nitrite/nitrate values in response to LPS/IFN-gamma are indicative of inducible NOS induction as well as functional transcriptional/translational control mechanisms after LPS/cytokine treatment. Table I gives the response of RAW 264.7 and RES macrophages to LPS (10 µg/ml)/IFN-gamma (100 units/ml) with or without NMMA addition. Both RES and RAW parent cells showed a massive 24 h accumulation of NOx values after the LPS/IFN-gamma challenge. These effects are suppressed by inducible NOS inhibition.

Table I.

Nitrite/nitrate accumulation in the cell supernatant of RAW 264.7 and RES macrophages in response to endogenously generated nitric oxide

RAW 264.7 and RES macrophages (4 × 105 cells/assay) were cultured for 24 h without any addition (control), with 10 µg/ml LPS and 100 units/ml IFN-gamma (LPS + IFN-gamma ), or with both agonists in the presence of 1 mM NMMA (LPS + IFN-gamma  + NMMA). Nitrite/nitrate was determined using the Griess reaction as described under "Experimental Procedures." The figure gives mean values ± S.E. of at least four separate experiments.
Nitrite
Nitrate
RAW RES RAW RES

µM µM
Control 1  ± 1 2  ± 1 NDa ND
LPS/IFN-gamma 18  ± 4 17  ± 5 19  ± 8 33  ± 9
LPS/IFN-gamma 3  ± 2 2  ± 1 ND ND
NMMA

a ND, not detectable.

RES cells, in general, produced less nitrite, however, the difference is not significant. Furthermore, we analyzed the media for the presence of nitrate by culturing cells in Dulbecco's modified Eagle's medium, correcting for low levels of naturally occurring nitrate. Under resting conditions no nitrate was detectable, neither in RAW nor in RES cells. After the agonist challenge, RAW macrophages produced less nitrate compared with RES cells although differences never reached statistically different levels.

Superoxide Production and Apoptotic Cell Death in RES Macrophages

Suspecting that macrophages produce not only reactive nitrogen species but also reactive oxygen species upon activation, we determined the rate of superoxide production or more correctly, the release of Obardot 2 to the cell exterior in both cell systems. RAW 264.7 macrophages released 0.03 ± 0.01 nmol of Obardot 2/106 cells × min under resting conditions. Unexpectedly, RES cells released nearly 2-3 times the amount of cytochrome c reductive material under resting conditions (Table II). As a control, we showed complete inhibition of cytochrome c reduction by 500-1000 units/ml superoxide dismutase (data not shown). To determine superoxide formation in RAW 264.7 and RES macrophages upon agonist addition, we included NMMA to prevent the possible interference of ONOO- with our detection system. In agreement with published data (22) we noticed an interference of the cytochrome c reduction method, when allowing the simultaneous generation of nitric oxide. As proposed, NO formation and ONOO- production led to an underestimation of rates of superoxide production (22).

Table II.

Rates of superoxide and nitrite production in agonist-stimulated RAW 264.7 and RES macrophages

RAW 264.7 and RES macrophages (4 × 105 cells/assay) were cultured for 15 h without any additions (control), with a combination of 10 µg/ml LPS, 100 units/ml IFN-gamma , (for nitrite determination), and with the further addition of 1 mM NMMA (LPS, IFN-gamma , NMMA; for superoxide determination). Subsequently, superoxide was determined using the cytochrome c reduction assay, while nitrite was analyzed using the Griess reaction as described under "Experimental Procedures." Mean values ± S.E. of at least four separate experiments are given.
Superoxide
Nitrite
RAW RES RAW RES

nmol/106 cells × min nmol/106 cells × min
Control 0.03  ± 0.01a 0.1  ± 0.02a NDb ND
Agonists 0.08  ± 0.03a,c 0.22  ± 0.04a,c 0.08  ± 0.03c 0.05  ± 0.03c

a Significant difference between RAW and RES cells.
b ND, not detectable.
c Statistically significant different values compared with individual controls.

After stimulation with LPS (10 µg/ml)/IFN-gamma (100 units/ml) in the presence of 1 mM NMMA, RAW 264.7 and RES macrophages responded with increased superoxide release. Similar to the control situation, agonist-activated RES cells produced significantly more superoxide compared to RAW 264.7 macrophages. Superoxide generation, under control and stimulated conditions, was roughly doubled in RES compared to RAW 264.7 cells (Table II). For comparison, we also determined the rate of nitrite formation. Determinations were carried out in the linear phase of radical production, between 15 and 18 h post-agonist challenge.

Considering the increased superoxide formation to be related to decreased NO·-mediated cellular toxicity, we probed for the apoptotic response of unstimulated RES macrophages in the presence of the NO donor S-nitrosoglutathione (GSNO) and the redox cycler DMNQ (Table III). RES cells, when exposed to exogenously derived NO·, showed substantial DNA fragmentation, which was quantified using the diphenylamine assay. DNA fragmentation became obvious using concentrations of 300-750 µM of the NO donating compound. In an additional, supportive observation, GSNO induced p53 accumulation in RES cells (data not shown). Redox cycling quinones like DMNQ, depending on the concentration used, are reported to result in either cell proliferation or apoptotic or necrotic cell death. Employing DMNQ at a concentration of 1 or 2 µM resulted in neither apoptotic nor necrotic RES cell death. Using the superoxide producing DMNQ in combination with GSNO lowered the apoptotic inducing ability of GSNO significantly.

Table III.

Apoptotic response of RAW 264.7 and RES macrophages exposed to S-nitrosogluthathione and the redox-cycler DMNQ

RAW 264.7 and RES macrophages (4 × 105 cells/assay) were exposed for 8 h to S-nitrosoglutathione, DMNQ, or a combination of GSNO and DMNQ. DNA fragmentation was quantitated using the diphenylamine reaction as outlined under "Experimental Procedures." The table shows the percentage of fragmentation as mean values ± S.D. of at least four separate experiments.
RES cells
RAW cells
Control GSNO (300 µM) GSNO (500 µM) GSNO (750 µM) GSNO (500 µM) Spermine-NO (500 µM)

Control 4  ± 2 24  ± 4 29  ± 3 35  ± 2 35  ± 4 43  ± 3
DMNQ (1 µM) 5  ± 2 18  ± 1a 22  ± 3a 24  ± 5a 26  ± 5 38  ± 4
DMNQ (2 µM) 5  ± 2 16  ± 2a 14  ± 2a 11  ± 2a 22  ± 2a 23  ± 3a

a Statistically significant different values compared with individual controls.

During our studies we carefully examined for, and discriminated between, apoptosis and necrosis by microscopy. The decreased apoptotic response therefore refers to inhibition of apoptosis without compensating cellular necrosis. Low levels of the redox cycling compound suppressed DNA fragmentation in combination with all tested GSNO concentrations. Although substantial reduction of GSNO-induced apoptosis became apparent, we never decreased the signal to control values. With increasing concentrations of DMNQ (>= 5 µM) we noticed necrotic RES cell death. Assuming that increased superoxide formation in the presence of a NO donor is protective, this also should apply for RAW 264.7 macrophages. Therefore, experiments were subsequently performed with parent RAW 264.7 cells. Again coincubation of GSNO and DMNQ resulted in decreased macrophage apoptosis (Table III). Similar results were obtained using the NO·-releasing compound spermine-NO. Spermine-NO induced more than 40% DNA fragmentation in RAW cells. Values were decreased to nearly 20% in the presence of DMNQ (Table III). Overall these experiments suggest that macrophage apoptotic cell death in response to exogenous NO·, derived from GSNO or spermine-NO, is partially decreased in the presence of low concentrations of a redox cycling compound like DMNQ. DMNQ releases approximately 260 pmol of Obardot 2/106 cells × min under these experimental conditions.

In further experiments we assayed for RES cell apoptosis after GSNO addition under both resting and stimulated conditions (Fig. 3). When RES macrophages were incubated with increasing concentrations of GSNO, dose-dependent DNA fragmentation was observed.


Fig. 3. Preactivation of RES cells blocks GSNO-induced apoptosis. RES macrophages (4 × 105 cells/assay) were cultured for 15 h with vehicle or 10 µg/ml LPS, 100 units/ml IFN-gamma , and 1 mM NMMA, followed by the addition of increasing concentrations of GSNO. After a total incubation period of 23 h DNA fragmentation was assayed using the diphenylamine test. The figure gives mean values ± S.D. of at least three separate experiments.
[View Larger Version of this Image (20K GIF file)]


In contrast, stimulation of RES cells for 15 h with LPS, IFN-gamma , and NMMA, followed by an 8-h exposure to GSNO, totally blocked DNA cleavage. Obviously, preactivation of RES cells confers resistance to exogenously added and otherwise apoptogenic GSNO.


DISCUSSION

Apoptosis elicited by endogenous or exogenously derived NO· may play a role in pathologic conditions ranging from neurodegeneration to pancreatic beta cell death and activated macrophage self-destruction (12-15). Apoptosis is an active process with specific defined morphological and biochemical features. RAW 264.7 macrophages in cell culture respond to endogenously generated NO· with characteristic apoptotic cell death (15, 18). Although many cells respond to LPS and/or cytokine treatment with NOS induction and massive NO generation, not all of them are susceptible to NO toxicity. Whereas RAW 264.7 macrophages, RINm5F cells, and authentic rat beta cells are responsive to NO·-mediated apoptosis, hepatocytes are spared the toxic effects of endogenous NO· formation (7). Similar differences in toxicities hold for exogenously supplied peroxynitrite. Cerebrocortical cultures show apoptotic cell death in response to low concentrations of ONOO- (10 µM) and respond with necrotic death at 100 µM ONOO- (23). PC12 cells undergo apoptosis in response to 850 µM ONOO- (24), whereas some endothelial cell lines are completely insensitive to ONOO--induced apoptosis (25). These observations are consistent with the notion that in the brain, NO-producing cells are selectively spared the toxic effects of NO· (26).

Not only does cellular susceptibility to NO· differ among cells, the ultimate damaging molecule remains ambiguous. Although exogenously added ONOO- causes, depending on the concentration used, apoptosis or necrosis, at the same time it is also considered a biological mediator exhibiting physiological NO·-related reactions (20, 27, 28). By analogy, the production of NO· is not always associated with cellular toxicity. Increased NO· formation protects against conditions like ischemia-reperfusion, peroxide-induced toxicity, lipid peroxidation, and myocardial injury (29-32).

Our strategy of subculturing RAW 264.7 macrophages that survived LPS/IFN-gamma treatment allowed us to select for macrophages with resistance to NO·-mediated apoptosis. Resistance was complete when NO· was endogenously generated, but was barely detectable after exogenous GSNO addition (Table III). This substantiated a specific agonist-inducible protective cellular response. Decreased inducible NOS induction cannot account for cellular resistance, as both RES and RAW 264.7 macrophages clearly responded to these agonists. Consistent with published observations (33, 34), RAW 264.7 macrophages produced approximately equal amounts of both nitrite and nitrate. The increased production, which is not statistically significant, of nitrate in RES cells over RAW 264.7 macrophages might be related to increased ONOO- formation, as the decomposition of ONOO- under cellular conditions is rather complex, depending upon the production rate of both Obardot 2 and NO· radicals. Both nitrite and nitrate are expected decomposition products of ONOO- (33, 34). Unexpectedly, RES cells consistently produced more superoxide under both non-stimulated and stimulated conditions. Formation of Obardot 2 in RES cells, under stimulated conditions with inducible NOS being inhibited, is in the range of approximately 0.2 nmol of Obardot 2/106 cells per min. Previous reports for RAW 264.7 macrophages indicated that the formation of both nitrite and nitrate was in the range of 0.1-0.25 nmol NObardot 2 and NObardot 3/106 cells per min (Refs. 32 and 35, and references cited therein). These findings are in agreement with our measurements in RAW and RES cells (Tables I and II). As the formations of both Obardot 2 and NO (as assayed from the sum of NObardot 2/NObardot 3) are approximately equal, one would assume efficient signaling by modulating the relative production rate of one of the radicals.

To substantiate a link between increased steady-state production of superoxide and protection against NO·-derived toxicity, we chose to incubate a redox cycler in the presence of a NO-liberating agent while assaying for apoptotic cell death. Although the correlation may or may not demonstrate cause, supporting evidence for protection against GSNO-induced apoptosis came from a coculture with the redox cycler DMNQ. As expected, this experimental setting protected both RES and RAW 264.7 macrophages from the effects of exogenously generated NO·. Similar results were obtained with spermine-NO/DMNQ. To further strengthen the idea of inducible resistance toward NO· we treated RES cells with agonists in the presence of NMMA, followed by the addition of GSNO. This conferred complete protection of RES cells toward exogenously added NO (GSNO). Further investigations are necessary to relate protection from apoptosis to increased Obardot 2 under these experimental conditions.

We suggest that modulating the flux ratio of Obardot 2 relative to NO· interferes with the otherwise damaging potency of NO·. NO· and superoxide produce ONOO- in a nearly diffusion-controlled reaction (36, 37). Although ONOO- might be formed under our experimental conditions, a low production rate of ONOO- does not automatically contribute to the initiation of apoptosis. However, in agreement with published data, a higher rate of ONOO- production results in substantial cell death (23, 24) indicating severe cellular damage in response to massive NO·/Obardot 2 production. It is most likely that, under these conditions, cellular rescue/repair mechanisms are overwhelmed, either by superoxide formation, NO production, or a combination of product generation.

Our results describe cellular resistance to NO·-mediated apoptotic cell death and p53 accumulation through low level superoxide generation. A steady-state level of superoxide generation may channel NO· toxicity through pathways that allow cellular defense mechanisms to cope with an otherwise lethal insult. This seems especially relevant for systems that are equipped to produce NO· and superoxide under inflammatory conditions, such as astrocytes, endothelial cells, mesangial cells, or macrophages. The situation seen in RES cells is analogous to chondrocyte apoptosis (38). Chondrocytes treated with sodium nitroprusside undergo apoptosis, whereas endogenous NO· formation in response to IL-1 failed to do so, because endogenous production of oxygen radicals seemed to cause protection. Our results show that cells may acquire a defense mechanism in order to protect themselves against NO toxicity. Cellular resistance of NO·-producing cells relative to NO· target cells may therefore be linked to compensating Obardot 2 formation.

As described for Obardot 2, the controlled production of the radical might allow cells to proliferate, inhibit growth, or to enter the apoptotic/necrotic pathway depending on the concentration and probably the flux rate of its formation (39). A similar scenario might apply for the NO signaling system. The final outcome of biological reactions of NO· will be determined by the rate of NO· and Obardot 2 production. The generation of superoxide relative to NO· compensates for an otherwise NO·-mediated apoptotic insult. Cellular protection may be due to the elimination of adverse effects associated with reactive nitrogen species due to Obardot 2-dampening effects. It may well be that RES cells have been selected on the basis of their increased superoxide formation after repetitive stimulation. Therefore, RES cell sensitivity to NO donors like GSNO may be due to the lack of compensating Obardot 2 formation. Consistent with this, GSNO or spermine-NO in the presence of chemically generated superoxide produces less apoptotic cell death.

Decreased apoptosis in response to endogenously generated NO is also observed after Bcl-2 gene transfer (40). However, protection afforded by Bcl-2 still allowed accumulation of p53, whereas down-regulated apoptosis in RES cells correlated with a suppressed tumor suppressor response. Further studies will examine whether this points to a redirection of the DNA-damaging species in RES cells normally accounting for the positive p53 response.

Increased superoxide production has also been linked to the development of nitrate tolerance (41) and emphasizes the nondestructive mechanisms of NO· formation under conditions of enlarged superoxide generation. In the macrophage system, the simultaneous formation of NO· and Obardot 2 protect, at least in part, cells from NO-induced apoptosis. This is clearly the case when looking at apoptotic signals like chromatin condensation or p53 accumulation. The controlled formation of reactive nitrogen species and reactive oxygen species may play a fundamental role in controlling human diseases by determining the apoptogenic activity of endogenously generated radicals.


FOOTNOTES

*   Financial support was received from Deutsche Forschungsgemeinschaft and the European Community.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.
Dagger    To whom correspondence should be addressed: University of Erlangen-Nürnberg, Faculty of Medicine, Loschgestrasse 8, 91054 Erlangen, Federal Republic of Germany. Tel.: 49-9131-856311; Fax: 49-9131-859202.
1   The abbreviations used are: NO, nitric oxide; NOS, nitric-oxide synthase; GSNO, S-nitrosoglutathione; LPS, lipopolysaccharide; IFN-gamma , interferon-gamma ; NMMA, NG-monomethyl-L-arginine; DMNQ, 2,3-dimethoxy-1,4-naphthoquinone; TBS, Tris-buffered saline.

Acknowledgment

We thank Prof. P. Nicotera for providing DMNQ.


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