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Sustained Nitric Oxide Delivery Delays Nitric Oxide-Dependent Apoptosis in Macrophages: Contribution to the Physiological Function of Activated Macrophages ¹

Instituto de Bioquímica (Centro Mixto Consejo Superior de Investigaciones Científicas-Universidad Complutense) and Centro Nacional de Investigaciones Cardiovasculares, Facultad de Farmacia, Universidad Complutense, Madrid, Spain

	Abstract

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Treatment of the macrophage cell line RAW 264.7 with the short-lived NO donor S-nitrosoglutathione triggers apoptosis through the release of mitochondrial mediators. However, continuous supply of NO by long-lived NO donors protected cells from apoptosis through mechanisms that involved the maintenance or an increase in the levels of the inhibitor of apoptosis proteins (IAPs) cIAP-1, cIAP-2, and xIAP and decreases in the accumulation of p53 and in the levels and targeting of Bax to the mitochondria. As a result of these changes, the activation of caspases 9 and 3 was notably delayed, expanding the time of viability of the macrophages. Moreover, inhibition of NO synthase 2 activity after 8 h of stimulation of RAW 264.7 cells with LPS and IFN- accelerated apoptosis via an increase in the processing and activation of caspases. These data suggest that NO exerts an important role in the autoregulation of apoptosis in macrophages.

	Introduction

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Macrophage activation plays a central role in innate and acquired immunity, and this process is subjected to a fine regulation to respond and adapt to the specific necessities of the inflamed area (1, 2). Among other functions, macrophages participate actively in host defense against pathogen infection through phagocytosis and pathogen killing and in inflammatory reactions. In the course of activation by pathogens and proinflammatory cytokines, macrophages express a series of genes involved in host defense through the release of proinflammatory mediators such as bioactive lipids (i.e., prostanoids and other arachidonic acid-derived metabolites), reactive oxygen and nitrogen species, and matrix metalloproteinases to accomplish tissue remodeling (1, 3). Elevated NO synthesis after NO synthase-2 (NOS-2)³ expression by activated macrophages is one of the main cytostatic, cytotoxic, and proapoptotic mechanisms participating in the innate response, at least in rodents (4, 5, 6, 7). The proapoptotic activity of NO has been well documented (4, 5, 6); it involves the release of cytochrome c from the mitochondria (7), the rise in the tumor suppressor p53 (8, 9), and the accumulation and targeting of proapoptotic members of the Bcl-2 family, in particular Bax (10). However, most physiological macrophage functions, such as phagocytosis, require preservation of cell integrity, in particular an attenuation of the autoapoptotic response. Indeed, it has been reported that NO plays a dual role, favoring cell viability or inducing apoptotic death depending on the cell type and amount of NO produced (11, 12, 13, 14, 15). For example, moderate NO synthesis inhibits basal apoptosis in B lymphocytes (16, 17) or the TGF--induced apoptosis in hepatocytes (18).

Regarding the protective mechanisms mediated by NO, several targets have been identified, including the regulation of caspase activation (19, 20) and the expression of antiapoptotic genes of the inhibitor of apoptosis protein (IAP) family (21, 22). Caspases are a family of proteases that catalyze specific protein cleavage through a cysteine/histidine catalytic mechanism, but require intermolecular processing to be active (23, 24). Because the active site of the enzyme and the procaspase contain a cysteine residue susceptible to S-nitrosylation by NO, it has been proposed that this could be a physiological mechanism of apoptotic regulation (25, 26, 27, 28).

In studies of apoptosis induced by treatment of macrophages with an NO donor, such as S-nitrosoglutathione (GSNO), we realized that the appearance of characteristic hallmarks of apoptosis was very rapid compared with those observed with molecules that release NO in a sustained way or after NOS-2 expression via LPS and IFN- challenge of the cells. In addition to this, a clear cell-specific susceptibility to NO-dependent apoptosis has been observed, and in general, macrophages appear to be more resistant than other cells, such as neurons or T and B lymphocytes. In view of these observations we analyzed the possibility that high output NO concentrations would contribute to delay the occurrence of apoptosis. Using NO donors with different half-lives of NO generation and pharmacological inhibitors of NOS-2 activity, we characterized a dual role for NO in macrophage viability; although NO rapidly triggers the release of early mitochondrial mediators of apoptosis (cytochrome c), sustained NO levels paradoxically prevent caspase processing and activity and favor the accumulation of IAPs, delaying the action of the downstream apoptotic pathway and contributing to cell viability and integrity.

	Materials and Methods

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Chemicals

Reagents were obtained from Roche (Mannheim, Germany), Calbiochem (Darmstadt, Germany), Bachem (Bubendorf, Switzerland), and Sigma-Aldrich (St. Louis, MO). Fluorescent probes and Abs were purchased from BD PharMingen (San Diego, CA) and Santa Cruz Biotechnology (Santa Cruz, CA). Culture media were obtained from BioWhittaker (Verviers, Belgium).

Cell culture conditions

The murine macrophage cell line RAW 264.7 was maintained in RPMI 1640 medium supplemented with 10% FCS, L-glutamine, and antibiotics, as previously described (5, 6).

Flow cytometric analysis of apoptosis

Propidium iodide (PI) staining was performed after incubation of the cells with 0.005% PI, following a previously described protocol (6, 7, 16). Cells were carefully resuspended and run in a FACScan cytometer (BD Biosciences, San Jose, CA) equipped with a 25-mW argon laser. Quantification of the percentage of apoptotic cells was performed using a dot plot of the forward scatter against the PI fluorescence. Apoptotic and viable cells were sorted, and the integrity of the DNA was analyzed in agarose gels to confirm the criteria of gating (6) and by TUNEL (29).

NO measurement

Real-time NO levels were determined in the culture medium using an NO-selective amperometric sensor (World Precision Instruments, Sarasota, FL). The electrode was calibrated following the decomposition of S-nitroso-N-acetyl-DL-penicillamine using Cu⁺ as catalyst, following the instructions of the supplier.

Preparation of cytosolic and total protein extracts

RAW 264.7 cells were washed twice with ice-cold buffer A (10 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM EGTA, 10 mM KCl, 1 mM DTT, 0.5 mM PMSF, 2 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml N-tosyl-lys-chloromethyl ketone, 5 mM NaF, 1 mM NaVO₄, and 10 mM Na₂MoO₄) containing 120 mM NaCl and scraped off the plate. Cells were lysed at 4°C with 0.2 ml of buffer A supplemented with 0.5% Nonidet P-40 and under continuous shaking. After centrifugation of the cell lysate, the supernatant was stored at -80°C (cytosolic extract). The presence of cytochrome c in the cytosol was determined by Western blotting cell extracts obtained by controlled lysis of the plasma membrane as previously described (6, 30). Total cell extracts were prepared after homogenization of the cells with buffer A supplemented with 0.5% 3-[(3-cholamidopropyl)dimethylamonio]-1-propane sulfonate. Protein content was assayed using the Bio-Rad protein reagent (Hercules, CA). All steps of cell fractionation were conducted at 4°C.

Western blot analysis of proteins

Protein extracts were size-separated in 10–12% SDS-PAGE. The gels were blotted onto a Hybond-P membrane (Amersham Pharmacia Biotech, Arlington Heights, IL) and incubated with the following Abs: anti-IAPs (R&D Systems, Minneapolis, MN); anti-p53, anti-Bax, an anti-caspases 3 and 9 (Santa Cruz Biotechnology; sc-6243, sc-7148, and sc526, respectively); and anti-cytochrome c Ab (BD Biosciences). The blots were submitted to sequential reprobing with Abs after treatment with 100 mM 2-ME and 2% SDS in TBS and heated at 60°C for 30 min. The blots were revealed by ECL following the manufacturer’s instructions (Amersham Pharmacia Biotech).

In vitro and in vivo caspase assays

For in vitro caspase assays, cell extracts were prepared in 10 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM EGTA, 120 mM NaCl, 1 mM DTT, 0.5 mM PMSF, 2 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml N-tosyl-lys-chloromethyl ketone, 5 mM NaF, 1 mM NaVO₄, 10 mM Na₂MoO₄, and 0.5% Nonidet P-40. After centrifugation of the cell lysate the supernatant was stored at -80°C (cytosolic extract), and protein content was assayed using the Bio-Rad protein reagent. The DEVDase (corresponding mainly to caspases 3 and 7) and caspase 9 activities were determined in cell lysates using N-acetyl-DEVD-7-amino-4-trifluoromethylcoumarin and N-acetyl-LEHD-7-amino-4-trifluoromethylcoumarin as fluorogenic substrates and following the instructions of the supplier (Calbiochem, La Jolla, CA). The corresponding peptide aldehyde and Z-VAD.fmk were used to inhibit caspase activity in vivo and to ensure the specificity of the reaction in the in vitro assay. The caspase activities were linear over a 30-min reaction period. In vivo activation of caspase 3 was measured by confocal microscopy after labeling the cells with PhyPhyLux-G1D2 (a derivative of GDEVDGI that yields a fluorescent peptide (green) after caspase excision; OncoImmunin, Gaithersburg, MD) following the instructions of the supplier. Plates were visualized using an MRC-1024 confocal microscope (Bio-Rad), and the fluorescence was acquired and electronically evaluated. Staining with 40 nM chloromethyl X-rosamine was also performed to label the mitochondria (red).

Statistical analysis

The data shown are the mean ± SEM (n = 3–4). Statistical significance was estimated with Student’s t test for unpaired observations. A value of p < 0.05 was considered significant. In studies of Western blot analysis, a linear correlation between increasing amounts of input protein and signal intensity was observed (correlation coefficients, >0.84).

	Results

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Sustained NO synthesis delays apoptosis in macrophages

Treatment of macrophages with the short-lived NO donor GSNO induced apoptosis, followed by the appearance of hypodiploid DNA 6 h after addition of this molecule. However, when macrophages were incubated simultaneously with GSNO and a sustained NO donor such as diethylenetriamine NONOate (DETA-NO), the percentage of cells exhibiting hypodiploid DNA was significantly reduced, and only after 18 h in culture was a significant increase in the percentage of apoptotic cells evidenced. To explain this distinct behavior when sustained NO release occurs, the kinetics of NO concentration in the culture medium were determined using an amperometric NO electrode. As Fig. 1B shows, the release of NO in cells treated with GSNO showed a rapid decrease, with a half-life of 40 min. However, DETA-NO maintained sustained levels of NO for at least 5 h. In addition to this, an evaluation of NO levels in vivo was deduced by measuring the nitrosylation pattern of proteins using the Jaffrey method of selective oxidation of S-nitrosylated groups (31). One of the most intense bands corresponded to a protein of 14 kDa, which was identified as cytochrome c. This band was evidenced at short times, whereas it disappeared after longer periods of time (4 h). Moreover, the sustained release of NO influenced the profile of proteins controlling apoptosis. As Fig. 1D shows, the expression levels of the antiapoptotic proteins cIAP-1, cIAP-2, and xIAP correlated with the severity of the induced apoptotic response. When the accumulation of cytochrome c in the cytoplasm was determined by Western blot as a marker of the release of mitochondrial-dependent apoptotic mediators, the levels were notably lower in the presence of DETA-NO than in the GSNO-only counterparts.

View larger version (34K): [in this window] [in a new window]   FIGURE 1. Sustained NO synthesis delays NO-dependent apoptosis in macrophages. RAW 264.7 cells were incubated with 0.5 mM GSNO and 0.1 mM DETA-NO and at the indicated times the percentage of apoptotic cells was determined (A). The levels of NO in the culture medium of cells incubated with 0.1 mM GSNO or DETA-NO were determined with a NO-selective amperometric electrode (B). The time course of S-nitrosylated cytochrome c (14 kDa) was determined by Western blot after immunoprecipitation with a specific Ab, using the selective modification of thiolic groups as described by Jaffrey et al. (31 ) (C). The levels of IAP proteins and the accumulation of cytochrome c in the cytosol were determined by Western blot (D). Results show the mean ± SEM of four experiments (A) and a representative measurement or blot of three performed (B–D). *, a, p < 0.01 vs the control or in the absence of DETA-NO, respectively (A).

View larger version (56K): [in this window] [in a new window]   FIGURE 2. Sustained NO synthesis impairs Bax processing and DNA degradation. RAW 264.7 macrophages were treated as indicated in Fig. 1, and at 0, 4, and 8 h samples were obtained to determine the protein levels of IAPs and p53 as well as those of Bax in different subcellular localizations. Analysis of DNA laddering was conducted in agarose gels. Results show a representative experiment of four performed.

NO-dependent apoptosis involves the release of mitochondrial cofactors (7, 29). Therefore, to evaluate the effect of continuous supply of NO by DETA-NO on proteins involved in the apoptotic pathway, the time course of activation of procaspases 9 and 3, p53 accumulation, and release of cytochrome c from mitochondria to cytosol were measured. As Fig. 3 shows, caspase activity using LEHD as substrate (a preferred caspase 9 and 7 substrate) increased after 4 h of treatment of cells with GSNO, whereas in the presence of DETA-NO this activity was notably impaired. Similar results were obtained with DEVD as substrate. Moreover, when cells were loaded with PhyPhyLux, a permeable fluorescent substrate of caspase 3, the percentage of positive cells was 70% in macrophages treated for 8 h with GSNO, but only 20% when cells were maintained under continuous NO supply. In addition to these parameters, the amount of p53 and the release of cytochrome c to the cytoplasm were notably attenuated when DETA-NO was present. Analysis of the processing of procaspase 9 and 3 showed that in the presence of DETA-NO, caspase processing was significantly inhibited.

View larger version (30K): [in this window] [in a new window]   FIGURE 3. Caspase activation is delayed by continuous NO supply. Macrophages were treated with 0.5 mM GSNO in the absence or the presence of 0.1 mM DETA-NO, and the LEHDase and DEVDase activities were measured in cell extracts prepared at the indicated times. When cells were labeled with the permeable caspase 3 fluorescent substrate PhyPhyLux (green) and the mitotracker chloromethyl X-rosamine (red), the percentage of caspase 3-positive cells was determined and plotted as a function of time in culture. A representative picture corresponding to the distribution of fluorescence at 6 h is shown. The levels of p53, the release of cytochrome c to the cytosol, and the processing of caspases 9 and 3 were analyzed by Western blot. The results show the mean of three experiments (left panels) and a representative blot of three performed.

View larger version (36K): [in this window] [in a new window]   FIGURE 4. Prolonged incubation with an NO donor exerts protective effects against NO-dependent apoptosis. RAW 264.7 cells were pretreated for different periods of time (from -20 to 0 h) with 0.1 mM DETA-NO; at time zero the medium was replaced, cells were challenged with 0.5 mM GSNO, and apoptosis was determined at 18 h (A). The levels of IAPs and Bax associated with the mitochondria were analyzed at 18 h by Western blot (B). Results show the mean ± SEM (n = 4) or a representative value of four (B). *, p < 0.005 vs in the absence of DETA-NO (A).

Inhibition of NO synthesis accelerates apoptosis in LPS- and IFN--activated macrophages

To evaluate the contribution of NO to the appearance of the apoptotic phenotype in activated macrophages, cells were treated with LPS and IFN-, and at various times the selective NOS-2 inhibitor N-(3-aminomethylbenzyl)acetamidine (1400W) was added to the culture medium. As Fig. 5 shows, when 1400W was added before NOS-2 expression, the percentage of apoptotic cells determined after 18 h of incubation was significantly reduced with respect to that of NO-synthesizing cells. However, when 1400W was added 8 h after activation, the percentage of apoptotic cells determined at 18 h was notably increased, indicating that endogenous NO contributes to regulation of the kinetics of apoptosis. Indeed, when caspase activity was measured using LEHD and DEVD as substrates, the inhibition of NOS-2 at short times, when the expression levels of the enzyme are negligible, resulted in a low caspase activity compared with LPS- and IFN--treated cells. However, the inhibition of NOS-2 at 8 h resulted in a marked increase in caspase 3 activity.

View larger version (32K): [in this window] [in a new window]   FIGURE 5. Late suppression of NO synthesis in LPS- and IFN--activated macrophages accelerates apoptosis. Cells were activated with 200 ng/ml of LPS and 50 U of mIFN-, and at the indicated times the NOS-2-selective inhibitor 1400W was added. The percentage of apoptotic cells and the activities of caspases 9 and 3, evaluated with LEHD-FMC and DEVD-FMC as substrates, were determined at 18 h. The protein levels of NOS-2 were determined by Western blot at 18 h. Results show the mean ± SD of four experiments. *, p < 0.05; **, p < 0.01 (vs LPS and IFN- activation, respectively, in the absence of 1400W).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

NO is a short-lived mild oxidant molecule that acts as an intra- and intercellular messenger mediating several physiologic and pathologic processes, including the regulation of cell viability (i.e., through the inhibition of stimulus-dependent apoptosis), apoptosis, and necrotic death (1, 4, 5, 7, 32). Furthermore, high output NO synthesis, such as occurs after the expression of NOS-2, contributes to the induction of apoptosis in inflammatory cells and therefore to the resolution of inflammation (5). In addition to the effects observed with elevated concentrations of NO, it has been described in neurons, which are very susceptible to NO-dependent apoptosis, that low, but prolonged, NO supply via NOS-1 activation through excitotoxicity mechanisms is sufficient to promote apoptotic death in these cells (33, 34). The pathways involved in NO-dependent apoptosis have been described in some detail, and there are specific patterns dependent on the cell type (6). In macrophages, for example, NO induces apoptosis in the absence of apoptotic volume decrease and promotes the release of mitochondrial mediators, such as cytochrome c and apoptosis-inducing factor, in the absence of a decrease in the mitochondrial inner membrane potential (7). NO also promotes a rapid up-regulation of the levels of p53 preceding cytochrome c release (8), followed by an activation of caspases and changes in the expression of proteins of the Bcl-2 family. In this way, monocytes express relatively high levels of the antiapoptotic protein Bcl-x_L (8), whereas once differentiated to macrophages, they loose this antiapoptotic protein at the time that the levels of Bax increase and the protein is targeted to the mitochondria (this work and Ref. 10).

In contrast to these proapoptotic effects of NO, this molecule can contribute to maintain cell viability, and this capacity appears to be dependent on the cell type considered, the amount of NO released, and the activation status of the cell (20, 35, 36, 37). Indeed, antiapoptotic effects of NO have been described in many cell types, such as macrophages, mesangial cells, hepatocytes, endothelial cells, and B and T lymphocytes (16, 17, 18, 21, 27, 38). In macrophages, several mechanisms have been described to protect against activation-dependent apoptosis. For example, chaperones of the heat shock protein 70 family inhibit efficiently apoptosis (39, 40), and signaling through the membrane receptor Ron, a Met family member tyrosine kinase receptor that is activated by macrophage-stimulating protein and negatively regulates the activation process in response to LPS, inhibits apoptosis not only in response to LPS, but also after incubation of macrophages with GSNO (41).

In addition to these mechanisms, NO appears to be very effective in inhibiting the process of caspase activation (19, 28, 37), probably through S-nitrosylation modifications. The incorporation of up to three molecules of NO in the p17 and p12 subunits of caspase 3 has been observed, but not when the protein was mutated in the cysteine residue present in the active site of the enzyme (26). In addition to NO, other molecules, such as the H₂O₂ released by activated macrophages, might also contribute in vivo to the reversible inhibition of caspases 3 and 8 (42). Moreover, it has been recently described that NO donors reduce the efficiency of formation of the apoptosis protease-activating factor 1/procaspase 9 apoptosome through a deficient assembly of the subunits, which impairs later steps of apoptosome activation through the caspase-recruitment domain motifs of Apaf 1 and procaspase 9 (20). Furthermore, the effects of modifications of cytochrome c, via nitrosylation of tyrosine residues, on the assembly and function of the apoptosome cannot be excluded (7).

In addition to the effects of NO on procaspase processing and activity, our data show that IAPs are up-regulated under conditions of continuous supply of NO. IAPs were identified in insect cells infected with baculovirus and then were described in mammalian cells, showing a high degree of conservation among species in two domains: the baculovirus inhibitory repeat and the C-terminal, zinc-binding motif. Several IAPs have been described, including cIAP-1, cIAP-2, and xIAP (43, 44, 45). These proteins bind to and potently inhibit caspases 9, 3, and 7 (with affinity constants in the nanomolar concentration range). Moreover, the IAPs can be recruited to the complexes activated in response to proinflammatory signaling, such as TNF-, redirecting signaling toward the activation of NF-B and the generation of additional survival signals through this pathway (22).

In summary, our data describe a mechanism by which NO delays apoptosis in the macrophage, allowing the cell to maintain its viability for longer periods of time, but primed to accomplish apoptosis as soon as the synthesis of NO decreases, a characteristic of the resolution step of inflammation, or when the balance favoring the accumulation of proapoptotic factors (mainly Bax and p53) and activation of caspases dominates.

	Acknowledgments

 
We thank Aleksandra Stpiczynska for secretarial help.

	Footnotes

 
¹ This work was supported by Grant SAF2002-00783 from Ministerio de Ciencia y Tecnología and Grant 08.3/0008/2001 from Comunidad de Madrid (Spain).

² Address correspondence and reprint requests to Dr. Lisardo Boscá, Instituto de Bioquímica, Facultad de Farmacia, 28040 Madrid, Spain. E-mail addresses: boscal{at}farm.ucm.es or lbosca{at}cnic.es

³ Abbreviations used in this paper: NOS-2, NO synthase-2; AFC, 7-amino-4-trifluoromethylcoumarin; DETA-NO, diethylenetriamine NONOate; GSNO, S-nitrosoglutathione; IAP, inhibitor of apoptosis protein; PI, propidium iodide; 1400W, N-(3-aminomethylbenzyl)acetamidine.

Received for publication April 2, 2003. Accepted for publication September 17, 2003.

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