Activation of the macrophage cell line RAW 264.7 with LPS and IFN-γ induces apoptosis through the synthesis of high concentrations of NO due to the expression of NO synthase-2. In addition to NO, activated macrophages release other molecules involved in the inflammatory response, such as reactive oxygen intermediates and PGs. Treatment of macrophages with cyclopentenone PGs, which are synthesized late in the inflammatory onset, exerted a negative regulation on cell activation by impairing the expression of genes involved in host defense, among them NO synthase-2. However, despite the attenuation of NO synthesis, the percentage of apoptotic cells increased with respect to activated cells in the absence of cyclopentenone PGs. Analysis of the mechanisms by which these PGs enhanced apoptosis suggested a potentiation of superoxide anion synthesis that reacted with NO, leading to the formation of higher concentrations of peroxynitrite, a more reactive and proapoptotic molecule than the precursors. The effect of the cyclopentenone 15-deoxy-Δ12,14-PGJ2 on superoxide synthesis was dependent on p38 mitogen-activated protein kinase activity, but was independent of the interaction with peroxisomal proliferator-activated receptor γ. The potentiation of apoptosis induced by cyclopentenone PGs involved an increase in the release of cytochrome c from the mitochondria to the cytosol and in the nitration of this protein. These results suggest a role for cyclopentenone PGs in the resolution of inflammation by inducing apoptosis of activated cells.

Activation of the host immune system by Gram-negative bacteria can be reproduced in vitro by incubation of cells with LPS and proinflammatory cytokines. Macrophages participate actively in the onset of inflammation and immune system activation by releasing cytokines that amplify the initial inflammatory stimulation, bioactive lipids (e.g., PGs and leukotrienes), reactive oxygen intermediates (ROI)3 and reactive nitrogen intermediates (RNI) that exert cytotoxic effects against pathogens and tumor cells (1, 2, 3, 4). Several early signaling pathways have been identified in response to LPS triggering of macrophages, including mitogen-activated protein kinases p42/p44 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase, and p38/stress-activated protein kinase (5, 6, 7), as well as various members of the Src family of protein tyrosine kinases and Vav (8, 9). Some of these pathways have been related to the synthesis of proinflammatory cytokines, participating in the amplification loop due to the initial LPS signaling through CD14, a 55-kDa glycosylphosphatidylinositol-linked protein that is the main LPS surface receptor in the macrophage, and the Toll-like receptor 2, a transmembrane protein that interacts with LPS and CD14 in these cells (10, 11). As result of this activation sequence, macrophages express enzymes involved in inflammation, such as NO synthase-2 (NOS-2), cyclooxygenase-2 (COX-2; the enzyme responsible for the high output synthesis of PGs), and matrix metalloproteinases, and they exhibit an enhancement of the synthesis of ROI (4, 12, 13, 14).

Resolution of inflammation is accomplished by the presence of anti-inflammatory cytokines (e.g., IL-4, IL-10, and IL-13) (8) and by the negative regulation of the activation process exerted by some of the effector molecules released by activated macrophages, in particular ROI, RNI, and cyclopentenone PGs (15, 16, 17, 18, 19). At the end of the inflammatory response, the cells that have participated in the process are removed by apoptosis (12, 20, 21, 22, 23). Induction of apoptosis in activated macrophages has been recognized as a physiological and altruistic mechanism that helps to reduce the inflammatory stress and to avoid the establishment of chronic persistent infection by intracellular pathogens (3, 13, 24). The contribution of NO to trigger apoptosis in macrophages has been well established (12, 23). The elevated synthesis of NO due to the expression of NOS-2 releases mitochondrial mediators that initiate caspase activation, leading to a characteristic apoptotic death (25, 26, 27). However, in several in vivo models of inflammation the synthesis of anti-inflammatory PGs has been described, namely 15-deoxy-Δ12,14-PGJ2 (15dPGJ2) and related cyclopentenone PGs (17), that exert their effects through the inhibition of IκB kinase (IKK2) and activation of the peroxisomal proliferator-activated receptor γ (PPARγ) (15, 19, 28, 29). According to these data, in the presence of 15dPGJ2 the synthesis of NO is significantly reduced, and therefore, the intrinsic apoptotic capacity of NO might be compromised. To address this apparent controversy we have investigated in this work the action of anti-inflammatory PGs on the apoptotic response in macrophages activated with LPS/IFN-γ. Under these conditions, an important inhibition of NOS-2 expression was observed due to the presence of 15dPGJ2. However, the percentage of apoptotic cells increased significantly. As our data show, the likely mechanism by which cyclopentenone PGs favor apoptosis is through an important increase in the synthesis of ROI that react immediately with NO, leading to the formation of peroxynitrite, a more potent and efficient inductor of apoptosis than NO.

Reagents were obtained from Roche (Mannheim, Germany), Calbiochem (Darmstadt, Germany), and Sigma (St. Louis, MO). Peptides were purchased from Bachem (Bubendorf, Switzerland). Materials and chemicals for electrophoresis were obtained from Bio-Rad (Richmond, CA). Potential-sensitive fluorescent probes were purchased from Molecular Probes (Eugene, OR). Abs were obtained from PharMingen (San Diego, CA) and New England Biolabs (Beverly, MA). Culture media were obtained from BioWhittaker (Verviers, Belgium).

The murine macrophage cell line RAW 264.7 was cultured (6–8 × 104 cells/cm2) in RPMI 1640 medium supplemented with 2 mM glutamine, 10% FCS, and antibiotics (50 μg/ml of penicillin, streptomycin, and gentamicin). After 2 days, the cell layers were washed with PBS, and the culture medium was replaced with phenol red-free RPMI 1640 containing 0.5 mM arginine and 0.5% FCS and stimulated (30). PGs and rosiglitazone were added 5 min before activation with LPS and IFN-γ.

Propidium iodide (PI) staining was performed after incubation of the cells with 0.005% PI, following a previously described protocol (30, 31). Cells were carefully resuspended and run in a FACScan cytometer (Becton Dickinson, 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. Cell sorting and analysis of viable and apoptotic populations were performed to confirm the criteria of gating (30).

Cells were incubated for 15 min at 37°C in the presence of the potential-sensitive probes chloromethyl X-rosamine, 3,3′-dihexyloxacarbocyanine iodide, tetrachlorotetraethylbenzimidazolylcarbocyanine iodide, and rhodamine 123, all at 40 nM (25), followed by analysis in a FACScan flow cytometer. The fluorescence in the presence of 10 μM of the uncoupling agent m-chlorophenylhydrazone carbonylcyanide was considered as 100%, and values were calculated as the percent change in fluorochrome fluorescence.

Cells challenged for the indicated periods of time with different stimuli were incubated for 15 min with 10 μM 2′,7′-dichlorofluorescein diacetate (DCFH) or hydroethidine (HE); and the fluorescence corresponding to the oxidized probes was followed by analysis in a flow cytometer as previously described (25, 32). Simultaneous incubation of the cells with the probes and 50 μM t-butyl hydroperoxide was used as a positive control of ROI release.

Peroxynitrite (OONO) was synthesized by reaction of hydrogen peroxide with nitrous acid as previously described (33, 34). Briefly, a 0.6 M solution of sodium nitrite was mixed vigorously with an equal volume of 0.7 N HCl and 0.6 M H2O2 and immediately stabilized with 1.2 N NaOH. Unreacted H2O2 was removed by passing the solution through an MnO2 column. Appropriate controls were conducted to ensure that after decomposition of peroxynitrite at pH 6.0, the reaction mixture did not affect cell viability. The concentration of peroxynitrite was determined before use by measuring the absorbance at 302 nm (ε = 1670 M−1 cm−1).

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 Tosyl-l-phenylalanine chloromethyl ketone, 5 mM NaF, 1 mM NaV04, and 10 mM Na2MoO4) containing 120 mM NaCl and scraped off the plate. Lysis of the cells was performed 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), and the pellet was resuspended in 50 μl of buffer A supplemented with 20% glycerol and 0.4 M KCl and gently shaken for 30 min at 4°C. Nuclear protein extracts were obtained by centrifugation at 13,000 × g for 15 min, and the supernatants were stored at −80°C. Protein content was assayed using the Bio-Rad protein reagent. All steps of cell fractionation were conducted at 4°C.

The presence of cytochrome c in the cytosol was determined in cell extracts prepared as previously described (26). To evaluate the nitration of cytochrome c, equal amounts of supernatant protein (50 μg) were immunoprecipitated with anti-NO-Y Ab (a gift from J. S. Beckman, University of Alabama, Birmingham, AL) and revealed by Western blot with anti-cytochrome c mAb (clone 7H8.2C12;PharMingen) following the instructions of the Ab supplier. The levels of IκBα, IκBβ, and phosphorylated or dephosphorylated p38, c-Jun N-terminal kinase (JNK), and extracellular- regulated kinase ERK) 1 and ERK2 were determined by Western blot using cytosolic extracts and specific commercial Ab (Santa Cruz Biotechnology (Santa Cruz, CA) and New England Biolabs, respectively). The proteins were size-separated in 12% SDS-PAGE, transferred to a polyvinylidene difluoride membrane and incubated with the indicated Ab. The bands recognized were visualized by the ECL technique.

The consensus sequence of NF-κB (5′-TGCTAGGGGGATTTTCCCTCTCTCTGT-3′, corresponding to nucleotides −978 to −952 of the murine NOS-2 promoter) was used (19). Aliquots of 50 ng of annealed oligonucleotide were end-labeled with Klenow enzyme fragment in the presence of 50 μCi of [α-32P]dCTP and the other unlabeled dNTPs in a final volume of 50 μl. For each binding assay 3 μg of nuclear protein was incubated for 15 min at 4°C with the DNA (5 × 104 dpm) and 2 μg of poly(dI:dC), 5% glycerol, 1 mM EDTA, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, and 10 mM Tris-HCl (pH 7.8) in a final volume of 20 μl. The DNA-protein complexes were separated on native 6% polyacrylamide gels in 0.5% Tris-borate-EDTA buffer. Supershift assays were conducted after incubation of the nuclear extracts with 2 μg of Ab (anti-p50, anti-c-Rel, and anti-p65) for 1 h at 4°C, followed by EMSA (data not shown).

The DEVDase activity (corresponding mainly to caspases 3 and 7) was determined in cell lysates using N-acetyl-DEVD-7-amino-4-methylcoumarin as fluorogenic substrate and following the instructions of the supplier (PharMingen). The corresponding peptide aldehyde and Z-VAD fluoromethyl ketone were used to inhibit caspase activity in vivo and to ensure the specificity of the reaction in the in vitro assay. The linearity of the caspase assay was determined over a 30-min reaction period.

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

Induction of apoptosis in LPS/IFN-γ-activated cells was dependent on the synthesis of NO (26, 30), since the selective NOS-2 inhibitor N-(3-aminomethylbenzyl)acetamidine (1400W) abolished this effect (Fig. 1,A). Treatment of cells with up to 2 μM 15dPGJ2 did not affect cell viability, but significantly increased the apoptosis induced by LPS/IFN-γ (100 ng/ml and 5 U/ml, respectively). However, the dose-dependent apoptosis elicited by 15dPGJ2 in LPS/IFN-γ-activated cells was in parallel with an inhibition of NOS-2 expression and, therefore, of NO synthesis (Fig. 1, B and C). Apoptosis was measured by flow cytometry (Fig. 1 D). Moreover, this apoptosis remained dependent on the synthesis of NO, since it was suppressed efficiently by 1400W. These results suggest that in activated cells treated with 15dPGJ2 apoptosis was dependent on NO synthesis and on another molecule produced in response to PG challenge. Similar results were obtained using elicited peritoneal macrophages.

FIGURE 1.

15dPGJ2 increased apoptosis in activated macrophages. RAW 264.7 cells were incubated for 18 h with 5 U/ml of IFN-γ and the indicated concentrations of LPS (A) or with 100 ng/ml of LPS, 5 U/ml of IFN-γ, and the indicted concentration of 15dPGJ2 (B). The percentage of apoptotic cells and the synthesis of NO were measured. The effect of 15dPGJ2 on the levels of NOS-2 in LPS/IFN-γ-activated cells was determined by Western blot (C). Apoptosis was determined by flow cytometric analysis after PI staining of the cells and is expressed as the percentage of cells in R2 and R3 regions of the dot plot distribution (D). Cells gated in R4 and R2 plus R3 were sorted, and the extranuclear DNA was analyzed by electrophoresis in agarose gels. GSNO was used at 500 μM. Results show the mean ± SEM of four experiments or a representative blot of three (C).

FIGURE 1.

15dPGJ2 increased apoptosis in activated macrophages. RAW 264.7 cells were incubated for 18 h with 5 U/ml of IFN-γ and the indicated concentrations of LPS (A) or with 100 ng/ml of LPS, 5 U/ml of IFN-γ, and the indicted concentration of 15dPGJ2 (B). The percentage of apoptotic cells and the synthesis of NO were measured. The effect of 15dPGJ2 on the levels of NOS-2 in LPS/IFN-γ-activated cells was determined by Western blot (C). Apoptosis was determined by flow cytometric analysis after PI staining of the cells and is expressed as the percentage of cells in R2 and R3 regions of the dot plot distribution (D). Cells gated in R4 and R2 plus R3 were sorted, and the extranuclear DNA was analyzed by electrophoresis in agarose gels. GSNO was used at 500 μM. Results show the mean ± SEM of four experiments or a representative blot of three (C).

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Inhibition of endogenous COX-2 activity with NS398 promoted a moderate, although significant, increase in NO synthesis in LPS/IFN-γ-activated cells. However, the percentage of apoptotic cells increased under these conditions, suggesting a minor contribution of endogenous 15dPGJ2 to resolution of inflammation (Table I).

Table I.

Effect of COX-2 inhibition with NS398 on NO synthesis and apoptosisa

TreatmentNO Synthesis (% vs LPS/IFN-γ)TreatmentApoptosis (%)
+ 10 μM NS398+ 20 μM NS398+ 10 μM NS398+ 20 μM NS398
None 4.5 ± 0.3 2.1 ± 0.1 2.9 ± 0.1 None 11 19 16 
15dPGJ2 1.1 ± 0.1 1.4 ± 0.1 1.0 ± 0.1 15dPGJ2 13 16 20 
LPS/IFN-γ 100 115 ± 9 135 ± 9 LPS/IFN-γ 33 41 43 
15dPGJ2+ LPS/IFN-γ 32 ± 2 37 ± 3 40 ± 3 15dPGJ2+ LPS/IFN-γ 62 65 63 
TreatmentNO Synthesis (% vs LPS/IFN-γ)TreatmentApoptosis (%)
+ 10 μM NS398+ 20 μM NS398+ 10 μM NS398+ 20 μM NS398
None 4.5 ± 0.3 2.1 ± 0.1 2.9 ± 0.1 None 11 19 16 
15dPGJ2 1.1 ± 0.1 1.4 ± 0.1 1.0 ± 0.1 15dPGJ2 13 16 20 
LPS/IFN-γ 100 115 ± 9 135 ± 9 LPS/IFN-γ 33 41 43 
15dPGJ2+ LPS/IFN-γ 32 ± 2 37 ± 3 40 ± 3 15dPGJ2+ LPS/IFN-γ 62 65 63 
a

RAW 264.7 cells were incubated for 18 h with combinations of LPS (100 ng/ml), IFN-γ (5 U/ml), 15dPGJ2 (2 μM), and the indicated concentration of COX-2 inhibitor. At the end of the incubation period, the synthesis of NO (considering 100% that of LPS/IFN-γ treated cells; 64 ± 4 nmol/mg of protein) and the percentage of apoptotic cells were measured. Results show the mean ± SEM of three experiments. ∗, p < 0.01 vs the condition in the absence of inhibitor.

Evaluation of changes in fluorescence of probes sensitive to the mitochondrial inner transmembrane potential (chloromethyl X-rosamine, tetrachlorotetraethylbenzimidazolylcarbocyanine iodide, 3,3′-dihexyloxacarbocyanine iodide, and rhodamine 123) showed an increase in fluorescence in apoptotic cells (LPS/IFN-γ plus 15dPGJ2 condition) regardless of the probe used (Fig. 2,A), confirming previous results in these cells (26). Moreover, in cells treated with 15dPGJ2 and LPS/IFN-γ a 3.7-fold increase in cytochrome c in the cytosol was measured with respect to cells incubated with LPS/IFN-γ (Fig. 2 B). Analysis of NO modifications of cytosolic cytochrome c showed the presence of nitrotyrosine in activated cells incubated with 15dPGJ2.

FIGURE 2.

Determination of mitochondrial potential and cytosolic cytochrome c in activated cells. RAW 264.7 cells were stimulated for 8 h with combinations of LPS (100 ng/ml), IFN-γ (5 U/ml), and 15dPGJ2 (2 μM), and after labeling with the indicated probes the fluorescence was determined by flow cytometry. Depolarization with m-chlorophenylhydrazone carbonylcyanide (10 μM) was used to normalize the changes in fluorescence (A). The amount of cytochrome c in the cytosol was determined by Western blot after 18 h of incubation with the indicated stimuli. Alternatively, cytosolic extracts were immunoprecipitated with anti-NO-Y Ab and revealed with anti-cytochrome c mAb (B). Results show the mean ± SEM of three experiments. ∗, p < 0.01 vs the corresponding condition in the absence of 15dPGJ2.

FIGURE 2.

Determination of mitochondrial potential and cytosolic cytochrome c in activated cells. RAW 264.7 cells were stimulated for 8 h with combinations of LPS (100 ng/ml), IFN-γ (5 U/ml), and 15dPGJ2 (2 μM), and after labeling with the indicated probes the fluorescence was determined by flow cytometry. Depolarization with m-chlorophenylhydrazone carbonylcyanide (10 μM) was used to normalize the changes in fluorescence (A). The amount of cytochrome c in the cytosol was determined by Western blot after 18 h of incubation with the indicated stimuli. Alternatively, cytosolic extracts were immunoprecipitated with anti-NO-Y Ab and revealed with anti-cytochrome c mAb (B). Results show the mean ± SEM of three experiments. ∗, p < 0.01 vs the corresponding condition in the absence of 15dPGJ2.

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The apoptosis observed in the presence of 15dPGJ2 and LPS/IFN-γ involved a 2.2-fold increase in DEVDase activity, presumably due to caspase 3 activation, that was inhibited efficiently after treatment with aldehyde (DEVD.CHO). Indeed, a more general caspase inhibitor such as z-VAD prevented apoptosis to similar levels as did DEVD.CHO (Fig. 3).

FIGURE 3.

15dPGJ2-dependent apoptosis was prevented by DEVDase inhibitors. Cells were incubated for 18 h with LPS/IFN-γ, 15dPGJ2, and the caspase inhibitors DEVD.CHO (20 μM) and z-VAD fluoromethyl ketone (30 μM). The percentage of apoptotic cells and the DEVDase activity in cell extracts were determined. Results show the mean ± SEM of three experiments. ∗, p < 0.005 vs the corresponding condition in the absence of 15dPGJ2.

FIGURE 3.

15dPGJ2-dependent apoptosis was prevented by DEVDase inhibitors. Cells were incubated for 18 h with LPS/IFN-γ, 15dPGJ2, and the caspase inhibitors DEVD.CHO (20 μM) and z-VAD fluoromethyl ketone (30 μM). The percentage of apoptotic cells and the DEVDase activity in cell extracts were determined. Results show the mean ± SEM of three experiments. ∗, p < 0.005 vs the corresponding condition in the absence of 15dPGJ2.

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15dPGJ2 and related cyclopentenone PGs have been described as agonists of PPARγ (15, 35, 36). To determine whether the effect of 15PGJ2 was due to the engagement of this nuclear receptor, the actions of several PGs and structurally unrelated PPARγ ligands were investigated. As Fig. 4,A shows, among the PGs assayed, only 15dPGJ2 and PGA2 potentiated apoptosis. Treatment of activated RAW 264.7 cells with 10 μM of the PPARγ ligand rosiglitazone did not exert any effect on apoptosis, suggesting that cyclopentenone PGs were acting through a mechanism distinct from PPARγ ligation. Moreover, the RAW cells used failed to express PPARγ upon challenge with LPS/IFN-γ as did cultured peritoneal macrophages under similar experimental conditions (Fig. 4 B).

FIGURE 4.

Cyclopentenone PGs enhanced apoptosis through a PPARγ-independent mechanism in RAW cells. Cells were incubated with 2 μM of the indicated bioactive lipids or with 10 μM of the PPARγ ligand rosiglitazone. The percentage of apoptotic cells was determined by flow cytometry (A). The levels of PPARγ in cell extracts were determined by Western blot (B). Results show the mean ± SEM of three experiments. ∗, p < 0.05 with respect to the condition in the absence of lipids.

FIGURE 4.

Cyclopentenone PGs enhanced apoptosis through a PPARγ-independent mechanism in RAW cells. Cells were incubated with 2 μM of the indicated bioactive lipids or with 10 μM of the PPARγ ligand rosiglitazone. The percentage of apoptotic cells was determined by flow cytometry (A). The levels of PPARγ in cell extracts were determined by Western blot (B). Results show the mean ± SEM of three experiments. ∗, p < 0.05 with respect to the condition in the absence of lipids.

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To further investigate the mechanisms by which 15dPGJ2 enhanced apoptosis in LPS/IFN-γ-activated cells, we analyzed the release of ROI and RNI by following the fluorescence corresponding to the oxidation of HE and DCFH. As Fig. 5,A shows, the oxidation of HE, reflecting mainly the synthesis of O2, increased moderately in LPS/IFN-γ-activated cells treated with 15dPGJ2. When NO synthesis was transiently suppressed after inhibition of NOS-2 with 1400W, a further increase in HE oxidation was observed, indicating an overproduction of O2. However, the ability O2 to oxidize HE was lost in the presence of NO, probably due to the rapid reaction between both reactive species producing peroxynitrite (see Discussion). Regarding DCFH fluorescence, a time-dependent increase was observed in cells treated with 15dPGJ2 and LPS/IFN-γ, with an apparent steady state rate of oxidation after 8 h of stimulation (Fig. 5 B). These kinetics paralleled the rate of NO synthesis due to the expression of NOS-2.

FIGURE 5.

15dPGJ2 increased ROI synthesis in activated macrophages. RAW cells were incubated with combinations of 100 ng/ml LPS, 5 U/ml of IFN-γ, 2 μM 15dPGJ2, and 50 μM 1400W, and the changes in fluorescence of HE (14 h) and DCFH (time course) were determined. 1400W was added 1 h before HE challenge. t-butyl hydroperoxide was used as a positive control of HE oxidation. Results show the mean ± SEM of three experiments (A) or the mean of two experiments (B). ∗, p < 0.05 with respect to the condition in the absence of 15dPGJ2; a,p < 0.01 with respect to the condition without 1400W.

FIGURE 5.

15dPGJ2 increased ROI synthesis in activated macrophages. RAW cells were incubated with combinations of 100 ng/ml LPS, 5 U/ml of IFN-γ, 2 μM 15dPGJ2, and 50 μM 1400W, and the changes in fluorescence of HE (14 h) and DCFH (time course) were determined. 1400W was added 1 h before HE challenge. t-butyl hydroperoxide was used as a positive control of HE oxidation. Results show the mean ± SEM of three experiments (A) or the mean of two experiments (B). ∗, p < 0.05 with respect to the condition in the absence of 15dPGJ2; a,p < 0.01 with respect to the condition without 1400W.

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The nature of the molecules involved in the oxidation of DCFH was investigated by using reagents that interfere with the synthesis of ROI and RNI (Fig. 6). Treatment of cells with ebselen, which reacts with ROI and NO, abrogated DCFH oxidation and apoptosis (37). Inhibition of NOS-2 with 1400W significantly decreased the oxidation of DCFH and prevented apoptosis. Incubation of cells with 2,3-dimethoxy-1,4-naphtoquinone (DMNQ), which released O2 in solution, enhanced the fluorescence of DCFH in LPS/IFN-γ-activated cells, but not in resting macrophages, suggesting that an O2-derived metabolite was the main molecule responsible for DCFH oxidation (Fig. 6,A). When cells were incubated with Mn3+-tetrakis(4-benzoic acid)porphyrin, a permeant porphyrine that reduces O2, a significant, but not complete, decrease in DCFH fluorescence was observed (data not shown). These results together with the observation of an increase in cytochrome c nitration (Fig. 2,B) as well as previous reports on DCFH oxidation (38, 39) suggest that peroxynitrite was the main contributor to the changes in DCFH fluorescence in activated macrophages treated with 15dPGJ2. In agreement with these data, incubation of RAW 264.7 cells with substances that release NO and O2 in solution enhanced DCFH oxidation. As Fig. 6,B shows, incubation of cells with S-nitrosogluthathione (GSNO) and DMNQ significantly increased the oxidation of DCFH and promoted apoptosis. Indeed, when cells were treated with the radical scavenger ebselen, the changes in fluorescence of DCFH and apoptosis were abolished. Moreover, incubation of cells with peroxynitrite promoted dose-dependent parallel changes in DCFH fluorescence and apoptosis (Fig. 6 C).

FIGURE 6.

15dPGJ2 increased the synthesis of peroxynitrite. RAW 264.7 cells were incubated for 14 h with combinations of 100 ng/ml LPS, 5 U/ml of IFN-γ, 2 μM 15dPGJ2, 1 mM ebselen, 50 μM 1400W, and 200 μM DMNQ. The changes in DCFH fluorescence and the percentage of apoptotic cells were determined (A). When cells were incubated with 500 μM GSNO (B) or the indicated concentrations of peroxynitrite (C), the changes in DCFH and the percentage of apoptotic cells were determined after 8 h of treatment. Results show the mean ± SEM of three experiments.

FIGURE 6.

15dPGJ2 increased the synthesis of peroxynitrite. RAW 264.7 cells were incubated for 14 h with combinations of 100 ng/ml LPS, 5 U/ml of IFN-γ, 2 μM 15dPGJ2, 1 mM ebselen, 50 μM 1400W, and 200 μM DMNQ. The changes in DCFH fluorescence and the percentage of apoptotic cells were determined (A). When cells were incubated with 500 μM GSNO (B) or the indicated concentrations of peroxynitrite (C), the changes in DCFH and the percentage of apoptotic cells were determined after 8 h of treatment. Results show the mean ± SEM of three experiments.

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Since addition of 15dPGJ2 4 h or more later than LPS/IFN-γ challenge did not affect the percentage of apoptotic cells (data not shown), we investigated potential early signaling pathways involved in the enhancement of O2 synthesis elicited by this PG, among them the phosphorylation state of members of the MAPK family and the activation of NF-κB. As Table II shows, inhibition of the p38 MAPK with SB203580 or SB202190 significantly reduced DCFH oxidation and apoptosis (65 and 75%, respectively), whereas the inactive analogue SB202474 failed to affect these parameters. Inhibition of the MAP/ERK kinase/ERK pathway with PD98059 (50 μM) did not modify these parameters. Moreover, the phosphorylation state of p38, ERK1 and 2, and JNK was investigated by Western blot. Treatment of LPS/IFN-γ-activated cells with 15dPGJ2 did not affect ERK1 and 2 phosphorylation, but exerted a moderate inhibition of p38 (31%) and JNK (17%) phosphorylation (Fig. 7,A). The other likely candidate to be affected by 15dPGJ2 in LPS/IFN-γ activated cells was NF-κB (18, 19, 28). As Fig. 7 B shows, activated RAW 264.7 cells exhibited a significant reduction of NF-κB activity when incubated with 15dPGJ2 and a complete inhibition in the presence of ebselen. The cytosolic levels of IκBα and IκBβ inversely paralleled the activation of NF-κB as determined by EMSA.

Table II.

Effect of MAPK inhibitors on apoptosis and DCFH fluorescencea

TreatmentApoptosis (%)DCFH Fluorescence (AU)b
LPS/IFN-γ++++
15dPGJ2++
None 33 ± 4 77 ± 9 34 ± 3 84 ± 7 
SB203580 (10 μM) 24 ± 2* 28 ± 3* 21 ± 2* 38 ± 4* 
SB202190 (10 μM) 22 ± 2* 25 ± 2* 20 ± 1* 35 ± 2* 
SB202474 (10 μM) 35 ± 4 75 ± 8 35 ± 4 87 ± 9 
PD98059 (50 μM) 35 ± 2 74 ± 5 34 ± 5 84 ± 10 
TreatmentApoptosis (%)DCFH Fluorescence (AU)b
LPS/IFN-γ++++
15dPGJ2++
None 33 ± 4 77 ± 9 34 ± 3 84 ± 7 
SB203580 (10 μM) 24 ± 2* 28 ± 3* 21 ± 2* 38 ± 4* 
SB202190 (10 μM) 22 ± 2* 25 ± 2* 20 ± 1* 35 ± 2* 
SB202474 (10 μM) 35 ± 4 75 ± 8 35 ± 4 87 ± 9 
PD98059 (50 μM) 35 ± 2 74 ± 5 34 ± 5 84 ± 10 
a

Raw 264.7 cells were incubated for 18 h with combinations of LPS (100 ng/ml), IFN-γ (5 U/ml), 15dPGJ2 (2 μM), and the indicated concentration of inhibitors. At the end of the incubation period, the percentage of apoptotic cells and the fluorescence corresponding to the oxidation of DCFH were measured. Results show the mean ± SEM of three experiments per triplicate. ∗, p < 0.001 vs the condition in the absence of inhibitor.

b

AU, Arbitrary units.

FIGURE 7.

Effect of 15dPGJ2 on MAPK and NF-κB activation. Cells were stimulated with LPS/IFN-γ (100 ng/ml and 5 U/ml, respectively), 1 mM ebselen, and 2 μM 15dPGJ2, and at the indicated times cell extracts were prepared to determine the phosphorylation state of ERK, p38, and JNK by Western blot, using Ab specific for the phosphoenzyme and for the total enzyme (cytosolic extracts, left panel). NF-κB activity was determined by EMSA in nuclear extracts obtained at 30 min. The proteins present in the retained bands were determined by supershift assays. The levels of IκBα and IκBβ proteins were determined in the corresponding cytosolic extracts. Results show representative blots of three.

FIGURE 7.

Effect of 15dPGJ2 on MAPK and NF-κB activation. Cells were stimulated with LPS/IFN-γ (100 ng/ml and 5 U/ml, respectively), 1 mM ebselen, and 2 μM 15dPGJ2, and at the indicated times cell extracts were prepared to determine the phosphorylation state of ERK, p38, and JNK by Western blot, using Ab specific for the phosphoenzyme and for the total enzyme (cytosolic extracts, left panel). NF-κB activity was determined by EMSA in nuclear extracts obtained at 30 min. The proteins present in the retained bands were determined by supershift assays. The levels of IκBα and IκBβ proteins were determined in the corresponding cytosolic extracts. Results show representative blots of three.

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We investigated the contribution of 15dPGJ2 and related cyclopentenone PGs to the resolution of inflammation via triggering apoptosis in activated macrophages. Previous reports have shown that incubation of resident or elicited peritoneal macrophages and macrophage cell lines with LPS and proinflammatory cytokines promoted apoptosis through both NO-dependent and -independent pathways (12, 23, 26, 40). At low concentrations of LPS and IFN-γ, apoptosis was very dependent on the synthesis of NO, probably because the synergism between both stimuli resulted in efficient NOS-2 expression and a high output of NO, minimizing the contribution of other proapoptotic pathways. Under these conditions, cyclopentenone PGs significantly inhibited NOS-2 expression due to the inactivation of IKK2 and the transcriptional inhibition dependent on PPARγ activation (15, 19, 28). In RAW 264.7 cells, however, the effects of 15dPGJ2 cannot be attributed to PPARγ activity, since this protein was undetectable by Western blot, and rosiglitazone, a PPARγ ligand, failed to mimic the action of cyclopentenone PGs. Moreover, in other cell types, 15dPGJ2 and PPARγ ligands exerted apoptotic effects independent of proinflammatory stimulation; in human monocyte-derived macrophages activation of PPARγ, but not PPARα, was sufficient to induce apoptosis, although the activation of caspases (in particular, caspase 3) was more efficient when cells were treated with TNF-α and IFN-γ (41). In human vascular endothelial cells, PPARγ activation by 15dPGJ2 or ciglitizone was also sufficient to induce apoptosis (42). Contrary to this situation, exogenously added 15dPGJ2 induced proliferation of COX-2-depleted colorectal cancer cells (HCA-7) (43), indicating that the effects of these molecules are very dependent on the cell type. Regarding the mechanism by which 15dPGJ2 enhanced apoptosis in RAW 264.7 cells, we analyzed the changes in ΔΨm and the release of cytochrome c to the cytosol under these conditions. Our data show that ΔΨm was maintained or even increased after treatment with 15dPGJ2, indicating the involvement of a pathway similar to that described for the apoptosis dependent on high concentrations of NO (26). Interestingly, the amount of nitrated cytochrome c increased upon treatment with PGs, suggesting an improved efficiency in the synthesis of peroxynitrite. Indeed, previous studies indicated that nitration of cytochrome c was an early event in the release of this protein from the mitochondria and preceded the changes in ΔΨm (26, 44).

The remarkable potentiation of DCFH oxidation elicited by 15dPGJ2 in LPS/IFN-γ activated cells provides an important clue to understand the observed enhancement of apoptosis. The pathways involved in the increase in O2 synthesis and the mechanisms used by the cells to sense oxidative stress are a subject of current research (4). Recently, it has been reported that p66shc, a protein containing a Src homology 2 domain and involved in the transmission of mitogenic signals from membrane receptors to Ras, acts as a sensor of oxidative stress, becoming phosphorylated in Ser and initiating an as yet uncharacterized signaling pathway that regulates oxidative stress and life span in mammalian cells (45). In addition to this, various reports describe a cross-talk between ROI signaling through the MAPK pathways and activation of transcription factors implicated in early gene regulation (NF-κB and AP-1), the balance of them influencing cell viability (4, 18, 46). The enzymes of the MAPK pathways, p38, ERK1 and 2, and JNK, are transiently activated in LPS-stimulated macrophages (5, 6). Using inhibitors of some of these MAPK pathways, we identified an important contribution of p38 activity to the synthesis of ROI that participate in the induction of apoptosis in RAW 264.7 cells. Our data show a moderate inhibitory effect by 15dPGJ2 (<36%) on LPS/IFN-γ-dependent activation of p38 and JNK, but not on the ERK1 and 2 activities. In the same vein, in cardiac myocytes deficient of MEK kinase it has been shown that p38 constitutes an important step for apoptotic ROI signaling (47). Alternatively, overexpression of catalase and superoxide dismutase or decomposition of O2 with permeant porphyrines protected cells from ROS injury (48). Therefore, the potential regulatory effects of O2 on the balance between viability and apoptosis of the macrophage are complex. Priming of RAW 264.7 cells with low concentrations (5 μM) of the O2 donor DMNQ protected against NO-dependent cell death when the apoptotic stimulation was performed 15 h after DMNQ challenge. In this case, the mechanism involved an impaired activation of NF-κB and AP-1 compared with nonprimed cells (49). The experiments reported in this work provide the characterization of an additional mechanism of potentiation of apoptosis in activated macrophages through the reaction of NO and O2 to yield the potent oxidant peroxynitrite (14, 50, 51). Regarding NF-κB activation by ROI as a sensor of oxidative stress in macrophages (4, 18), a paradoxical situation appears when the synthesis of O2 is accomplished by cyclopentenone PGs; the inhibitory effect of these lipids on IKK activity, and therefore on IκB phosphorylation, results in an impaired NF-κB activation.

Moreover, in the presence of a potent radical scavenger such as ebselen, ROI synthesis and NF-κB activity were inhibited, and apoptosis was completely abrogated. Although these data suggest that inhibition of NF-κB activity is not sufficient to promote apoptosis in cells treated with ebselen, it cannot be excluded that the impairment of NF-κB activity exerted by 15dPGJ2 might contribute to some extent to apoptosis. To summarize our data, a schematic representation of the concerted mechanism involving the synthesis of peroxynitrite from NO and O2 is shown in Fig. 8.

FIGURE 8.

Schematic representation of peroxynitrite-dependent apoptosis in RAW 264.7 cells stimulated with 15dPGJ2 and LPS/IFN-γ. Activated macrophages release NO through the expression of NOS-2 and release PGs through the expression of COX-2. 15dPGJ2 is released late in the activation process and promotes the synthesis of high amounts of O2. NO and O2 react immediately, producing the potent apoptogenic molecule peroxynitrite. The release of ROI and RNI can be controlled pharmacologically, resulting in a modulation of apoptosis.

FIGURE 8.

Schematic representation of peroxynitrite-dependent apoptosis in RAW 264.7 cells stimulated with 15dPGJ2 and LPS/IFN-γ. Activated macrophages release NO through the expression of NOS-2 and release PGs through the expression of COX-2. 15dPGJ2 is released late in the activation process and promotes the synthesis of high amounts of O2. NO and O2 react immediately, producing the potent apoptogenic molecule peroxynitrite. The release of ROI and RNI can be controlled pharmacologically, resulting in a modulation of apoptosis.

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We analyzed the intracellular localization of the fluorescence of DCFH by confocal microscopy to determine whether a gradient of ROI synthesis could be established in the cell. However, a broad diffusion of fluorescence covering the cell and without a clear pattern of subcellular distribution was observed, suggesting a high diffusion of the substrate (peroxynitrite) and/or the oxidized DCFH (data not shown).

The physiopathological relevance of the synthesis of cyclopentenone PGs can be deduced from models of carrageenin-induced inflammation in which a two-phase PG release has been described after expression of COX-2. PGE2 synthesis predominates during the early inflammatory step, whereas 15dPGJ2 substitutes PGE2 formation at the end of the process, coincident with the accumulation of macrophages (16, 17). Therefore, cyclopentenone PGs, in addition to their characterization as anti-inflammatory molecules, can be viewed as important factors in the resolution of the inflammatory process through the potentiation of apoptosis (24). Attempts to determine the contribution to apoptosis of the endogenous synthesis of 15dPGJ2 by activated RAW 264.7 cells treated with the COX-2 inhibitor NS398 resulted in an enhancement of cell death due to an up-regulation of NOS-2 expression. Moreover, these data reveal a moderate contribution of macrophages to the synthesis of 15dPGJ2 during the resolution of inflammation, suggesting a paracrine, rather than autocrine, regulatory loop for this PG.

In summary, our data stress the relevance of cooperative signaling leading to the synthesis of superoxide and NO to favor apoptosis. This mechanism is especially relevant at the end of the inflammatory process, when cyclopentenone PGs are produced and the synthesis of NO decreases due to the action of negative regulatory pathways (among them NO itself) and to arginine exhaustion. In this way, pharmacological modulation of the NOS-2 and COX-2 pathways can provide additional tools to regulate the balance between apoptosis and cell viability and, therefore, to control the inflammatory process.

We thank O. G. Bodelón for technical support, Dr. M. J. M. Díaz-Guerra for helpful discussion, Dr. M. A. Moro for help in the synthesis of peroxynitrite, and E. Lundin for critical reading of this manuscript.

1

This work was supported by Grants PM98-0120 and 2FD97-1432 from Comisión Interministerial de Ciencia y Tecnología (Spain).

3

Abbreviations used in this paper: ROI, reactive oxygen intermediates; RNI, reactive nitrogen intermediates; 1400W, N-(3-aminomethylbenzyl)acetamidine; COX-2, cyclooxygenase-2; 15dPGJ2, 15-deoxy-Δ12,14-PGJ2; DCFH, 2′,7′-dichlorofluorescein diacetate; DMNQ, 2,3-dimethoxy-1,4-naphtoquinone; ERK, extracellular-regulated kinase; HE, hydroethidine; IKK, IκB kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NOS-2, NO synthase-2; PPARγ, peroxisomal proliferator-activated receptor γ; PI, propidium iodide; ΔΨm, mitochondrial transmembrane potential; DEVD.CHO, aldehyde; GSNO, S-nitrosogluthathione.

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