Induction of Heme Oxygenase-1 Expression in Murine Macrophages Is Essential for the Anti-inflammatory Effect of Low Dose 15-Deoxy-{Delta}12,14-prostaglandin J2*

Tzong-Shyuan Lee, Hui-Ling Tsai and Lee-Young Chau {ddagger}

From the Division of Cardiovascular Research, Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, Republic of China

Received for publication, January 16, 2003 , and in revised form, March 14, 2003.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
15-Deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2), a cyclopentenone prostaglandin, displays a potent anti-inflammatory effect at micromolar concentrations (>2 µM) through direct inhibition of nuclear factor (NF)-{kappa}B activation. Here we show that at submicromolar concentrations (0.1–0.5 µM) 15d-PGJ2 retains the ability to suppress the production of tumor necrosis factor-{alpha} (TNF-{alpha}) and nitric oxide (NO) in lipopolysaccharide (LPS)-activated murine J774 macrophages under the conditions of a prolonged incubation (>12 h). Western blot analysis revealed that the expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1), was induced and coincident with the anti-inflammatory action of 15d-PGJ2. Inhibition of HO-1 activity or scavenging carbon monoxide (CO), a byproduct derived from heme degradation, significantly attenuated the suppressive activity of 15d-PGJ2. Furthermore, LPS-induced NF-{kappa}B activation assessed by the inhibitory protein of NF-{kappa}B(I{kappa}B) degradation and p50 nuclear translocation was diminished in cells subjected to prolonged treatment with the low concentration of 15d-PGJ2. Treatment of cells with the protein synthesis inhibitor, cycloheximide, or the specific p38 MAP kinase inhibitor, SB203580, blocked the induction of HO-1 and suppression of LPS-induced I{kappa}B degradation mediated by 15d-PGJ2. Likewise, HO inhibitor and CO scavenger were effective in abolishing the inhibitory effects of 15d-PGJ2 on NF-{kappa}B activation induced by LPS. The functional role of CO was further demonstrated by the use of a CO releasing molecule, tricarbonyldichlororuthenium(II) dimer, which significantly suppressed LPS-induced nuclear translocation of p50 as assessed by confocal immunofluorescence. Collectively, these data suggest that even at submicromolar concentrations 15d-PGJ2 can exert an anti-inflammatory effect in macrophages through a mechanism that involves the action of HO/CO.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
15-Deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2),1 a dehydration product of prostaglandin D2, has been shown to be present in the inflammatory exudates and is increased during the resolution phase of inflammation (1). The biological effects of 15d-PGJ2 have attracted considerable interest in recent years. It was initially identified as a high affinity natural ligand for the peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) and shown to exert several effects through binding to this nuclear receptor (2, 3, 4, 5). More recently, several recent studies have shown that 15d-PGJ2 exhibits a potent anti-inflammatory effect by attenuating the expression of proinflammatory mediators in activated monocytes/macrophages mainly through the inhibition of NF-{kappa}B-dependent transcription of inflammatory genes (6, 7). In addition to antagonizing the NF-{kappa}B activity through PPAR{gamma}-dependent mechanism (6, 7, 8), 15d-PGJ2 can directly inhibit the signaling steps leading to NF-{kappa}B activation (9, 10, 11, 12). It has also been shown that the {alpha},{beta}-unsaturated carbonyl group of 15d-PGJ2 can act as an electrophile to react covalently with specific cysteine residues located in the activation loop of I{kappa}B kinase-{beta} and the DNA binding domains of NF-{kappa}B subunits, in combination leading to the suppression of NF-{kappa}B-mediated transactivation (9, 10, 11, 12). By studying PPAR{gamma}-deficient embryonic stem cells, Chawla et al. (13) demonstrated that PPAR{gamma} is required for the effect of 15d-PGJ2 on the modulation of macrophage lipid metabolism but not for its inhibitory effect on the production of inflammatory cytokines, suggesting that the PPAR{gamma}-independent mechanism may account for the major anti-inflammatory action of 15d-PGJ2.

Heme oxygenase-1 (HO-1) is a stress-inducible enzyme, catalyzing the degradation of heme to liberate free iron, carbon monoxide (CO), and biliverbin in mammalian cells (14, 15). Over the past few years, numerous studies have revealed the important function of HO-1 as a cytoprotective defense mechanism against oxidative insults through the antioxidant activities of biliverdin and its metabolite, bilirubin, as well as the anti-inflammatory action of CO (16). It has been shown that 15d-PGJ2 induces in vitro HO-1 expression in various cell types (17, 18, 19). Furthermore, the repression of inducible nitric-oxide synthase (iNOS) expression by 15d-PGJ2 in macrophages is correlated with the induction of HO-1 (17). Nevertheless, to date it is not clear if HO-1 expression is functionally related to the anti-inflammatory function of 15d-PGJ2.

In view of its potent anti-inflammatory activity, 15d-PGJ2 has been suggested to play a feedback regulatory role in the resolution phase of inflammation and is considered as a potential therapeutic target for treating inflammatory diseases (20). However, some recent studies have revealed that 15d-PGJ2 at concentrations (>2 µM) required to elicit direct inhibition of NF-{kappa}B activation would induce the expression of proinflammatory cytokines, such as interleukin-8, and potentiate the inflammatory response (21, 22, 23). To resolve this apparent contradiction, it is important to explore the possibility that 15d-PGJ2 at lower concentrations can exert anti-inflammatory function through alternate mechanisms. In the present study, we assessed the differential effects of 15d-PGJ2 at 5 µM and at a submicromolar concentration (0.5 µM) on the production of inflammatory mediators as well as the NF-{kappa}B activation in lipopolysaccharide (LPS)-activated J774 macrophages. We found that 0.5 µM 15d-PGJ2 was capable of preventing the production of tumor necrosis factor-{alpha} (TNF-{alpha}) and nitric oxide (NO) in LPS-activated cells incubated in culture for >12 h. Furthermore, LPS-induced NF-{kappa}B activation was significantly suppressed by the prolonged treatment with this low concentration of 15d-PGJ2. The delayed anti-inflammatory effect was paralleled by the induction of heme oxygenase-1 (HO-1). Additional experiments provide convincing evidence to support the involvement of HO-1 in the anti-inflammatory function of 15d-PGJ2 in macrophages at submicromolar concentrations.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—LPS from Escherichia coli (serotype 055:B5) was from Difco. 15d-PGJ2 was obtained from Cayman and Biomol. Zinc protoporphyrin IX (ZnPP), cobalt protoporphyrin IX (CoPP), and copper protoporphyrin IX (CuPP) were from Porphyrin Products Inc. SB203580 and PD98059 were from Calbiochem. Ruthenium chloride and tricarbonyldichlororuthenium(II) dimer were obtained from Sigma. Rabbit polyclonal HO-1 antibody was from StressGen. Rabbit polyclonal iNOS antibody, rabbit polyclonal I{kappa}B{alpha} antibody, and goat polyclonal p50 antibody were purchased from Santa Cruz.

Cell Culture—Murine J774 macrophages (ATCC, TIB-67) were sub-cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (HyClone), 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells at 60% confluence were changed to serum-free medium containing 0.1% bovine serum albumin and subjected to various treatments.

Determination of TNF-{alpha} and Nitrite Concentrations—The concentration of TNF-{alpha} in culture medium was determined using an EIA kit (Assay Designs). Accumulated nitrite, a stable breakdown product of NO, in culture medium was determined using the Griess reagent. Briefly, an aliquot of cell culture medium was mixed with an equal volume of Griess reagent and then incubated at room temperature for 15 min. The azo dye production was determined by the absorbance at 540 nm. Sodium nitrite was used as a standard.

Western Blot Analysis—Cells were rinsed once with ice-cold phosphate-buffered saline (PBS) and lysed with PBS containing 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 1 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride on ice for 10 min. After sonication for 30 s using microprobe sonicator, crude extracts were subjected to centrifugation at 5000 x g for 5 min at 4 °C. The supernatants (cell lysates) were collected and protein concentrations were determined by Bio-Rad protein assay. To prepare nuclear extracts, cells were lyzed in 10 mM Hepes, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, and 0.5% Nonidet P-40, 1 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride on ice for 5 min. Nuclei were pelleted at 13,000 x g for 5 min, resuspended in 20 mM Hepes, pH 7.9, 400 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride and incubated on ice for 10 min. After centrifugation at 13,000 x g for 5 min, the supernatants (nuclear extracts) were collected and stored at -70 °C until use. Fifty µg of cell lysates or nuclear extracts was electrophoresed on 8 or 12% SDS-polyacrylamide gel and then transblotted onto the ImmobilonTM-P membrane (Millipore, Bedford, MA). The Western blots were carried out as described previously (24).

Confocal Immunofluorescence—Cells grown on cover slips and subjected to various treatments were subsequently washed with PBS and fixed with 4% paraformaldehyde at room temperature for 30 min. Cells were washed three times with ice-cold PBS and permeabilized with 70% alcohol for 2 h at 4 °C. Nonspecific binding of the fixed cells was blocked in PBS containing 2% bovine serum albumin at 37 °C for 30 min, followed by incubation with goat anti-p50 antibodies (1:50) at 37 °C for 1 h. After three washes with PBS, cells were incubated with anti-goat biotin-conjugated antibody (1:100) at 37 °C for another 1 h. After washing, cells incubated with FITC-conjugated streptoavidin (1:100) at 4 °C overnight. After washing with PBS, the coverslips were mounted on slide with 80% glycerol, and the fluorescence was visualized using a laser confocal microscope (Bio-Rad MRC-1000) coupled with an image analysis system.

Statistical Analysis—Each experiment was duplicated at least three-times. Results were expressed as mean ± S.D. Data were analyzed by Student's t test with p < 0.05 considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Differential Effects of 15d-PGJ2 at High and Low Concentrations on LPS-induced TNF-{alpha} Production—To explore the possible differential effects of 15d-PGJ2 on the production of inflammatory cytokines induced by LPS in macrophages at high and low micromolar concentrations, we treated J774 cells with 15d-PGJ2 at 0.5 and 5 µM, respectively, at various time intervals relative to the addition of LPS in culture. As shown in Fig. 1A, 5 µM 15d-PGJ2 added prior to or concurrently with LPS completely blocked TNF-{alpha} production induced by LPS. The inhibitory effect, however, was gradually lost when it was added after LPS treatment. In experiments with 0.5 µM 15d-PGJ2, the inhibition on LPS-induced TNF-{alpha} production was not observed until cells were incubated with 15d-PGJ2 for at least 12 h prior to LPS addition. Western blot analysis demonstrated that the expression of HO-1 was evident at 3 and 12 h after treatment with 5 and 0.5 µM of 15d-PGJ2, respectively. 15d-PGJ2 at 5 µM was effective in suppressing LPS-induced TNF-{alpha} production even at the time period that HO-1 expression was not detectable. In contrast, when cells treated with 0.5 µM 15d-PGJ2, the suppression on TNF-{alpha} production was not observed until the time at which HO-1 was induced. At submicromolar concentrations, the inhibitory effect of 15-PGJ2 on LPS-induced TNF-{alpha} production was closely correlated with HO-1 induction in a dose-dependent manner (Fig. 1C).



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FIG. 1.
Effects of 15d-PGJ2 at high and low micromolar concentrations on TNF-{alpha} production in LPS-activated J774 cells. 15d-PGJ2 at 5 µM (A) or 0.5 µM (B) was added before (18–0.5 h), at the same time (0 h), or after (-0.5 and -1 h) the addition of 1 µg/ml of LPS in culture. C, cells were incubated with indicated concentrations of 15d-PGJ2 (0.1–0.5 µM) in culture for 18 h prior to LPS stimulation. The TNF-{alpha} levels in culture medium were determined at 2 h after LPS treatment. The cell lysates were prepared and the expression levels of HO-1 and {beta}-actin were examined by Western blot analysis. *, p < 0.005 compared with cells treated with LPS alone.

 

HO-1 Mediates the Suppressive Effect of a Submicromolar Concentration of 15d-PGJ2 on the Production of TNF-{alpha} and NO in LPS-activated Macrophages—To assess the potential role of HO-1 on the inhibition of LPS-induced TNF-{alpha} production in cells after prolonged treatment with a low concentration of 15d-PGJ2, J774 cells were pretreated with 0.5 µM 15d-PGJ2 for 14 h, followed by addition of a HO competitive inhibitor, ZnPP, or an inactive compound, CuPP, for 4 h prior to LPS challenge. As shown in Fig. 2A, ZnPP treatment significantly reversed the inhibitory effect of 15d-PGJ2 on TNF-{alpha} production, whereas CuPP showed no effect, suggesting HO-1 mediates the suppressive effect of 15d-PGJ2. As CO derived from heme degradation by HO-1 has been shown to exert potent anti-inflammatory effect (25), additional experiments were carried out to examine the effect of hemoglobin, a scavenger for CO, on TNF-{alpha} production under the same experimental setting. The results clearly show that hemoglobin effectively reversed the inhibition on LPS-induced TNF-{alpha} production by prolonged treatment with 0.5 µM 15d-PGJ2 in a dose-dependent manner (Fig. 2B). Similar experiments were performed to examine whether the production of NO, another important inflammatory mediator induced by LPS in macrophages (26), might also be affected by the treatment with low concentration of 15d-PGJ2. As shown in Fig. 3A, prolonged treatment of J774 cells with submicromolar concentrations of 15-PGJ2 dose-dependently resulted in reduced iNOS expression and NO production following LPS challenge. Prolonged treatment with 0.5 µM 15d-PGJ2 completely blocked the LPS-induced expression of iNOS and NO production, whereas cotreatment with ZnPP or hemoglobin again significantly attenuated the inhibitory effect of 15d-PGJ2 (Fig. 3, B and C)



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FIG. 2.
HO-1 mediates the inhibition of LPS-induced TNF-{alpha} production in J774 cells following prolonged treatment with 0.5 µM 15d-PGJ2. A and B, cells were incubated without or with 0.5 µM 15d-PGJ2 for 14 h, followed by treatment without or with ZnPP (1 µM), CuPP (1 µM), or hemoglobin (Hb) (2.5–20 µM) for 4 h prior to LPS (1 µg/ml) challenge for 2 h. The TNF-{alpha} levels in culture medium were determined by enzyme-linked immunosorbent assay. *, p < 0.005 compared with cells treated with 15d-PGJ2/LPS.

 


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FIG. 3.
HO-1 mediates the suppression of LPS-induced iNOS expression and NO production in J774 cells following prolonged treatment with submicromolar concentrations of 15d-PGJ2. A, cells were incubated without (control) or with 1 µg/ml LPS in the absence or presence of indicated concentrations of 15-PGJ2 (0.1–0.5 µM) in culture for 18 h. *, p < 0.25 compared with cells treated with LPS alone. B and C, cells were incubated without or with 0.5 µM 15d-PGJ2 and 1 µg/ml LPS for 12 h, followed by treatment without or with ZnPP (1 µM), CuPP (1 µM), or hemoglobin (Hb) (2.5–20 µM) for additional 6 h. Cell lysates were prepared, and the expression levels of iNOS and {beta}-actin were examined by Western blot analysis. The nitrites accumulation in culture medium was determined with Griess reagent. *, p < 0.005 compared with control cells treated with 15d-PGJ2/LPS.

 

HO-1 Mediates the Inhibitory Effect of Low Submicromolar Concentration of 15d-PGJ2 on LPS-induced NF-{kappa}B Activation—Earlier studies have shown that 15d-PGJ2 at high micromolar concentrations inhibits NF-{kappa}B activation through direct alkylation of specific cysteine residue of I{kappa}B kinase, which in turn leads to reduced phosphorylation and degradation of I{kappa}B{alpha}, a key step in NF-{kappa}B activation (27). As shown in Fig. 4, pretreatment of cells with 5 µM 15d-PGJ2 for 30 min significantly suppressed the degradation of I{kappa}B{alpha} as well as the increase of nuclear level of NF-{kappa}B p50 subunit following LPS stimulation. Under these conditions, HO-1 was not yet detectably induced and ZnPP treatment did not influence the effect of 15d-PGJ2, indicating that HO-1 was not involved. To determine whether prolonged treatment of cells with 0.5 µM 15-PGJ2 also affects the NF-{kappa}B activation, we studied I{kappa}B{alpha} degradation as well as level of nuclear p50 in cells pretreated with 0.5 µM of 15d-PGJ2 for 18 h followed by LPS challenge. As shown in Fig. 5A, both I{kappa}B{alpha} degradation and the increase of nuclear p50 induced by LPS were substantially reduced in cells pretreated with 0.5 µM 15d-PGJ2. Furthermore, ZnPP cotreatment significantly reduced the inhibitory effect, suggesting HO-1 mediates the suppression of NF-{kappa}B activation by 15d-PGJ2. To further explore the effect of HO-1 on NF-{kappa}B activation, additional experiments were performed using CoPP, a HO-1 inducer. Similar to the effect of 0.5 µM 15 d-PGJ2, pretreatment of cells with CoPP suppressed NF-{kappa}B activation induced by LPS (Fig. 5B). The suppressive effect of CoPP was inhibited by cotreatment with ZnPP as shown in the same figure. To further confirm the importance of HO-1 induction in the suppression of NF-{kappa}B activation, cells were pretreated with 0.5 µM 15d-PGJ2 for 6 h, followed by cycloheximide, the protein synthesis inhibitor, for 12 h prior to LPS stimulation. As shown in Fig. 6, HO-1 expression induced by 15d-PGJ2 was substantially reduced. Conversely, the LPS-induced I{kappa}B{alpha} degradation was not significantly affected under the same conditions. In a separate experiment, we found that the induction of HO-1 by 15d-PGJ2 was inhibited by SB 203580, a specific inhibitor for p38 MAP kinase (28) (Fig. 7A). Again, SB203580 treatment blocked the inhibitory effect of 0.5 µM 15d-PGJ2 on I{kappa}B{alpha} degradation induced by LPS (Fig. 7B).



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FIG. 4.
Effect of 5 µM 15d-PGJ2 on LPS-induced NF-{kappa}B activation in J774 cells. Cells were pretreated with 5 µM 15d-PGJ2 for 30 min, followed by treatment with 1 µg/ml LPS for 30 min in the absence or presence of 1 µM ZnPP. The cell lysates and nuclear extracts were prepared and the cytoplasmic level of I{kappa}B{alpha} and nuclear level of p50 were determined by Western blot analysis.

 


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FIG. 5.
HO-1 mediates the suppressive effect of 0.5 µM 15d-PGJ2 on LPS-induced NF-{kappa}B activation. Cells were incubated with or without 0.5 µM 15d-PGJ2 (A) or 1 µM CoPP (B) for 14 h, followed by treatment with or without 1 µM ZnPP for 4 h prior to LPS (1 µg/ml) stimulation for 30 min. The levels of nuclear p50 and cytoplasmic I{kappa}B{alpha} and HO-1 were determined by Western blot analysis.

 


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FIG. 6.
Effect of cycloheximide on LPS-induced I{kappa}B{alpha} degradation in cells following prolonged treatment with 0.5 µM 15d-PGJ2. Cells were preincubated with 0.5 µM 15d-PGJ2 for 6 h, followed by treatment with 2 µg/ml of cycloheximide for 12 h prior to LPS (1 µg/ml) stimulation for 30 min. The expression levels of I{kappa}B{alpha}, HO-1, and {beta}-actin were examined by Western blot analysis.

 


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FIG. 7.
Effect of p38 MAP kinase inhibitor on HO-1 expression and LPS-induced I{kappa}B{alpha} degradation in cells following prolonged treatment with 0.5 µM 15d-PGJ2. A, cells were pretreated with indicated concentrations of SB203580 or PD98059 for 1 h, followed by treatment with 0.5 µM 15d-PGJ2 for 18 h. B, cells were pretreated with 10 µM SB203580, followed by treatment with 0.5 µM 15d-PGJ2 for 18 h prior to LPS (1 µg/ml) stimulation for 30 min. Expression levels of HO-1 and I{kappa}B{alpha} were assessed by Western blot analysis.

 

CO Mediates the Inhibition on NF-{kappa}B Activation—To determine whether CO mediates the effect of HO-1 on NF-{kappa}B activation, the influence of hemoglobin was examined. As illustrated in Fig. 8, A and B, hemoglobin treatment significantly reversed the inhibition of LPS-induced I{kappa}B{alpha} degradation as well as increase of nuclear p50 by 15-PGJ2 or CoPP. The involvement of HO/CO in mediating the inhibition of NF-{kappa}B activation was also revealed by p50 confocal immunofluoresence. The 15d-PGJ2-mediated suppression on LPS-induced p50 nuclear translocation was substantially attenuated by ZnPP or hemoglobin treatment (Fig. 9). Furthermore, pretreatment of cells with tricarbonyldichlororuthenium(II) dimer, a CO releasing compound (29), for 30 min exhibited similar suppressive effects on LPS-induced p50 nuclear translocation (Fig. 10).



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FIG. 8.
CO mediates the suppressive effect of 0.5 µM 15d-PGJ2 on LPS-induced NF-{kappa}B activation. Cells were preincubated with or without 0.5 µM 15d-PGJ2 (A) or 1 µM CoPP (B) for 14 h, followed by treatment with or without 10 µM hemoglobin (Hb) for 4 h prior to LPS (1 µg/ml) stimulation for 30 min. Cell lysates and nuclear extracts were prepared, and the levels of nuclear p50, cytoplasmic I{kappa}B{alpha}, and HO-1 were determined by Western blot analysis.

 


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FIG. 9.
Inhibition of LPS-induced nuclear localization of p50 by prolonged treatment with 0.5 µM 15d-PGJ2. Cells were preincubated with or without 0.5 µM 15d-PGJ2 for 12 h, followed by treatment with or without 1 µM ZnPP for 6 h prior to LPS (1 µg/ml) stimulation for 30 min. Cells were then fixed, and the immunofluorescence staining with antibody against p50 was performed and visualized with confocal microscope.

 


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FIG. 10.
Inhibition of LPS-induced nuclear localization of p50 by prolonged treatment with CO releasing compound. Cells were pretreated without or with 100 µM [Ru(CO)3Cl2]2 or RuCl3 for 30 min prior to LPS (1 µg/ml) stimulation for 30 min. Cells were then fixed and the immunofluorescence staining with antibody against p50 was performed and visualized with confocal microscope.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Within the past few years, considerable evidence has been produced that supports the important role of HO-1 in the cytoprotective defense response against oxidative insults both in vitro and in vivo (16). In addition to its pivotal role in regulating iron homeostasis, HO-1 has been shown to display potent anti-oxidant and anti-inflammatory functions through the actions of end products derived from heme degradation, bilirubin and CO, respectively. Most recently, a study from our laboratory has demonstrated that HO-1 is induced by interleukin-10 and mediates the anti-inflammatory effect of this cytokine (30). A study by Scapagnini et al. (31) has also shown that two polyphenolic natural products with anti-inflammatory properties, curcumin and caffeic acid phenyl ester, are HO-1 inducers, although the relevance of HO-1 expression to their anti-inflammatory actions remains to be determined. 15d-PGJ2, an anti-inflammatory prostaglandin produced at the sites of inflammation, is also capable of up-regulating the expression of HO-1 (17, 18, 19). Although earlier studies had shown that 15d-PGJ2 exhibits potent anti-inflammatory effect by directly inhibiting I{kappa}B kinase and NF-{kappa}B through covalent modification (9, 10, 11, 12), the concentrations required are much higher than those detected in the inflammatory fluids (1). Furthermore, at such high concentrations, 15d-PGJ2 can induce the expression of proinflammatory cytokines (21, 22, 23), challenging the notion that 15d-PGJ2 has a feedback inhibitory function in the resolution of inflammation. Nevertheless, several recent studies using animal disease models have demonstrated in vivo the beneficial effect of 15d-PGJ2 (19, 32, 33). As much interest has been drawn on its therapeutic potentials, we are interested in elucidating the possibility whether 15d-PGJ2 can exert anti-inflammatory effect through other mechanism(s). In the present study, we show that 15d-PGJ2 at concentration of 5 µM elicited a profound inhibition on TNF-{alpha} production even when it was added at 30 min after LPS challenge in J774 cells. In contrast, the suppressive effect of 0.5 µM 15d-PGJ2 was observed only after a prolonged incubation period (12–18 h) prior to LPS treatment, indicating that differential mechanisms mediate the anti-inflammatory effects of 15d-PGJ2 at high and low concentrations. Since the time course experiment indicated a close correlation between HO-1 expression and the suppression on LPS-induced TNF-{alpha} production in cells treated with 0.5 µM 15d-PGJ2, it provoked our interest to examine the potential role of HO-1 in the anti-inflammatory effect of 15d-PGJ2 at the low submicromolar concentrations. In addition to the inhibitory effect on TNF-{alpha} production, prolonged treatment of cells with 0.5 µM 15d-PGJ2 also resulted in the reduced iNOS expression and NO production following LPS stimulation. To investigate whether the NF-{kappa}B activation, a key signaling pathway leading to iNOS gene expression (34), is an upstream target for the suppressive effect of low concentration of 15d-PGJ2, we further examined the extent of I{kappa}B{alpha} degradation and the nuclear abundance of NF-{kappa}B subunit, p50, in these cells. Our results clearly demonstrate that both I{kappa}B{alpha} degradation and nuclear translocation of p50 induced by LPS are significantly attenuated in cells after prolonged treatment with 0.5 µM 15d-PGJ2. Similar to the suppressive effects on TNF-{alpha} and NO production, the inhibitory effect on NF-{kappa}B activation was also diminished by HO/CO inhibitors. Furthermore, blockade of HO-1 induction by the protein synthesis inhibitor, cycloheximde, or the p38 MAP kinase inhibitor, SB203580, completely abolished the suppressive effect of the low concentration 15d-PGJ2 on NF-{kappa}B activation in J774 cells. Together, these data strongly support the assertion that HO-1 in macrophages mediates the anti-inflammatory effect of 15d-PGJ2 at least at low submicromolar concentrations. Consistent with our observations, a recent study on a rat model of acute myocardial infarction also supports the role of HO-1 in the cardioprotective effect of 15d-PGJ2 (19).

The finding that CO mediates the inhibition on NF-{kappa}B activation is novel. It has been shown that CO exerts the anti-inflammatory effect through modulating the activation of p38 MAP kinase, although the detailed mechanism is not yet clear. In the present study, we demonstrate that the CO releasing compound, tricarbonyldichlororuthenium(II) dimer, effectively suppresses the nuclear translocation of the p50 as revealed by immunofluorescence staining. During the preparation of this manuscript, a work published by Sarady et al. (35) also demonstrated that CO suppresses LPS-induced expression of granulocyte macrophage colony-stimulating factor through inhibiting NF-{kappa}B activation. As the molecules involved in the signaling pathway leading to NF-{kappa}B activation do not contain heme moiety, the identity of the direct target for CO action remains to be determined. Very recently, a report by Brouard et al. (36) showed that HO/CO did not induce NF-{kappa}B activation, but the ability of CO to protect endothelial cells from TNF-{alpha}-induced apoptosis required the expression of NF-{kappa}B-dependent anti-apoptotic genes. The potential effect of CO-mediated suppression on NF-{kappa}B activation in their experimental settings is not clear. Nevertheless, they found that quiescent endothelial cells have basal nuclear NF-{kappa}B activity and express basal levels of NF-{kappa}B-dependent anti-apoptotic genes. It is speculated that CO did not affect the basal NF-{kappa}B activity required to support the protective effect of CO. Furthermore, p38 MAP kinase activation has also been implicated in the anti-apoptotic effect of CO (36). It is envisioned that the protective function of CO in TNF-{alpha}-treated endothelial cells is influenced by the interaction between NF-{kappa}B and p38 MAP kinase signaling pathways. In any event, the diverse effects of endogenous CO derived from heme degradation on cellular signaling pathways in various cell types warrant further investigation. In summary, here we provide convincing evidence to support the essential role of HO-1 in the anti-inflammatory function of 15d-PGJ2 at low submicromolar concentrations. NF-{kappa}B appears to be one of the important targets for the action of HO/CO, although further studies are required to elucidate the underlying mechanisms.


    FOOTNOTES
 
* This work was supported by grants from the National Science Council of Taiwan (NSC-91-2320-B-001-028) and the Institute of Biomedical Sciences, Academia Sinica, Taiwan, Republic of China. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Division of Cardiovascular Research, Inst. of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, ROC. Tel.: 886-2-2652-3931; Fax: 886-2-2785-8847; E-mail: lyc{at}ibms.sinica.edu.tw.

1 The abbreviations used are: 15-PGJ2, 15-deoxy-{Delta}12,14-prostaglandin J2; HO-1, heme oxygenase-1; LPS, lipopolysaccharide; TNF-{alpha}, tumor necrosis factor-{alpha}; iNOS, inducible nitric-oxide synthase; NO, nitric oxide; CO, carbon monoxide; PPAR{gamma}, peroxisome proliferator-activated receptor-{gamma}; NF-{kappa}B, nuclear factor {kappa}B; I{kappa}B, inhibitory protein of NF-{kappa}B; ZnPP, zinc protoporphyrin IX; CoPP, cobalt protoporphyrin IX; CuPP, copper protoporphyrin IX; PBS, phosphate-buffered saline; MAP, mitogen-activated protein. Back


    ACKNOWLEDGMENTS
 
We thank Dr. C Weaver for editorial assistance and Dr. F. Bach for comments.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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