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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 739-745, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.4816

Carbon Monoxide Modulates Endotoxin-Induced Production of Granulocyte Macrophage Colony-Stimulating Factor in Macrophages

Judit K. Sarady, Sherrie L. Otterbein, Fang Liu, Leo E. Otterbein and Augustine M. K. Choi

Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Address correspondence to: Leo E. Otterbein, Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh School of Medicine, 3459 5th Avenue, MUH628, Pittsburgh, PA 15213. E-mail: otterbeinl{at}msx.upmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The stress-inducible gene heme oxygenase-1 (HO-1) provides protection against oxidative stress. Although the mechanisms by which HO-1 exerts its cytoprotection are not clearly understood, it has been speculated that carbon monoxide (CO), a catalytic byproduct following heme catabolism by HO-1, may mediate cellular cytoprotection via its anti-inflammatory properties. Granulocyte macrophage colony-stimulating factor (GM-CSF) is a potent cytokine generated in response to bacterial endotoxin (lipopolysaccharide [LPS]) to stimulate proliferation, maturation, and effector functions of leukocytes, contributing to the proinflammatory responses to LPS. We hypothesized that HO-1 and/or CO could regulate the expression and production of GM-CSF. HO-1 overexpression, as well as exposure to a low concentration of CO, inhibited LPS-induced GM-CSF production in macrophages. Furthermore, CO inhibited LPS-induced GM-CSF induction via inhibition in the activation of the transcription factor NF-{kappa}B. CO inhibited LPS-induced activation of NF-{kappa}B, which has been shown to regulate GM-CSF transcription, by preventing the phosphorylation and degradation of the regulatory subunit I{kappa}B-{alpha}. These data raise the intriguing possibility that CO at low concentrations may play an important role in inflammatory disease states and thus has potential therapeutic implications.

Abbreviations: bronchoalveolar lavage fluid, BALF • carbon monoxide, CO • enzyme-linked immunosorbent assay, ELISA • electrophoretic mobility shift assay, EMSA • granulocyte macrophage–colony-stimulating factor, GM-CSF • heme oxygenase-1, HO-1 • lipopolysaccharide, LPS • mitogen-activated protein, MAP • nuclear factor-{kappa}B, NF-{kappa}B • parts per million, ppm • sodium dodecyl sulfate, SDS


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Heme oxygenase (HO) is the first and rate-limiting enzyme in the oxidative degradation of heme (1). It cleaves the {alpha}-mesocarbon bridge of the heme molecule to yield equimolar quantities of biliverdin IXa, carbon monoxide (CO), and free iron. Biliverdin is subsequently converted to bilirubin via the action of biliverdin reductase, whereas the iron released is sequestered into ferritin (1). Currently, three isoforms of HO have been identified. HO-2 (2) and HO-3 (3) are constitutively synthesized primarily in the brain and testes, whereas the expression of HO-1, the inducible isoform, is increased in response to numerous stimuli, including but not limited to heme, hyperoxia, hypoxia, endotoxin, and heavy metals (4). Under physiologic conditions HO activity is highest in the spleen, where erythrocytes are sequestered and metabolized, but its activity has been observed in all systemic organs (1). HO-1 induction has been shown to play a role in cellular and tissue defense against oxidative stress possessing potent anti-inflammatory properties (1, 2, 5, 6). Its activity increases as the inflammatory state progresses, with the highest expression occurring during resolution of the inflammation (7). Recently, Otterbein and coworkers demonstrated that one of the byproducts of heme catabolism, CO, exhibits potent anti-inflammatory effects otherwise observed with HO-1 in models of endotoxin shock (8), acute lung injury in rats (9), xenotransplantation (10, 11), and most recently a murine model of aeroallergen-induced asthma (12).

In chronic inflammatory diseases or in sepsis there is an excessive stimulation of immune cells, combined with a vast production of pro- and anti-inflammatory cytokines. The outcome of the disease depends on the balance of these cells, the mediators they generate, and ultimately the endogenous response. Lipopolysaccharide (LPS), a constituent of the gram-negative bacterial cell wall, can induce many of the pathophysiologic changes observed in sepsis following administration to cells or animals (13, 14). LPS binds to the CD14 and toll-like receptor 2 or 4 at the cell surface, resulting in the activation of a diverse range of intracellular signaling cascades ultimately leading to the transcription of inflammatory gene expression (14). One such signaling pathway that has been well described following LPS administration is the nuclear factor (NF)-{kappa}B transcription factor that has been shown to regulate the expression of numerous stress response genes, including cytokines (15–17). One cytokine that is potently induced during sepsis and endotoxic shock is granulocyte macrophage–colony-stimulating factor (GM-CSF).

GM-CSF is a 23-kD glycoprotein, initially identified by its ability to promote in vitro proliferation and differentiation of hematopoietic progenitors into neutrophils and macrophages, as well as to modulate the generation of eosinophils and erythrocytes. It can also regulate the function of mature hematopoietic cells enhancing antigen presentation, increased complement- and antigen-mediated phagocytosis, and augmentation of antitumor immunity (18). This cytokine is secreted constitutively in humans in the low pg/ml range, but its level is increased following bacterial infection or administration of lipopolysaccharide. The concentration of GM-CSF in bronchoalveolar lavage fluid (BALF) can be increased in some inflammatory lung conditions such as sarcoidosis, asthma, chronic obstructive pulmonary disease, and interstitial pneumonitis (19). GM-CSF is produced by many cells, including fibroblasts, endothelial cells, macrophages, and T cells. Although its principal function may be to increase macrophage proliferation, it can also enhance secretion of other inflammatory mediators, such as superoxide anion, prostaglandin E, arachidonic acid, interleukin-1, plasminogen activator, interferon-{gamma}, tumor necrosis factor-{alpha}, and other colony-stimulating factors (CSF) (20).

Although the functions of GM-CSF in humans are not clearly understood, there are several animal studies and reported human cases which suggest that defects in GM-CSF gene expression or a defect in the GM-CSF receptor ß-subunit can result in pulmonary alveolar proteinosis (PAP) (21, 22). PAP is a rare but potentially deadly disease characterized by an accumulation of surfactant in the alveolar air spaces, which ultimately results in impaired gas exchange. Expression of GM-CSF in the lung corrected the catabolic defect in alveolar macrophages, but not in peritoneal macrophages. This suggests that GM-CSF is acting as a differentiation or maturation factor rather than as a direct activator of macrophage function, and that local production of GM-CSF is required to correct the metabolic defect (21). Although the pathophysiology of GM-CSF has been well described, relatively little is known regarding the signaling pathways that control its expression in response to endotoxin.

In this present study, we tested our hypothesis that HO-1 and CO modulate expression and production of the proinflammatory cytokine GM-CSF, contributing to the anti-inflammatory effects of HO-1 and CO. HO-1 overexpression in RAW 264.7 macrophages, as well exposure to a low concentration of CO (250 ppm), decreased LPS-induced GM-CSF expression. We show that one of the mechanisms by which HO-1 and/or CO can exert its effects is via attenuation of LPS-induced phosphorylation of I{kappa}B-{alpha}, resulting in inhibition of NF-{kappa}B activation and subsequent upregulation of GM-CSF expression and production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture and Reagents
RAW 264.7 mouse peritoneal macrophages and MHS cells (purchased from the American Tissue Cell Culture, Rockville, MD), Neo RAW 264.7 (transfected with ß-galactosidase gene) or HO-1 overexpressing RAW 264.7 macrophages (transfected using a plasmid with full-length HO-1 cDNA driven by pSFFV-long terminal repeat), were maintained and grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 0.1% gentamycin, and stimulated with 10 µg/ml LPS (Escherischia coli serotype 0127:B8; Sigma Chemical Co., St. Louis, MO). Neo and HO-1 overexpressing RAW 264.7 macrophages were generated as previously reported (8). Briefly, RAW 264.7 cells were plated (1 x 106/10 cm plate) and 16 h later, transfected with 15 µg of pSFFV/neo or pSFFV/HO-1 (pSFFV was kindly provided by Stanley Korsmeyer, Harvard Medical School, Boston, MA). Cells were exposed to the DNA-CaPO4 precipitate for 6 h, shocked by a 1-min treatment with glycerol in phosphate-buffered saline, and cultured for an additional 24 h in complete medium before addition of G418. Transfectants were selected over a 3-wk period in the presence of G418 (up to 80 µg/ml), and individual clones were isolated by limited dilution. The experiments were performed in subconfluent cell culture conditions. SN50 and SN50 M (control peptide for SN50) were obtained from Calbiochem (La Jolla, CA) and used at a concentration of 50 and 100 µg/ml.

CO Exposures
For cell culture experiments, CO at a concentration of 1% (10,000 ppm) in compressed air was mixed with compressed air containing 5% CO2 in a stainless steel mixing cylinder before being delivered into the exposure chamber. Flow into the chamber was at a rate 2 liters/min. The chamber was humidified and maintained at 37°C. A CO analyzer (Interscan Corporation, Chatsworth, CA) was used to measure CO levels permanently in the chamber and there were no fluctuations in the CO concentrations after the chamber had equilibrated.

Measurement of GM-CSF and cGMP
The concentration of GM-CSF released by LPS-stimulated macrophages into the culture supernatant was measured by enzyme-linked immunosorbent assay (ELISA) as described by the manufacturer (R&D Systems, Minneapolis, MN). Cellular levels of cGMP were quantified using a commercially available immunoassay (Biomol, Plymouth Meeting, PA). HASMC were incubated in the presence or absence of CO (250 ppm) and cell lysates were analyzed for cGMP content, as suggested by the vendor.

Adenovirus Administration
The recombinant adenovirus Ad5-I{kappa}B super-repressor and the control Ad5-LacZ (generous gift from Dr. D. A. Brenner, University of North Carolina at Chapel Hill, Chapel Hill, NC), were grown and purified as previously described. For infections, 1,000,000 RAW 264.7 cells were infected with 100 pfu/cell Ad5-I{kappa}B or control viral stock into serum-free media. Culture dishes were rocked continuously for 4 h, after which time the media was supplemented with 10% fetal bovine serum. Cells were then cultured for an additional 24–48 h before they were used for experiments.

Electrophoretic Mobility Gel Shift Assay
Nuclear protein extracts from RAW 264.7 macrophages were prepared using NE-PER nuclear and cytoplasmic extraction reagents (CER; Pierce, Rockford, IL), according to the manufacturer's instructions. Ten micrograms of nuclear proteins were incubated in binding buffer (Promega, Madison, WI) with 3.5 pmol of double-stranded DNA oligonucleotide containing an NF-{kappa}B consensus binding sequence (5'-AGT TGA GGG GAC TTT CCC AGG C-3') which was labeled with {gamma}-32P-ATP using T4 polynucleotide kinase (Promega). Binding reactions were completed by incubating reaction solution for 30 min at room temperature. Protein–DNA complexes were separated from the unbound DNA probe by electrophoresis through 5% native polyacrylamide gels containing 0.5x Tris-Borate-EDTA. The gel was dried and exposed to Biomax MR film (Kodak, Rochester, NY).

Western Blot Analyses
Mouse monoclonal antibodies against phosphorylated I{kappa}B-{alpha} was purchased from Cell Signaling (Beverly, MA) and were used following the manufacturer's instructions. Rabbit polyclonal antibodies were purchased from Pierce. At various times after LPS administration, cells were rinsed with cold PBS followed by addition of 100 µl sodium dodecyl sulfate (SDS) sample buffer containing 62.5 mM Tris HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, and 0.1% bromophenol blue to each plate. Cells were scraped from the plates and sonicated for 15–20 s. Each sample was then boiled for 5 min. Twenty microliters of each sample was separated by 12% PAGE at 125 V for 90 min in Tris-glycine-SDS running buffer. The gel was transferred overnight at 20 V onto nitrocellulose membrane. Membranes were then incubated for 1 h with blocking buffer (5% nonfat dry milk in TTBS [10% Tween in Tris-buffered saline]), washed with TTBS, and then incubated overnight in rabbit polyclonal primary antibody in 1:1,000 dilution against phosphorylated I{kappa}B-{alpha}. After incubation with primary antibody the membranes were washed three times for 5 min in TTBS. Membranes were then incubated and visualized with an appropriate anti-rabbit secondary antibody (1:2,000 dilution) and detected using Enhanced Chemiluminescence Assay (Pierce) according to the manufacturer's instructions.

RNA Extraction and Northern Blot Analysis
Total RNA was isolated using the Trizol Method, with homogenization of the cells in Trizol Lysis buffer followed by chloroform extraction (Life Technologies, Gaithersburg, MD). Ten micrograms of total RNA was separated by 1% agarose gel electrophoresis and then transferred to GeneScreen Plus membrane (NEN, Boston, MA) by capillary action. Ethidium bromide staining of the gel was used to confirm the integrity and equal loading of the RNA. The nylon membrane was then prehybridized at 65°C for 3 h in hybridization buffer (25 ml Church and Gilberts buffer with 125 µL Salmon sperm), followed by incubation at 65°C overnight with hybridization buffer containing 32P-labeled mouse GM-CSF cDNA. The cDNA was labeled with 32P-CTP using the random primer kit from Amersham (Arlington Heights, IL). Nylon membranes were then washed 25 min twice each at 65°C in wash buffer A (0.5% bovine serum albumin, 5% SDS, 40 mM phosphate buffer, pH 7.0, and 1 mM EDTA) followed by washes for 25 min three times each at 65°C in buffer B (1% SDS, 40 mM phosphate buffer, pH 7.0, 1.0 mM EDTA) and exposed to X-ray film.

Statistical Analysis
Data are presented as mean ± SD. Statistical analysis was made by using Student's t test. Results were considered statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of LPS on GM-CSF Production in RAW 264.7 Macrophages
The basal amount of GM-CSF in RAW 264.7 mouse peritoneal macrophages was below detection limits of the assay. LPS administration induced a time- and dose-dependent increase in the production in GM-CSF in RAW 264.7 macrophages assessed by ELISA (Figures 1A and 1B). Initial increase of GM-CSF production occurs at 8 h, and peaks at 16 h of LPS treatment. Later time points showed no further increase in the levels of GM-CSF. This increase of GM-CSF protein production correlated with increased GM-CSF mRNA levels by Northern blot analysis (Figure 1B, inset).



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  		Figure 1. (A) Dose-dependent increase of GM-CSF production after LPS treatment in macrophages. RAW 264.7 macrophages were treated with LPS at the indicated doses for 16 h, and media was collected for determination of GM-CSF levels by ELISA (*P < 0.05 versus control PBS). Data represent the mean value ± SE of samples from three independent experiments. (B) Kinetics of LPS-induced GM-CSF production and expression in macrophages. RAW 264.7 macrophages were treated with LPS (10 µg/ml), and media was collected at the indicated times and analyzed for GM-CSF levels by ELISA (*P < 0.05 versus control). Data represent the mean value ± SE of samples from three independent experiments. Inset: RAW 264.7 macrophages were treated with LPS (10 µg/ml) and total RNA were collected at the indicated times and analyzed for GM-CSF mRNA expression by Northern blot analysis. The blot is representative of three independent experiments.

 
Effect of HO-1 and CO on LPS-Induced GM-CSF Generation in RAW 264.7 Macrophages
To examine whether HO-1 can modulate LPS-induced GM-CSF production, we generated RAW 264.7 macrophages overexpressing HO-1 as described in MATERIALS AND METHODS. We confirmed increased HO-1 mRNA and protein levels in these overexpressing cells by Northern blot and Western blot analyses, respectively, with densitometric analyses showing a 3.8- to 4.9-fold increased expression over neomycin control transfected cells in four separate clones tested (data not shown). Two different clones were tested and showed similar results on LPS-induced GM-CSF production. Clone #4 is presented in Figure 2. The RAW 264.7 macrophages overexpressing HO-1 produced significantly less GM-CSF after LPS treatment versus the neomycin and wild-type control cells (Figure 2). The HO-1 overexpressing cells had a 47% reduction in LPS-induced GM-CSF production. We then exposed wild-type RAW 264.7 macrophages to LPS in the presence or absence of 250 ppm CO and measured the GM-CSF production in the media. Cells exposed to CO (250 ppm) exhibited significant attenuation of LPS-induced GM-CSF expression by Western blot analysis (Figure 3A) and GM-CSF production by ELISA (Figure 3B). We observed similar inhibitory effects of CO on GM-CSF production in another macrophage cell line, MHS cells (Figure 3C).



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  		Figure 2. Effects of overexpression of HO-1 in macrophages on LPS-induced GM-CSF production. Stable transfection, selection, and clonal isolation of RAW 264.7 macrophages were performed as described in MATERIALS AND METHODS and our previous report (8). Neo transfected or HO-1–overexpressing cells were treated with LPS (10 µg/ml) and media was collected at 16 h for GM-CSF determination by ELISA (*P < 0.05 versus neo/LPS). Data represent the mean value ± SE of samples from three independent experiments.

 


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  		Figure 3. (A) CO inhibits LPS-induced GM-CSF protein expression. RAW 264.7 macrophages were pretreated with 250 ppm CO before treatment with LPS (10 µg/ml) and cell lysates were collected at 16 h and analyzed for GM-CSF protein expression by Western blot analysis. Data are representative of three independent experiments. (B) CO inhibits LPS-induced GM-CSF production. RAW 264.7 macrophages were pretreated with 250 ppm CO before treatment with LPS (10 µg/ml) and media was collected at 16 h for GM-CSF protein production by ELISA (*P < 0.05 versus Air/LPS). Data represent the mean value ± SE of samples from three independent experiments. (C) CO inhibits LPS-induced GM-CSF production in MH-S cells. MH-S macrophages were pretreated with 250 ppm CO before treatment with LPS (10 µg/ml) and media was collected at 16 h for GM-CSF protein production by ELISA (*P < 0.05 vs Air/LPS). Data represent the mean value ± SE of samples from three independent experiments.

 
CO Prevented I{kappa}B-{alpha} Phosphorylation following LPS Treatment
RAW 264.7 macrophages were treated with LPS and protein was extracted at different time points and analyzed via immunoblotting for the expression of phosphorylated I{kappa}B-{alpha}. Maximal phosphorylation of the I{kappa}B-{alpha} was observed by 10 min following LPS administration. To evaluate the role of CO on I{kappa}B-{alpha} phosphorylation, we pretreated RAW 264.7 macrophages for 3 h with 250 ppm CO before LPS and then assayed for I{kappa}B-{alpha} activation at the indicated time point. Western blot analysis showed a marked decrease in I{kappa}B-{alpha} phosphorylation (Figure 4A). The bottom panel in Figure 4A depicts levels of total I{kappa}B-{alpha} in RAW 264.7 macrophages after LPS in the presence of CO treatment. We also measured cGMP production in RAW 264.7 cells after CO treatment. We first exposed airway smooth muscle cells, used as positive control, to CO (250 ppm for 8 h) and observed a 5-fold induction of cGMP production (0.1 pmol/mg protein in control cells; 0.5 pmol/mg protein in CO-treated cells). Interestingly, however, the RAW 264.7 cells exposed to CO (250 ppm for 8 h) did not exhibit increase in cGMP levels.



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  		Figure 4. (A) Effect of CO on LPS-induced I{kappa}B-{alpha} expression. RAW 264.7 macrophages were pretreated with 250 ppm CO before treatment with LPS (10 µg/ml) and cell lysates were collected at the indicated times (min) and analyzed for I{kappa}B-{alpha} phosphorylation and total I{kappa}B-{alpha} by Western blot analysis. Data are representative blots from three independent experiments. (B) Effect of CO on LPS-induced NF-{kappa}B activation. RAW 264.7 macrophages were pretreated with 250 ppm CO before treatment with LPS (10 µg/ml) and nuclear extracts were collected at 1 h for NF-{kappa}B activation by EMSA. Data are representative blots from four independent experiments.

 
Effect of CO on LPS-Induced NF-{kappa}B Activation
In order for NF-{kappa}B to be functional, it must dissociate from I{kappa}B, which occurs following phosphorylation of I{kappa}B-{alpha} by IKK (23). Based on our observation above that CO can attenuate I{kappa}B-{alpha} activation, we then hypothesized that CO would attenuate LPS-induced NF-{kappa}B activation. Macrophages were treated with LPS in the presence or absence of 250 ppm CO (pretreated of 3 h) and nuclear extracts were obtained 1 h later and analyzed for NF-{kappa}B binding by electrophoretic mobility shift assay (EMSA). Those cells treated with LPS showed a predicted and reproducible increase in NF-{kappa}B binding, whereas those treated with LPS in the presence of CO showed a marked reduction in NF-{kappa}B binding.(Figure 4B).

Effect of SN 50, a cell-permeable chemical inhibitor of NF-{kappa}B on LPS-induced GM-CSF production. RAW 264.7 macrophages were pretreated for 1 h with SN50 or the inactive analog (SN 50 M) at 50 or 100 µg/ml, which has previously been shown to block NF-{kappa}B activation, and then treated with LPS (10 µg/ml). After 16 h, GM-CSF levels were significantly less in the cells pretreated with the NF-{kappa}B inhibitor versus cells treated with the inactive peptide (Figure 5A). With this observation that chemical inhibition of NF-{kappa}B activation resulted in attenuation of GM-CSF production, we then genetically inhibited NF-{kappa}B activation by using overexpression of I{kappa}B. RAW 264.7 macrophages were infected with Ad5-I{kappa}B super-repressor or Ad5-LacZ control virus and then treated with LPS. After 16 h, the GM-CSF concentration in the cells infected with Ad5-I{kappa}B super-repressor virus were significantly decreased when compared with Ad-Gal–infected cells (Figure 5B).



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  		Figure 5. (A) Effect of SN50 on LPS-induced GM-CSF production. RAW 264.7 macrophages were pretreated with SN50, an inhibitor peptide of NF-{kappa}B, as described in MATERIALS AND METHODS, and then analyzed for GM-CSF production by ELISA 8 h following LPS treatment (10 µg/ml) (*P < 0.05 versus LPS). LPS-treated cells were pretreated with SN50 M, an inactive peptide, as controls for SN 50 treatment (*P < 0.001 versus PBS; #P < 0.05 versus LPS). Data represent the mean ± SE of samples from five independent experiments. (B) Effect of Ad-I{kappa}B on LPS-induced GM-CSF production. RAW 264.7 macrophages were infected with Ad-I{kappa}B super-repressor virus as described in MATERIALS AND METHODS, and then analyzed for GM-CSF production by ELISA 8 h after LPS treatment (10 µg/ml) (*P < 0.001 versus PBS and Ad-I{kappa}B controls; #P < 0.05 versus LPS). Data represent the mean value ± SE of samples from three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Over the past few years growing attention has been given to a well described, but perhaps in some respects mislabeled, gas: CO. Although the toxicity of CO is well established, recent studies demonstrating important anti-inflammatory and cytoprotective effects of CO in maintaining cellular and tissue homeostasis against oxidative stress have renewed the interest of many investigators (8, 10, 24). CO is one of the by-products of heme catabolism by HO-1. Although the induction of HO-1 has been shown by numerous laboratories over the past 10 yr to be cytoprotective, the mechanism by which it does so has remained elusive until recently, when one potential mechanism has been uncovered through experimentation using low concentrations of CO. Otterbein and Brouard (8, 25) have suggested that one possible mechanism by which CO elicits its cytoprotective and anti-inflammatory function is through selective activation of the mitogen-activated protein kinase (MAPK). In the studies presented here, we have uncovered another signaling pathway by which CO mediates its anti-inflammatory effects. We observed that CO can modulate the proinflammatory cytokine GM-CSF via the NF-{kappa}B pathway. In the studies presented here, we show that LPS induces a time- and dose-dependent increase in GM-CSF production in RAW 264.7 macrophages (Figures 1A and 1B). Overexpression of HO-1 in these same macrophages resulted in a remarkable decrease in the expression of GM-CSF in response to LPS treatment versus neomycin-transfected controls (Figure 2). Similarly, wild-type RAW 264.7 cells when treated with LPS in the presence of a low concentration of CO show a decrease in GM-CSF production in the media (Figure 3). In efforts to examine the mechanism by which CO was inhibiting GM-CSF, we chose to examine one signaling pathway by which LPS modulates GM-CSF production. The NF-{kappa}B family of transcription factors controls the expression of many genes, including genes encoding cytokines, cytokine receptors, immunoregulatory molecules, and anti-apoptotic proteins. One such mediator is GM-CSF (31, 32). Because NF- {kappa}B has been shown to directly regulate GM-CSF and becomes activated in response to LPS, we hypothesized that this signaling pathway would be involved in the mechanism by which CO inhibited GM-CSF expression. Using a selective chemical inhibitor (SN50) of NF-{kappa}B we show an inhibition in LPS-induced GM-CSF expression (Figure 5A). CO prevented I{kappa}B phosphorylation measured by Western blot as well as NF-{kappa}B binding as measured by EMSA (Figures 4A and 4B), indicating that the interference of CO on NF-{kappa}B activation was a mechanism by which inhibition of GM-CSF production was suppressed. Consistent with previous studies (26) LPS administration resulted in a rapid phosphorylation of I{kappa}B-{alpha} and subsequent activation of NF-{kappa}B as detected by EMSA. In its inactive state, NF-{kappa}B dimers of the Rel family e.g., C-Rel, p50, and p65, are constitutively present in the cytoplasm bound to members of the I{kappa}B family (I{kappa}B-{alpha}, I{kappa}B-ß, and I{kappa}B-{epsilon}). Activation occurs via phosphorylation of I{kappa}B, by the I{kappa}B kinase (IKK) at serine 32 and 36, resulting in the release and nuclear translocation of NF-{kappa}B (27). I{kappa}B-{alpha} phosphorylation and resulting Rel-dependent transcription are activated by a highly diverse group of extracellular signals, including endotoxin, inflammatory cytokines, growth factors, and LPS (28, 29). Because phosphorylation of I{kappa}B at Ser32 is essential for release of active NF-{kappa}B, phosphorylation at this site is an excellent marker of NF-{kappa}B activation (30). The precise mechanism by which CO inhibits I{kappa}B is not clear at this time. The inability of CO to increase cGMP levels in RAW 264.7 cells in our study perhaps suggest that the cGMP is not involved in this effect.

Although previous work from this laboratory using this cellular model showed the involvement of the MAPKs in regulation of TNF-{alpha} following LPS (8), this study elucidates a second pathway (NF-{kappa}B) by which CO can signal to transduce anti-inflammatory effects. In a recent study (26) it was described that in human monocytes the signaling pathways involved in the generation of GM-CSF are the p38 and ERK MAPK pathways, whereas, unexpectedly, NF-{kappa}B was not involved in the regulation of GM-CSF expression in human peripheral blood monocytes. Other laboratories have clearly demonstrated that NF-{kappa}B does regulate expression of GM-CSF in human epithelial cells (33) and Jurkat cells (34), suggesting that the regulation may be cell type and stimulus specific. It is certainly plausible that CO can mediate its anti-inflammatory effects via both MAPK and NF-{kappa}B pathways, as recent findings have begun to suggest that there is most likely an interaction between these two pathways (35, 36).

In conclusion, our study supports the previous hypothesis that a low concentration of CO suppresses the proinflammatory response in RAW 264.7 macrophages in a model of LPS-induced inflammation. In the near future, perhaps this once-touted toxic gas will assist in the treatment of inflammatory diseases.


   Acknowledgments
 
The authors thank Jawed Alam, who provided the HO-1 cDNA, and Emeka Ifedigbo for technical support. The work by A.M.K.C. was supported in part by the NIH HL60234, and NIH AI42365. L.O. was supported by the AHA Grant in Aid.

Received in original form January 11, 2002

Received in final form July 26, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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