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Circulation Research. 2005;97:305-313
Published online before print July 21, 2005, doi: 10.1161/01.RES.0000177893.24574.13
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(Circulation Research. 2005;97:305.)
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Molecular Medicine

Interleukin-10 Suppresses Tissue Factor Expression in Lipopolysaccharide-Stimulated Macrophages via Inhibition of Egr-1 and a Serum Response Element/MEK-ERK1/2 Pathway

Motohiro Kamimura, Christiane Viedt, Alexander Dalpke, Michael E. Rosenfeld, Nigel Mackman, David M. Cohen, Erwin Blessing, Michael Preusch, Christian M. Weber, Jörg Kreuzer, Hugo A. Katus, Florian Bea

From the Departments of Internal Medicine III (M.K., C.V., E.B., M.P., C.M.W., J.K., H.A.K., F.B.) and Hygiene and Medical Microbiology (A.D.), University of Heidelberg, Germany; the Department of Pathobiology and Interdisciplinary Graduate Program in Nutritional Sciences (M.E.R.), University of Washington, Seattle; The Scripps Research Institute (N.M.), La Jolla, Calif; and the Department of Nephrology and Hypertension (D.M.C.), Oregon Health and Science University and the Portland V.A. Medical Center, Portland, Ore.

Correspondence to Florian Bea, MD, Innere Medizin III, Universität Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany. E-mail florian.bea{at}med.uni-heidelberg.de


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Atherosclerosis is considered to be an inflammatory disease. Tissue factor (TF), a prothrombotic molecule expressed by various cell types within atherosclerotic plaques, is thought to play an essential role in thrombus formation after atherosclerotic plaque rupture. Recent studies suggest that the antiinflammatory cytokine interleukin-10 (IL-10) has many antiatherosclerotic properties. Therefore, the effects of IL-10 on TF expression in response to inflammation were investigated. Mouse macrophages were stimulated with lipopolysaccharide (LPS) in the presence or absence of IL-10. Pretreatment with IL-10 resulted in a 50% decrease in TF mRNA expression and TF promoter activity. Binding of early growth response gene-1 (Egr-1) to the consensus DNA sequence, a key transcriptional activator of TF expression in response to inflammation, and the expression of Egr-1 mRNA were also inhibited by IL-10. This inhibition was independent of the induction of suppressor of cytokine signaling protein-3 by IL-10. Macrophages that had been transfected with luciferase reporter constructs containing the murine Egr-1 5'-flanking sequence exhibited reduced reporter gene activity in response to LPS stimulation with IL-10 pretreatment. Studies with deletion constructs of the Egr-1 promoter identified the proximal serum response element SRE3 as a potential regulatory site for the IL-10 mediated suppression of Egr-1 expression. Furthermore, activation of the upstream signal-transduction elements, such as mitogen-activated protein kinase kinase (MEK) 1/2, extracellular signal-regulated kinase 1/2, and Elk-1 were also inhibited by IL-10 pretreatment. Taken together, these results demonstrate a pathway for the IL-10 mediated inhibition of TF expression during inflammation and may explain the antiatherosclerotic effects of IL-10.


Key Words: atherosclerosis • tissue factor • interleukin • early growth response gene • macrophages


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Atherosclerosis is thought to be an inflammatory disease of the artery wall.1 In particular, inflammatory processes are seminal events during the progression of an early stable atherosclerotic plaque into an advanced unstable plaque prone to rupture.2 After plaque rupture, the procoagulant protein tissue factor (TF) plays a crucial role in initiating blood coagulation and consecutive occlusive thrombus formation in patients with acute coronary syndromes.3 Macrophages are one of the prime sources of TF within the atherosclerotic plaque.4 Recent studies have identified the transcription factor early growth response gene-1 (Egr-1) as an important transcriptional activator for TF expression in response to inflammatory and infectious stimuli in monocytes and macrophages.5,6 Egr-1 is expressed in human atherosclerotic plaques as well as in atherosclerotic lesions of hyperlipidemic mice.7–10 Egr-1 acts as a key regulator of genes such as platelet derived growth factor-A,11 platelet derived growth factor-B,12 transforming growth factor-ß1,13 fibroblast growth factor-2,14 membrane type 1 matrix metalloproteinase,15 and intercellular adhesion molecule-1.16 Egr-1 is activated by proatherosclerotic inflammatory mediators, such as lipopolysaccharide (LPS), CD40, tumor necrosis factor (TNF-{alpha}), and Chlamydia pneumoniae.5,6,17,18 Furthermore, hyperlipidemic mice deficient of Egr-1 have decreased atherosclerosis and vascular inflammation.19

Clinical trials as well as experimental studies have identified interleukin-10 (IL-10) as a potent antiinflammatory cytokine produced by lymphocytes, mast cells, and macrophages. Elevated serum levels of IL-10 are an important favorable prognostic determinant in patients with acute coronary syndromes.20,21 Hyperlipidemic mice overexpressing leukocyte-derived IL-10 have less atherosclerosis,22 whereas hyperlipidemic mice deficient in IL-10 have increased atherosclerosis and thrombosis.23 The observed effects on atherosclerosis may be explained by the IL-10 mediated inhibition of interferon-{gamma} activation,23 metalloproteinases production,24 and TF expression.25 Inhibition of TF expression by IL-10 has been demonstrated in monocytes after stimulation with LPS.26 However, the molecular mechanisms underlying this inhibition remain unclear. Thus, the present study was designed to investigate possible mechanistic pathways for the IL-10 mediated inhibition of TF and Egr-1 expression.


*    Materials and Methods
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 up arrowIntroduction
 *Materials and Methods
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Cell Culture
A murine macrophage cell line, RAW 264.7 cells (ATCC, Manassas, Va), was grown in DMEM (Gibco BRL, Karlsruhe, Germany) supplemented with 10% heat-inactivated FCS, glutamine (10 U/mL), penicillin (10 U/mL)/ streptomycin (10 mg/mL). RAW 264.7 cells stably overexpressing suppressor of cytokine signaling (SOCS)-3 were used as previously described.27 Before the experiments, cells were grown in serum-free medium (0.1% BSA in DMEM) for 24 hours. Mouse peritoneal macrophages (106 cells per animal on average) were obtained through peritoneal lavage with ice-cold PBS from C57BL/6J mice 4 days after intraperitoneal injection of thioglycolate (72 µg/mouse). The C57BL/6J mice were purchased from the Jackson Laboratories (Bar Harbor, Me) and used according to a protocol approved by the University of Heidelberg, Germany. LPS (from Escherichia. coli serotype 0111: B4) and murine IL-10 were obtained from Sigma (Seelze, Germany).

RNA-Isolation and Quantitative RT-PCR
Total RNA was extracted using TRIZOL according to the manufacturer’s protocol (Life Technologies SRL). Specific cDNA was reverse transcribed from 1 µg RNA. Expression levels of TF, Egr-1, SOCS-1, -3, and ß-actin were detected and quantified with LightCycler FastStart DNA Master SYBR Green I and Light Cycler system and with Taqman ABI Prism 7700 Sequence Detection System (PE Biosystems) as previously described.6,19,27

Preparation of Nuclear Extracts
Nuclear protein extracts were prepared according to the method of Schreiber et al28 with minor modifications. Cells were scraped into 400 µL hypotonic buffer (10 mmol/L Hepes, pH 7.9, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 2 mmol/L dithiothreitol (DTT)) supplemented with 5 µg/mL E 64, 1 mmol/L NaF, 0.2 mmol/L Na3VO4, 0.5 mg/mL Pefabloc, incubated for 15 minutes on ice, after which 25 µL of 10% NP-40 was added. The nuclei were recovered by centrifugation (14 000 rpm, 1 minute, 4°C). The nuclear pellets were resuspended in 50 µL ice-cold buffer (20 mmol/L HEPES, 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 2 mmol/L DTT supplemented with 5 µg/mL E64, 1 mmol/L NaF, 0.2 mmol/L Na3VO4, 0.5 mg/mL Pefabloc). Following centrifugation (14 000 rpm, 5 minutes, 4°C), the supernatants containing nuclear proteins were collected and stored at –80°C.

Preparation of Whole Cell Lysates
Cells were lysed with lysis buffer (4°C, 20 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L Na3VO4, 1 µg/mL leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride), scraped, sonicated, and centrifuged (13 000 rpm, 4°C, 10 minutes). Supernatants were either used immediately or stored at –80°C.

Electrophoretic Mobility Shift Assay
Nuclear extracts (10 µg) were incubated with labeled double-stranded oligonucleotide probes. The sequences of the oligonucleotides used in the present study were as follows: consensus Egr-1, 5'-GGATCCAGCGGGGGCGAGCGGGGGCGA-3', mutant Egr-1, 5'-GGATCCAGCTAGGGCCAGCTAGGGCGA -3', SRE3, 5'-AGCACCTTATTTGGAGTGGCCGGATATGGCCCGGCGCTTCC-3', SRE4, 5'-AGGATCCCCCGCCGGAACAACCCTTATTTGGGCAG-3', SRE5, 5'-TGCGACCCGGAAATGCCATATAAGGAGCAGGAAGGATCCCCT-3', NFkB, 5'-AGTTGAGGGGACTTTCCCAGGC-3', AP-1, 5'-CTGGGGTGAGTCATCCCTT-3'. The oligonucleotides were labeled with [{gamma}32P]-ATP by using T4 polynucleotide kinase. Binding reactions were resolved on a 4% native polyacrylamide gel and exposed to x-ray film for 12 to 24 hours. For supershift assays the following antibody was used: Egr-1 (rabbit polyclonal IgG, Santa-Cruz).

Western Blotting
Whole cell lysates (20 µg per lane) or nuclear extracts (10 µg per lane) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany). Membranes were blocked over night at 4°C in 5% nonfat milk powder dissolved in TBST, and then incubated with the primary antibody at room temperature (RT) for 1 hour, followed by incubation with the secondary antibodies (1 hour, RT). The proteins were visualized with ECL (Amersham), recorded on Hyperfilm (Amersham) and quantified by use of a densitometer (Bio-Rad). The following antibodies were used: Egr-1 (rabbit polyclonal IgG, Santa-Cruz, Santa Cruz, Calif), pElk-1 (mouse monoclonal IgG, Cell-Signaling, Beverly, Mass), Elk-1 (rabbit polyclonal IgG, Santa-Cruz), pERK1/2 (rabbit polyclonal IgG, Cell-Signaling), ERK1/2 (rabbit polyclonal IgG, Cell-Signaling), pMEK1/2 (rabbit polyclonal IgG, Cell-Signaling), MEK1/2 (rabbit polyclonal IgG, Santa-Cruz).

Plasmids and Transfections
Luciferase reporter constructs containing the murine Egr-1 promotor and plasmids containing the rat TF promoter have been described elsewhere.6,29,30 RAW cells were transfected as previously described.6 After transfection, cells were incubated in standard growth medium for 24 hours. Cells were stimulated with LPS in the presence or absence of IL-10 pretreatment for 2 hours after transfection with the Egr-1 plasmid and for 12 hours after transfection with the TF plasmid. These time points demonstrated maximum luciferase activity as evaluated through time course experiments (data not shown). In all experiments, cells were cotransfected with pRL-CMV (Promega, Munich, Germany), which expresses Renilla luciferase to compensate for variation in transfection efficiencies (<10%). Cell lysates were assayed using Dual Luciferase Reporter System (Promega) according to the manufacturer’s protocol.

Statistics
Statistical analysis was performed using the unpaired Student t test. Data are presented as mean±SEM and values of P<0.05 were considered statistically significant. All experiments were performed at least 3 times and representative results are shown.


*    Results
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 up arrowMaterials and Methods
 *Results
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Il-10 Pretreatment Inhibits LPS-Induced TF mRNA Expression and Reporter Gene Activity of the TF Promoter
RAW cells were stimulated with LPS for various time points and TF mRNA expression was determined by quantitative RT-PCR. LPS (100 ng/mL) treatment increased TF mRNA expression by 6.5 fold in comparison to untreated cells. Maximum induction was observed after 2 hours (Figure 1A). RAW cells were pretreated with or without IL-10 (50 ng/mL) for 24 hours before stimulation with LPS (100 ng/mL for 2 hours). TF mRNA expression was reduced by more than 50% with IL-10 pretreatment (Figure 1B). To determine whether the reduced TF mRNA expression after IL-10 pretreatment was attributable to reduced de novo transcription, the RAW cells were transiently transfected with a reporter construct of the proximal rat TF promoter containing the serum response region (TF-143). This highly conserved region contains 3 Sp1 and 1 Egr-1 binding site and mediates induction of the TF promoter in response to LPS stimulation.5,30 LPS alone (100 ng/mL) induced a 2.8-fold increase in luciferase reporter activity compared with nonstimulated control cells. IL-10 pretreatment (50 ng/mL for 24 hours) reduced the LPS-stimulated luciferase activity by 50% (Figure 1C).



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  		Figure 1. IL-10 inhibits LPS-induced TF mRNA expression and reporter gene activity of the TF promoter. A, RAW macrophages were stimulated with LPS (100 ng/mL) for the indicated time (hours), and TF mRNA expression was quantified with quantitative RT-PCR as described. B, RAW cells were pretreated with or without IL-10 (50 ng/mL) for 24 hours followed by stimulation with LPS (100 ng/mL) for 2 hours, and mRNA expression of TF was evaluated by RT-PCR. *P<0.05 vs LPS-stimulated cells. C, After transient transfection with plasmids containing the proximal rat TF promoter, RAW cells were pretreated with or without IL-10 (50 ng/mL) for 24 hours and stimulated with LPS (100 ng/mL) for 12 hours. Luciferase activity in cell lysates was determined. *P<0.05 vs LPS-stimulated cells.

IL-10 Pretreatment Inhibits LPS-Induced Egr-1 mRNA and Protein Expression, Nuclear Binding, and Egr-1 Promoter Activity
In RAW 267.4 mouse macrophages, LPS induced maximum Egr-1 mRNA expression 1 hour after treatment (data not shown). Maximum Egr-1 protein expression and DNA binding activity occurred 2 hours after treatment (data not shown). Before treatment with IL-10 (50 ng/mL) inhibited the LPS-induced mRNA expression by 50% and inhibited protein expression and DNA binding activity up to 80% (Figure 2). The use of a mutant oligonucleotide for Egr-1 (Figure 2B, lane 7) and an antibody against Egr-1, (Figure 2B, lane 6), confirmed the specificity of the reaction. Interleukin-10 inhibited LPS-induced Egr-1 binding activity and expression in a dose-dependent manner (Figure 3A and 3B; online Figure I available in the online data supplement at http://circres.ahajournals.org) Furthermore, time course experiments revealed that at least 12 hours of pretreatment with IL-10 were necessary to inhibit Egr-1 binding activity and expression with a maximum observed for 24 hours of pretreatment (Figure 3C and 3D; online Figure II).



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  		Figure 2. LPS-induced mRNA expression, DNA binding activity, and protein expression of Egr-1 is inhibited by IL-10. RAW cells were pretreated with or without IL-10 (50 ng/mL) for 24 hours before treatment with LPS (100 ng/mL). A, Messenger RNA expression of Egr-1 was determined after stimulation for 1 hour using RT-PCR. *P<0.05 vs LPS-stimulated cells. B, EMSA with the consensus sequence for Egr-1 was performed after 2 hours stimulation with LPS as described. Lane 5, addition of 50-fold excess of unlabeled Egr-1 oligonucleotide; lane 6, use of an Egr-1 specific antibody; lane 7, use of a mutant oligonucleotide against Egr-1. C, Western blots of RAW cells stimulated for 2 hours with LPS using anti–Egr-1 antibody.



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  		Figure 3. Inhibition of LPS-induced DNA binding activity and protein expression of Egr-1 by IL-10 is dose- and time-dependent. RAW cells were pretreated with differing IL-10 doses for 24 hours as indicated (ng/mL) before treatment with LPS (100 ng/mL) for 2 hours. A, EMSA with the consensus sequence for Egr-1. B, Western blots of RAW cells using anti–Egr-1 antibody. For time course experiments RAW cells were pretreated with IL-10 (50 ng/mL) for different time points before treatment with LPS (100 ng/mL) as indicated. C, EMSA with the consensus sequence for Egr-1. D, Western blots using anti–Egr-1 antibody.

Effects of Interleukin-10 on Other Transcriptional Activators Within the TF Promotor
LPS stimulation resulted in a strong induction of binding of nuclear proteins to the consensus sequence for NFkB, which was inhibited by IL-10 pretreatment by 40% (Figure 4A). Binding of nuclear proteins to the consensus sequence for AP-1 demonstrated a weak induction attributable to the stimulation with LPS, which was not affected by IL-10 pretreatment (Figure 4B).



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  		Figure 4. Effects of interleukin-10 on other transcriptional activators within the TF promotor. RAW cells were pretreated with or without IL-10 (50 ng/mL) for 24 hours before treatment with LPS (100 ng/mL). EMSA experiments with the consensus sequence for NFkB (A) and AP-1 (B) were performed after 2 hours stimulation with LPS as described.

Localization of IL-10 Responsive Elements Within the Egr-1 Promotor
As shown in Figure 5A, the inhibition of expression and DNA-binding of Egr-1 was attributable to reduced transcription as demonstrated with a luciferase reporter construct of the proximal murine Egr-1 promoter.



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  		Figure 5. Localization of IL-10 responsive elements within the Egr-1 promotor. RAW cells were transiently transfected with various plasmids (A through V) containing a 5' deletion series of the proximal Egr-1 promotor. After transfection, cells were pretreated with IL-10 (50 ng/mL) and stimulated for 2 hours with LPS followed by measuring luciferase activity. A, Luciferase activity of the 1.2kb proximal Egr-1 promotor in fold increase. *P<0.05 vs LPS-stimulated cells. B, Luciferase activity of various deletion constructs displayed in relative luciferase activity. C, Nuclear extracts were prepared from RAW cells with or without pretreatment of IL-10 (50 ng/mL) before stimulation with LPS (100 ng/mL) for 30 minutes and EMSA with an oligonucleotide for SRE3 were performed. Lane 4, addition of 50-fold excess of unlabeled SRE3 oligonucleotide. NS indicates, nonspecific binding reaction.

Deletion of the 2 upstream AP-1 binding sites within the Egr-1 promoter had no significant effect on LPS induction and IL-10 mediated inhibition (Figure 5B, plasmid C), although the absence of the putative SRE sites 1 to 5 (Figure 5B, plasmid B) decreased luciferase activity comparable to levels seen with a minimal Egr-1 promotor (Figure 5B, plasmid G). Deletion of the 2 proximal SRE sites reduced the constitutive luciferase activity (Figure 5B, plasmid D). However, LPS increased luciferase activity 2-fold, and IL-10 pretreatment resulted in a strong reduction of luciferase activity, which was comparable to results shown with the wild-type construct (Figure 5B, plasmid A). In contrast, deletion of the region with the cluster containing the 3 distal SRE sites 3 to 5 diminished constitutive and inducible Egr-1 activity to baseline levels (Figure 5B, plasmid E and F). Furthermore, pretreatment of the cells with IL-10 had no effect on Egr-1 promotor activity in these constructs. These results suggest that the SRE 3 to 5 sites mediate inhibition of LPS-induced Egr-1 activity by IL-10.

IL-10 Inhibits LPS-Induced Binding of Nuclear Proteins to the SRE3
To further determine the functional role of these SRE, we conducted electrophoretic mobility shift assays with SRE specific oligonucleotides. As demonstrated in Figure 5C, LPS induced protein binding to the SRE3 oligonucleotides. Addition of excess unlabeled oligonucleotides successfully competed away the binding of the nuclear proteins, indicative of binding specificity in this reaction (lane 4). Pretreatment with IL-10 before LPS stimulation resulted in reduced binding of nuclear proteins to the SRE3 oligonucleotides.

Effect of IL-10 on Expression of Transcription Factors Which Bind to the SRE
Guha et al, previously demonstrated that the induced protein complex in LPS-stimulated cells contains serum response factor (SRF) and p-Elk-1.5 We therefore used antibodies against SRF and p-Elk-1 and performed Western blots to study the expression patterns of these transcription factors in response to IL-10 pretreatment. Phosphorylation of Elk-1 after LPS treatment was induced in a time-dependent manner, showing maximum phosphorylation after 30 minutes (data not shown). Pretreatment of the cells with IL-10 resulted in a marked reduction of Elk-1 phosphorylation (Figure 6A). In contrast, there was weak induction of SRF expression after 15 minutes LPS stimulation, which was not inhibited by IL-10 pretreatment (Figure 6B).



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  		Figure 6. Effects of IL-10 on expression of transcription factors which bind to the SRE and on the phosphorylation of MEK-1/2 and ERK-1/2 in LPS-stimulated macrophages. Western blots were performed from nuclear extracts of RAW cells, which have been stimulated with LPS (100 ng/mL) for 30 minutes with or without pretreatment with IL-10 for 24 hours (50 ng/mL). A, Western blot using antibodies specific for pElk-1 and Elk-1, respectively. B, Western blot analysis using antibodies against SRF. RAW cells were stimulated with LPS (100 ng/mL) for 15 minutes with or without pretreatment with IL-10 (50 ng/mL), and whole cell lysates were harvested as described. C, Immunoblotting for phosphorylated and nonphosphorylated MEK-1/2. D, Immunoblotting for phosphorylated and nonphosphorylated ERK-1/2.

IL-10 Inhibits Phosphorylation of the MEK–ERK-1/2 Pathway
Elk-1 is phosphorylated by upstream MAPK kinases including the MEK–ERK-1/2 pathway. LPS induced phosphorylation of ERK-1/2 and MEK1/2 occurred within 15 minutes of LPS treatment (data not shown). Phosphorylation of both ERK1/2 and MEK1/2 was inhibited by IL-10 pretreatment (Figure 6C and 6D).

Suppressor of Cytokine Signaling Proteins Are not Involved in Interleukin-10–Mediated Inhibition of LPS-Induced TF and Egr-1 Expression
To determine whether the antiinflammatory effects of IL-10 occur via intermediate expression of SOCS molecules, time course experiments were conducted with quantitative real-time RT-PCR. Treatment of RAW cells with IL-10 resulted in induction of SOCS-3 expression. (Figure 7A); whereas no significant induction in SOCS-1 expression could be observed (data not shown). To further elucidate the role of SOCS-3 in IL-10 related inhibition of TF and Egr-1, we performed a series of experiments using RAW 267.4 cells stably overexpressing SOCS-3. As shown in Figure 7, overexpression of SOCS-3 had no significant effect on LPS-mediated Egr-1 and TF expression with and without IL-10 pretreatment (Figure 7B and 7C).



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  		Figure 7. SOCS-3 is not involved in the IL-10 mediated inhibition of TF and Egr-1 expression on LPS stimulation. A, RAW264.7 macrophages were stimulated for the indicated time period with IL-10 (100 ng/mL). SOCS-3 expression was measured by quantitative RT-PCR. B, SOCS-3 overexpressing RAW cells or mock transfected RAW cells (EV) were pretreated with or without IL-10 (50 ng/mL) for 24 hours before treatment with LPS (100 ng/mL). Messenger RNA expression of TF was determined after stimulation for 2 hours using RT-PCR. C, Messenger RNA expression of Egr-1 was determined after stimulation for 1 hour using RT-PCR.

IL-10 also Inhibits TF and Egr-1 Expression and Binding of Nuclear Proteins to Egr-1 Consensus Sequence in Mouse Peritoneal Macrophages
As shown in Figure 8, IL-10 inhibited the LPS-induced increase in TF (Figure 8A) and Egr-1 (Figure 8B) mRNA expression in mouse peritoneal macrophages. Furthermore, IL-10 also prevented binding of nuclear proteins to the Egr-1 consensus oligonucleotide in LPS-stimulated cells (Figure 8C).



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  		Figure 8. IL-10 also inhibits mRNA expression of TF and Egr-1 as well as binding of nuclear proteins to Egr-1 consensus sequence in peritoneal macrophages. Mouse peritoneal macrophages were stimulated with LPS (100 ng/mL) for 2 hours (A) or 1 hour (B) with or without IL-10 pretreatment for 24 hours (50 ng/mL), and mRNA expression was determined using real-time RT-PCR for TF (A) and Egr-1 (B). *P<0.05 vs LPS-stimulated cells. C, EMSA with peritoneal macrophages using the consensus sequence for Egr-1 was performed after 2 hours stimulation with LPS as described. Lane 5, addition of 50-fold excess of unlabeled Egr-1 oligonucleotide; lane 6, use of a mutant oligonucleotide against Egr-1


*    Discussion
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 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
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Inflammation and coagulation are closely linked in the atherogenic process. Inflammatory mediators such as LPS, Chlamydia pneumoniae, and CD40 have all been demonstrated to induce the expression of TF in macrophages.5,6,18 Inhibition of inflammation by cytokines such as IL-10 may therefore be a promising therapeutic approach to reduce atherosclerosis and thrombosis.20,31

In this study, pretreatment of mouse macrophages with IL-10 resulted in a robust decrease of TF mRNA expression. This is consistent with several previous studies that demonstrated an IL-10 mediated reduction of TF activity, protein, and mRNA expression.25,32,33 We extended those studies to evaluate the molecular pathways underlying the inhibitory effects of IL-10 on TF expression. The classical pathway of TF induction by LPS involves binding of LPS to the Toll-like receptor 4 (TLR4), followed by the activation of several intracellular pathways. These include phosphorylation of MEK1 and ERK1/2. Phosphorylated ERK1/2 rapidly phosphorylates Elk-1 in a complex of pElk-1 and serum response factor. This ternary complex binds to and activates serum response elements within the promoter of Egr-1. Egr-1 in association with AP-1 and NFkB activates the TF promoter.34 Using luciferase reporter constructs of the serum response region within the TF promoter, we showed that the inhibition of LPS induction of TF by IL-10 occurs via inhibition of gene transcription. However, IL-10 inhibits only the LPS mediated induction of TF expression, because treatment with IL-10 alone did not reduce luciferase activity or mRNA expression below baseline levels.

Recent studies suggest that Egr-1 is the key regulatory element within the TF promoter that responds to inflammatory stimuli.5,6 We clearly demonstrated that IL-10 strongly reduces DNA binding of LPS activated Egr-1 by up to 80% as well as expression of both Egr-1 mRNA and protein. Furthermore, inhibition of the Egr-1 promoter by IL-10 in response to LPS stimulation also occurred on a transcriptional level, which could be demonstrated by using luciferase reporter contructs. However, because AP-1 and NFkB can also activate the TF promoter and previous studies have clearly demonstrated the antiinflammatory properties of IL-10 related inhibition on NFkB activation,35 we performed electrophoretic mobility shift assay (EMSA) to determine which transcription factors might be involved in IL-10 mediated inhibition of TF. We did not see any inhibition of AP-1 binding activity by IL-10 in macrophages. On the other hand, pretreatment of macrophages with IL-10 (50 ng/mL for 24 hours) before stimulation with LPS caused {approx}40% reduction in NFkB binding activity. This is consistent with the observations of Clarke et al and Denys et al that suggest that IL-10 mediated inhibition of NFkB binding activity is only partially responsible for the antiinflammatory effects of IL-10.36,37 Taken together, these data suggest that inhibition of LPS-induced Egr-1 activation and expression by IL-10 is one of the underlying mechanisms by which IL-10 inhibits TF expression.

To our knowledge, this is the first report demonstrating IL-10 mediated inhibition of Egr-1. Because Egr-1 is involved in the induction of many proatherosclerotic genes, such as TNF{alpha}, intercellular adhesion molecule-1, and fibroblast growth factor-2, the inhibition of Egr-1 induction by IL-10 might have a much broader effect then simply inhibiting TF induction and helps explain the antiatherosclerotic effects of IL-10 in vivo and in vitro.

The 1.2 proximal kilobases of the Egr-1 promoter contains putative enhancers, such as an AP-1 site, a Sp1 site, and 5 SRE sites.29 Studies in monocytes and endothelial cells using LPS and thrombin have identified these SRE sites as important transcriptional activators in response to inflammation.5,38 In a series of experiments with 5' deletion constructs of the Egr-1 promoter, we were able to identify a promoter region which might be involved in mediating the antiinflammatory effects of IL-10. This small region contains the SRE sites 3 to 5. Our data demonstrate that it is the SRE3 site that mediates the IL-10 inhibition of LPS-induced DNA binding. SRE4 and SRE5 demonstrated only a weak induction after LPS stimulation (data not shown).

Previous studies identified phosphorylated Elk-1 and SRF as proteins that form ternary complexes and lead to the activation of serum response elements in the Egr-1 and c-fos promoters.39 Our data support a role for Elk-1, as we showed that IL-10 inhibits the phosphorylation of Elk-1, although the mild induction of SRF in response to LPS treatment was not reduced by IL-10.

Phosphorylation of Elk-1 is mediated by the MEK-ERK1/2 pathway. Activation of MEK-1/2 and ERK1/2 caused by LPS stimulation occurred as early as 30 minutes after treatment. Pretreatment with IL-10 led to a robust reduction of phosphorylation of MEK-1/2 and ERK1/2. This is consistent with the observations of Niiro et al, who reported inhibition of phosphorylation of ERK2 by IL-10 in human monocytes.40

It has been shown that LPS activates the intracellular MEK-ERK1/2 pathway via activation of TLR4. Inhibition of the MEK-ERK1/2 pathway followed by inhibition of Egr-1 and TF by IL-10, as shown in this study, could simply be attributable to reduced expression of TLR4. However, Tamadl et al demonstrated that treatment of human monocytes with recombinant IL-10 had no effect on TLR4 mRNA and surface expression.41

On binding of IL-10 to its receptor, receptor-bound Janus kinase 1 is activated and phosphorylates the receptor thereby creating docking and activation sites for signal transducer and activator of transcription (STAT) factors, such as STAT3. After translocation to the nucleus, STAT3 can bind to IL-10 responsive genes including SOCS proteins. In turn SOCS proteins act as feedback inhibitors limiting further cytokine receptor signaling. Previous studies have suggested the involvement of SOCS proteins, in particular SOCS-3, in the antiinflammatory effects of IL-10 on LPS signaling.42 However, studies from Baetz et al did not find a role for SOCS-3 in LPS signal transduction.27 Also, Lang et al and Yasukawa et al using macrophages from SOCS-3 –/– mice demonstrated that SOCS-3 is not an essential mediator in the antiinflammatory effects of IL-10.43,44 Our data confirmed these results. Treatment of SOCS-3 overexpressing macrophages with LPS did not lead to a reduction in Egr-1 and TF expression, although SOCS-3 expression levels in these cells were even higher than mock transfected cells treated with IL-10 (data not shown). Furthermore, IL-10 pretreatment before LPS stimulation led to the same reduction in Egr-1 and TF expression as seen in mock transfected cells.

In summary, results of this study have demonstrated one possible molecular pathway for the IL-10 mediated reduction in TF expression after stimulation with LPS, a classical inflammatory stimulus. We show that IL-10 inhibits phosphorylation of ERK1/2, MEK-1/2, and Elk-1, leading to a reduced activation of the serum response element 3 within the Egr-1 promotor and reduced TF expression. We further demonstrate that this inhibition is independent of SOCS-3. This data might help explain how IL-10 contributes to reduced coagulatory and inflammatory activity in atherosclerosis and thrombosis.


*    Acknowledgments
 
This work was supported by grants from the Deutsche Forschungsgemeinschaft Be3188/2-1, Da592/1, and Da592/2. The authors thank Annette Buttler for technical assistance.


*    Footnotes
 
Original received March 7, 2005; revision received June 20, 2005; accepted July 7, 2005.


*    References
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 *References
 

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