doi: 10.1152/ajplung.00257.2011302:L785-L792, 2012. First published 13 January 2012;Am J Physiol Lung Cell Mol Physiol 

Jamie K. Patel, Rachel L. Clifford, Karl Deacon and Alan J. Knoxhuman airway smooth muscle cellschemotactic protein-1 (MCP-1/CCL2) secretion from

-induced monocyteβ- and IL-1αCiclesonide inhibits TNF

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Ciclesonide inhibits TNF�- and IL-1�-induced monocyte chemotacticprotein-1 (MCP-1/CCL2) secretion from human airway smooth muscle cells

Jamie K. Patel,* Rachel L. Clifford,* Karl Deacon, and Alan J. KnoxDivision of Respiratory Medicine and Nottingham Respiratory Biomedical Research Unit, University of Nottingham,Nottingham, United Kingdom

Submitted 2 August 2011; accepted in final form 10 January 2012

Patel JK, Clifford RL, Deacon K, Knox AJ. Ciclesonide inhibitsTNF�- and IL-1�-induced monocyte chemotactic protein-1 (MCP-1/CCL2) secretion from human airway smooth muscle cells. Am JPhysiol Lung Cell Mol Physiol 302: L785–L792, 2012. First pub-lished January 13, 2012; doi:10.1152/ajplung.00257.2011.—Mono-cyte chemotactic protein-1 (MCP-1) is a member of the CC family ofcytokines. It has monocyte and lymphocyte chemotactic activity andstimulates histamine release from basophils. MCP-1 is implicated inthe pathogenesis of inflammatory diseases, including asthma. Theairway smooth muscle (ASM) layer is thickened in asthma, andthe growth factors and cytokines secreted by ASM cells play a role inthe inflammatory response of the bronchial wall. Glucocorticoids and�2-agonists are first-line drug treatments for asthma. Little is knownabout the effect of asthma treatments on MCP-1 production fromhuman ASM cells. Here, we determined the effect of ciclesonide (aglucocorticoid) and formoterol (a �2-agonist) on MCP-1 productionfrom human ASM cells. TNF� and IL-1� induced MCP-1 secretionfrom human ASM cells. Formoterol had no effect on MCP-1 expres-sion, while ciclesonide significantly inhibited IL-1�- and TNF�-induced MCP-1. Furthermore, ciclesonide inhibited IL-1�- andTNF�-induced MCP-1 mRNA and IL-1�- and TNF�-induced MCP-1promoter and enhancer luciferase reporters. Western blots showed thatciclesonide had no effect on I�B degradation. Finally, ciclesonideinhibited an NF-�B luciferase reporter. Our data show that ciclesonideinhibits IL-1�- and TNF�-induced MCP-1 production from humanASM cells via a transcriptional mechanism involving inhibition ofNF-�B binding.

glucocorticoid; nuclear factor-�B; inflammation

MONOCYTE CHEMOTACTIC PROTEIN (MCP)-1 (CCL-2) is a memberof the CC family of cytokines. It is a three-exon gene, regulatedby a distal enhancer and a proximal promoter region separatedby 2.2 kb of DNA (18). The proximal promoter region is justupstream of the transcription start site and contains a GC boxthat binds Sp1, NF-�B, and nuclear factor (NF)-1 binding sites,in addition to two activator protein (AP)-1 sites. The distalenhancer contains two NF-�B binding sites (3).

MCP-1 has monocyte and lymphocyte chemotactic activityand can stimulate histamine release from basophils (13). It isimplicated in a number of inflammatory diseases, includingasthma. Asthma is a chronic inflammatory disease character-ized by increased lamina reticularis, smooth muscle cell hy-perplasia, airway hyperreactivity, and airway inflammation(15). Staining of asthmatic and nonasthmatic bronchial biop-sies shows the presence of MCP-1 in the bronchial epithelium,

subepithelial macrophages, blood vessels, and bronchial smoothmuscle, with stronger MCP-1 staining in the asthmatic epitheliumand subepithelial layer (24). MCP-1 is also present in bronchoal-veolar lavage fluid (BALF) from healthy and atopic asthmapatients, with higher concentrations in the asthmatic BALF.Furthermore, asthmatic levels of BALF MCP-1 inversely cor-relate with measures of pulmonary function, namely, forcedexpiratory volume in 1 s and forced expiratory flow at 50% offorced vital capacity (13). Allergen challenge can furtherincrease BALF MCP-1 levels (12). Serum MCP-1 levels arealso increased in asthmatic compared with nonasthmatic sam-ples, and asthmatic levels increase further during an acuteasthma attack (4). Expression of MCP-1 in murine lungscoincides with leukocyte infiltration in an ovalbumin model ofinduced lung allergic inflammation. Blockade of MCP-1 in theovalbumin model reduces migration of macrophages, mono-cytes, T lymphocytes, and eosinophils to the lung in responseto ovalbumin and the induction of bronchial hyperreactivity(10). MCP-1 blockade also reduces ovalbumin-induced levelsof leukotriene B4, PGE2, thromboxane B2, IL-5, and IL-4,suggesting that MCP-1 is involved in B and T cell activation(10). Interestingly, a polymorphism in the enhancer region ofthe MCP-1 gene is associated with asthma in children (26).

The grossly thickened airway smooth muscle (ASM) is animportant cellular source of chemokines and growth factors inasthma (9). Smooth muscle cells are considered to play acentral role in orchestration of the inflammatory responsewithin the bronchial wall (6). MCP-1 secretion from humanASM cells was first described in 1998 (28), and since then itsregulation by various stimuli has been investigated. IL-1�,TNF�, and endothelin-1 stimulate MCP-1 mRNA productionand protein secretion from human ASM cells (16, 25, 28).Pharmacological inhibitor studies suggest that IL-1�-inducedMCP-1 expression is dependent on p38 MAPK, JNK kinase,p42/44 ERK, and the transcription factor NF-�B (29). Detailedcharacterization of endothelin-1-induced MCP-1 expressionshows a requirement for p38 and p42/44 MAPK signaling anda transcriptional mechanism involving binding of the AP-1c-Jun subunit and the NF-�B p65 subunit to the MCP-1promoter, with little effect on the MCP-1 enhancer (25). Themechanism of TNF�-induced MCP-1 expression in humanASM cells has not been described. However, in NIH 3T3fibroblasts, TNF�-induced MCP-1 requires a sequential seriesof events that links the proximal promoter and distal enhancerregions. TNF� induction of NF-�B binding to the distal en-hancer recruits the histone acetyl transferase p300/CREB bind-ing protein, which acetylates the distal enhancer- and proximalpromoter-associated histones. Proximal histone acetylation al-

* J. K. Patel and R. L. Clifford contributed equally to this work.Address for reprint requests and other correspondence: A. J. Knox. Division

of Respiratory Medicine and Nottingham Respiratory Biomedical ResearchUnit, Univ. of Nottingham, Clinical Sciences Bldg., City Hospital, NottinghamNG5 1PB, UK (e-mail: alan.knox@nottingham.ac.uk).

Am J Physiol Lung Cell Mol Physiol 302: L785–L792, 2012.First published January 13, 2012; doi:10.1152/ajplung.00257.2011.

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lows access for Sp1 binding to the proximal promoter andsubsequent transcription (27).

Glucocorticoids and �2-agonists are commonly used forasthma therapy because of their anti-inflammatory and bron-chodilatory effects, respectively. The glucocorticoid dexa-methasone inhibits cytomix (combination of IL-1�, TNF�, andIFN�)-induced MCP-1 mRNA and protein secretion (21),while fluticasone inhibits TNF�-induced MCP-1 via an unde-fined posttranscriptional mechanism (16). The �2-agonist sal-meterol has no effect on TNF�-induced MCP-1 expression(16). There is no further information regarding MCP-1 regu-lation by glucocorticoids or �2-agonists. The purpose of thisstudy was to investigate the effect of the glucocorticoidciclesonide and the �2-agonist formoterol on IL-1�- andTNF�-induced MCP-1 expression from human ASM cells. Wefound that ciclesonide inhibited TNF�- and IL-1�-inducedMCP-1 expression, while formoterol had no effect. Theciclesonide effect was mediated via the MCP-1 promoter andenhancer regions and regulation of NF-�B binding.

MATERIALS AND METHODS

Materials. Formoterol fumarate dehydrate, 5-[(p-fluorophenyl)-2-ureido]thiophene-3-carboxamide (TPCA-1), DMEM, L-glutamine,amphotericin B, penicillin, streptomycin, and trypsin-EDTA werepurchased from Sigma (Poole, Dorset, UK). Ciclesonide was a giftfrom Altana Pharma (Konstanz, Germany). Recombinant human IL-1�, recombinant human TNF�, and the DuoSet ELISA developmentsystem for human CCL2/MCP-1 were purchased from R & D Systems(Abingdon, Oxfordshire, UK). All reagents for the firefly luciferaseassay system were obtained from Promega (Southampton, Hampshire,UK). FuGene 6 transfection reagent was purchased from RocheMolecular Biochemicals (Lewes, East Sussex, UK). Rainbow-coloredprotein molecular weight marker, ECL Western blot detection re-agent, and Hyperfilm-ECL were obtained from Amersham (Bucking-hamshire, UK). Nitrocellulose membrane was purchased from Bio-Rad (Hemel Hemstead, Hertfordshire, UK). Polyclonal goat anti-rabbit antibody conjugated to horseradish peroxidase was obtainedfrom DakoCytomation (Ely, Cambridge, UK). Phosphorylated I�B-�(Ser32) and I�B-� were purchased from Cell Signaling/New EnglandBiolabs (Hitchin, Hertfordshire, UK). The NucleoSpin RNA II kit wasobtained from Fisher Scientific (Loughborough, Leicestershire, UK).Moloney’s murine leukemia virus (MMLV) RT, RNase inhibitor,(dT)15 primer, dNTPs, and MMLV RT buffer were purchased fromPromega. TaKaRa SYBR Premix Ex Taq was obtained from Lonza(Basel, Switzerland). MB-120L was a kind gift from GlaxoSmithK-line.

Cell culture. Human ASM cells from human tracheas were ob-tained from postmortem examinations, as previously described (18a).The study was approved by the North Nottinghamshire ResearchEthics Committee. Primary normal human ASM cells were grown inDMEM supplemented with 4 mM L-glutamine, 2.5 �g/ml amphoter-icin B, 100 U/ml penicillin, 100 �g/ml streptomycin, and 10%heat-inactivated fetal bovine serum in humidified 5% CO2-95% air at37°C. Cells at passage 6–7 were used for all experiments.

Experimental protocols. Once fully confluent, the cells weregrowth-arrested in DMEM serum-free medium and incubated at 37°Cfor 24 h prior to treatment. Cells were then treated with IL-1� (0–10ng/ml) or TNF� (0–100 ng/ml) for 24 h in concentration-responseexperiments. The supernatants were assayed for MCP-1. In inhibitorstudies, cells were preincubated for 30 min and then treated for 24 hwith IL-1� or TNF�. Vehicle (DMSO) was added to control wells atequivalent concentration (0.1% maximum concentration).

MCP-1 ELISA. The MCP-1 DuoSet ELISA kit was used to measureMCP-1 concentrations in cell culture supernatants according to the

manufacturer’s protocol. All cell supernatants were diluted 1:50 or1:200 in reagent diluent, so that all concentrations were within thestandard curve.

RNA isolation and reverse transcription. Total RNA was isolatedusing the NucleoSpin RNA II kit following the manufacturer’sprotocol: 5 �l of RNA was reverse-transcribed in a total volume of25 �l, including 132 U of MMLV RT, 26.4 U of RNase inhibitor,0.6 �g of (dT)15 primer, dNTPs at 2 �M, and 1� MMLV RTbuffer. The resulting RT products were used for real-time PCRamplification.

Quantitative real-time PCR. Total human MCP-1 expression wasdetermined using the primer sequences 5=-GCTCAGCCAGATG-CAAT-3= (forward) and 5=-GCTTGTCCAGGTGGTCCATG-3= (re-verse). For GAPDH, which was used as the housekeeping gene, theprimers were as follows: 5=-CCACCCATGGCAAAATTCCATG-3=(forward) and 5=-TCTAGACGGCAGGTCAGG-3= (reverse). Forreal-time PCR of 1 �l of reverse-transcribed cDNA, TaKaRa SYBRPremix Ex Taq and the Mx3000P quantitative PCR system (Strat-agene) were used. Each reaction consisted of 1� SYBR Premix ExTaq, sense and antisense primers at 0.2 �M, 1 �l of DNA, and waterto a final volume of 25 �l. Thermal cycler conditions includedincubation at 95°C for 30 s followed by 50 cycles of 95°C for 5 s,60°C for 30 s, and 72°C for 15 s. Integration of the fluorescent SYBRGreen into the PCR product was monitored after each annealing step.Amplification of one specific product was confirmed by melting curveanalysis.

Vectors and transient transfections. MCP-1 enhancer and promotervectors consisted of the pGL3-basic plasmid vector containing thewild-type human MCP-1 enhancer or promoter regulatory sequencesdriving a luciferase reporter gene. The enhancer construct harbors twoNF-�B sites and is contained in the region �2802 to �2573 bprelative to the translation start codon. The MCP-1 promoter constructharbors a number of different transcription factor binding sites andcontains the proximal section of the wild-type human MCP-1 pro-moter region �161 to �1 bp relative to the translation start codon.These constructs have been previously described in detail (16, 25).The NF-�B reporter construct 6NF�Btkluc contains six copies of theNF-�B binding site, which is upstream of a minimal thymidine kinasepromoter driving a luciferase gene. The NF-�B reporter was a giftfrom Dr. Robert Newton (University of Calgary, Calgary, AB, Can-ada).

FuGene 6 transfection reagent was used to carry out all transienttransfections according to the manufacturer’s protocol. Human ASMcells were seeded out into 24-well plates at 2.5 � 104 cells/ml inDMEM-supplemented medium. At 50–60% confluence, cells weregrowth-arrested for 8 h in serum-free, antibiotic-free DMEM contain-ing 4 mM L-glutamine. Fresh serum-free, antibiotic-free DMEMcontaining 4 mM L-glutamine was changed after 8 h, and cells weretransfected with 0.4 �g of DNA and 3 �l of FuGene 6 per well. After16 h of transfection, the cells were pretreated for 30 min with orwithout drugs and then stimulated for 6 h with IL-1� (10 ng/ml) orTNF� (10 ng/ml). Cells were then lysed, and firefly and Renillaluciferase activity was determined. For data analysis, the firefly valueswere divided by the Renilla values, and the data are expressed as theratio of firefly to Renilla luciferase activity.

Western blot. Western blot was performed to assess the phosphor-ylation of I�B-� in response to TNF�/IL-1� and in the presence of theglucocorticoid ciclesonide. The medium was aspirated, and the cellswere washed with ice-cold PBS and incubated with phospho-lysisbuffer (20 mM Tris·HCl, pH 7.4, 1% Triton X-100, 137 mM NaCl, 2mM EDTA, 25 mM �-glycerophosphate, 10% glycerol, 1 mMNa3VO4, 1 mM PMSF, 10 �g/ml leupeptin, 1 mM DTT, and 0.1 U/mlprotease inhibitor cocktail). Samples were collected, and proteinconcentration was determined by Bradford assay. The BSA standardcurve was used to convert the values to protein concentrations (�g/ml). Cell protein (30 �g per lane) was subjected to electrophoresis ona 10% SDS-polyacrylamide gel and electroblotted to a polyvinylidene

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difluoride membrane. After the membrane was blocked [5% milk inTris-buffered saline � 0.1% Tween 20 (TBST)], it was incubatedovernight at 4°C with phosphorylated (Ser32) I�B-� (1:1,000 dilution)or I�B-� (1:1,000 dilution) primary antibody in 5% BSA in 1�TBST. The membrane was washed (1� TBST) and then incubatedwith a horseradish peroxidase-conjugated secondary antibody (goatanti-rabbit, 1:2,000 dilution in 5% BSA in 1� TBST) for 1 h at roomtemperature. The membrane was incubated with ECL Western blot-ting detection reagent and exposed to Hyperfilm-ECL. Subsequently,blots were scanned, and densitometry was performed using AdobePhotoshop.

Statistical analysis. The mean values of replicate wells for MCP-1and luciferase levels were calculated and expressed as fold increase.Where MCP-1 absolute values are presented (concentration-responseexperiments), the mean of all the replicates is shown. Each experimentwas performed in cells from three different tracheas, each obtainedfrom a different human donor. Three biological replicates wereperformed per donor cell line. Values are means SE. The data fromthe experiments were subjected to statistical analysis to determinestatistical significance using GraphPad Prism version 4.00 for Win-dows (Graphpad Software, San Diego, CA). One-way analysis ofvariance of the raw data followed by a Dunnett’s post test was used tocompare all groups with unstimulated control. One-way analysis ofvariance of the raw data followed by a Tukey’s post test was used tocompare stimulated samples with drug-treated samples. P 0.05 wasconsidered statistically significant.

RESULTS

TNF� and IL-1� induce MCP-1 protein release. We firststudied the effect of TNF� and IL-1� on the release of MCP-1from human ASM cells. Confluent, growth-arrested humanASM cells were treated for 24 h with increasing concentrationsof TNF� (�100 ng/ml) or IL-1� (�10 ng/ml). TNF� stimu-lated MCP-1 release in a concentration-dependent manner(Fig. 1A) above control levels, with a significant increase inMCP-1 at 1–100 ng/ml TNF�. This was also true for IL-1�stimulation, with a significant increase in MCP-1 at 0.1–10ng/ml IL-1� compared with control (Fig. 1B).

Differential effects of formoterol and ciclesonide on IL-1�-and TNF�-induced MCP-1. Formoterol is a long-acting �2-adrenoceptor agonist. Long-acting �2-adrenoceptor agonistsare frequently used to treat asthma. Formoterol’s effects in-clude bronchodilation and inhibition of mediator release frommast cells and monocytes. Serum-deprived, confluent humanASM cells were preincubated with formoterol and then stim-ulated for 24 h with TNF� or IL-1�. Formoterol had no effecton TNF�- or IL-1�-induced MCP-1 release (Fig. 2, A and B).

Glucocorticoids are a first-line asthma treatment. Theirmechanism of action involves inhibitory effects on proinflam-matory cytokine production, cell migration, and lymphocyteactivation. Ciclesonide is an inhaled glucocorticoid with onlymild side effects (20). We tested the effect of ciclesonide onTNF�- and IL-1�-stimulated MCP-1 release from humanASM cells. Serum-deprived human ASM cells were pretreatedfor 30 min with ciclesonide and then stimulated for 24 h withTNF� or IL-1�. Ciclesonide at 10�12–10�5 M significantlyinhibited TNF�-stimulated MCP-1 production (Fig. 2C). IL-1�-stimulated MCP-1 release was also significantly inhib-ited in a concentration-dependent (10�12–10�5 M) manner(Fig. 2D).

When ciclesonide and formoterol were added in combina-tion, no inhibition beyond that elicited by ciclesonide alonewas observed (Fig. 2, E and F).

Ciclesonide inhibits MCP-1 mRNA production and MCP-1 pro-moter and enhancer activity. To establish whether ciclesonidewas acting transcriptionally to inhibit MCP-1, we used quan-titative real-time PCR to measure the MCP-1 mRNA level inresponse to ciclesonide. Confluent serum-starved human ASMcells were preincubated for 30 min with 10�7 M ciclesonideand then stimulated for 8 h with 0.1 ng/ml TNF� or IL-1�. TNF�and IL-1� induced MCP-1 mRNA levels (Fig. 3, A and B).Ciclesonide inhibited TNF�- and IL-1�-induced MCP-1 (Fig. 3, Aand B).

Subsequently, we transfected luciferase reporter constructsfor the MCP-1 promoter and enhancer into human ASM cellsto establish whether ciclesonide affected promoter or enhanceractivity. The promoter region construct harbors an NF-�B, anSp1, and an NF-1 binding site, in addition to two AP-1 sites,while the enhancer region construct harbors 2 NF-�B bindingsites. Human ASM cells were transfected with the MCP-1promoter reporter for 16 h and preincubated for 30 min with10�7 M ciclesonide before incubation with TNF� (10 ng/ml)or IL-1� (10 ng/ml) for 2 h. TNF� and IL-1� significantlyincreased MCP-1 promoter activity (Fig. 3, C and D).Ciclesonide inhibited MCP-1 promoter activity in the presenceof either cytokine.

Human ASM cells were then transfected with the MCP-1enhancer reporter. When stimulated with TNF� (10 ng/ml)or IL-1� (10 ng/ml), MCP-1 enhancer activity was signifi-cantly increased compared with control cells, and this ac-tivity was inhibited by nearly twofold with ciclesonide (Fig.3, E and F).

Fig. 1. TNF� and IL-1� induce monocytechemotactic protein (MCP)-1 secretion. A andB: human airway smooth muscle (ASM) cellswere treated for 24 h with TNF� (0–100 ng/ml) or IL-� (0–10 ng/ml), and MCP-1 secre-tion was measured by ELISA. Values aremeans SE (n � 3). ��P 0.01 vs. control.

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I�B kinase 2 activity is required for TNF�- and IL-1�-induced MCP-1 expression. Inasmuch as the MCP-1 promoterand enhancer regions contain NF-�B binding sites, we hypoth-esized that ciclesonide was acting on the NF-�B pathway.Briefly, TNF� and IL-1� signaling can activate I�B kinase 2(IKK-2). IKK-2 phosphorylates I�B, resulting in its degrada-tion. I�B inhibits NF-�B signaling when they are associated.Initially, we incubated cells with TPCA-1 to investigatewhether IKK-2 was required for TNF�- and IL-1�-stimulatedMCP-1 production. TPCA-1 is an inhibitor of IKK2 and, thus,inhibits NF-�B signaling by preventing I�B degradation.TPCA-1 dose dependently inhibited TNF�- and IL-1�-stimu-lated MCP-1 protein release (Fig. 4, A and B). A comparableresponse was observed with a second IKK2 inhibitor, MB-120B (Fig. 4, C and D). The same effect was seen withTPCA-1 at the mRNA level (Fig. 4, E and F).

Ciclesonide inhibits binding of NF-�B to its consensus sequence.Having shown that TNF� and IL-1� require IKK2 activity for

MCP-1 production, we investigated whether ciclesonide affected theIKK2-I�B-NF-�B pathway. Initially, we performed Westernblots to determine whether ciclesonide was able to prevent I�Bdegradation. A representative blot is shown in Fig. 5A. TNF�and IL-1� induced phosphorylation and subsequent degrada-tion of I�B, but this was not inhibited in the presence ofciclesonide (Fig. 5, B and C).

Finally, using a luciferase reporter containing six copiesof the NF-�B binding site upstream of the luciferase gene,we established the effect of ciclesonide on the transcrip-tional activity of NF-�B. Human ASM cells were transientlytransfected with NF-�B reporter constructs, pretreated withciclesonide (10�7 M), and stimulated for 6 h with TNF� (10ng/ml) or IL-1� (10 ng/ml). TNF� caused a significantincrease (Fig. 5D) in NF-�B reporter activity, which wasinhibited by ciclesonide. IL-1� also caused a significant in-crease (Fig. 5E) in NF-�B reporter activity, which was inhib-ited by ciclesonide.

Fig. 2. Differential effects of ciclesonide andformoterol on TNF�- and IL-1�-inducedMCP-1 secretion. A and B: human ASM cellswere pretreated for 30 min with formoterol(10�7–10�6 M) and then stimulated for 24 hwith TNF� (0.1 ng/ml) or IL-1� (0.1 ng/ml).MCP-1 released into the culture medium wasmeasured by ELISA. Values are means SE(n � 4). C and D: human ASM cells werepreincubated for 30 min with ciclesonide(10�12–10�5 M) and then stimulated for 24 hwith TNF� or IL-1� (0.1 ng/ml). MCP-1 re-leased into the culture medium was measuredby ELISA. Values are means SE (n � 3). Eand F: human ASM cells were pretreated for30 min with formoterol (Form, 10�7 M) orciclesonide (Cicl, 10�8 M) or both and thenstimulated for 24 h with TNF� (0.1 ng/ml) orIL-1� (0.1 ng/ml). MCP-1 released into theculture medium was measured by ELISA. Val-ues are means SE (n � 3). ���P 0.001vs. control. *P 0.05, **P 0.01, ***P 0.001 vs. stimulated cells.

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Together, these data show that ciclesonide inhibits TNF�-and IL-1�-induced MCP-1 expression by reducing the poten-tial of NF-�B to associate with its binding site.

DISCUSSION

This study is the first to demonstrate that the glucocorticoidciclesonide inhibits TNF�- and IL-1�-induced MCP-1 expres-sion from human ASM cells via a transcriptional mechanisminvolving the MCP-1 proximal promoter and distal enhancerand the transcription factor NF-�B.

Initially, we confirmed observations made previously, thatMCP-1 is secreted basally from human ASM cells and thatTNF� and IL-1� can concentration dependently increaseMCP-1 in the culture supernatant of these cells. Then weshowed that the �2-agonist formoterol had no effect on TNF�-

or IL-1�-induced MCP-1 secretion. These findings are inagreement with previous data from our laboratory showing thatsalmeterol has no effect on TNF�-induced MCP-1 expression(16). Contrary to this, cAMP-elevating agents can inhibitMCP-1 secretion from ASM cells (30). However, �2-agonistsare also ineffective against IL-1�-induced IL-8 secretion fromASM cells, while cAMP-elevating agents have an effect, sug-gesting that �2-agonists act via a different mechanism to directcAMP elevators (14). We also show that formoterol has noeffect when added in combination with the glucocorticoidciclesonide. However, ciclesonide alone did inhibit TNF�- andIL-1�-induced MCP-1 secretion over a concentration range.Ciclesonide also had a small effect on basal MCP-1 expression(data not shown); however, the effect on stimulated levels wasmore dramatic and reproducible. This is the first report of any

Fig. 3. Ciclesonide inhibits MCP-1 mRNAproduction and MCP-1 promoter and enhanceractivity. A and B: human ASM cells werepreincubated for 30 min with 10�7 Mciclesonide and then stimulated for 8 h withTNF� (0.1 ng/ml) or IL-1� (0.1 ng/ml). Cellswere lysed, RNA was extracted and reversetranscribed, and cDNA levels were determinedby quantitative PCR. Values are means SE(n � 3). C and D: 50–60% confluent humanASM cells were transfected for 16 h withMCP-1 promoter plasmid (0.4 �g/well), pre-treated for 30 min with ciclesonide (10�7 M),and then incubated for 6 h with TNF� (10ng/ml) or IL-1� (10 ng/ml). Firefly and Renillaluciferase activity was assayed, and the ratio offirefly to Renilla luciferase activity was calcu-lated to represent activity of the reporter. Val-ues are means SE (n � 3). E andF: 50–60% confluent human ASM cells weretransfected for 16 h with MCP-1 enhancerplasmid (0.4 �g/well), pretreated for 30 minwith ciclesonide (10�7 M), and then incubatedfor 6 h with TNF� (10 ng/ml) or IL-1� (10ng/ml). Firefly and Renilla luciferase activitywas assayed, and the ratio of firefly to Renillaluciferase activity was calculated to representactivity of the reporter. Values are means SE (n � 3). �P 0.05, ���P 0.001 vs.control. *P 0.05, **P 0.01 vs. stimulatedalone.

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ciclesonide effect on human ASM cells. Pype et al. (21)showed that dexamethasone can inhibit cytomix-inducedMCP-1 secretion from human ASM cells. They also showedthat dexamethasone reduced cytomix-induced MCP-1 mRNAlevels, suggesting that dexamethasone was acting transcrip-tionally, but no mechanism was established. Conversely, Nie etal. (16) showed that fluticasone inhibited TNF�-inducedMCP-1 protein secretion without affecting mRNA levels. Fur-thermore, fluticasone did not affect TNF�-induced MCP-1mRNA stability, and Nie et al. concluded that fluticasone wasacting via an undefined posttranscriptional mechanism. Dha-wan et al. (8) showed that platelet-derived growth factor-induced MCP-1 secretion from arterial smooth muscle cells isinhibited by dexamethasone via glucocorticoid receptor-dependent destabilization of MCP-1 mRNA. Hypoxia-in-duced MCP-1 is also inhibited by dexamethasone, in partvia effects on mRNA stability (7). IL-1�-induced MCP-1inhibition by any glucocorticoid is not reported. It seems

clear that the effects of glucocorticoids on MCP-1 expres-sion are diverse and mediated by mechanisms targeting eachstep of MCP-1 production.

To determine whether ciclesonide was acting transcription-ally in our system, we measured MCP-1 mRNA levels inresponse to TNF�, IL-1�, and ciclesonide. In agreement withprevious publications (16, 28, 30), TNF� and IL-1� inducedMCP-1 mRNA expression. This was inhibited by ciclesonide,suggesting that ciclesonide was acting transcriptionally.

The MCP-1 gene is under the control of a distal enhancerregion (�2802 to �2573 bp relative to the translation startcodon) and a proximal promoter region (�161 to �1 bprelative to the translation start codon). We used luciferasereporters for these regions and determined that IL-1� andTNF� stimulate the enhancer and promoter reporters. Thepromoter and enhancer region reporter activities were inhibitedby ciclesonide. As the enhancer and promoter regions haveNF-�B binding sites in common, we investigated further the

Fig. 4. I�B kinase 2 (IKK-2) activity is requiredfor TNF�- and IL-1�-induced MCP-1 secre-tion. A–D: human ASM cells were pretreatedfor 30 min with 5-[(p-fluorophenyl)-2-ureido]thiophene-3-carboxamide (TPCA-1, A and B)or MB-120B and then incubated for 24 h withTNF� (0.1 ng/ml) or IL-1� (0.1 ng/ml). Valuesare means SE (n � 3). E and F: human ASMcells were preincubated for 30 min with 10�5

M TPCA-1 and then stimulated for 8 h withTNF� (0.1 ng/ml) or IL-1� (0.1 ng/ml). Cellswere lysed, RNA was extracted and reversetranscribed, and cDNA levels were determinedby quantitative PCR. Values are means SE(n � 3). �P 0.05, ���P 0.001 vs.control. *P 0.05, **P 0.01, ***P 0.001vs. stimulated alone.

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role of upstream NF-�B signaling on IL-1�- and TNF�-induced MCP-1 expression. In order for NF-�B to translocateto the nucleus and be transcriptionally active, it must bereleased from the inhibitory cytoplasmic protein I�B. Releasefrom I�B requires I�B degradation. IKK phosphorylates I�Bon two serine residues, which mark the protein for ubiquitina-tion and degradation by the proteasome. Thus we used inhib-itors of IKK2, namely, TPCA-1 and MB-120B, to determine ifupstream NF-�B signaling was involved in IL-1�- and TNF�-induced MCP-1 expression. MCP-1 protein secretion was in-hibited by TPCR and MB-120B. MCP-1 mRNA levels werereduced in the presence of TPCA-1. We finally tested whetherciclesonide could affect I�B degradation or NF-�B binding toits consensus binding sequence. We found that ciclesonide hadno effect on I�B degradation but did inhibit NF-�B binding.This is contrary to a previous report that pancreatitis-associatedascitic fluid-induced MCP-1 in pancreatic acinar cells is inhib-ited by dexamethasone via prevention of I�B degradation (22).Prevention of I�B degradation prevents NF-�B translocationand, subsequently, reduces the amount available to bind DNA.Similarly, prednisolone inhibits platelet-derived growth factor-stimulated NF-�B translocation in human pulmonary arterysmooth muscle cells (17). In our system, I�B degradation wasnot changed by ciclesonide, suggesting that the change in

NF-�B reporter activity is due to modulation of NF-�B bindingas opposed to changes in nuclear NF-�B levels. Interestingly,Amrani et al. (1) showed that dexamethasone cannot inhibitTNF�-induced activation of the NF-�B reporter in ASM cells,suggesting that different glucocorticoids may modulate TNF�signaling differently. Furthermore, Park et al. (19) showed thatdexamethasone inhibition of TNF�-induced MCP-1 expres-sion from human glomerular endothelial cells was independentof I�B degradation and NF-�B binding. Studies investigatinggenes other than MCP-1 have, however, shown effects similarto those seen in the current study. For example, inhibition ofTNF�-induced IL-1� expression by dexamethasone in rheu-matoid arthritis synovial cells occurs via a change in NF-�Bbinding ability that is not dependent on I�B degradation or achange in nuclear NF-�B p65 levels (11). There are reports ofglucocorticoid-mediated inhibition of NF-�B Ser276 phosphor-ylation and histone H3 Ser10 phosphorylation (2). Abolishmentof NF-�B Ser276 phosphorylation prevents its binding to DNA(23), and phosphorylated (Ser10) histone H3 is associated withopen chromatin and active transcription and is suggested as amarker for NF-�B recruitment (5). It is possible that ciclesonide isinhibiting upstream kinase signaling to modify NF-�B or thechromatin environment to regulate MCP-1 expression; however,we failed to see any effect of ciclesonide on NF-�B Ser536

Fig. 5. Ciclesonide does not affect I�B degrada-tion but does inhibit NF-�B binding to its consen-sus sequence. A: human ASM cells were prein-cubated for 30 min with ciclesonide and thenincubated for 5 and 15 min with or withoutTNF� (0.1 ng/ml) or IL-1� (0.1 ng/ml). Cellswere lysed and run on a Western blot. Mem-brane was probed for phosphorylated andtotal I�B. A representative blot of 3 indepen-dent experiments in 3 donor cell lines isshown. B and C: Western blots for phosphor-ylated and total I�B were scanned, and den-sitometry was performed for TNF�- and IL-1�-stimulated samples. Values are means SE (n � 3). D and E: 50–60% confluenthuman ASM cells were transfected for 16 hwith NF-�B reporter plasmid (0.4 �g/well),pretreated for 30 min with ciclesonide (10�7

M), and then incubated for 6 h with TNF�(10 ng/ml) or IL-1� (10 ng/ml). Firefly andRenilla luciferase activity was assayed, andthe ratio of firefly to Renilla luciferase activ-ity was calculated to represent activity of thereporter. Values are means SE (n � 3).��P 0.01, ���P 0.001 vs. control. *P 0.05 vs. stimulated alone.

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phosphorylation and were unable to detect a signal for NF-�BSer276 phosphorylation.

In conclusion, we have made the novel observation thatinhibition of TNF�- and IL-1�-induced MCP-1 expressionfrom human ASM cells by the glucocorticoid ciclesonide ismediated via a transcriptional mechanism that it not dependenton I�B degradation but, rather, on the ability of TNF�- andIL-1�-induced NF-�B p65 to bind its consensus binding site.The effect of glucocorticoids on MCP-1 expression and NF-�Bsignaling is diverse, and this study provides new insight into acomplex field.

GRANTS

This work was supported by the National Institute for Health ResearchRespiratory Biomedical Research Unit, a Wellcome Trust Programme Grant,and an unrestricted grant from Altana Pharma.

AUTHOR CONTRIBUTIONS

J.K.P., R.L.C., and K.D. designed and performed the experiments, R.L.C.drafted the manuscript, A.J.K. initiated and directed the study.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

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