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Am J Physiol Lung Cell Mol Physiol 291: L11-L18, 2006. First published February 17, 2006; doi:10.1152/ajplung.00488.2005
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TRANSLATIONAL PHYSIOLOGY

Downregulation by a long-acting beta2-adrenergic receptor agonist and corticosteroid of Staphylococcus aureus-induced airway epithelial inflammatory mediator production

Konstantina Fragaki,1 Claire Kileztky,1 Chantal Trentesaux,2 Jean-Marie Zahm,1 Odile Bajolet,1 Malcolm Johnson,3 and Edith Puchelle1

INSERM, Unité Mixte de Recherches 514, Institut Fédératif de Recherche (IFR) 53, Centre Hospitalier Universitaire Maison Blanche, Reims, France; 2Laboratoire d’Onco-Pharmacologie, Jeune Equipe 2428, IFR 53, Unité de Formation et de Recherche de Pharmacie, Université de Reims Champagne-Ardenne, Reims, France; and 3GlaxoSmithKline Research and Development, Middlesex, United Kingdom

Submitted 17 November 2005 ; accepted in final form 14 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although Staphylococcus aureus is a major cause of pulmonary infection, the role played by this bacterium in the induction of inflammation of human airway epithelial cells (HAEC) is poorly understood. In this study, we investigated the inflammatory response of HAEC to S. aureus soluble virulence factors and demonstrate that the combination of a long-acting beta2-adrenergic receptor agonist (salmeterol) with a glucocorticoid (fluticasone propionate) has an anti-inflammatory effect on HAEC. First, we demonstrate increased expression at both the mRNA and protein levels of interleukin (IL)-8, IL-6, and tumor necrosis factor (TNF)-{alpha} following incubation of HAEC in the presence of S. aureus soluble virulence factors and the increase of 1) the free nuclear factor-{kappa}B (NF-{kappa}B) and activator protein-1 (AP-1) activities and 2) the phosphorylated (P-) extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2), the P-c-Jun NH2-terminal kinase (JNK), and the P-isoform-{alpha} of the NF-{kappa}B inhibitor (I{kappa}B{alpha}). Next, when HAEC were preincubated with the combination of salmeterol and fluticasone propionate, the inflammatory response of HAEC was markedly attenuated in that levels of IL-8, IL-6, TNF-{alpha}, NF-{kappa}B, AP-1, P-ERK1/ERK2, P-JNK, and P-I{kappa}B{alpha} decreased significantly. These data emphasize the deleterious effect of S. aureus soluble virulence factors and suggest that the combination of a beta2-adrenergic receptor agonist with a glucocorticoid may attenuate the associated airway epithelial inflammation.

airway inflammation; proinflammatory cytokines; nuclear fator-{kappa}B and activator protein-1 activation; glucocorticoid; bacterial virulence factors

STAPHYLOCOCCUS AUREUS is one of the most common gram-positive bacterial pathogens, involved in airway infections, either primary or subsequent to viral diseases (17). S. aureus is also a major cause of hospital-acquired lower respiratory tract infections and is often implicated in early infectious airway disease in cystic fibrosis patients (2, 5, 6, 22). S. aureus expresses several potential virulence factors (VF) that may induce airway epithelial injury, inactivate host defense mechanisms, and impair the epithelial wound/repair process. The increasing emergence of methicillin-resistant S. aureus requires a better understanding of staphylococcal pathogenesis in infectious and inflammatory airway diseases. S. aureus surface proteins such as adhesins and protein A are produced during exponential growth phase, whereas most exoproteins, including toxins, hemolysins, and tissue-degrading enzymes, are secreted during the stationary phase. Alpha-toxin, one of the major soluble VF of S. aureus, is able to induce transient apoptosis followed by the necrosis of human airway epithelial cells (HAEC) (9). We have also recently shown that live S. aureus and more predominantly soluble VF in contact with HAEC induce marked alterations in the transcriptional expression profile of HAEC associated with activation of the NF-{kappa}B and activator protein (AP)-1 pathway, upregulation of PGE2 and cyclooxygenase (COX)-2 expression and proinflammatory cytokine release such as IL-1beta and IL-8 (23). The role of the airway inflammation in the pathogenesis of chronic obstructive pulmonary diseases is well known, but the specific effects of S. aureus VF on the regulation of the inflammation have not been well defined.

We recently reported that airway epithelial integrity is protected from Pseudomonas aeruginosa VF by a long-acting beta2-adrenergic receptor (beta2-AR) agonist (salmeterol), which is able to induce a time-dependent increase in zonula occludens (ZO)-1 protein (8) and that beta2-AR activation regulates cystic fibrosis transmembrane conductance regulator chloride channel expression, which is critical for normal functioning of epithelial cells (27). Moreover, it has been clearly demonstrated that corticosteroids control genes encoding inflammatory cytokines and adhesion molecules in respiratory epithelial cells (3, 7, 16, 26). Fluticasone propionate (FP) attenuates airway epithelial inflammation through its capacity to reduce NF-{kappa}B deoxyribonucleic binding, activated by Pseudomonas aeruginosa lipopolysaccharide (LPS) stimulation (13), but it is not known whether this is also the case for S. aureus.

We therefore explored the effect of the S. aureus VF on airway epithelial inflammation, and we analyzed the activity of a beta2-AR agonist (salmeterol) and a glucocorticoid (FP) either alone or in combination on HAEC, in basal conditions and after stimulation by soluble S. aureus VF. We found that S. aureus VF induce a strong upregulation of proinflammatory cytokines and activation of NF-{kappa}B, AP-1, phosphorylated (P)-ERK1/ERK2, P-JNK, and P-I{kappa}B{alpha}. Moreover, we demonstrate, for the first time, that the combination of salmeterol and FP induces downregulation of proinflammatory cytokines and a reduction of 1) NF-{kappa}B and AP-1 transcription and 2) P-ERK1/ERK2, P-JNK, and P-I{kappa}B{alpha}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Culture of airway epithelial cells. The transformed human tracheal gland cell line MM-39 (21) was cultured at 37°C under 5% CO2 atmosphere on culture plates coated with 2% type I collagen (cells were seeded at a density of 105 cells/cm2). Cells were grown in Dulbecco's modified Eagle's medium (DMEM)/F-12 (Sigma Aldrich, St. Louis, MO) supplemented with 1% Ultroser G serum substitute (Biosepra; Villeneuve, La Garenne, France), glucose (10 g/l), sodium pyruvate (0.33 g/l), penicillin (100 IU/ml), streptomycin (100 µg/ml), and amphotericin B (2 µg/ml). Experiments were performed at confluence.

Preparation of bacterial culture supernatant. Staphylococcus aureus strain 8325-4, a wild-type laboratory strain [fibronectin-binding protein (FnBP) A+ and FnBPB+, NCTC 8325 cured of prophages], was a generous gift from T. J. Foster (Department of Microbiology, Trinity College, Dublin, Ireland). Bacterial supernatant was prepared by growing bacteria in tryptocasein soja medium (TSB; AES Laboratoire, Bruz, France) for 16–18 h at 37°C under agitation (120 rpm). Supernatant of 5 x 108 cfu/ml at stationary phase was obtained by centrifugation at 960 g for 10 min at 4°C, followed by filtration through a 0.2-µm filter (Pall Gelman Science, Ann Arbor, MI). The supernatant corresponding to S. aureus soluble VF was diluted at 20% in DMEM/F-12. TSB medium was used as control at a 20% dilution in DMEM/F-12.

Preparation of salmeterol hydroxynaphthoate. Salmeterol hydroxynaphthoate (Sal), a generous gift from GlaxoSmithKline (Uxbridge, UK), was dissolved in a minimum amount of glacial acetic acid (30 µl), then diluted at a concentration of 2 x 10–4 M in phosphate-buffered saline (PBS), and kept at –20°C. The stock solution was used at a final concentration of 2 x 10–7 M in DMEM/F-12 previously defined as optimal for inducing airway epithelial cytoprotection (12). Solutions were buffered to a pH of 7.4.

Preparation of FP. A stock solution of FP generously provided by GlaxoSmithKline was prepared (1 x 10–5 M) in 1 mM ethanol (Merck Eurolab, Darmstadt, Germany). FP was further diluted with DMEM/F-12 medium to final concentration of 1 x 10–8 M previously defined as relevant for inducing anti-inflammatory effects in bronchial epithelial cells (13).

Protein immunolocalization by immunofluorescence. MM-39 cells were seeded onto glass slides coated with 2% of type I collagen. For beta2-AR immunolocalization, MM-39 monolayers at confluence were fixed with methanol for 10 min at –20°C. Coverslips were then saturated for 30 min with 3% bovine serum albumin (BSA) in PBS. Cells were successively (after PBS wash) incubated for 1 h with anti-beta2-AR (1:10; Santa Cruz Biotechnology, Santa Cruz, CA), biotinylated donkey anti-rabbit antibody (1:50; Amersham, Aylesbury, UK), and Alexa Fluor 594-conjugated streptavidin (1:100; Molecular Probes, Eugene, OR). After incubation with the different antibodies, cells were incubated for 5 min with chromomycin A3 (1:10, Sigma Aldrich) for nucleus staining and then mounted with Aquapolymount antifading solution (Polysciences, Warrington, PA) onto glass slides. beta2-AR-stained slides were observed under an Axiophot fluorescence microscope (Zeiss, Le Pecq, France) at a magnification of x40.

For the glucocorticoid receptor (GR) immunolocalization, MM-39 monolayers were fixed in 2% paraformaldehyde, washed in PBS, treated with 3% BSA, washed in PBS, and incubated overnight with a 1:8,000 dilution of an anti-GR specific antibody (Ab57), a generous gift from J. A. Cidlowski (Laboratory of Signal Transduction, National Institute of Environmental Health Sciences). The following day, the slides were incubated overnight at 4°C with an Alexa Fluor 488 goat anti-rabbit IgG antibody (Molecular Probes) (24). Cells were incubated for 10 min with 4',6-diamidino-2-phenylindole (1 µg/ml) for nucleus staining and then mounted with Aquapolymount antifading solution (Polysciences) onto glass slides. GR-stained slides were observed under an Axiophot fluorescence microscope (Zeiss) at x40 magnification. Negative controls were obtained either by using specific IgG as the primary antibody or by omitting the primary antibody incubation.

Fluorescence images of the GR and nucleus staining were recorded with a charge-coupled device video camera (Coolsnap; Roper Scientific, Tucson, AZ). The same view of the preparation was used for both images, which were acquired at 488 and 360 nm excitation wavelength for the GR receptor staining and the nucleus staining, respectively. Metamorph (Universal Imaging, Sunnyvale, CA) software was used to quantify regions of overlap of the two fluorescent probes. Both source images were thresholded, and we calculated the areas of overlap of the nuclear staining and of the GR receptor staining. We determined the areas of overlap by calculating the number of pixels of the nuclear staining overlapping with the GR receptor staining. Data are expressed in percentage of pixel overlap.

RNA extractions and RT-PCR analyses. RNA extraction of MM-39 cells was performed with the High Pure RNA isolation kit as recommended by the manufacturer (Roche Diagnostics).

RT-PCR was performed with 10 ng of total RNA using the GeneAmp Thermostable RNA PCR Kit (Perkin Elmer, Foster City, CA) and three pairs of oligonucleotides (Eurogentec, Seraing, Belgium). Forward and reverse primers for human IL-8, IL-6, TNF-{alpha}, and 28S were designed as follows: IL-8 primers, forward 5'-gccaaggagtgctaaagaacttag-3', reverse 5'-gaattctcagccctcttcaaaaac-3'; IL-6 primers, forward 5'-gccagagctgtgcagatgagta-3', reverse 5'-gctacatttgccgaagagccct-3'; TNF-{alpha} primers, forward 5'-cagcctcttctccttcctga-3', reverse 5'-tgaggtacaggccctctgat-3'; and 28 S primers, forward 5'-gttcacccactaatagggaacgtga-3', reverse 5'-ggattctgacttagaggcgttcagt-3'. For the IL-8 PCR, an initial denaturation at 95°C for 2 min was followed by 25 amplification cycles (denaturation at 94°C for 15 s, annealing at 60°C for 20 s, elongation at 72°C for 10 s, and a final 2-min elongation at 72°C). For the IL-6 PCR, an initial denaturation at 95°C for 2 min was followed by 23 amplification cycles (denaturation at 94°C for 15 s, annealing at 60°C for 20 s, and elongation at 72°C for 10 s) and a final 2-min elongation at 72°C. For the TNF-{alpha} PCR, the conditions were as follows: initial denaturation (94°C, 2 min), 29 amplification cycles (denaturation 94°C, 30 s; annealing 59°C, 30 s; and elongation 72°C, 30 s; and final elongation 72°C, 7 min). For the 28S PCR, the conditions were as follows: initial denaturation (95°C, 2 min), 13 amplification cycles (denaturation 94°C, 15 s, annealing 66°C, 20 s, and elongation 72°C, 10 s), final elongation (72°C, 2 min). The expected sizes of the transcripts of IL-8, IL-6, TNF-{alpha}, and 28S were 222, 223, 302, and 212 bp, respectively. RT-PCR products were separated by acrylamide gel electrophoresis, stained with SYBR gold (Molecular Probes), and visualized by fluorimetric scanning (Fuji, LAS-1,000, Raytest). The IL-8, IL-6, and TNF-{alpha}, mRNA values were normalized to 28S mRNA values. Results represent means ± SD of three independent experiments performed in duplicate.

Enzyme-linked immunoabsorbent assays and total protein extractions. The enzyme-linked immunoabsorbent assays (ELISA) for IL-8, IL-6, and high-sensitivity TNF-{alpha} detection in the culture supernatants of MM-39 cells were performed by following the manufacturer's instructions (R&D Systems Europe, Abington, UK). Data were expressed as pg/ml. Results represent means ± SD of five independent experiments performed in duplicate. According to the manufacturer's manual, the limits of detection for IL-8, IL-6, and TNF-{alpha} are 3.5, 0.70, and 0.12 pg/ml, respectively.

The ELISA test for the detection of the intracellular P-ERK1/ERK2 and P-JNK were performed following the manufacturer's instructions (R&D Systems Europe) in total MM-39 cell protein extracts, prepared by scraping the MM-39 cells into radioimmunoprecipitation assay buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% Igepal (vol/vol), 1% sodium deoxycholate (wt/vol), 1% Triton X-100 (vol/vol), 5 mM iodoacetamide, and 0.1% sodium dodecyl sulfate (SDS, wt/vol)] containing a complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Protein was quantified by BC assay protein quantitation kit (Interchim, Montluçon, France). Data are expressed as ng·ml–1·total proteins–1. Results represent means ± SD of three independent experiments.

The ELISA test for the detection of the intracellular P-I{kappa}B{alpha} was performed following the manufacturer's instructions (Biosource International, Camarillo, CA) in total MM-39 cell protein extracts. Data are expressed as units·ml–1·total proteins–1. Results represent means ± SD of three independent experiments.

NF-{kappa}B and AP-1 family-activated transcription factors assays. Nuclear protein extracts of MM-39 cells were prepared by a procedure slightly modified from that of Schreiber et al. (25). In brief, 4 x 106 cells were pelleted by centrifugation at 1,500 g for 5 min, washed four times with 10 ml of PBS, and then resuspended in 400 µl of cold buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, and 1 mM DTT) containing a complete protease inhibitor cocktail (Roche Diagnostics) by gentle pipetting. The cells were then incubated on ice for 15 min; thereafter 25 µl of a 10% solution of Nonidet P-40 (Fluka) was added, and the tube was vigorously vortexed for 10 s. The homogenate was centrifuged at 10,000 g for 30 s. The nuclear pellet was resuspended in 50 µl of ice-cold buffer B (20 mM HEPES pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 1 mM DTT) containing a complete protease inhibitor cocktail, and the tube was vigorously rocked at 4°C for 15 min on a shaking platform. The nuclear extract was centrifuged at 12,000 g for 5 min at 4°C, and the supernatant (55 µl) was frozen in 5-µl aliquots at –80°C.

Detection and quantitation of the activated subunits of the NF-{kappa}B family (p65 and p50) and of the AP-1 family (c-Fos and JunB) were conducted by specific transcription factor assays, performed following the manufacturer's instructions (Active Motif, Rixensart, Belgium). In brief, we incubated nuclear extracts (2 µg, analyzed by BC assay protein quantitation kit; Interchim, Montluçon, France) for 1 h with mild agitation in a 96-well plate on which oligonucleotide containing 1) the NF-{kappa}B consensus site 5'-gggactttcc-3' and 2) the TRE 5'-TGAGTCA -3' had been immobilized. Only the active forms of NF-{kappa}B and AP-1, respectively, contained in nuclear extracts specifically bind to the oligonucleotides. After three washes with 1x final volume-wash buffer, primary antibodies (1:1,000 diluted) were added for 1 h to detect specific epitopes on 1) p65 and p50, which are accessible only when NF-{kappa}B is activated and bound to its target DNA and 2) c-Fos and JunB proteins upon AP-1 DNA binding. After three washes, a horseradish peroxidase-conjugated antibody (1:1,000 diluted) was added for 1 h; then, after four washes, colorimetric reaction was effectuated by adding developing solution. Absorbance was read on a spectrophotometer at 450 nm after stopping of the reaction. Controls were obtained by replacing nuclear extracts by cell-lysis buffer. Data are expressed as 450-nm optical density. Results represent means ± SD of three independent experiments.

Cell viability. The viability of MM-39 cells, either in basal conditions or after incubation with Sal, FP, and S. aureus soluble VF, was determined by the dimethylthiazole-2,5-diphenyltetrazolium bromide (MTT) viability assay (10). Cells were incubated with 1 mg/ml MTT in culture medium for 1 h. Supernatants were then removed, and cells were treated with isopropanol to dissolve the formazan crystals formed in alive metabolically active cells. The percentage of viability of S. aureus-treated epithelial cells was calculated by determining the absorbance of treated and untreated cells with an automatic microplate scanning spectrophotometer (Xenius Safas, Monaco). Results represent means ± SD of three independent experiments performed in triplicate.

Statistical analyses. Results are expressed as means ± SD. Each data point was confirmed in duplicate at least, and each cell culture experiment was performed at least three times. Differences in IL-8, IL-6, TNF-{alpha}, NF-{kappa}B, and AP-1 family subunits, P-ERK1/ERK2, P-JNK, and P-I{kappa}B{alpha}, as well as GR staining, were analyzed by the Student's t-test and by a one-way ANOVA. A P value <0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of S. aureus soluble VF on gene expression and protein release of inflammatory cytokines. To evaluate the effect of S. aureus VF on inflammation, we measured IL-8, IL-6, and TNF-{alpha} using a specific semiquantitative RT-PCR, on mRNA of MM-39 cells, either unstimulated or stimulated by S. aureus VF for 0.5, 1, 2, and 3 h. No difference in the IL-8, IL-6, and TNF-{alpha} transcript expression was detected after 0.5 h of interaction between unstimulated and S. aureus-stimulated cells. As early as 1 h of interaction with S. aureus VF, IL-8, IL-6, and TNF-{alpha} mRNA expression was significantly (P < 0.001) increased (IL-8 +1,281%, IL-6 +885%, and TNF-{alpha} +515%) in MM-39 cells compared with unstimulated cells (Fig. 1, A, B, and C, respectively). This increased expression was maintained up to 3 h.


Figure 1
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  		Fig. 1. RT-PCR semiquantitative analysis and ELISA analysis of IL-8, IL-6, and TNF-{alpha} mRNA in MM-39 airway epithelial cells after stimulation with Staphylococcus aureus soluble virulence factors (VF). A significant increase in IL-8 (A), IL-6 (B), and TNF-{alpha} (C) mRNA levels was observed after 1-h stimulation with 20% S. aureus soluble VF compared with control (unstimulated) cell values (C). IL-8, IL-6, and TNF-{alpha} mRNA values were normalized to 28S mRNA values. Results represent means ± SD of 3 independent experiments performed in duplicate. A significant increase in IL-8 (D), IL-6 (E), and TNF-{alpha} (F) protein release was observed after 3-h stimulation with 20% S. aureus soluble VF compared with control (unstimulated) cell values. Data are expressed as pg/ml. Results represent means ± SD of 5 independent experiments performed in duplicate. Differences in IL-8, IL-6, and TNF-{alpha} were analyzed by the Student's t-test and by a 1-way ANOVA. **P < 0.01, ***P < 0.001 compared with control (unstimulated) cells.

 
We then investigated the release of IL-8, IL-6, and TNF-{alpha} by ELISA test on culture supernatants of MM-39 cells, either unstimulated or stimulated by S. aureus VF for 0.5, 1, 2, and 3 h. We observed a progressive increase in IL-8, IL-6, and TNF-{alpha} release as early as 2 h of interaction that was maximal after 3 h (Fig. 1, D, E, and F, respectively). IL-8, IL-6, and TNF-{alpha} release was significantly (IL-8, IL-6 P < 0.001; TNF-{alpha} P < 0.01) higher in MM-39 cells with S. aureus VF (IL-8 +2,046%; IL-6 +3,034%; TNF-{alpha} +4,140%) compared with unstimulated cells.

Effect of S. aureus soluble VF on NF-{kappa}B and AP-1 activities and on P-ERK1/ERK2, P-JNK, and P-I{kappa}B{alpha}. We then addressed the question whether S. aureus VF had an effect on NF-{kappa}B and AP-1 activities. We first studied the early (5 min) activation by S. aureus VF of the p65 and p50 subunits of NF-{kappa}B by specific transcription factor assays. A 5-min exposure of MM-39 cells with S. aureus VF led to a significant upregulation of p65 (+441%, P < 0.001) and p50 (+1,183%, P < 0.001) activities compared with unstimulated cells (Fig. 2, A and B, respectively), which was still observed after 3 h (data not shown). We also studied the early (5 min) activation by S. aureus VF of the c-Fos and JunB subunits of AP-1 by specific transcription factor assays. A 5-min exposure of MM-39 cells with S. aureus VF led to a significant upregulation of c-Fos (+1,212%, P < 0.001) and JunB (+352%, P < 0.001) activities compared with unstimulated cells (Fig. 2, C and D, respectively), which was still observed after 3 h (data not shown).


Figure 2
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  		Fig. 2. NF-{kappa}B (p65 and p50) and activator protein (AP)-1 (c-Fos and JunB) binding activities and ELISA analysis of phosphorylated (P-) ERK1/ERK2, P-JNK, and P-I{kappa}B{alpha} in MM-39 airway epithelial cells after stimulation with S. aureus soluble VF. A significant increase in p65 (A), p50 (B), c-Fos (C), and JunB (D) binding activities was observed after 5-min stimulation with 20% S. aureus soluble VF compared with the control (unstimulated) cell values. Transcription factor assay data (A–D) are expressed as 450 nm optical density (OD), and results represent means ± SD of 4 independent experiments. A significant increase in P-ERK1/ERK2 (E), P-JNK (F), and P-I{kappa}B{alpha} (G) was observed after 5-min stimulation with 20% S. aureus soluble VF compared with the control (unstimulated) cell values. P-ERK1/ERK2 and P-JNK data are expressed as ng·ml–1·total proteins–1. P-I{kappa}B{alpha} data are expressed as units·ml–1·total proteins–1. Results represent means ± SD of 3 independent experiments. Differences in p65, p50, c-Fos, JunB, P-ERK1/ERK2, P-JNK, and P-I{kappa}B{alpha} were analyzed by Student's t-test and by 1-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control (unstimulated) cells.

 
To examine the signaling pathways leading to the NF-{kappa}B and AP-1 activation, we performed specific ELISA tests detecting the P-ERK1/ERK2, P-JNK, and P-I{kappa}B{alpha} on total protein extracts of MM-39 cells, either unstimulated or stimulated by S. aureus VF for 5 min. We observed a significant increase of the P-ERK1/ERK2 (+3,411%, P < 0.05), P-JNK (+624%, P < 0.01), and P-I{kappa}B{alpha} (+190%, P < 0.01) compared with unstimulated cells (Fig. 2, E, F, and G, respectively), which was still observed after 3 h (data not shown).

Distribution of glucocorticoid and beta2-AR on MM-39 cells under basal conditions and after Sal and FP preincubation. We examined the localization and distribution of glucocorticoid and beta2-AR in MM-39 cells by in situ immunolabeling on the entire cell culture seeded onto glass slides coated with collagen. The pattern of staining of the GR (green staining) was essentially cytoplasmic in unstimulated and 16-h Sal (2 x 10–7 M)-stimulated cells (Fig. 3, A and B, respectively). In contrast, the GR was essentially nuclear after 16 h of FP (1 x 10–8 M) alone (+35.0% compared with unstimulated cells, P < 0.05), and nuclear staining was more intense when FP was combined with Sal (+52.4% compared with unstimulated cells, P < 0.001) (Fig. 3, C and D, respectively). beta2-AR distribution revealed bright red staining over the cell membrane surface, suggesting that the receptor was mainly localized in the plasma membrane (Fig. 3, E) in unstimulated cells. No modification of its expression was observed after a 16-h preincubation with Sal and FP either alone or in combination (data not shown).


Figure 3
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  		Fig. 3. Localization and distribution of beta2-adrenergic receptors (AR) and glucocorticoid receptors (GR) in MM-39 airway epithelial cells. The GR (green staining) localization was mainly cytoplasmic and slightly nuclear in unstimulated and in 16-h salmeterol hydroxynaphthoate (Sal, 2 x 10–7 M)-stimulated cells (A and B, respectively). In contrast, the GR was essentially nuclear in 16-h fluticasone propionate (FP, 1 x 10–8M) alone, and the nuclear labeling was homogenous and more marked when FP was combined with Sal (C and D, respectively). beta2-AR distribution revealed bright red staining over the cell membrane surface (E) in unstimulated cells, and cell nuclei were identified with chromomycin A3 (green). F: Nomarski picture of MM-39 unstimulated cells. The results are representative of 3 independent experiments. Differences in GR staining were analyzed by Student's t-test and by 1-way ANOVA. Bars = 17 µm.

 
Effect of Sal and FP on gene expression of inflammatory cytokines after S. aureus soluble VF stimulation. To analyze whether Sal and FP could inhibit the epithelial inflammation due to S. aureus VF, we performed a 16-h pretreatment of Sal and FP, either alone or combined, of MM-39 cells, followed by 0.5-, 1-, 2-, and 3-h S. aureus VF stimulation. We then carried out an IL-8-, IL-6-, and TNF-{alpha}-specific semiquantitative RT-PCR on mRNA of MM-39 cells. No difference in IL-8, IL-6, and TNF-{alpha} transcript expression was detected after 0.5 h of interaction between unstimulated or S. aureus-stimulated cells.

As shown in Fig. 4, A–C, after 1 h of interaction Sal alone did not significantly alter S. aureus VF-induced IL-8, IL-6, and TNF-{alpha} mRNA expression. FP alone significantly reduced S. aureus VF-induced IL-8 (–62%, P < 0.01) and IL-6 (–57%, P < 0.001) mRNA expression (Fig. 4, A and B, respectively). Interestingly, the combination of Sal and FP significantly reduced S. aureus VF-induced IL-6 (–57%, P < 0.001) and TNF-{alpha} (–46%, P < 0.05) mRNA expression (Fig. 4, B and C, respectively). This effect was synergistic for IL-8 (–72%, P < 0.001) mRNA expression (Fig. 4A).


Figure 4
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  		Fig. 4. RT-PCR semiquantitative analysis of IL-8, IL-6, and TNF-{alpha} mRNA expression in MM-39 airway epithelial cells before treatment and pretreated with Sal and FP and then incubated with S. aureus soluble VF. When Sal was administered alone (16 h, 2 x 10–7 M), we observed no significant decrease of the 1-h VF-induced IL-8, IL-6, and TNF-{alpha} mRNA expression (A, B, and C, respectively). FP alone (16 h, 1 x 10–8 M) significantly reduced VF-induced IL-8 and IL-6 mRNA expression (A and B, respectively). Sal and FP in combination significantly reduced VF-induced IL-6 and TNF-{alpha} (B and C, respectively) and induced a synergistic decrease of IL-8 mRNA expression (A). IL-8, IL-6, and TNF-{alpha} mRNA values were normalized to 28S mRNA values. Results represent means ± SD of 3 independent experiments performed in duplicate. Differences in IL-8, IL-6, and TNF-{alpha} were analyzed by Student's t-test and by 1-way ANOVA. The treatment effects were analyzed by reference to the VF-stimulated cells (*P < 0.05, **P < 0.01, ***P < 0.001).

 
Effect of Sal and FP on protein release of inflammatory cytokines after S. aureus soluble VF stimulation. To complete our study on the anti-inflammatory effect of Sal and FP on HAEC after 0.5-, 1-, 2-, and 3-h S. aureus stimulation, we analyzed IL-8, IL-6, and TNF-{alpha} by a specific ELISA test on culture supernatants of MM-39 cells. We observed a progressive decrease of these three chemokines after 2 h of interaction that was maximal after 3 h.

Figure 5, A–C, shows the results obtained after a 3-h period of S. aureus incubation. No significant alteration was observed after Sal treatment alone in S. aureus VF-induced IL-8, IL-6, and TNF-{alpha} release, respectively. In contrast, FP alone significantly reduced S. aureus VF-induced IL-8 (–50%, P < 0.01) and TNF-{alpha} (–74%, P < 0.05) release (Fig. 5, A and C, respectively). Interestingly, the Sal and FP combination significantly reduced S. aureus VF-induced IL-8 (–53%, P < 0.01) release and induced a synergistic decrease in IL-6 (–52%, P < 0.05) and an additive decrease in TNF-{alpha} (–98%, P < 0.01) release (Fig. 5, A, B, and C, respectively). TNF-{alpha} release after Sal/FP combined pretreatment exhibits a value similar to control cells, demonstrating the marked anti-inflammatory effect of the Sal/FP combination (Fig. 5C).


Figure 5
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  		Fig. 5. ELISA analysis of IL-8, IL-6, and TNF-{alpha} protein release in MM-39 airway epithelial cells before treatment and pretreated with Sal and FP and then incubated with S. aureus soluble VF. When Sal was administered alone, we observed no significant decrease of the 3-h VF-induced IL-8, IL-6, and TNF-{alpha} protein release (A, B, and C, respectively). FP alone significantly reduced the VF-induced IL-8 (A) and TNF-{alpha} (C) protein release. Sal and FP in combination significantly reduced VF-induced IL-8 and induced a synergistic decrease in IL-6 and an additive decrease in TNF-{alpha} (A, B, and C, respectively). Data are expressed as pg/ml. Results represent means ± SD of 5 independent experiments performed in duplicate. Differences in IL-8, IL-6, and TNF-{alpha} were analyzed by Student's t-test and by 1-way ANOVA. The treatment effects were analyzed by reference to the VF-stimulated cells (*P < 0.05, **P < 0.01).

 
Effect of Sal and FP on NF-{kappa}B and AP-1 activities and on P-ERK1/ERK2, P-JNK, and P-I{kappa}B{alpha}. To analyze whether the Sal and FP anti-inflammatory effect could be regulated by NF-{kappa}B and/or AP-1, we studied the following in MM-39 cell nuclear protein extracts: 1) the p65 and p50 subunits of NF-{kappa}B family activities and 2) the c-Fos and JunB subunits of AP-1 family activities, using specific transcription factor assays.

Figure 6, A–C, shows the results obtained after a 5-min period of S. aureus VF incubation in p65, p50, and JunB activities, respectively. No significant alteration in S. aureus VF-induced p65, p50, and JunB was observed after Sal or FP treatment alone. In contrast, Sal and FP in combination significantly and synergistically reduced the 5-min S. aureus VF-induced p65 (–21%, P < 0.01, Fig. 6A) and p50 (–26%, P < 0.05, Fig. 6B) activities, which were still observed after 3 h (data not shown). The Sal/FP combination also significantly reduced the 5-min S. aureus VF-induced JunB activity (–30%, P < 0.05, Fig. 6C). No significant alteration of 5-min S. aureus VF-induced c-Fos activity was observed following Sal and FP treatment either alone or used in combination (data not shown).


Figure 6
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  		Fig. 6. NF-{kappa}B (p65 and p50) and AP-1 (JunB) binding activities and ELISA analysis of P-ERK1/ERK2, P-JNK, and P-I{kappa}B{alpha} in MM-39 airway epithelial cells before treatment and pretreated with Sal and FP and then incubated with S. aureus soluble VF. When Sal and FP were administered alone, we observed no significant decrease of the 5-min VF-induced p65, p50, and JunB transcription factor activities (A, B, and C, respectively). Sal and FP in combination significantly reduced the 5-min VF-induced p65, p50, and JunB transcription factor activities (A, B, and C, respectively). Data are expressed as 450-nm OD, and results represent means ± SD of 4 independent experiments. When Sal and FP were administered alone, we observed no significant decrease of the 5-min VF-induced P-ERK1/ERK2, P-JNK, and P-I{kappa}B{alpha} (D, E, and F, respectively). Sal and FP in combination significantly reduced the 5-min VF-induced P-ERK1/ERK2 and P-I{kappa}B{alpha} (D and F, respectively) and induced a synergistic decrease of the 5-min VF-induced P-JNK (E). P-ERK1/ERK2 and P-JNK data are expressed as ng·ml–1·total proteins–1. P-I{kappa}B{alpha} data are expressed as units·ml–1·total proteins–1. Results represent means ± SD of 3 independent experiments. Differences in p65, p50, JunB, P-ERK1/ERK2, P-JNK, and P-I{kappa}B{alpha} were analyzed by Student's t-test and by 1-way ANOVA. The treatment effects were analyzed by reference to the VF-stimulated cells (*P < 0.05, **P < 0.01).

 
To get more mechanistic information about the underlying mechanisms leading to the Sal- and FP-dependent NF-{kappa}B and AP-1 decrease after early (5 min) S. aureus stimulation, we analyzed P-ERK1/ERK2, P-JNK, and P-I{kappa}B{alpha} on total protein extracts of MM-39 cells by specific ELISA tests (Fig. 6, D, E, and F, respectively). No significant alteration was observed after Sal or FP treatment alone in 5-min S. aureus VF-induced P-ERK1/ERK2, P-JNK, and P-I{kappa}B{alpha}, respectively. In contrast, the Sal and FP combination significantly reduced 1) the 5-min S. aureus VF-induced P-ERK1/ERK2 (–98%, P < 0.05, Fig. 6D), which was still observed after 3 h (data not shown), and 2) the 5-min S. aureus VF-induced P-I{kappa}B{alpha} (–39%, P < 0.01, Fig. 6F). The Sal and FP combination also reduced significantly and synergistically the 5-min S. aureus VF-induced P-JNK (–48%, P < 0.05, Fig. 6E).

In parallel, we studied the Sal/FP anti-inflammatory effect after a longer (up to 3 h) S. aureus VF activation (data not shown). FP alone shows a significant downregulation of P-JNK (–28%) after 1 h of S. aureus activation and P-I{kappa}B{alpha} (–20%), P-ERK1/ERK2 (–74%), and NF-{kappa}B p65 subunit (–15%) after 3 h.

Cell viability. Cell viability after 16 h of treatment with Sal, FP, or their combination followed or not by a 0.5-, 1-, 2-, or 3-h treatment with 20% S. aureus soluble VF was consistently >95% (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although S. aureus is a major human pathogen associated with diverse types of local and systemic infections characterized by inflammation dominated by polymorphonuclear leukocyte (14), its role on the activation of proinflammatory molecule expression and release in the airway epithelium is poorly documented. On the basis of earlier studies comparing the transcription profile of airway epithelial cells, after contact either with live S. aureus or with their soluble VF present in the stationary-phase culture supernatants (23), we chose to select a 20% S. aureus supernatant bacterial stimulation condition that has been shown to be able to enhance transcription of proinflammatory molecules.

In the present study, we first demonstrate that S. aureus VF induce a strong upregulation of proinflammatory molecules such as IL-8, IL-6, and TNF-{alpha} at mRNA and protein levels and early (5 min) activations of 1) NF-{kappa}B and AP-1 transcription and 2) P-ERK1/ERK2, P-JNK [members of the mitogen-activated protein (MAP) kinase superfamily], and P-I{kappa}B{alpha} that persist up to 3 h. The transcriptional regulation of proinflammatory cytokine expression in lung epithelial cells is complex. The transcriptional regulation of IL-8 expression has been described as involving NF-{kappa}B, AP-1, and NF for IL-6 (NF-IL-6) promoter sequences (19, 20). TNF-{alpha}-induced transcription from the IL-8 promoter activates the classical NF-{kappa}B pathway in the A549 airway cell line (4).

Activation of NF-{kappa}B in airway epithelial cells with upregulation of proinflammatory molecules such as IL-8, IL-6, and TNF-{alpha} has been reported after epithelial cell interaction with several types of bacteria, either gram positive or gram negative (3, 11, 15, 26). These cytokines may be stimulated by NF-{kappa}B and are also potent NF-{kappa}B inducers that may amplify the inflammatory response and lead to chronic inflammation (interestingly, the NF-{kappa}B DNA-binding persists up to 3 h after infection). An important question is how S. aureus signals reach the nucleus and activate target genes. We suggest that 1) the NF-{kappa}B complex (especially the p65 and p50 subunits) is activated by the JNK and IKK pathway and 2) the AP-1 complex (especially the c-Fos and JunB subunits) is stimulated by a process mediated by ERK1 and two MAP kinases. Both the activated NF-{kappa}B and AP-1 then enhance the upregulation of inflammatory molecule genes, leading to their extracellular release.

Taking our results together, we anticipate that upregulation of inflammatory cytokines following S. aureus infection could benefit from the combination of a beta2-AR agonist (Sal) and a glucocorticoid (FP). FP has been shown to exert an anti-inflammatory effect by blocking NF-{kappa}B activation via a reduction of IKK after P. aeruginosa LPS stimulation activity (13) and is widely used clinically as an anti-inflammatory immunosuppressive drug (28). Sal has been recently reported to exert an anti-inflammatory effect in the pulmonary compartment of healthy volunteers after inhalation of LPS (18). It has also been shown to have a cytoprotective effect by increasing the protein expression of ZO-1 and to reduce the damage of respiratory epithelium after P. aeruginosa infection (8). Furthermore, there is a complementary effect of these drugs since long-acting beta2-AR agonists are capable of activating the GR through an MAP kinase cascade (1). It has been also reported that glucocorticoids induce an increased beta2-AR expression associated with an increased secretion of glandular serous origin antibacterial proteins such as lysozyme and lactoferrin (1). We confirm, in the present study, that the combination of Sal and FP induced a marked increase in the nuclear GR expression of airway epithelial cells, and we demonstrate that this drug combination induces a significant decrease of IL-8, IL-6, and TNF-{alpha}, at both transcriptional and translational levels (this effect is synergistic for TNF-{alpha} mRNA expression and IL-6 protein release), even though the effect may be somewhat more marked at the protein level, particularly for TNF-{alpha}. We suggest that Sal and FP reduce the NF-{kappa}B (p65 and p50 subunits) and AP-1 (JunB subunit) activity via 1) the reduced activation of ERK1/ERK2 and JNK pathway and 2) the reduced phosphorylated form of I{kappa}B{alpha}, the initial event targeting I{kappa}B{alpha} for degradation leading to NF-{kappa}B nucleus binding inhibition, as previously described (29).

In summary, the present data emphasize the proinflammatory effect of S. aureus soluble VF on airway epithelial cells. Moreover, we demonstrate, for the first time, that the combination of two types of drugs, a long-acting beta2-AR agonist and a corticosteroid, induces a downregulation of S. aureus-induced airway epithelial inflammation via the NF-{kappa}B and AP-1 pathway, suggesting that the combination of these two drugs could allow better control of S. aureus-driven airway inflammatory exacerbations.


   ACKNOWLEDGMENTS
 
We thank the Institut National de la Santé et de la Recherche Médicale (INSERM), GlaxoSmithKline (UK), and the Association Vaincre la Mucoviscidose (France) for their support. We also thank Dr. Azzzaq Belaaouaj (INSERM, UMRS 514, Reims) for careful reading of the manuscript and important constructive recommendations.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Puchelle, INSERM UMRS 514, Centre Hospitalier Universitaire Maison Blanche, 45, rue Cognacq Jay, 51092 Reims Cedex, France (e-mail: edith.puchelle{at}univ-reims.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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E. Puchelle, J.-M. Zahm, J.-M. Tournier, and C. Coraux
Airway Epithelial Repair, Regeneration, and Remodeling after Injury in Chronic Obstructive Pulmonary Disease
Proceedings of the ATS, November 1, 2006; 3(8): 726 - 733.
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