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Published ahead of print on June 28, 2007, doi:10.1165/rcmb.2006-0404OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 37, pp. 606-616, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0404OC

Effect of Dexamethasone and ACC on Bacteria-Induced Mucin Expression in Human Airway Mucosa

Hans-Peter Hauber1, Torsten Goldmann2, Ekkehard Vollmer2, Barbara Wollenberg3 and Peter Zabel1

1 Medical Clinic, and 2 Department of Pathology, Research Center Borstel, Borstel, Germany; and 3 ENT Department, University Hospital Schleswig Holstein Campus Lübeck, Lübeck, Germany

Correspondence and requests for reprints should be addressed to Hans-Peter Hauber, M.D., Medical Clinic, Research Center Borstel, Parkallee 35, 23845 Borstel, Germany. E-mail: hphauber{at}web.de


   Abstract
Top
 Abstract
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 References
 
Gram-negative bacteria can stimulate mucin production, but excessive mucus supports bacterial infection and consequently leads to airway obstruction. Therefore, the effect of dexamethasone (DEX) and the antioxidant acetyl-cysteine (ACC) on bacteria-induced mucus expression was investigated. Explanted human airway mucosa and mucoepidermoid cells (Calu-3) were stimulated with lipopolysaccharide (LPS) or PAM3 (a synthetic lipoprotein). DEX or ACC were added to either LPS- or PAM3-stimulated airway mucosa or Calu-3 cells. Mucin mRNA expression (MUC5AC) and total mucus glycoconjugates (mucin protein) were quantified using real-time PCR and periodic acid Schiff staining. LPS and PAM3 significantly increased mucin expression in airway mucosa and Calu-3 cells (P < 0.05). DEX alone had no significant effect on mucin expression in airway mucosa or Calu-3 cells (P > 0.05). In contrast, DEX significantly reduced LPS- and PAM3-induced mucin expression in explanted mucosal tissue and mucin expression in Calu-3 cells (P < 0.05). In explanted human airway mucosa ACC alone significantly increased mucin expression (P < 0.05). In contrast, ACC significantly decreased LPS- and PAM3-induced mucin expression (P < 0.05). In Calu-3 cells ACC alone had no significant effect on mucin expression (P > 0.05). ACC decreased LPS- and PAM3-induced mucin expression, but this effect was not significant (P > 0.05). These data suggest that DEX can effectively reduce bacteria-induced mucin expression in the airways. ACC alone may increase mucin expression in noninfected mucosa, but it decreased bacteria-induced mucin expression. Further studies are warranted to evaluate whether the effect of DEX or ACC is clinically relevant.

Key Words: acetyl-cysteine • dexamethasone • lipoprotein • lipopolysaccharide • mucin

Chronic inflammatory lung diseases (e.g., bronchial asthma, chronic obstructive pulmonary disease [COPD], and cystic fibrosis) are often associated with excessive mucus production, especially in cases of bacterial exacerbation. Mucus hypersecretion is also found in acute pulmonary infection as seen in acute bronchitis or pneumonia.

Bacteria can stimulate mucin expression via inflammation and inflammatory mediators. On the other hand, bacterial products like lipopolysaccharide (LPS) have been demonstrated to directly induce mucin gene expression in cell culture systems and animal models (1, 2). Although mucins are part of the innate immunity and help to clear bacteria from the lungs, excessive mucus production can have deleterious effects. Mucus plugs can lead to pulmonary obstruction and support bacterial colonization and infection. Moreover, in clinical practice patients often complain about coughing and mucous secretions.

Gram-negative bacteria such as Haemophilus influenzae or Pseudomonas aeruginosa can be frequently isolated from airway secretions of patients with COPD or pneumonia with exacerbations (3, 4). Previous studies in man have described signaling pathways by which LPS from gram-negative bacteria can induce mucin gene expression (5–7). In contrast, other parts of the outer cell membrane of gram-negative bacteria such as lipoproteins are less well characterized for their ability to induce mucin expression.

At present, there is no specific and good mucin-regulating agent available. In clinical practice, glucocorticosteroids are used to decrease mucus production by attenuating inflammation in the airways (8, 9). Studies have confirmed the direct inhibition of mucin gene expression by dexamethasone (DEX) in cell culture and tissue culture experiments as well (10–12). However, information on the direct effect of glucocorticosteroids on bacteria-induced mucin expression is sparse.

Acetyl-cysteine (ACC) is a commonly used mucolytic that may perhaps act as an anti-inflammatory drug and an antioxidant (13, 14). A recent study has shown ACC to be ineffective in reducing the rate of exacerbations of COPD (15). However, previous studies have shown that generation of reactive oxygen species (ROS) increased mucin gene expression by stabilization of mRNA (7, 16, 17). Therefore, we hypothesize that antioxidants may also reduce mucin expression.

The first aim of the present study was to investigate the effect of LPS and lipoprotein stimulation on mucin expression. We used explanted human airway mucosa as an ex vivo model and cell culture as an in vitro model. Our second aim was to evaluate the effect of DEX and ACC on LPS- and lipoprotein-induced mucin expression.


   MATERIAL AND METHODS
Top
 Abstract
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 References
 
Tissue Culture
Upper airway mucosa (sinus) specimens were obtained from a total of 40 nonallergic and allergic individuals (16 male, 24 female; mean age, 36 yr). Allergic tissue was taken outside the allergen season, and these patients did not receive any glucocorticosteroids. No presurgical corticosteroids were given. Tissue was resected from patients undergoing sinus surgery, who had given informed consent before the procedure, and was rinsed in medium before culture. Serial sections of tissue were placed on 0.4-µm well inserts (Millipore, Bedford, MA) in 2 ml of defined medium as described previously (18, 19) and incubated in 5% CO2/95% air. Tissue was stimulated for 24 hours as described below. After culture, tissue was fixed using the HOPE (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) technique (20).

Cell Culture
Mucus producing Calu-3 cells were grown in Eagle's Minimal Essential medium with Earl's BBS and 2 mM L-glutamine (EMEM) containing 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 1.5 g/L sodium bicarbonate, supplemented with 10% fetal bovine serum in 5% CO2/95% air. For in vitro cell culture experiments, 100,000 cells were put in 2 ml of medium per each well in 6-well plates. After reaching confluence, cells were starved for 24 hours in medium without serum and then stimulated for another 24 hours. After incubation, cells were placed on cytospins for periodic acid Schiff (PAS) staining or lysed for RNA extraction.

Stimulation with LPS and PAM3
Tissue samples and Calu-3 cells were cultured for 24 hours in medium alone and in the presence of either LPS (LPS from Salmonella friedenau H: 909; kindly provided by Dr. H. Brade) or the synthetic lipoprotein analog PAM3 (N-palmitoyl-S-(2, 3-bis(palmitoyloxy)-(2RS)-propyl)-(R)-cysteinyl-(S)-seryl(S)-lysyl-(S)-lysyl-(S)-lysyl-(S)-lysine x 3HCl; EMC Microcollections GmbH, Tübingen, Germany). For LPS, a concentration of 10 ng/ml was used for tissue samples and concentrations of 10, 50, and 200 ng/ml for cell culture experiments. For PAM3, concentrations of 50 and 200 nM were used in tissue and cell culture experiments.

Stimulation with DEX or ACC
Concentrations of DEX (Merck, Darmstadt, Germany) were 0.1, 0.4, 1.0, 4.0, 40, and 400 µM. Concentrations of ACC (Hexal, Holzkirchen, Germany) were 0.3, 3.0, and 30 mM. These concentrations were based on data from the literature (10, 22, 23). DEX and ACC were dissolved in sterile, physiologic saline solution. To investigate the possible effect of the solvent, DEX was also dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, Taufkirchen, Germany). A stock solution of 0.1 M DEX in DMSO was made. The drug was then diluted 1:1,000 in buffer. Appropriate DMSO controls (e.g., DMSO alone and stimulation with LPS and PAM3 in the presence of DMSO) were performed. Explanted mucosal tissue was stimulated with LPS (10 ng/ml) or PAM3 (200 nM) with or without DEX (n = 23) or ACC (n = 13). Calu-3 cells (n = 3) were stimulated with LPS (200 ng/ml) or PAM3 (200 nM) without or in the presence of either DEX or ACC. Moreover, tissue and Calu-3 cells were preincubated with DEX alone at concentrations of 0.1, 0.4, 1.0, 4.0, and 40 µM (and 400 µM for Calu-3 cells) for 16 hours and LPS or PAM3 were added for another 8 hours of stimulation. DEX and ACC were also applied to unstimulated mucosa and Calu-3 cells. The glucocorticoid receptor blocker RU486 (Sigma-Aldrich) was added to explanted mucosal tissue and Calu-3 cells in the presence of LPS/PAM3 and DEX. RU486 was used at a concentration of 1.0 µM as indicated by the manufacturer and data from the literature.

Immunohistochemistry
HOPE fixed tissue sections of 2 µm were cut and embedded in paraffin. Slides were stained with PAS to identify mucus glycoconjugates and mucus-producing goblet cells. Immunostaining was performed with the previously described horseradish peroxidase–labeled streptavidin-biotin technique (LSAB2; Dako, Hamburg, Germany) technique (20) to assess inflammatory cells (anti–neutrophil elastase for neutrophils, anti-CD3 for lymphocytes, and anti-CD68 for monocytes/macrophages, anti-EG2 for eosinophils; Dako).

RNA Extraction and Reverse Transcription
RNA from whole tissue samples and Calu-3 cells was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription was performed with 0.5 to 1.0 µg of RNA per reaction using Superscript II reverse transcriptase (RT, 200 U per reaction; Invitrogen, Karlsruhe, Germany) and oligo-dT in the presence of an RNase inhibitor (RNase Out; Invitrogen). The RNA was reverse transcribed in 30 µl of total volume at 65°C for 10 minutes, at 42°C for 60 minutes, and at 100°C for 1 minute. The resultant first-strand complementary DNA (cDNA) was used as template for PCR.

Quantitative Real-Time PCR
Quantitative real-time PCR (QRTPCR) was performed using a LightCycler system (Roche Diagnostics, Mannheim, Germany). MUC5AC was selected to measure mucin gene expression because it encodes for the most important secreted mucin in human airways (21). MUC5AC mRNA expression was quantified using QRTPCR. Delta-aminolevulinate synthase-1 (ALAS-1) was used as housekeeping gene. Primers were based on published mRNA sequences for MUC5AC, and ALAS-1 (GenBank Data Library accession numbers: AJ001402-MUC5AC, NM000688-ALAS-1) and were designed to span at least two exons to avoid binding to genomic DNA. Specific amplification using these primers was confirmed by ethidium bromide staining of the predicted size of the PCR products on an agarose gel. PCR was performed using the QuantiTect SYBR Green PCR Kit (Qiagen) with the appropiate primers and samples according to the manufacturer's protocol. In brief, 1 µl of cDNA was added to 10 µl of 2x QuantiTect SYBR Green PCR master mix, 8 µl of RNase-free water, and 0.5 µl of each primer (20 µM), resulting in a total volume of 20 µl. All PCR experiments were performed in triplicate.

Data Analysis and Quantification
Mucin expression in surface epithelial cells of explanted airway mucosal tissue was quantified as percentage of mucin-expressing (PAS-positive) epithelial cells/total epithelium. In Calu-3 cells, mucin protein expression was quantified as percentage of PAS-positive cells/total cells. PAS-positive epithelial or Calu-3 cells were counted by two experienced, blinded observers. The within-observer coefficient of variation for repeated measures was less than 5%. The numbers of immunoreactive inflammatory cells in the explanted mucosal tissue were counted per high-powered field in the submucosa (magnification x200). At least four nonoverlapping randomly selected fields of submucosa were quantified, and means were calculated. Results were expressed as the mean number of positive cells per field. For QRTPCR, the amount of mRNA was determined using standard curves of the gene of interest. Ratios of target gene/house keeping gene were calculated and values were normalized to medium alone.

Statistics
An overall ANOVA, followed by multiple testing with the Bonferroni correction, was performed. Differences between conditions were assessed by means of post hoc pairwise comparison with the Dunnet test. A P value of less than 0.05 was considered statistically significant. All values are given as means ± SEM if not otherwise stated.


   RESULTS
Top
 Abstract
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 References
 
LPS and PAM3 Induce Mucin Expression In Vitro and Ex Vivo
LPS increased MUC5AC mRNA expression in Calu-3 cells in a dose-dependent manner (Figure 1A). The effect was significant at a concentration of 50 ng/ml (3-fold; P < 0.05). To ensure adequate stimulation, an LPS concentration of 200 ng/ml (5-fold) was used for subsequent stimulation experiments with DEX and ACC (see below). Mucin protein expression in Calu-3 cells was also increased in a dose-dependent manner after stimulation with LPS (Figure 1B). This effect was significant at a concentration of 10 ng/ml (60.8 ± 2.6% versus 33.5 ± 5.6%; P < 0.05). In explanted human airway mucosa, LPS (10 ng/ml) significantly increased MUC5AC mRNA (3-fold; P < 0.05) (Figure 1C) and mucin protein expression (55.0 ± 8.8% versus 37.5 ± 8.7%; P < 0.05) (Figure 1D).


Figure 1
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  		Figure 1. Effect of LPS and PAM3 on mucin mRNA and protein expression in vitro and ex vivo. LPS and PAM3 increase mucin (MUC5AC) mRNA (A) and protein (B) expression in a dose-dependent manner in Calu-3 cells (n = 3). In explanted mucosal tissue, LPS and PAM3 significantly increase mucin mRNA (C) and protein (D) expression at concentrations of 10 ng/ml and 200 nM, respectively (n = 6). Incubation time was 24 hours. Concentrations of LPS is shown in ng/ml. Concentrations of PAM3 is shown in nM. MA: medium alone. Results shown as mean values ± SEM. *P < 0.05 versus MA.

 
Stimulation with the synthetic lipoprotein PAM3 caused a dose-dependent increase in expression of MUC5 mRNA in Calu-3 cells. This effect was significant at 200 nM (4-fold; P < 0.05) (Figure 1A). PAM3 also significantly increased mucin protein expression in Calu-3 cells at 200 nM (47.0 ± 10.9% versus 33.5 ± 5.6%; P < 0.05) (Figure 1B). In explanted mucosal tissue, PAM3 significantly increased MUC5AC mRNA (2-fold; P < 0.05) (Figure 1C) and mucin protein expression using 200 nM (52.5 ± 8.8% versus 37.5 ± 8.7%; P < 0.05) (Figure 1D).

Effect of LPS and PAM3 on Inflammatory Cells in Airway Submucosa
There was no significant change in the numbers of inflammatory cells in the submucosa of explanted human airways after stimulation with either LPS or PAM3 alone (P > 0.05).

Effect of DEX on LPS- and PAM3-Induced Mucin Expression in Human Airway Mucosa
Stimulation with different concentrations of DEX (0.1, 0.4, 1.0, 4.0, 40, and 400 µM) alone had no significant effect on epithelial mucin expression in explanted human airway mucosa (P > 0.05) (Figure 2A). In contrast, DEX significantly reduced LPS- and PAM3-induced mucin expression in a dose-dependent manner when given at the same time. The effect was significant at concentrations >= 1.0 µM after stimulation with LPS (45.8 ± 4.2% versus 57.5 ± 4.4%; P < 0.05) or PAM3 (40.6 ± 7.9% versus 47.5 ± 5.5%; P < 0.05) (Figures 2B and 2C). This inhibition is seen in original histology photographs as shown in Figures 2D–2H.


Figure 2
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  		Figure 2. Effect of DEX on LPS- and PAM3-induced mucin expression in explanted mucosal tissue. DEX alone has no significant effect (A), whereas DEX decreases LPS- and PAM3-induced mucin expression in a dose-dependent manner (B, C). (D–H) Original histology showing PAS staining (epithelial cells) in tissue cultured with medium alone (D), LPS 10 ng/ml alone (E), LPS + DEX 400 µM (F), PAM3 200 nM alone (G), or PAM3 + DEX 400 µM (H) (magnification: x200) (n = 17). MA: medium alone. Results shown as mean values ± SEM. *P < 0.05 versus MA. **P < 0.05 versus +LPS. +P < 0.05 versus MA. ++P < 0.05 versus +PAM3.

 
Pre-stimulation with DEX at concentrations ranging from 0.1 to 40 µM resulted in decreased ability of LPS and PAM3 to induce epithelial mucin expression compared with stimulation with LPS or PAM3 alone. Attenuation of LPS- or PAM3-induced mucin expression was dose dependent. The effect was statistically significant at DEX concentrations of >= 1.0 µM for LPS (43.8 ± 2.5% versus 56.3 ± 3.5%; P < 0.05) and PAM3 (31.3 ± 3.5% versus 43.8 ± 3.5%; P < 0.05) (Figure 3).


Figure 3
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  		Figure 3. Effect of LPS and PAM3 on mucin expression in explanted mucosal tissue after pre-incubation with different concentrations of DEX. LPS- (A) and PAM3-induced (B) mucin expression is attenuated by pre-incubation with DEX in a dose-dependent manner. MA: medium alone. Results shown as mean values ± SEM. *P < 0.05 versus MA. **P < 0.05 versus +LPS. +P < 0.05 versus MA. ++P < 0.05 versus +PAM3.

 
Effect of DEX on LPS- and PAM3-Induced Mucin Expression In Vitro
Stimulation with different concentrations of DEX alone (0.1–400 µM) had no significant effect on MUC5AC mRNA and mucin protein expression in Calu-3 cells (P > 0.05). DEX significantly decreased LPS- and PAM3-induced MUC5AC mRNA expression in dose-dependently (Figures 4A and 4C). The effect was significant concentrations >= 0.4 µM (P < 0.05). Moreover, DEX reduced LPS- and PAM3-induced mucin protein expression in a dose-dependent manner (Figures 4B and 4D). This effect was statistically significant at >= 0.4 µM for LPS stimulation (34.0 ± 1.1% versus 39.4 ± 1.0%; P < 0.05) and >= 1.0 µM for PAM3 stimulation (28.0 ± 0.5% versus 35.2 ± 7.2%; P < 0.05).


Figure 4
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  		Figure 4. Effect of DEX on LPS- and PAM3-induced mucin (MUC5AC) mRNA and protein expression in Calu-3 cells. DEX significantly decreased LPS- (A, B) and PAM3-induced (C, D) mucin mRNA (A, C), and protein (B, D) expression. MA: medium alone. Results shown as mean values ± SEM. *P < 0.05 versus MA. **P < 0.05 versus +LPS. +P < 0.05 versus MA. ++P < 0.05 versus +PAM3.

 
Pre-stimulation with DEX (0.1–400 µM) reduced the ability of LPS and PAM3 to up-regulate epithelial MUC5AC mRNA, and mucin protein expression compared with stimulation with LPS or PAM3 alone (Figure 5). Attenuation of LPS- or PAM3-induced MUC5AC and mucin protein expression was dose-dependent. The effect was statistically significant at DEX concentrations >= 1.0 µM for LPS-induced MUC5AC mRNA expression and mucin protein expression (27.8 ± 6.5% versus 38.6 ± 1.1%; P < 0.05) (Figures 5A and 5C). PAM3-induced MUC5AC mRNA expression was significantly decreased by pre-incubating with DEX concentrations >= 0.4 µM, whereas mucin protein expression was significantly reduced at DEX concentrations of >= 1.0 µM (27.5 ± 1.9% versus 34.3 ± 3.8%; P < 0.05) (Figures 5B and 5D).


Figure 5
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  		Figure 5. Effect of LPS and PAM3 on mucin (MUC5AC) mRNA and protein expression in Calu-3 cells following pre-stimulation with different concentrations of DEX. LPS- (A, B) and PAM3-induced (C, D) mucin mRNA (A, C) and protein (B, D) expression is attenuated by pre-incubation with DEX. MA: medium alone. Results shown as mean values ± SEM. *P < 0.05 versus MA. **P < 0.05 versus +LPS. +P < 0.05 versus MA. ++P < 0.05 versus +PAM3.

 
Using DMSO as a diluent for DEX did not significantly affect its potency. DEX decreased LPS- and PAM3-induced MUC5AC mRNA and mucin protein expression in a dose-dependent manner (Figures 6A–6D). This effect was significant at concentrations of >= 1.0 µM (P < 0.05).


Figure 6
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  		Figure 6. Effect of DEX dissolved in DMSO on LPS- and PAM3-induced mucin (MUC5AC) mRNA and protein expression in Calu-3 cells. DEX significantly decreased LPS- (A, B) and PAM3-induced (C, D) mucin mRNA (A, C) and protein (B, D) expression. MA: medium alone. Results shown as mean values ± SEM. *P < 0.05 versus MA. **P < 0.05 versus +LPS. +P < 0.05 versus MA. ++P < 0.05 versus +PAM3.

 
Effect of RU486 on DEX-Induced Attenuation of Mucin Expression in Human Airway Mucosa and In Vitro
Addition of RU486 (1.0 µM) partly inhibited the suppressive effect of DEX on LPS- and PAM3-induced mucin expression in human airway mucosa (Figures 7A–7B). This effect was significant for stimulation with LPS and DEX (66.7 ± 4.2% versus 45.8 ± 4.3%; P < 0.05) and for PAM3 and DEX (41.7 ± 4.2% versus 29.3 ± 4.3%; P < 0.05).


Figure 7
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  		Figure 7. Effect of RU486 (1.0 µM) on DEX (1.0 µM)-mediated suppression of LPS (10 ng/ml)- (A) and PAM3 (200 nM)-induced mucin expression (B) in explanted mucosal tissue. DEX significantly decreased LPS- and PAM3-induced mucin expression. This effect was significantly inhibited by addition of RU486. MA: medium alone. Results shown as mean values ± SEM. *P < 0.05 versus MA. **P < 0.05 versus +LPS. ***P < 0.05 versus +LPS+DEX. +P < 0.05 versus MA. ++P < 0.05 versus +PAM3. +++P < 0.05 versus +PAM3+DEX.

 
In Calu-3 cells, RU486 also inhibited in part DEX-mediated suppression of LPS- (Figures 8A and 8B) and PAM3-induced (Figures 8C and 8D) MUC5AC mRNA (Figures 8A and 8C) and mucin protein expression (Figures 8B and 8D). It significantly attenuated the effect of DEX on LPS- and PAM3-induced MUC5AC mRNA expression (~ 2-fold increase; P < 0.05). RU486 also significantly increased LPS- and PAM3-induced mucin protein expression in the presence of DEX (55.0 ± 5.0% versus 39.0 ± 1.0% and 45.5 ± 4.3% versus 32.5 ± 5.4%, respectively; P < 0.05).


Figure 8
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  		Figure 8. Effect of RU486 (1.0 µM) on DEX (1.0 µM)-mediated suppression of LPS (200 ng/ml)- (A, B) and PAM3 (200 nM)-induced mucin (MUC5AC) mRNA and protein expression (C, D) in Calu-3 cells. DEX significantly decreased LPS- and PAM3-induced mucin mRNA (A, C) and protein expression (B, D). This effect was significantly inhibited by addition of RU486. MA: medium alone. Results shown as mean values ± SEM. *P < 0.05 versus MA. **P < 0.05 versus +LPS. ***P < 0.05 versus +LPS+DEX. +P < 0.05 versus MA. ++P < 0.05 versus +PAM3. +++P < 0.05 versus +PAM3+DEX.

 
Effect of ACC on LPS- and PAM3-Induced Mucin Expression in Human Airway Mucosa
Stimulation with different concentrations of ACC (at 0.3, 3.0, and 30.0 mM, respectively) alone increased epithelial mucin expression in explanted human airway mucosa in a dose-dependent manner (Figure 9A). This effect was statistically significant at >= 3.0 mM (60.0 ± 6.0% versus 33.3 ± 2.8%; P < 0.05). However, ACC inhibited the LPS-induced mucin protein expression in a dose-dependent manner. This effect was significant at all used concentrations (58.3 ± 4.3% versus 37.5 ± 7.3%, 35.0 ± 7.3%, and 31.3 ± 12.0%, respectively; P < 0.05) (Figure 9B). ACC also significantly decreased PAM3-induced mucin protein expression at similar concentrations (50.0 ± 9.3% versus 31.3 ± 6.3%; P < 0.05) (Figure 9C). Original histology photographs are shown in Figures 9D–9G.


Figure 9
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  		Figure 9. Effect of ACC on LPS- and PAM3-induced mucin expression in explanted mucosal tissue. ACC increased epithelial mucin expression dose-dependently (A). In contrast, ACC reduced LPS- (B) and PAM3-induced (C) mucin expression. (D–G) Original histology showing PAS staining (epithelial cells) in medium alone (D), after stimulation with 30 mM ACC (E), after stimulation with 10 ng/ml LPS alone (F), and in the presence of 30 mM ACC (G) (original magnification: x200) (n = 13). MA: medium alone. Results shown as mean values ± SEM. *P < 0.05 versus MA. +P < 0.05 versus MA. ++P < 0.05 versus +LPS. §P < 0.05 versus MA. §§P < 0.05 versus +PAM3.

 
Effect of ACC on LPS- and PAM3-Induced Mucin Expression In Vitro
In Calu-3 cells, stimulation with different concentrations of ACC alone had no significant effect on MUC5AC mRNA and mucin protein expression (P > 0.05). However, ACC decreased both LPS- and PAM3-induced MUC5AC mRNA expression by approximately one third, but this effect was not significant (P > 0.05) (Figures 10A and 10C). In contrast, LPS-induced mucin protein expression was significantly decreased at 0.3 mM ACC (25.8 ± 6.7% versus 53.5 ± 6.7%; P < 0.05), whereas higher concentrations of ACC had no significant effect (53.3 ± 6.7% versus 38.1 ± 5.7%, and 41.9 ± 5.9%, respectively; P > 0.05) (Figure 10B). PAM3-induced mucus protein expression was not significantly reduced in the presence of ACC at all used concentrations (48.5 ± 4.5% versus 34.4 ± 12.8%, 35.2 ± 6.9%, and 38.7 ± 11.9%, respectively; P > 0.05) (Figure 10D).


Figure 10
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  		Figure 10. Effect of ACC on LPS and PAM3 induced mucin mRNA (MUC5AC) and protein expression in Calu-3 cells. ACC decreased LPS- (A, B) and PAM3-induced (C, D) mucin mRNA (A, C) and protein (B, D) expression. MA: medium alone. Results shown as mean values ± SEM. *P < 0.05 versus MA. **P < 0.05 versus +LPS. +P < 0.05 versus MA.

 

   DISCUSSION
Top
 Abstract
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
References
 
In the present study the effect of DEX and ACC on LPS- and PAM3-induced mucin expression were investigated. The rationale for this study was that these drugs are commonly used in clinical practice to reduce and ameliorate mucus hypersecretion. However, studies looking at the cellular level of action are sparse.

LPS has been shown previously to increase mucin gene expression and to induce mucous metaplasia (1, 2). Our data agree with those findings. We also found a dose-dependent increase in MUC5AC mRNA and mucin protein expression both in nasal mucosal tissue and in mucus-producing cells.

Since other parts of the outer membrane wall of Gram-negative bacteria such as lipoproteins may also stimulate mucin expression, PAM3 was used as a synthetic lipoprotein. However, there is no specific information available on the effect of PAM3 on mucin expression in the lungs. A previous study found increased mucin expression in an animal model of mycoplasma-infected mice (24). In that study, lipoprotein from mycoplasma induced mucin expression via TLR2-signaling. In our study, PAM3 also increased MUC5AC mRNA and mucin protein expression ex vivo and in vitro in dose-dependent manner; however, its effect was weak compared with stimulation with LPS. Higher concentrations of PAM3 than those used in this study (> 200 nM) did not have a stronger effect (data not shown).

Both LPS and PAM3 had no significant effect on the total numbers of monocytes, lymphocytes, neutrophils, or eosinophils in the submucosa of explanted mucosal tissue. This is not surprising, since the explanted tissue had no circulation and limited possibility of inflammatory cells to migrate. Another reason may be that incubation time (24 h) was too short for proliferation or apoptosis of cells to occur. In our model LPS and PAM3 may therefore directly increase epithelial mucin expression via induction of cytokines or other mediators from inflammatory cells. Expression of Toll-like receptor (TLR)-2 (for lipoprotein) and TLR-4 (for LPS) has been shown in epithelial as well as in inflammatory cells (25, 26).

Most previous studies have investigated the effect of DEX on unstimulated epithelial cells or tissue (10–12, 22). Information regarding the effect of DEX on bacterial-induced mucin expression is sparse despite its use in clinical practice. For this reason, we investigated the effect of DEX on bacteria-induced mucus production. Since glucocorticoid therapy is known to reduce airway inflammation in vivo (8), it is reasonable to envisage that glucocorticoids reduce inflammation-induced mucus hypersecretion. Beclomethasone dipropionate has previously been reported to significantly reduce inflammation and mucin production in patients with cystic fibrosis (9). In vitro studies have shown that DEX transcriptionally mediates repression of MUC5AC gene expression (10, 11, 22) and in cultured human airways DEX decreases the basal rate of mucus secretion (12). Such studies to date have predominantly used A549 and NCI-H292 cells. The disadvantage of these studies is that A549 cells are derived from type 2 pneumocytes and may therefore not be the best cells to investigate mucin expression, while NCI-H292 cells, which are commonly used to examine mucin expression (27), have high baseline or constitutive mucin production. In our study we used Calu-3 cells, a mucoepidermoid cell line that can be easily stimulated to express mucin. Interestingly, in this cell line we did not observe reduced mucin production at protein level in the presence of DEX as previously described (12). These data suggest that the effect of DEX may vary in different cell lines. Our results also support the notion that data from one cell line may not apply to what is seen in a clinical setting.

DEX significantly decreased LPS- and PAM3-induced MUC5AC mRNA and mucin protein expression in vitro. These data agree with previous findings that show direct inhibition of mucin gene expression by glucocorticosteroids (10, 11, 21).

Interestingly, tissue and cells pre-stimulated with DEX did not have a stronger inhibitory effect on LPS- and PAM3-induced MUC5AC mRNA and mucin protein expression compared with stimulation done at the same time. In a previous study by Chen and colleagues, MUC5AC mRNA expression in A549 cells was significantly decreased by DEX at a concentration of 100 nM from 6 hours after stimulation (10). In our experiments tissue and Calu-3 cells were pre-incubated with DEX for 16 hours before LPS or PAM3 was added. However, pre-stimulation with DEX significantly decreased MUC5AC mRNA expression at a concentration of 400 nM (0.4 µM). Mucin protein expression in explanted tissue was significantly decreased at a concentration of 1,000 nM (1.0 µM). These data indicate that higher concentrations of DEX may be required to reduce mucin expression in an ex vivo model and in different cell lines (as stated above). To ensure that the effect of DEX was not influenced by the solvent, we also tested the effect of DEX dissolved in DMSO. However, DMSO had no significant effect on the potency of DEX to attenuate mucin expression.

Another possibility is that the effect of DEX is not mediated by binding to the glucocorticoid receptor alone. Moreover, our findings suggest that DEX may not exclusively decrease mucus production by suppressing mucin gene expression alone, but that DEX may also have inhibitory effects on mucin protein production and secretion. This, however, remains to be further investigated. In our experiments the glucocorticoid receptor inhibitor RU486 had a significant effect on DEX-induced attenuation of mucin expression. RU486 blocked most of the suppressive effect of DEX on LPS- and PAM3-induced mucin expression. This finding indicates that in fact the greatest part of the effect of DEX is mediated via the glucocorticoid receptor.

An increase in reactive oxygen species (ROS) has been shown to stimulate mucin gene expression in cell culture (7, 16, 17). Since ACC has potent antioxidative properties, we investigated the effect of ACC on bacteria-induced mucus expression. In contrast to our expected results in explanted human airway mucosal tissue, ACC alone increased mucin expression, but LPS- or PAM3-induced mucin expression was decreased. It seems that ACC alone can act as a strong secretagogue and may therefore be beneficial in patients to enhance expectoration and improve mucociliary clearance. On the other hand, ACC may also function as an antioxidant that decreases LPS- or PAM3-induced generation of ROS and mucin expression. In a previous study a protective role of ACC in a rat model of bleomycin-induced lung fibrosis was described (28). In that study, ACC decreased bleomycin-induced mucin protein expression. However, a small, non–statistically significant increase in mucin protein expression could be noted with ACC alone. This finding supports the notion that ACC alone can increase mucin protein expression. Interestingly, in that study no change in Muc5AC gene expression was observed with ACC alone. It is possible that ACC acts on the post-transcriptional level. This remains to be further studied.

The most recent study to date looked at the effect of high concentrations of ACC in endothelial cell culture (29). In that study, TNF-{alpha}–generated oxidized glutathione levels were higher after treatment with ACC at a concentration of 30 mM compared with pretreatment with ACC at 3 mM. These data indicate that excess of antioxidative agents may cause further stress with generation of ROS. In that study, high concentrations of ACC caused glutathionylation of IKK{alpha}, thereby inhibiting its activity, which in turn enhanced NF-{kappa}B activation (29). In our study we used ACC concentrations between 0.3 mM and 30 mM; therefore, ROS may be generated in unstimulated explanted mucosal tissue, leading to increased mucin expression. Unfortunately we did not measure glutathione levels in this study, as this was beyond the scope of our study.

The inhibition of ACC on LPS- and PAM3-induced mucin expression in cell culture experiments failed to reach statistical significance, but this may be due to the small number of experiments. However, ACC had a significant effect in explanted human airway mucosa (ex vivo), thereby suggesting that ACC may also be effective in vivo.

Experiments using different types of LPS (e.g., derived from Pseudomonas aeruginosa or Haemophilus influenzae) showed LPS-induced MUC5AC mRNA and mucin protein expression that could be attenuated by DEX or ACC (data not shown). Therefore, DEX and ACC may be effective in bacterial infection with different strains.

Although DEX and ACC are commonly used in clinical practice, the effect of these drugs on bacteria-induced mucus expression has not been fully investigated. In the present study we used an ex vivo model to investigate the effect of DEX and ACC. This model is close to the in vivo situation. However, there is no systemic circulation and inflammatory cells are limited in their ability to migrate. Nevertheless, our study provides a link from basic research to clinical work.

In summary, in the present study we demonstrated stimulation of mucin expression by gram-negative bacterial membrane products in explanted human airway mucosal tissue. Both DEX and ACC decreased epithelial mucin expression. From these data it seems that DEX had a stronger effect than ACC. However, we did not perform direct comparison of these effects. DEX and ACC may be used to reduce mucus hypersecretion in case of bacterial infection or exacerbation. Although patients may have a clinical benefit (less mucus, less coughing) it remains to be further investigated whether this can affect prognosis.


   Acknowledgments
 
The authors thank Carmen Schöne, Simone Ross, and Jessica Hofmeister for excellent technical assistance. The authors also thank Dr. M. K. Tulic for carefully reading this manuscript.


   Footnotes
 
Originally Published in Press as DOI: 10.1165/rcmb.2006-0404OC on June 28, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form October 30, 2006

Accepted in final form June 6, 2007


   References
Top
 Abstract
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 References
 

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