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Bronchial epithelial spheroids: an alternative culture model to investigate epithelium inflammation-mediated COPD.

Deslee G, Dury S, Perotin JM, Al Alam D, Vitry F, Boxio R, Gangloff SC, Guenounou M, Lebargy F, Belaaouaj A - Respir. Res. (2007)

Bottom Line: The ciliary beat frequency of ciliated cells was not significantly different between the two groups.BES released the inflammatory mediators IL-8, PGE2 and LTB4 constitutively and following exposure to LPS.This study provides for the first time a compelling evidence that the BES model provides an unaltered bronchial surface epithelium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Service des Maladies Respiratoires, Hôpital Maison Blanche, CHU de REIMS, France. gdeslee@chu-reims.fr

ABSTRACT

Background: Chronic obstructive pulmonary disease (COPD) is characterized by abnormal lung inflammation that exceeds the protective response. Various culture models using epithelial cell lines or primary cells have been used to investigate the contribution of bronchial epithelium in the exaggerated inflammation of COPD. However, these models do not mimic in vivo situations for several reasons (e.g, transformed epithelial cells, protease-mediated dissociation of primary cells, etc.). To circumvent these concerns, we developed a new epithelial cell culture model.

Methods: Using non transformed non dissociated bronchial epithelium obtained by bronchial brushings from COPD and non-COPD smokers, we developed a 3-dimensional culture model, bronchial epithelial spheroids (BES). BES were analyzed by videomicroscopy, light microscopy, immunofluorescence, and transmission electron microscopy. We also compared the inflammatory responses of COPD and non-COPD BES. In our study, we chose to stimulate BES with lipopolycaccharide (LPS) and measured the release of the pro-inflammatory mediators interleukin-8 (IL-8) and leukotriene B4 (LTB4) and the anti-inflammatory mediator prostaglandin E2 (PGE2).

Results: BES obtained from both COPD and non-COPD patients were characterized by a polarized bronchial epithelium with tight junctions and ciliary beating, composed of basal cells, secretory cells and ciliated cells. The ciliary beat frequency of ciliated cells was not significantly different between the two groups. Of interest, BES retained their characteristic features in culture up to 8 days. BES released the inflammatory mediators IL-8, PGE2 and LTB4 constitutively and following exposure to LPS. Interestingly, LPS induced a higher release of IL-8, but not PGE2 and LTB4 in COPD BES (p < 0.001) which correlated with lung function changes.

Conclusion: This study provides for the first time a compelling evidence that the BES model provides an unaltered bronchial surface epithelium. More importantly, BES represent an attractive culture model to investigate the mechanisms of injuring agents that mediate epithelial cell inflammation and its contribution to COPD pathogenesis.

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Release of IL-8, PGE2 and LTB4 by bronchial epithelial spheroids in function of LPS dose and time. (A,B,C) BES from non-COPD (open bars) and COPD smokers (filled bars) were exposed to various concentrations of LPS for 24 h. Data are expressed as fold increase of IL-8, PGE2 and LTB4 by comparison to basal levels. The findings are illustrative of 5 independent experiments. (D,E,F) LPS treatment (10 μg/ml) of COPD and non-COPD smokers BES for 1, 4, 8 and 24 h. Data of IL-8, PGE2 and LTB4 protein levels are expressed in pg.mg total protein-1. The findings are illustrative of 4 independent experiments.
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Figure 4: Release of IL-8, PGE2 and LTB4 by bronchial epithelial spheroids in function of LPS dose and time. (A,B,C) BES from non-COPD (open bars) and COPD smokers (filled bars) were exposed to various concentrations of LPS for 24 h. Data are expressed as fold increase of IL-8, PGE2 and LTB4 by comparison to basal levels. The findings are illustrative of 5 independent experiments. (D,E,F) LPS treatment (10 μg/ml) of COPD and non-COPD smokers BES for 1, 4, 8 and 24 h. Data of IL-8, PGE2 and LTB4 protein levels are expressed in pg.mg total protein-1. The findings are illustrative of 4 independent experiments.

Mentions: Next, we determined the relevance of BES model to study the contribution of the bronchial surface epithelium to COPD airway inflammation. We obtained bronchial brushings from well-characterized COPD and non-COPD smokers. Freshly cultured spheroids were then exposed to LPS, an ubiquitous contaminant endotoxin. We incubated BES with different concentrations of LPS and examined their ability to induce the expression of the mediators IL-8, PGE2 and LTB4. Following LPS treatment for 24 h, IL-8, PGE2 and LTB4 levels increased in a LPS dose-dependent manner (0.1 to 10 μg/mL) in both COPD and non-COPD BES (Fig. 4A,B,C). These dose response and time course experiments found that 10 μg/mL LPS treatment resulted in a statistically significant increase of IL-8, but not LTB4 and PGE2, in COPD BES (p < 0.001) by comparison to non-COPD BES. Also, LPS-induced release of IL-8 and PGE2 from COPD and non-COPD BES increased progressively overtime and peaked by 24 h, whereas LTB4 release increased up to 4 h and remained constant thereafter (Fig. 4D,E,F). All the subsequent studies were carried out with 10 μg/mL LPS and 24 h culture time.


Bronchial epithelial spheroids: an alternative culture model to investigate epithelium inflammation-mediated COPD.

Deslee G, Dury S, Perotin JM, Al Alam D, Vitry F, Boxio R, Gangloff SC, Guenounou M, Lebargy F, Belaaouaj A - Respir. Res. (2007)

Release of IL-8, PGE2 and LTB4 by bronchial epithelial spheroids in function of LPS dose and time. (A,B,C) BES from non-COPD (open bars) and COPD smokers (filled bars) were exposed to various concentrations of LPS for 24 h. Data are expressed as fold increase of IL-8, PGE2 and LTB4 by comparison to basal levels. The findings are illustrative of 5 independent experiments. (D,E,F) LPS treatment (10 μg/ml) of COPD and non-COPD smokers BES for 1, 4, 8 and 24 h. Data of IL-8, PGE2 and LTB4 protein levels are expressed in pg.mg total protein-1. The findings are illustrative of 4 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2214730&req=5

Figure 4: Release of IL-8, PGE2 and LTB4 by bronchial epithelial spheroids in function of LPS dose and time. (A,B,C) BES from non-COPD (open bars) and COPD smokers (filled bars) were exposed to various concentrations of LPS for 24 h. Data are expressed as fold increase of IL-8, PGE2 and LTB4 by comparison to basal levels. The findings are illustrative of 5 independent experiments. (D,E,F) LPS treatment (10 μg/ml) of COPD and non-COPD smokers BES for 1, 4, 8 and 24 h. Data of IL-8, PGE2 and LTB4 protein levels are expressed in pg.mg total protein-1. The findings are illustrative of 4 independent experiments.
Mentions: Next, we determined the relevance of BES model to study the contribution of the bronchial surface epithelium to COPD airway inflammation. We obtained bronchial brushings from well-characterized COPD and non-COPD smokers. Freshly cultured spheroids were then exposed to LPS, an ubiquitous contaminant endotoxin. We incubated BES with different concentrations of LPS and examined their ability to induce the expression of the mediators IL-8, PGE2 and LTB4. Following LPS treatment for 24 h, IL-8, PGE2 and LTB4 levels increased in a LPS dose-dependent manner (0.1 to 10 μg/mL) in both COPD and non-COPD BES (Fig. 4A,B,C). These dose response and time course experiments found that 10 μg/mL LPS treatment resulted in a statistically significant increase of IL-8, but not LTB4 and PGE2, in COPD BES (p < 0.001) by comparison to non-COPD BES. Also, LPS-induced release of IL-8 and PGE2 from COPD and non-COPD BES increased progressively overtime and peaked by 24 h, whereas LTB4 release increased up to 4 h and remained constant thereafter (Fig. 4D,E,F). All the subsequent studies were carried out with 10 μg/mL LPS and 24 h culture time.

Bottom Line: The ciliary beat frequency of ciliated cells was not significantly different between the two groups.BES released the inflammatory mediators IL-8, PGE2 and LTB4 constitutively and following exposure to LPS.This study provides for the first time a compelling evidence that the BES model provides an unaltered bronchial surface epithelium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Service des Maladies Respiratoires, Hôpital Maison Blanche, CHU de REIMS, France. gdeslee@chu-reims.fr

ABSTRACT

Background: Chronic obstructive pulmonary disease (COPD) is characterized by abnormal lung inflammation that exceeds the protective response. Various culture models using epithelial cell lines or primary cells have been used to investigate the contribution of bronchial epithelium in the exaggerated inflammation of COPD. However, these models do not mimic in vivo situations for several reasons (e.g, transformed epithelial cells, protease-mediated dissociation of primary cells, etc.). To circumvent these concerns, we developed a new epithelial cell culture model.

Methods: Using non transformed non dissociated bronchial epithelium obtained by bronchial brushings from COPD and non-COPD smokers, we developed a 3-dimensional culture model, bronchial epithelial spheroids (BES). BES were analyzed by videomicroscopy, light microscopy, immunofluorescence, and transmission electron microscopy. We also compared the inflammatory responses of COPD and non-COPD BES. In our study, we chose to stimulate BES with lipopolycaccharide (LPS) and measured the release of the pro-inflammatory mediators interleukin-8 (IL-8) and leukotriene B4 (LTB4) and the anti-inflammatory mediator prostaglandin E2 (PGE2).

Results: BES obtained from both COPD and non-COPD patients were characterized by a polarized bronchial epithelium with tight junctions and ciliary beating, composed of basal cells, secretory cells and ciliated cells. The ciliary beat frequency of ciliated cells was not significantly different between the two groups. Of interest, BES retained their characteristic features in culture up to 8 days. BES released the inflammatory mediators IL-8, PGE2 and LTB4 constitutively and following exposure to LPS. Interestingly, LPS induced a higher release of IL-8, but not PGE2 and LTB4 in COPD BES (p < 0.001) which correlated with lung function changes.

Conclusion: This study provides for the first time a compelling evidence that the BES model provides an unaltered bronchial surface epithelium. More importantly, BES represent an attractive culture model to investigate the mechanisms of injuring agents that mediate epithelial cell inflammation and its contribution to COPD pathogenesis.

Show MeSH
Related in: MedlinePlus