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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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Relationship between LPS-induced IL-8 increase and clinical/functional parameters. (A,B,C) Correlations between LPS-induced IL-8 fold-increase and postbronchodilator FEV1/FVC ratio (Post-BD FEV1/FVC) (A), residual volume (RV) (B), and diffusing capacity for monoxide per liter alveolar volume (KCO) (C).
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Figure 6: Relationship between LPS-induced IL-8 increase and clinical/functional parameters. (A,B,C) Correlations between LPS-induced IL-8 fold-increase and postbronchodilator FEV1/FVC ratio (Post-BD FEV1/FVC) (A), residual volume (RV) (B), and diffusing capacity for monoxide per liter alveolar volume (KCO) (C).

Mentions: The LPS-enhanced secretion of IL-8 was inversely correlated with the level of obstruction (post-bronchodilator FEV1). No correlation was established between post bronchodilator FEV1 and PGE2 or LTB4 release (Fig. 5G,H,I). Because of the significant correlation between FEV1 and LPS-induced IL-8 release, other clinical and functional parameters were examined. In univariate analysis including all COPD and non-COPD smokers, basal levels of IL-8 release did not correlate with any clinical and functional parameters of the patients, and LPS-induced IL-8 release was not correlated with age, sex, weight, height and BMI (data not shown). Interestingly, a significant correlation was observed between LPS-induced IL-8 release and FEV1/FVC, RV and KCO (Fig. 6A,B,C). The smoking status (current/former) correlated with neither basal nor LPS-induced IL-8 release. In univariate analysis, the number of pack-years of smoking was correlated with LPS-induced IL-8 release (p < 0.01). Multivariate analysis demonstrated, however, that postbronchodilator FEV1, but not pack-years of smoking, correlated independently with LPS-induced IL-8 release (r2 = 0.35, p < 0.001). No correlation was observed between basal and LPS-induced release of PGE2 and LTB4 by BES and clinical and functional parameters of COPD and non-COPD patients (data not shown).


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)

Relationship between LPS-induced IL-8 increase and clinical/functional parameters. (A,B,C) Correlations between LPS-induced IL-8 fold-increase and postbronchodilator FEV1/FVC ratio (Post-BD FEV1/FVC) (A), residual volume (RV) (B), and diffusing capacity for monoxide per liter alveolar volume (KCO) (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Relationship between LPS-induced IL-8 increase and clinical/functional parameters. (A,B,C) Correlations between LPS-induced IL-8 fold-increase and postbronchodilator FEV1/FVC ratio (Post-BD FEV1/FVC) (A), residual volume (RV) (B), and diffusing capacity for monoxide per liter alveolar volume (KCO) (C).
Mentions: The LPS-enhanced secretion of IL-8 was inversely correlated with the level of obstruction (post-bronchodilator FEV1). No correlation was established between post bronchodilator FEV1 and PGE2 or LTB4 release (Fig. 5G,H,I). Because of the significant correlation between FEV1 and LPS-induced IL-8 release, other clinical and functional parameters were examined. In univariate analysis including all COPD and non-COPD smokers, basal levels of IL-8 release did not correlate with any clinical and functional parameters of the patients, and LPS-induced IL-8 release was not correlated with age, sex, weight, height and BMI (data not shown). Interestingly, a significant correlation was observed between LPS-induced IL-8 release and FEV1/FVC, RV and KCO (Fig. 6A,B,C). The smoking status (current/former) correlated with neither basal nor LPS-induced IL-8 release. In univariate analysis, the number of pack-years of smoking was correlated with LPS-induced IL-8 release (p < 0.01). Multivariate analysis demonstrated, however, that postbronchodilator FEV1, but not pack-years of smoking, correlated independently with LPS-induced IL-8 release (r2 = 0.35, p < 0.001). No correlation was observed between basal and LPS-induced release of PGE2 and LTB4 by BES and clinical and functional parameters of COPD and non-COPD patients (data not shown).

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