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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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Enhanced LPS-induced release of IL-8, but not PGE2 and LTB4 in COPD spheroids. (A,B,C) Levels of IL-8, PGE2 and LTB4 in untreated spheroids. Note, no significant differences could be detected between COPD and non-COPD BES. Results are expressed as pg of mediators per mg protein of BES. (D,E,F) Levels of IL-8, PGE2 and LTB4 after LPS stimulation (10 μg/ml LPS for 24 h). Results are expressed as fold increase by comparison to untreated conditions. (G,H,I) Correlations between LPS-induced IL-8, PGE2 and LTB4 fold-increase and postbronchodilator FEV1. Results are obtained from 16 COPD smokers (filled rhombs) and 13 non-COPD smokers (open rhombs). Mann-Whitney U test for comparisons between groups. Correlations between variables were calculated by means of the Spearman's rank correlation test.
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Figure 5: Enhanced LPS-induced release of IL-8, but not PGE2 and LTB4 in COPD spheroids. (A,B,C) Levels of IL-8, PGE2 and LTB4 in untreated spheroids. Note, no significant differences could be detected between COPD and non-COPD BES. Results are expressed as pg of mediators per mg protein of BES. (D,E,F) Levels of IL-8, PGE2 and LTB4 after LPS stimulation (10 μg/ml LPS for 24 h). Results are expressed as fold increase by comparison to untreated conditions. (G,H,I) Correlations between LPS-induced IL-8, PGE2 and LTB4 fold-increase and postbronchodilator FEV1. Results are obtained from 16 COPD smokers (filled rhombs) and 13 non-COPD smokers (open rhombs). Mann-Whitney U test for comparisons between groups. Correlations between variables were calculated by means of the Spearman's rank correlation test.

Mentions: Next, we investigated BES obtained from larger patients groups, 16 COPD smokers and 13 non-COPD smokers (Table 1). Bronchial brushes collected from COPD and non-COPD smokers displayed the same capacity to generate BES in vitro. BES responses were compared in the absence or presence of LPS. Untreated COPD and non-COPD BES released the same levels of IL-8, PGE2 and LTB4 (Fig. 5A,B,C). After LPS stimulation (10 μg/mL for 24 h), there was a 3-fold increase of IL-8 in COPD BES by comparison to non-COPD BES (Fig. 5D), whereas LPS-induced PGE2 and LTB4 release were similar in both COPD and non-COPD BES (Fig. 5E,F).


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)

Enhanced LPS-induced release of IL-8, but not PGE2 and LTB4 in COPD spheroids. (A,B,C) Levels of IL-8, PGE2 and LTB4 in untreated spheroids. Note, no significant differences could be detected between COPD and non-COPD BES. Results are expressed as pg of mediators per mg protein of BES. (D,E,F) Levels of IL-8, PGE2 and LTB4 after LPS stimulation (10 μg/ml LPS for 24 h). Results are expressed as fold increase by comparison to untreated conditions. (G,H,I) Correlations between LPS-induced IL-8, PGE2 and LTB4 fold-increase and postbronchodilator FEV1. Results are obtained from 16 COPD smokers (filled rhombs) and 13 non-COPD smokers (open rhombs). Mann-Whitney U test for comparisons between groups. Correlations between variables were calculated by means of the Spearman's rank correlation test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 5: Enhanced LPS-induced release of IL-8, but not PGE2 and LTB4 in COPD spheroids. (A,B,C) Levels of IL-8, PGE2 and LTB4 in untreated spheroids. Note, no significant differences could be detected between COPD and non-COPD BES. Results are expressed as pg of mediators per mg protein of BES. (D,E,F) Levels of IL-8, PGE2 and LTB4 after LPS stimulation (10 μg/ml LPS for 24 h). Results are expressed as fold increase by comparison to untreated conditions. (G,H,I) Correlations between LPS-induced IL-8, PGE2 and LTB4 fold-increase and postbronchodilator FEV1. Results are obtained from 16 COPD smokers (filled rhombs) and 13 non-COPD smokers (open rhombs). Mann-Whitney U test for comparisons between groups. Correlations between variables were calculated by means of the Spearman's rank correlation test.
Mentions: Next, we investigated BES obtained from larger patients groups, 16 COPD smokers and 13 non-COPD smokers (Table 1). Bronchial brushes collected from COPD and non-COPD smokers displayed the same capacity to generate BES in vitro. BES responses were compared in the absence or presence of LPS. Untreated COPD and non-COPD BES released the same levels of IL-8, PGE2 and LTB4 (Fig. 5A,B,C). After LPS stimulation (10 μg/mL for 24 h), there was a 3-fold increase of IL-8 in COPD BES by comparison to non-COPD BES (Fig. 5D), whereas LPS-induced PGE2 and LTB4 release were similar in both COPD and non-COPD BES (Fig. 5E,F).

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