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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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Related in: MedlinePlus

Functional analyses of bronchial epithelial spheroids. (A) Videomicroscopy shows ciliary beating associated with rolling of spheroids (see video online). (B). The ciliary beat frequency was similar in spheroids from both COPD and non-COPD smokers (n = 7) both at basal state (Basal) and after LPS stimulation (10 μg/ml LPS for 24 h) (LPS). (C) Representative micrographs of untreated (-LPS) and LPS-treated (+LPS) COPD spheroids showing LPS-enhanced expression of IL-8.
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Figure 3: Functional analyses of bronchial epithelial spheroids. (A) Videomicroscopy shows ciliary beating associated with rolling of spheroids (see video online). (B). The ciliary beat frequency was similar in spheroids from both COPD and non-COPD smokers (n = 7) both at basal state (Basal) and after LPS stimulation (10 μg/ml LPS for 24 h) (LPS). (C) Representative micrographs of untreated (-LPS) and LPS-treated (+LPS) COPD spheroids showing LPS-enhanced expression of IL-8.

Mentions: The BES rolling, as shown by videomicroscopy, was associated with ciliary beating (Fig. 3A, videomicroscopy). Interestingly, the ciliary beat frequency was not different between COPD and non-COPD BES (9.51 ± 1.34 Hz versus 9.22 ± 1.66 Hz, respectively) (Fig. 3B). Also, exposure of BES to LPS (10 μg/ml LPS for 24 h) resulted in slight but similar increase of ciliary beat frequency in both groups (Fig. 3B). Immunofluorescence staining for IL-8 found enhanced immunoreactivity following treatment of BES with LPS (Fig. 3C). All together, these data suggest that BES represent an intact bronchial epithelium that responds to stimulation.


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)

Functional analyses of bronchial epithelial spheroids. (A) Videomicroscopy shows ciliary beating associated with rolling of spheroids (see video online). (B). The ciliary beat frequency was similar in spheroids from both COPD and non-COPD smokers (n = 7) both at basal state (Basal) and after LPS stimulation (10 μg/ml LPS for 24 h) (LPS). (C) Representative micrographs of untreated (-LPS) and LPS-treated (+LPS) COPD spheroids showing LPS-enhanced expression of IL-8.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Functional analyses of bronchial epithelial spheroids. (A) Videomicroscopy shows ciliary beating associated with rolling of spheroids (see video online). (B). The ciliary beat frequency was similar in spheroids from both COPD and non-COPD smokers (n = 7) both at basal state (Basal) and after LPS stimulation (10 μg/ml LPS for 24 h) (LPS). (C) Representative micrographs of untreated (-LPS) and LPS-treated (+LPS) COPD spheroids showing LPS-enhanced expression of IL-8.
Mentions: The BES rolling, as shown by videomicroscopy, was associated with ciliary beating (Fig. 3A, videomicroscopy). Interestingly, the ciliary beat frequency was not different between COPD and non-COPD BES (9.51 ± 1.34 Hz versus 9.22 ± 1.66 Hz, respectively) (Fig. 3B). Also, exposure of BES to LPS (10 μg/ml LPS for 24 h) resulted in slight but similar increase of ciliary beat frequency in both groups (Fig. 3B). Immunofluorescence staining for IL-8 found enhanced immunoreactivity following treatment of BES with LPS (Fig. 3C). All together, these data suggest that BES represent an intact bronchial epithelium that responds to stimulation.

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