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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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Immunofluorescence staining of bronchial epithelium spheroids. I and II) Right panels of immunostained BES using antibodies specific to various cells types and intercellular junction proteins. Left panels show bright field images. (A) Anti-cytokeratin 13 antibody (CK13) for basal cells. (B) Anti-cytokeratin 18 (CK18) for ciliated cells. (C) Anti-mucin 5AC (MUC5AC) for secreted cells. (D,E,F) Antibodies against intercellular junction proteins zonula occludens-1 (ZO-1), Occludin (Occl) and E-Cadherin (E-Cad). Arrows depict staining for ZO-1 and Occludin in the intercellular junctions. Micrographs are representative of spheroids from COPD and non-COPD patients (n = 7).
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Figure 2: Immunofluorescence staining of bronchial epithelium spheroids. I and II) Right panels of immunostained BES using antibodies specific to various cells types and intercellular junction proteins. Left panels show bright field images. (A) Anti-cytokeratin 13 antibody (CK13) for basal cells. (B) Anti-cytokeratin 18 (CK18) for ciliated cells. (C) Anti-mucin 5AC (MUC5AC) for secreted cells. (D,E,F) Antibodies against intercellular junction proteins zonula occludens-1 (ZO-1), Occludin (Occl) and E-Cadherin (E-Cad). Arrows depict staining for ZO-1 and Occludin in the intercellular junctions. Micrographs are representative of spheroids from COPD and non-COPD patients (n = 7).

Mentions: Immunofluorescence microscopy analyses found that COPD and non-COPD BES comprised basal cells (CK13+), ciliated cells (CK18+), and few secretory cells (MUC5AC+) (Fig. 2A,B,C). Immunofluorescence staining of ZO-1, Occludin and E-Cadherin revealed distribution of these proteins in the intercellular junctions (Fig. 2D,E,F; arrows). A non-specific patchy staining could be observed on the apical (for ZO-1 and Occludin) and basal (for E-cadherin) sides of spheroids.


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)

Immunofluorescence staining of bronchial epithelium spheroids. I and II) Right panels of immunostained BES using antibodies specific to various cells types and intercellular junction proteins. Left panels show bright field images. (A) Anti-cytokeratin 13 antibody (CK13) for basal cells. (B) Anti-cytokeratin 18 (CK18) for ciliated cells. (C) Anti-mucin 5AC (MUC5AC) for secreted cells. (D,E,F) Antibodies against intercellular junction proteins zonula occludens-1 (ZO-1), Occludin (Occl) and E-Cadherin (E-Cad). Arrows depict staining for ZO-1 and Occludin in the intercellular junctions. Micrographs are representative of spheroids from COPD and non-COPD patients (n = 7).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Immunofluorescence staining of bronchial epithelium spheroids. I and II) Right panels of immunostained BES using antibodies specific to various cells types and intercellular junction proteins. Left panels show bright field images. (A) Anti-cytokeratin 13 antibody (CK13) for basal cells. (B) Anti-cytokeratin 18 (CK18) for ciliated cells. (C) Anti-mucin 5AC (MUC5AC) for secreted cells. (D,E,F) Antibodies against intercellular junction proteins zonula occludens-1 (ZO-1), Occludin (Occl) and E-Cadherin (E-Cad). Arrows depict staining for ZO-1 and Occludin in the intercellular junctions. Micrographs are representative of spheroids from COPD and non-COPD patients (n = 7).
Mentions: Immunofluorescence microscopy analyses found that COPD and non-COPD BES comprised basal cells (CK13+), ciliated cells (CK18+), and few secretory cells (MUC5AC+) (Fig. 2A,B,C). Immunofluorescence staining of ZO-1, Occludin and E-Cadherin revealed distribution of these proteins in the intercellular junctions (Fig. 2D,E,F; arrows). A non-specific patchy staining could be observed on the apical (for ZO-1 and Occludin) and basal (for E-cadherin) sides of spheroids.

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