An ex vivo model of severe asthma using reconstituted human bronchial epithelium

doi:10.1016/j.jaci.2012.01.073

Journal of Allergy and Clinical Immunology

Volume 129, Issue 5, May 2012, Pages 1259-1266.e1

https://doi.org/10.1016/j.jaci.2012.01.073 Get rights and content

Background

Structural changes to the airways are features of severe asthma. The bronchial epithelium facilitates this remodeling process. Learning about the changes that develop in the airway epithelium could improve our understanding of asthma pathogenesis and lead to new therapeutic approaches.

Objective

We sought to determine the feasibility and relevance of air-liquid interface cultures of bronchial epithelium derived from endobronchial biopsy specimens of patients with different severities of asthma for studying the airway epithelium.

Methods

Human bronchial epithelial cells derived from endobronchial biopsy specimens of patients with mild and severe asthma were maintained in culture for 21 days in an air-liquid interface to reproduce a fully differentiated airway epithelium. Initially, features of remodeling that included epithelial and subepithelial layers, as well as mucus production, were assessed in paraffin-embedded endobronchial biopsy specimens to evaluate morphologic characteristics of asthmatic patients’ epithelia. Ex vivo differentiated epithelia were then analyzed for morphology and function based on ultrastructural analysis, IL-8 release, lipoxin A₄ generation, mucin production, and lipoxygenase gene expression.

Results

Morphologic and inflammatory imbalances initially observed in endobronchial biopsy specimens obtained from patients with severe or mild asthma persisted in the air-liquid interface reconstituted epithelium throughout the differentiation process to 21 days. Epithelium from patients with severe asthma produced greater levels of mucin, released more IL-8, and produced lower levels of lipoxin A₄ than that from patients with mild asthma. Expression of 15-lipoxygenase 2 was increased in epithelium from patients with severe asthma, whereas expression levels of MUC5AC, MUC5B, 5-lipoxygenase, and 15-lipoxygeanse 1 were similar to those of patients with mild asthma.

Conclusion

Ex vivo cultures of fully differentiated bronchial epithelium from endobronchial biopsy specimens maintain inherent phenotypic differences specifically related to the severity of asthma.

Section snippets

Methods

Additional details are provided in the Methods section in this article’s Online Repository at www.jacionline.org.

Patients with mild (n = 7) and severe (n = 12) asthma were recruited at the Arnaud de Villeneuve Hospital, Montpellier, France. Asthma was diagnosed based on the presence of clinical features consistent with asthma and objective measures of variable airflow obstruction investigated 15 minutes before bronchoscopy (improvement in FEV₁ of ≥15% after inhalation of 200 μg of salbutamol).

Demographic data

Bronchial biopsy specimens were obtained from 19 subjects (7 with mild and 12 with severe asthma). Demographic characteristics are reported in Table I. Patients with severe asthma had greater airflow impairment (FEV₁ percentage) than those with mild asthma, and their symptoms were uncontrolled (daily symptoms and exacerbation rate) despite treatment. Biopsy specimens were analyzed from 5 control subjects (ie, no asthma, no allergies, nonsmokers, with normal results from chest radiographs and

Discussion

We developed air-liquid interface cultures of bronchial epithelial cells and demonstrated that they maintained characteristics specifically associated with mild and severe asthma. Functional and morphologic analyses of these cultures revealed epithelial differences associated with disease severity. Samples from patients with severe asthma differed in mucus production, cytokine production (sustained IL-8 release), and resolution mechanisms (a defect in LXA₄ production associated with

References (40)

A. Anzueto
Clinical course of chronic obstructive pulmonary disease: review of therapeutic interventions
Am J Med
(2006)
A. Bourdin et al.
Specificity of basement membrane thickening in severe asthma
J Allergy Clin Immunol
(2007)
P. Chanez et al.
Severe asthma in adults: what are the important questions?
J Allergy Clin Immunol
(2007)
J.V. Fahy
Goblet cell and mucin gene abnormalities in asthma
Chest
(2002)
P.G. Gibson et al.
Heterogeneity of airway inflammation in persistent asthma: evidence of neutrophilic inflammation and increased sputum interleukin-8
Chest
(2001)
I. Vachier et al.
Severe asthma is associated with a loss of LX4, an endogenous anti-inflammatory compound
J Allergy Clin Immunol
(2005)
C. Bonnans et al.
Lipoxin A(4) regulates bronchial epithelial cell responses to acid injury
Am J Pathol
(2006)
J.A. Voynow et al.
Mucins, mucus, and sputum
Chest
(2009)
B.D. Levy
Lipoxins and lipoxin analogs in asthma
Prostaglandins Leukot Essent Fatty Acids
(2005)
C.N. Serhan
Lipoxins and aspirin-triggered 15-epi-lipoxins are the first lipid mediators of endogenous anti-inflammation and resolution
Prostaglandins Leukot Essent Fatty Acids
(2005)

S.E. Wenzel et al.

Update in asthma 2005

Am J Respir Crit Care Med

(2006)

P. Godard et al.

Costs of asthma are correlated with severity: a 1-yr prospective study

Eur Respir J

(2002)

S.E. Wenzel et al.

Evidence that severe asthma can be divided pathologically into two inflammatory subtypes with distinct physiologic and clinical characteristics

Am J Respir Crit Care Med

(1999)

J. Bousquet et al.

Asthma. From bronchoconstriction to airways inflammation and remodeling

Am J Respir Crit Care Med

(2000)

O. Haworth et al.

Endogenous lipid mediators in the resolution of airway inflammation

Eur Respir J

(2007)

P. Chanez

Severe asthma is an epithelial disease

Eur Respir J

(2005)

J.V. Fahy

Remodeling of the airway epithelium in asthma

Am J Respir Crit Care Med

(2001)

L. Cohen et al.

Epithelial cell proliferation contributes to airway remodeling in severe asthma

Am J Respir Crit Care Med

(2007)

C.L. Ordonez et al.

Mild and moderate asthma is associated with airway goblet cell hyperplasia and abnormalities in mucin gene expression

Am J Respir Crit Care Med

(2001)

J.A. Voynow et al.

Regulation of mucin genes in chronic inflammatory airway diseases

Am J Respir Cell Mol Biol

(2006)

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: Disclosure of potential conflict of interest: P. Chanez is a consultant for Amgen, AstraZeneca, Centocor, Chiesi, GlaxoSmithKline, MSD, Novartis, and Nycomed and has received research support from Centocor. The rest of the authors declare that they have no relevant conflicts of interest.

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Asthma and lower airway diseaseAn ex vivo model of severe asthma using reconstituted human bronchial epithelium

Background

Objective

Methods

Results

Conclusion

Section snippets

Methods

Demographic data

Discussion

Am J Med

J Allergy Clin Immunol

J Allergy Clin Immunol

Chest

Chest

J Allergy Clin Immunol

Am J Pathol

Chest

Prostaglandins Leukot Essent Fatty Acids

Prostaglandins Leukot Essent Fatty Acids

Update in asthma 2005

Am J Respir Crit Care Med

Costs of asthma are correlated with severity: a 1-yr prospective study

Eur Respir J

Evidence that severe asthma can be divided pathologically into two inflammatory subtypes with distinct physiologic and clinical characteristics

Am J Respir Crit Care Med

Asthma. From bronchoconstriction to airways inflammation and remodeling

Am J Respir Crit Care Med

Endogenous lipid mediators in the resolution of airway inflammation

Eur Respir J

Severe asthma is an epithelial disease

Eur Respir J

Remodeling of the airway epithelium in asthma

Am J Respir Crit Care Med

Epithelial cell proliferation contributes to airway remodeling in severe asthma

Am J Respir Crit Care Med

Mild and moderate asthma is associated with airway goblet cell hyperplasia and abnormalities in mucin gene expression

Am J Respir Crit Care Med

Regulation of mucin genes in chronic inflammatory airway diseases

Am J Respir Cell Mol Biol

Asthma and lower airway disease
An ex vivo model of severe asthma using reconstituted human bronchial epithelium