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Published ahead of print on September 20, 2007, doi:10.1165/rcmb.2006-0453OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 38, pp. 276-282, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2006-0453OC

Epithelium Expression and Function of Retinoid Receptors in Asthma

Anne Druilhe*,1, Jean-Marie Zahm2, Laurent Benayoun{dagger},1, Delphine El Mehdi{ddagger},1, Martine Grandsaigne1, Marie-Christine Dombret3, Isabelle Mosnier4, Benoit Feger5, Joël Depondt6, Michel Aubier1,3 and Marina Pretolani1

1 Inserm, U700, Université Paris 7, Paris; 3 Assistance Publique des Hôpitaux de Paris, Groupe Hospitalier Universitaire Nord Bichat-Claude Bernard, Service de Pneumologie A, Paris; 6 Assistance Publique des Hôpitaux de Paris, Groupe Hospitalier Universitaire Nord Bichat-Claude Bernard, Service d'Oto-Rhino-Laryngologie, Paris; 2 Inserm, U514, Université Reims Champagne Ardenne, CHU Reims, Hôpital Maison Blanche, Reims; 4 Assistance Publique des Hôpitaux de Paris, Groupe Hospitalier Universitaire Nord Beaujon, Service d'Oto-Rhino-Laryngologie, Clichy; and 5 Clinique Pasteur, Brest, France

Correspondence and requests for reprints should be addressed to Marina Pretolani, Ph.D., Inserm Unité 700, Université Paris 7, Faculté de Médecine Denis Diderot, site Xavier Bichat, 16, rue Henri Huchard, 75018 Paris, France. E-mail: mpretol{at}bichat.inserm.fr


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Abnormal epithelial repair to damage participates in airway remodeling in asthma by the paracrine regulation of mesenchymal cell functions. Retinoids control epithelial functions through nuclear retinoic acid receptor (RAR) and retinoid X receptor (RXR) activation, yet their expression and contribution to epithelial repair and to airway remodeling in asthma are unknown. We determined the plasma levels of retinol and the immunohistochemical expression of retinoid receptors in damaged and repaired bronchial epithelium from 9 control subjects, 10 subjects with intermittent asthma, 8 subjects with mild-to-moderate asthma, and 8 subjects with severe asthma. In addition, the effect of the retinoid receptor ligands, all-trans-retinoic acid, and 9-cis retinoic acid, on the synthesis of 38 factors potentially involved in epithelial repair and in airway remodeling was determined in human cultured airway epithelial cells and correlated with cell migration and proliferation. Circulating retinol was similar in the three patient groups. In contrast, the epithelial expression of RAR{gamma}, RXR{alpha}, and RXR{gamma} was greater in subjects with severe asthma, as compared with patients with milder disease and to control subjects. Retinoid receptor expression correlated positively with the proportion of morphologically intact epithelium. In vitro, retinoids up-regulated the expression of the transcripts encoding transforming growth factor (TGF)-β1, metalloproteinase-9, β1-integrin, and hepatocyte growth factor receptor, and promoted wound repair and chemokinesis of human airway epithelial cells without altering proliferation. Cell treatment with an anti–TGF-β1 monoclonal antibody partially reduced retinoid-induced effects. Persistent interaction between retinoids and some of their receptors, which are overexpressed by the bronchial epithelium of individuals with severe asthma, may contribute to an abnormal repair and to airway remodeling, partly through TGF-β1 production.

Key Words: epithelium repair • airway remodeling • transforming-growth factor-β • metalloproteinase-9



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
This study improves the understanding of the role played by retinoid–receptor interaction in the aberrant repair of the airway epithelium in asthma and on their link with the onset of airway remodeling.

 
Damage of the bronchial epithelium, mucosal inflammation, and airway remodeling are cardinal features of asthma (1, 2).

Normal epithelium repair requires spreading and migration of epithelial cells adjacent to the damaged area into the wound to form a temporary squamous barrier consisting of poorly differentiated cells. This is followed by cell proliferation and differentiation toward a mucosecretory and ciliary phenotype, and by complete restoration of the barrier function (3). These stages involve a cyclic establishment and rupture of cell–cell contacts, of adherence to extracellular matrix, extracellular matrix remodeling by matrix metalloproteinases (MMPs) to accommodate cell spreading and migration, and up-regulation of growth factors and receptors that cooperate for ensuring migration, proliferation, and differentiation (3, 4). It has been postulated that an abnormal repair response of the bronchial epithelium to injury leads to an excessive extracellular matrix protein and MMP deposition in the stroma underlying the bronchial epithelium and to an overproduction of fibrogenic growth factors that cause functional disturbances of epithelial–mesenchymal interactions and ultimately contributes to airway remodeling in asthma (1, 2, 5).

Vitamin A and derivatives, including retinol and retinoic acid (RA), regulate several cell functions upon binding and activation of nuclear receptors from the steroid/thyroid receptor superfamily (6). Based on sequence homology and ligand binding specificity, retinoid receptors belong to two groups with three isotypes ({alpha}, β, and {gamma}) for each: the retinoid acid receptors (RAR), which are activated by both all-trans RA (ATRA) and 9-cis RA; and the retinoid X receptors (RXR), which recognize only 9-cis RA (6). The RARs/RXRs form homodimers and/or heterodimers that bind responsive elements of retinoid target genes and interact with a variety of coactivators and repressors (6).

Retinoids promote epithelial cell growth and differentiation during lung morphogenesis and, in adults, they play an essential role in the maintenance of mucociliary epithelium phenotype in the conducting airways (7, 8). In addition, retinoids regenerate rodent alveolar epithelium that has been injured by the intrapulmonary administration of dexamethasone or elastase (9–11). Consistently with these findings, retinoid deficiency in rodents is accompanied by extensive alterations of the epithelial structure, reminiscent of those seen in emphysema (12).

Retinoids have been shown to mediate transforming growth factor (TGF)-β–dependent hepatic fibrosis in vivo (13), a phenomenon that can be reversed by vitamin A deficiency (14). Although these observations support a regulatory role of retinoids in the development of fibrogenic processes, changes in their expression and function in damaged and repaired asthmatic epithelium, particularly in relation to airway remodeling, remain elusive.

To address this question, we examined the plasma levels of retinol and the distribution of RAR and RXR isotypes in the bronchial epithelium of individuals with asthma of different severity and with a variable extent of epithelial damage and of subepithelial fibrosis. To determine whether RA–retinoid receptor interaction mediates epithelium repair and to assess the relationship with airway remodeling, we studied the effect of ATRA and 9-cis RA on wound healing, chemokinesis, and proliferation of primary human airway epithelial cells and on the synthesis and release of several factors that potentially modulate epithelium repair and airway remodeling. These factors include adhesion molecules, extracellular matrix components, fibrogenic growth factors and their receptors, and MMPs and their tissue inhibitors. Finally, we determined the potential contribution of endogenously-produced TGF-β to retinoid-induced effects.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Subjects, Fiberoptic Bronchoscopy, Biopsy Processing, and Retinol Determination
Nine healthy volunteers, 10 subjects with intermittent asthma, 8 subjects with mild-to-moderate persistent asthma, and 10 subjects with severe persistent asthma fulfilling National Institutes of Health criteria (15) were recruited. A flow volume curve was performed in all subjects, and forced expiratory volume in one second (FEV1) was assessed before and after the inhalation of 400 µg of salbutamol.

Immunohistochemistry was performed on frozen bronchial biopsy sections, using anti-human RAR{alpha}, β, or {gamma}; RXR{alpha}, β, or {gamma}; or propyl-4-hydroxylase antibodies, followed by secondary antibodies conjugated to biotin, by avidine–alkaline phosphatase complex, by Fast Red staining and nuclear counterstaining. Epithelial cells expressing RAR{alpha}, β, and {gamma}, RXR{alpha}, β, and {gamma} (in %) were counted in intact areas of bronchial epithelium. Propyl-4-hydroxylase (fibroblasts/myofibroblasts)-positive cells were enumerated in the bronchial submucosa, in a zone 60 µm deep, along the length of the subepithelial basement membrane (16).

The proportion of epithelium with apparent normal morphology was assessed by morphometric analysis using computer-assisted image analysis on sections stained with Mayer's hematoxylin. Morphologically intact epithelium (in %) was defined as the total length of the subepithelial basement membrane with intact epithelium divided by the total length of the subepithelial basement membrane (17).

Retinol concentration in plasma from six control subjects, six steroid-untreated subjects with asthma, and eight steroid-treated subjects with asthma was measured by reverse-phase high-performance liquid chromatography (18).

Epithelial Cell Culture and Processing
Primary cultured human nasal epithelial cells, obtained from pronase-digested nasal polyps or turbinates and bronchial epithelial cells, were used. Circular wounds were made by adding a 1-µl drop of 1 N NaOH in the central area of confluent nasal epithelial cells, cultured in collagen I–coated wells in the absence of RA. Wounded cultures were stimulated over 72 hours with 0.01% dymethylsulphoxide (DMSO), or 1 µM ATRA, or 9-cis RA, 20 ng/ml epidermal growth factor (EGF), or 5 ng/ml TGF-β1 (19). Alternatively, nasal epithelial cells were cultured until confluence in a silicon ring placed in a collagen I–coated wells in medium without RA. At cell confluence the ring was removed and cells were stimulated as described above. The wounded area and the cell monolayer area (ring system) were measured every 24 hours for 72 hours using computer-assisted image analysis.

Proliferation was assessed at 24, 48, and 72 hours in NaOH-wounded and DMSO-, ATRA-, and 9-cis RA–stimulated cells by 3H-thymidine incorporation assay.

In separate experiments, wounded bronchial or nasal cell monolayers were incubated for 6 to 72 hours with 0.01% DMSO, 1 µM ATRA, 9-cis RA, 5 ng/ml TGF-β1 (with or without 1 µM dexamethasone), or 0.2 µg/ml neutralizing mouse anti–TGF-β monoclonal antibody or its control isotype. RNAs were extracted at 6, 24, and 48 hours, reverse transcribed using the Moloney Murine Leukemia Virus enzyme, amplified, and stained with Taq polymerase and SYBRGreen fluorochrome. The intensity of gene expression was calculated using cycle treshold less than 35, and it was normalized to the geometrical mean of the levels of transcripts encoding three housekeeping genes: ubiquitin C, succinate dehydrogenase A, and ribosomal protein L13a (21, 22).

The levels of pro–MMP-9 and TGF-β1 were quantified at 48 and 72 hours in cell supernatants by specific immunoassays.

Statistical Analysis
All analyses were performed using the StatView SE+Graphics program for Macintosh (Abacus Concepts, Berkeley, CA). P values <= 0.05 were considered significant.

Additional details concerning patients, methods, and statistics are provided in the online supplement.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Plasma Retinol Concentration
Plasma levels of retinol were similar in control subjects (median [25–75 percentiles] 1.97 µM [1.71–2.14], n = 6), in steroid-untreated subjects with asthma (1.85 µM [1.64–2.02], n = 6), and in steroid-treated subjects with asthma (2.03 µM [1.60–2.44], n = 8).

Morphologic and Immunohistochemical Analysis of Bronchial Biopsies
Changes in the proportion of bronchial epithelium layer with an intact structure were assessed by morphometry and computer-assisted image analysis in bronchial biopsies from control subjects and from subjects with asthma (Figure 1A). A significant decrease of the proportion of epithelium layer with a restored structure was seen in subjects with intermittent asthma, as compared with control subjects (Figure 1A). Subjects with mild-to-moderate and severe asthma showed a progressive and apparent epithelial restitution, and the extent of morphologically intact epithelium in this latter group was comparable to that measured in control individuals (Figure 1A).


Figure 1
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  		Figure 1. Changes in epithelial integrity and in the expression of retinoid receptors in normal and asthmatic bronchial epithelium. (A) The proportion of morphologically intact epithelium was determined by morphometry and image analysis on Mayer's hematoxylin-stained bronchial biopsies from control subjects/healthy individuals and from patients with intermittent, mild-to-moderate, and severe asthma, and it was defined as the length of the subepithelial basement membrane with intact epithelium divided by the total length of the subepithelial basement membrane. The expression of RAR{gamma} (B), RXR{alpha} (C), and of RXR{gamma} (D) was assessed by immunohistochemistry in acetone-fixed bronchial tissue cryosections. Results are expressed as the proportion of epithelial cells in intact epithelium areas expressing each receptor. The horizontal bars represent median values. (E) Representative RXR{alpha} immunolocalization in the bronchial epithelium from a control individual (left panel) and from a subject with severe persistent asthma (middle panel). The right panel illustrates the section sequential to the one shown in the middle panel that had been incubated with the control isotype. Note that epithelial integrity is morphologically similar in both sections. Bronchial biopsy sections were incubated with a rabbit anti-human RXR{alpha} polyclonal antibody, followed by anti-rabbit antibody conjugated to biotin, avidin–alkaline phosphatase complex, Fast red susbtrate (red deposit) and Mayer's hematoxylin blue counterstaining. Scale bars: 250 µm.

 
The number of fibroblasts/myofibroblasts (propyl-4-hydroxylase-positive cells) was comparable in the bronchial submucosa of control subjects (7.7% [6.7–12.7]) and of subjects with intermittent (9.8% [7.4–12.0]) and mild-to-moderate asthma (14.3% [10.4–29.5]). These numbers were higher in subjects with severe asthma (32.0% [29.0–43.0]), and they were significantly different in relation to control subjects and to subjects with intermittent and mild-to-moderate asthma (P = 0.0016, P = 0.0025, and P = 0.0321, respectively).

By immunohistochemistry, we established that RARs and RXRs were expressed at variable degrees by the bronchial epithelium from all patient groups (Figures 1B, 1C, and 1D and data not shown). The median proportion of cells expressing RAR{alpha}, β, and {gamma}, and RXR{alpha} and {gamma}, ranged between 7.0 and 28.0% (Figures 1B, 1C, and 1D and data not shown), whereas RXRβ was weakly detectable (median value < 1.0%, data not shown). No changes in the proportion of epithelial cells expressing RAR{alpha}, RARβ, and RXRβ were found among the four patient groups (data not shown). In contrast, RAR{gamma}, RXR{alpha}, and RXR{gamma} immunostaining was significantly increased in intact areas of bronchial epithelium from subjects with severe asthma (Figures 1B, 1C, and 1D), where it occurred uniformly throughout the epithelial layer. A representative RXR{alpha} immunostaining in a control individual and in a subject with severe persistent asthma is shown in Figure 1E. No staining was detected with control isotype (Figure 1E).

By univariate regression analysis, we found that RXR{alpha} and RXR{gamma} immunostaining in patients with asthma correlated with the proportion of structurally intact epithelium (r2 = 0.432, P = 0.028 and r2 = 0.430, P = 0.028, respectively).

Expression of Retinoid Receptors in Cultured Airway Epithelial Cells
We first verified that primary cultured airway epithelial cells isolated from nasal polyps or turbinates of 8 to 10 distinct donors expressed retinoid receptors. We found highly detectable amounts of the transcripts encoding RAR{alpha}, RARβ, RAR{gamma}, RXR{alpha}, and RXRβ in untreated epithelial cells, whereas RXR{gamma} mRNA levels were near the limit of the detection of our quantitative real-time PCR assay (cycle threshold [Ct] values >= 35, Table E2 in the online supplement). Highly comparable levels of retinoid receptor mRNA were found in epithelial cells isolated from polyps or turbinates (n = 3 and n = 7, respectively; data not shown), or in primary cultured human bronchial epithelial cells (Table E2).

A 48-hour exposure of airway epithelial cells to 1 µM dexamethasone failed to modify RAR and RXR gene expression (Table 2). Under the same conditions, ATRA and 9-cis RA (1 µM) increased respectively by 6.3- and 7.9-fold RARβ mRNA and decreased RAR{gamma} mRNA expression, without altering the levels of the transcripts encoding the other receptors analyzed (Table E2). Finally, TGF-β1 augmented by 2.1-fold and reduced by 0.4-fold the expression of RAR{alpha} and RAR{gamma}, respectively (Table E2).

Effect of Retinoids on Epithelial Wound Repair, Cell Migration, and Proliferation
We first determined whether retinoids promoted wound repair, using the NaOH-injured epithelial cell monolayer. Cell stimulation with ATRA, 9-cis RA, or TGF-β1 during 72 hours resulted in a decrease in the wounded area, as compared with DMSO-treated cells (Figure 2A). No significant changes in this area were seen at earlier time points (i.e., 24 and 48 hours [data not shown]). EGF induced a larger and significant wound repair, as compared with retinoids and with TGF-β1 (Figure 2A).


Figure 2
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  		Figure 2. Effect of retinoids and growth factors on epithelial wound repair and cell migration. (A) Confluent human airway epithelial cells were wounded by the addition of 1 µl of NaOH and, after washing, they were stimulated with 0.01% DMSO, or with 1 µM ATRA, 9-cis RA, 5 ng/ml TGF-β, or 20 ng/ml EGF. Changes in the surface of wounded area were assessed by computer-assisted image analysis 72 hours after the addition of stimuli. Data (in pixel2) are means ± SEM of five independent experiments. *P < 0.05, as compared with DMSO-treated cells (nonparametric Friedman test followed by Wilcoxon test for paired values). (B and C) Primary human airway epithelial cells were cultured in a silicon cylinder placed in a well of a 24-well plate. When cells reached 100% confluence, the cylinder was removed and cell monolayers were treated with 0.01% DMSO, or with 1 µM of ATRA or of 9-cis RA, with 5 ng/ml TGF-β, or with 20 ng/ml EGF. Changes in the surface of the cell monolayer were assessed by computer-assisted image analysis 72 hours after the addition of stimuli. The histograms in B represent the % increment of monolayer area over the increment measured in DMSO-treated cells. Data are means ± SEM of six independent experiments performed with epithelial cells from six distinct donors. *P < 0.05, as compared with DMSO-treated cells (nonparametric Friedman test followed by Wilcoxon test for paired values). (C) Representative phase contrast microphotographs of cell monolayers after 72 hours of treatment with DMSO, EGF, ATRA, or 9-cis RA (scale bar: 50 µm).

 
Similar findings were obtained in the ring culture system, in which ATRA, 9-cis RA, and TGF-β1 significantly increased epithelial monolayer area at 72 hours and EGF displayed the most potent effect (Figure 2B). Unlike the leading edge of the monolayer of DMSO-treated cells, where cells remained adherent to each other in a uniform sheet (Figure 2C), numerous isolated, flattened cells, with large lamellipodia and no apparent cell–cell contacts, characteristic of migrating cells (23), were found in ATRA-, 9-cis RA–, TGF-β1–, and EGF-treated cultures (Figure 2C and data not shown).

Finally, we found that ATRA and 9-cis RA failed to promote cell proliferation, as reflected by 3H-thymidine uptake. Indeed, at 72 hours, median cpm values (25–75 percentiles) for DMSO-, ATRA-, and 9-cis RA–treated cells were of 57495 (44,715–67,756, 52,169 (46,000–52,371) and 50,271 (46,000–51,073) (n = 5).

Effect of Retinoids on the Synthesis of Factors Involved in Epithelial Repair and in Airway Remodeling
To determine whether ligation of retinoids to their receptors expressed by the asthmatic bronchial epithelium would participate in epithelial cell migration, in epithelium repair and in airway remodeling, we examined the ability of ATRA and of 9-cis RA to influence the synthesis of a number of factors involved in these processes (Table E3). The effects of ATRA and of 9-cis RA were analyzed at 48 hours, a time-point selected on the basis of the above findings showing a significant retinoid-mediated wound repair and cell migration at 72 hours. By quantitative real-time PCR, we found detectable levels of the transcripts encoding MMP-1, -2, -9, and -12; tissue inhibitor of metalloproteinase-1, -2, -3, and -4; fibronectin; tenascin; β1-integrin; CD44; E-cadherin; β-catenin; EGF and its receptor; TGF-{alpha}, -β1, and -2 and their receptors I and II; keratinocyte growth factor and its receptor 2; platelet-derived growth factor {alpha} and β; insulin growth factor 1β and 2 and their receptor; endothelin-1; and hepatocyte growth factor receptor (HGF-R) (Table E3) in vehicle (DMSO)-stimulated human airway epithelial cells. The amounts of the transcripts of MMP-7, collagen type I{alpha}1, insulin growth factor 1{alpha}, and platelet-derived growth factor receptors {alpha} and β, were very low, and they were found in three or four out of the five cell preparations (Table E3). Finally, HGF and endothelin-1 receptors A and B were undetectable in all samples tested (Table E3).

Incubation of airway epithelial cells during 48 hours with 1 µM of ATRA or 9-cis RA augmented the levels of the transcripts encoding TGF-β1, MMP-9, HGF-R, and β1-integrin, without changes in the amounts of mRNA for the other factors analyzed (Table E3 and Figure 3). A similar stimulatory effect was noted in primary human bronchial epithelial cells (Table E3).


Figure 3
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  		Figure 3. Retinoid-induced changes in the expression of the transcripts encoding TGF-β1, MMP-9, HGF-R, and β1-integrin in primary cultured human airway epithelial cells. Nasal cells were incubated for 48 hours with 1 µM ATRA, 9-cis RA, or with their vehicle (0.01% DMSO), in the presence of 0.2 µg/ml neutralizing anti–TGF-β, or its control isotype, or with 5 ng/ml TGF-β1. The levels of mRNAs were assessed by quantitative real-time PCR. Results are expressed as the ratio of TGF-β1, MMP-9, HGF-R, or β1-integrin transcript expression over the geometric average of the expression of the housekeeping genes, ubiquitin C, succinate dehydrogenase A, and ribosomal protein L13a. Data are medians of five independent experiments performed with epithelial cells obtained from five distinct donors. Open bars, +control IgG; solid bars, +anti–TGF-β1. *P < 0.05, as compared with DMSO IgG-treated cells, {dagger}P < 0.05, as compared with anti–TGF-β1–untreated cells.

 
In addition, time-course studies showed that TGF-β1 and MMP-9, but not HGF-R and β1-integrin, mRNA, increased as early as 6 hours after airway epithelial cell stimulation and remained elevated at 24 and 48 hours (Table E4).

By enzyme-linked immunosorbent assay, we established that the transcriptional effects of retinoids resulted in proportionate changes in the production of the corresponding proteins, since increased levels of TGF-β1 and pro–MMP-9 were found at 48 and 72 hours in the supernatants of ATRA- and 9-cis RA– as compared with DMSO-stimulated cells (Figure 4).


Figure 4
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  		Figure 4. Effect of retinoids on the release of TGF-β1 and pro–MMP-9 from human cultured airway epithelial cells. Nasal cells were incubated during 48 or 72 hours with 1 µM ATRA (solid bars), or 9-cis RA (hatched bars), or with their vehiche (0.01% DMSO; open bars), and the levels of TGF-β1 (A) and pro–MMP-9 (B) in the supernatant were measured by specific immunoassays. Data are means ± SEM of 10 independent experiments. *P < 0.05, as compared with DMSO-treated cells.

 
To determine the involvement of endogenously-produced TGF-β in the synthesis of MMP-9, HGF-R, and β1-integrin, cells were stimulated with TGF-β1, or with retinoids, in the presence of a neutralizing anti–TGF-β monoclonal antibody. TGF-β1 significantly upregulated the levels of the mRNAs encoding MMP-9, HGF-R, and β1-integrin (Figure 3) and induced a 6-fold increase in the amounts of pro–MMP-9 in cell supernatants (n = 5, data not shown). TGF-β1 neutralization failed to inhibit ATRA- and 9-cis RA–induced HGF-R, β1 integrin, and TGF-β1 transcription (Figure 3), but significantly reduced the levels of MMP-9 mRNA (median inhibition of 58.3 and 39.5% in ATRA- and 9-cis RA–treated cells, respectively; Figure 3) and down-regulated pro–MMP-9 release in cell supernatants (median inhibition of 26.8 and 21.1% in ATRA- and 9-cis RA–treated cells, respectively; n = 5; data not shown).


   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
To determine whether retinoids are involved in epithelial repair and airway remodeling in asthma, we initially examined the levels of circulating retinol and the epithelial immunolocalization of their receptors in a group of control healthy individuals and in subjects with asthma of different severity. These patients had major differences in terms of epithelial integrity and of subepithelial fibrosis, since subjects with intermittent and mild-to-moderate asthma showed a loss of epithelial integrity but low numbers of fibroblasts/myofibroblasts beneath the subepithelial basement membrane, whereas in subjects with severe asthma the airway epithelium appeared fully restored and the number of fibroblasts/myofibroblasts was substantially augmented, as compared with patients with milder disease. These findings support previous observations showing that individuals with severe persistent asthma requiring high-dose long-term steroid therapy had an apparent morphologic restitution of their bronchial epithelium and that subepithelial fibrosis is one of the main bronchial wall changes seen in these patients (16, 24, 25).

Using this classification, we found comparable serum levels of retinol across the patient groups, extending previous observations in a large cohort of adults with asthma (26), but contradicting a recent report showing lower circulating vitamin A concentrations in children with asthma (27).

Immunohistochemical analyses of endobronchial biopsy samples showed a progressive increment in the expression of RAR{gamma}, RXR{alpha}, and RXR{gamma} in intact areas of bronchial epithelium of subjects with asthma in proportion with disease severity. In vitro studies have established that the expression of RA receptors is differentially regulated by a number of factors, including corticosteroids, the fibrogenic growth factor, TGF-β1, and retinoids themselves (28–32). However, using in vitro cultured human airway epithelial cells, we found that dexamethasone failed to modulate the levels of retinoid receptor transcripts, whereas TGF-β1 up-regulated the expression of RAR{alpha} mRNA, and ATRA and 9-cis RA augmented that of RARβ. Overall, these results suggest that these factors are not involved in RAR{gamma}, RXR{alpha}, and RXR{gamma} overexpression presently reported in bronchial epithelium of subjects with severe asthma. Nevertheless, these receptors might have been induced over a long period of time before biopsy sampling as a consequence of chronic steroid treatment, or of sustained generation of TGF-β1. Indeed, the expression of this growth factor persists elevated in the asthmatic airways despite steroid treatment (33–35), including in association with the bronchial epithelium (36).

The expression of RAR{gamma}, RXR{alpha}, and RXR{gamma} in morphologically restored epithelium of subjects with severe asthmas was observed throughout the epithelial layer, suggesting widespread functional changes in relation to the repair process. In support, univariate correlation analysis demonstrated that RXR{alpha} and RXR{gamma} imunostaining was positively related to the proportion of morphologically intact epithelium, suggesting that the expression of these receptors may represent a "reporter" of an ongoing repair process.

Interestingly, changes in the numbers of RAR{gamma}-, RXR{alpha}-, and RXR{gamma}-positive cells paralleled that of mucosal fibroblasts/myofibroblasts, suggesting that retinoid-mediated epithelial repair may be associated with functional abnormalities that directly contribute to subepithelial fibrosis. A similar concept has previously been illustrated by Puddicombe and colleagues (37), who showed that EGF-R was overexpressed in the asthmatic bronchial epithelium, even in patients with severe disease and treated by steroid therapy. The extent of EGF-R immunostaining correlated positively with subepithelial membrane thickening, used as a hallmark of airway remodeling. Using human bronchial epithelial cell monolayers, these authors also demonstrated that EGF-R signaling mediated both wound repair and TGF-β1 synthesis. Together, these observations have led to the postulation that persistent EGF-R expression would "lock" the airway epithelium into a repair phenotype in which it sustains airway remodeling by providing profibrogenic growth factors (37).

Using the NaOH-induced wound system, we show for the first time that retinoids promote epithelial repair in vitro, extending thus in vivo and in vitro studies demonstrating the ability of retinoids to promote epithelial regeneration after injury (9–11). The normal repair process of the bronchial epithelium involves cell migration, growth, and differentiation (4, 5). Since retinoids are known to prevent airway epithelial cell growth (38), and since in our hands ATRA and 9-cis RA fail to induce airway epithelial cell proliferation, regeneration may result either from migration or from differentiation, or from a combination of both events. The effects of a long-term (at least 7- to 14-d) exposure to retinoids on differentiation of airway epithelial cells toward a mucosecretory and ciliary phenotype are well documented (6, 7). We now demonstrate that exposure of airway epithelial cells to ATRA- and 9-cis RA–induced epithelial cell chemokinesis, as evidenced by the increment in the area of cell monolayers and by the acquisition of a morphology characteristic of migrating cells. Together, these results extend previous observations showing that retinoids promote migration of pro-myelocytic leukemia cells (39, 40).

We also found that a short-term (6- to 72-h) exposure of airway epithelial cells to ATRA and 9-cis RA selectively activated a pattern of factors (TGF-β1, MMP-9, HGF-R, and β1-integrin) that is known to participate in epithelium migration and repair, and in airway remodeling (4, 5, 20, 41–43). These results support previous observations showing that retinoids augmented the expression of TGF-β1, MMP-9, HGF-R, and β1-integrin in different cell types (44–47). TGF-β1 initiates the migratory response of airway epithelial cells by augmenting the surface expression of β1-integrin (48, 49), suggesting that the increase in β1-integrin transcript in response to retinoid stimulation presently described may be secondary to TGF-β1 synthesis. Our current findings demonstrating that the inhibition of endogenously produced TGF-β by a neutralizing antibody down-regulated MMP-9, but not HGF-R and β1 integrin, synthesis, suggest that the effects of retinoids involve both TGF-β1–dependent and -independent mechanism.

It is noteworthy that most of these factors contribute to and are reporter of lung remodeling. Thus, the levels of TGF-β and MMP-9 were shown to be up-regulated in the bronchial wall of subjects with asthma in relation with disease severity, chronicity, and airway structural abnormalities (1, 50, 51), and a greater expression of HGF-R was demonstrated in alveolar epithelial cells in association with fibrotic lung diseases (52).

In conclusion, the observations reported in the present study demonstrate that some RA receptor isotypes are overexpressed in the bronchial epithelium of subjects with severe asthma in relation to the extent of its morphologic restitution and in parellel with fibroblast/myofibroblast accumulation beneath the subepithelial basement membrane. Ligation of RA receptors by ATRA and 9-cis RA promotes wound repair and chemokinesis of airway epithelial cells and up-regulates the synthesis of TGF-β1, MMP-9, HGF-R, and β1-integrin that are known to regulate cell migration and to sustain tissue fibrosis. Hence, these findings suggest that persistent and exaggerated RA–receptor interaction results in an abnormal repair process, with the epithelium becoming a source of fibrogenic growth factors that participate in airway remodeling in severe asthma. Therefore, strategies aimed at normalizing RA receptor expression and function in the airway epithelium may represent a valuable tool to resolve airway remodeling and the associated respiratory dysfunction in this disease.


   Acknowledgments
 
The authors are indebted to the patients who participated in the study, to the staff of the Service d'Explorations Fonctionnelles of the Groupe Hospitalier Universitaire Nord Bichat Claude-Bernard, Paris, France, who performed lung function measurements, and Dr. Joëlle Le Moel (Service de Biochimie A, Groupe Hospitalier Universitaire Nord Bichat Claude-Bernard, Paris, France) for retinol determination. The authors also thank Dr. E. Puchelle (Inserm U514, Centre Hospitalier Universitaire Maison Blanche, Reims, France) for helpful discussion concerning this work and manuscript.


   Footnotes
 
This work was supported in part by the Agence Nationale de la Recherche (grant no. 0012405). L.B. was funded by the Association Nationale pour le Traitement à Domicile de l'Insuffisance Respiratoire chronique.

* Present affiliation: Inserm U845, Groupe Hospitalier Universitaire Sud Necker-Enfants Malades, Paris, France. Back

{dagger} Present affiliation: Service d'Anesthésie et Réanimation, Groupe Hospitalier Universitaire Nord Beaujon, Clichy-la-Garenne, France. Back

{ddagger} Present affiliation: Kosair Children's Hospital Research Institute, Louisville, KY. Back

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1165/rcmb.2006-0453OC on September 20, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form December 8, 2006

Accepted in final form July 26, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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Erratum: Epithelium Expression and Function of Retinoid Receptors in Asthma
Am. J. Respir. Cell Mol. Biol., November 1, 2008; 39(5): 628 - 629.
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