ARTICLE |
Correspondence to: Jean-François Bernaudin, Service d'HistologieBiologie Tumorale, Université Pierre et Marie Curie, Paris VI, Hôpital Tenon, 4 rue de la Chine, 75020 Paris, France. Fax:+ 33 1 56 01 78 53
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Summary |
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CFTR (cystic fibrosis transmembrane conductance regulator), MDR1 (multidrug resistance), and MRP1 (multidrug resistance-associated protein), members of the ABC transporter superfamily, possess multiple functions, particularly Cl-, anion, and glutathione conjugate transport and cell detoxification. They are also hypothesized to have a number of complementary functions. It is generally accepted that data obtained from nasal mucosa can be extrapolated to lower airway cell physiology. The aim of the present study was to investigate by immunohistochemistry the differential localization of CFTR, MDR1, and MRP1 in the normal mucosa of 10 human nasal turbinates. In ciliated epithelial cells, CFTR was inconstantly expressed at the apical cell surface, intense membranous labeling was observed for MDR1, and intense cytoplasmic labeling was observed for MRP1. In the glands, a higher level of expression was observed on serous cells, at the apical surface (for CFTR), on lateral membranes (for MDR1), and with an intracytoplasmic distribution (for MRP1). In conclusion, CFTR, MDR1 and MRP1 are expressed in the epithelium and glands of the nasal respiratory mucosa, but with different patterns of expression. These results suggest major roles for CFTR, MDR1, and MRP1 in serous glandular cells and a protective function for MDR1 and MRP1 in respiratory ciliated cells. (J Histochem Cytochem 48:12151222, 2000)
Key Words: CFTR, MDR1, MRP1, nasal mucosa, respiratory epithelium, immunohistochemistry
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Introduction |
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CFTR (cystic fibrosis transmembrane conductance regulator), MDR1 (multidrug resistance), and MRP1 (multidrug resistance-associated protein) are members of the ABC (ATP binding cassette) transporter superfamily, which use the energy of ATP hydrolysis to regulate or energize the transport of molecules as diverse as chloride, large organic anions, xenobiotics, or glutathione conjugates (
CFTR, the protein impaired in cystic fibrosis (CF), an inherited disease particularly affecting the conductive airways, is a chloride channel. MDR1 and MRP1 play a role in cell detoxification and multidrug resistance, and MRP1 is involved in glutathione conjugate transport. However, CFTR, MDR1, and MRP1 could share certain functions, such as control of heterologous chloride channels for CFTR and MDR1 (
The respiratory mucosa in nasal cavities and cartilaginous bronchial airways has a similar structure, consisting of respiratory pseudostratified epithelium with ciliated, mucus-secreting, and basal cells associated with glands, mainly composed of serous and mucus-secreting cells. It has therefore been widely accepted that respiratory cell biology data obtained from investigations performed on nasal mucosa can be extrapolated to lower airway cell physiology (
This study was therefore designed to precisely determine, by immunohistochemistry, the differential localization of CFTR, MDR1, and MRP1 transmembrane proteins in respiratory epithelium and in the glands of normal, non-polypous respiratory mucosa from human nasal turbinates.
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Materials and Methods |
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Nasal Mucosal Samples
Mucosal samples from the respiratory segment of the nasal cavity were obtained within 30 min of resection from nasal turbinates collected from patients who underwent surgery in the Oto-Rhino-Laryngology Department of Hôpital Tenon (Paris, France). The 10 patients included in the present study (seven men and three women) underwent turbinectomy for discomfort and nasal obstruction. The patients ranged in age from 25 to 73 years (mean 45 years). Five patients were current smokers and five were non-smokers. None of the patients presented with cancer and none had received prior chemotherapy or radiotherapy. This study was conducted in accordance with the rules of the National Recommendations for Medical Research.
Fresh respiratory nasal mucosal samples were immediately fixed for 24 hr in 80% ethanol at 4C and paraffin-embedded as previously reported (
Immunohistochemical Detection of CFTR, MDR1, and MRP1 Proteins
Antibodies.
Various specific monoclonal antibodies (MAbs) were used for immunodetection of CFTR, MDR1 and MRP1. MAb 24-1 (R&D Systems Europe; Abingdon, UK) is a mouse IgG2a directed against four C-terminal amino acids (14771480) of the CFTR protein (working dilution 1:200; 1 µg/ml) (
Negative controls were performed by replacing the primary antibody with an immunoglobulin of the same origin and isotype, i.e., a mouse IgG2a (Dako) for C494 and 24-1 and a rat IgG2a for MRPr1(Immunotech; Marseille, France) at the same protein concentration.
Immunohistochemical Procedure.
Four-micrometer paraffin sections were placed on slides pretreated with silane (3-amino-propyltriethoxysilane; Sigma Chemical, St Louis, MO) and kept overnight at 37C. The sections were dewaxed with toluene and rehydrated with distilled water through a series of alcohol solutions. Immunohistochemical visualization was performed using the sensitive Dako EnvisionTM+ system (
The sections were then rinsed in 1 x TBS0.1% Tween-20 (TBST; Sigma) and incubated with blocking serum containing 30% normal human AB serum (Sanofi Diagnostics Pasteur; MarneslaCoquette, France) in TBST for 1 hr at RT, followed by 1-hr incubation with the primary MAbs diluted in 3% TBSTbovine serum albumin (Sigma)10% normal human AB serum for 1 hr in a moist chamber at RT according to the different working solutions.
For C494 and 24-1, a secondary labeled polymer anti-mouse antibody (EnvisionTM+; Dako) was applied for 30 min at RT.
For the MRPr1 MAb, a second rabbit anti-rat antibody (Dako) was applied for 30 min at RT, followed by incubation with the labeled polymer anti-rabbit antibody.
The sections were then exposed to a working solution containing the diaminobenzidine chromogen (EnvisionTM+; Dako) for 5 min at RT according to the manufacturer's recommendations. Between each step, sections were rinsed three times for 5 min in TBST. The slides were counterstained with hematoxylin (Sigma) and mounted in Eukitt (Labonord; Villeneuve d'Ascq, France).
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Results |
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Histological examination of all samples did not reveal any inflammatory lesions or neoplastic changes. Normal pseudostratified surface epithelium, composed of ciliated cells, mucus-secreting cells, and one layer of basal cells in three samples, or with slight basal cell hyperplasia in the seven other samples, was observed (
Glands were a predominant feature of the nasal respiratory mucosa in all samples studied. They were composed of mucous, serous, and mixed secretory parts and rather short collecting ducts opening into the superficial respiratory epithelium layer.
Respiratory Epithelium
CFTR.
After incubation with the anti-human CFTR MAb 24-1, epithelial labeling was always much weaker than that observed on glands, and heterogeneous results were obtained. Focal staining of the apical cell surface of ciliated cells was observed in 4 / 10 samples (Fig 1A). Isolated weak intracytoplasmic labeling of the supranuclear region of ciliated cells was detected in 4 / 10 samples and was associated with apical cell membrane labeling in 2 / 10 samples (Fig 1B). No staining was observed in basal or mucus-secreting cells (Fig 1A and Fig 1B).
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MDR1. In contrast to the results obtained for CFTR, intense staining with a homogeneous pattern was observed on all samples after incubation with the anti-human MDR1 MAb C494 (Fig 1C). Dense apical and basolateral staining of the plasma membrane of ciliated cells was consistently observed and was consistently associated with intracytoplasmic staining. Labeling of the cell circumference of basal cells was observed except at points of contact with the basal lamina. No staining was detected over mucus-secreting cells.
MRP1. Homogeneous labeling, different from that described above for MDR1, was observed after incubation with the anti-human MRPr1 MAb. Granular cytoplasmic staining was observed in the apical part of all ciliated cells (Fig 1D). Weak labeling was detected in the cytoplasm of basal cells. No labeling was observed over mucus-secreting cells.
Glands
CFTR.
A consistent labeling pattern was observed in glands after incubation with the anti-human CFTR MAb, in contrast to that observed in the respiratory epithelium. Very dense staining was observed over all serous cells (Fig 1F1H), with intense labeling of the apical membrane and upper part of the intercellular space with invaginations (Fig 1G). Staining was also observed on the apical surface of ciliated cells and inside the lumen of collecting ducts (Fig 1H). No staining was detected over mucous cells, and only serous cells were labeled in mixed glands (Fig 1I).
MDR1. After incubation with the C494 anti-human MDR1 MAb, a consistent labeling pattern was observed. The cell membrane limiting the intercellular space between serous cells was always intensely stained (Fig 2A and Fig 2B), but labeling of the apical membrane was rarely observed (one sample of the 10 tested). Discrete granular cytoplasmic staining was consistently associated (Fig 2B). Lateral labeling of mucus-secreting cells was very rare (Fig 2A) and the great majority of these cells were negative, whereas myoepithelial cells were consistently stained (Fig 2C). Staining of collecting ducts was similar to that observed on serous acini. Dense staining of lymphocytes and the luminal side of endothelial cells was consistently observed (Fig 2A).
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MRP1. After incubation with the MRPr1 anti-human MRP1 antibody, dense intracellular granular labeling was consistently detected in all serous cells (Fig 2D). This staining was strongly reinforced in the apical part of the cell. In contrast, no peripheral staining of the cell membrane was present. Mucus-secreting cells were negative, whereas myoepithelial cells in contact with mucus-secreting cells displayed intense cytoplasmic immunoreactivity (Fig 2E). Cells of collecting ducts were negative. Lymphocytes and endothelial cells showed weak intracytoplasmic labeling.
Controls
Staining with control isotype-matched irrelevant antibodies was negative for all samples, both for respiratory epithelium (Fig 1E) and for glands (Fig 2F).
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Discussion |
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This study shows that immunohistochemistry clearly localizes CFTR, MDR1, and MRP1 in normal human nasal mucosa. In all samples studied, the respiratory epithelial layer had either a normal morphological appearance or exhibited only slight basal cell hyperplasia (
CFTR was inconsistently detected in ciliated cells of the respiratory epithelium, either on the apical surface or with an intracytoplasmic distribution. Previous studies either failed to demonstrate the presence of CFTR in these cells (
Glandular structures of the nasal mucosa are composed of mucous, serous, and mixed secretory units and myoepithelial cells (
We (
In the respiratory epithelium, intracellular granular staining for MRP1 was observed in all ciliated cells, particularly in the apical part of the cell. This result, at variance with the basolateral localization previously reported for bronchial respiratory cells (
In conclusion, although a potential crossreaction of the monoclonal antibodies used with as yet undiscovered ABC proteins cannot be formally excluded, the present study shows that CFTR, MDR1, and MRP1 are expressed in the epithelium and glands of human nasal respiratory mucosa, but with different patterns of expression. Unlike MDR1 and MRP1, CFTR is inconsistently expressed in ciliated epithelial cells. In contrast, all three ABC proteins are consistently and intensely expressed in serous cells of the glands. These results suggest major roles for CFTR, MDR1, and MRP1 in serous glandular cells, and at least a protective function for MDR1 and MRP1 in respiratory ciliated cells. The respective roles of these proteins in the various cells, particularly in serous glandular cells, either in the control of the composition of the bronchial superficial fluid layer or airway cell detoxification, need to be evaluated by cell function studies using in vitro models, such as organotypic cultures.
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Acknowledgments |
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Supported by Crédits Université Pierre et Marie Curie, Paris VI, and by grant R98018 from the Association Française de Lutte contre la Mucoviscidose (AFLM). M.-A. Wioland is an AFLM fellow.
We are greatly indebted to C. Danel, C. Guernier, L. Germain, C. Prengel, and S. Ricci for constant support and to V. Gerber for expert editorial assistance.
Received for publication December 28, 1999; accepted April 21, 2000.
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