ARTICLE |
Correspondence to: Peter H. Michelson, Div. of Pulmonary and Critical Care Medicine, Northwestern U. Medical School, 300 E. Superior, Tarry 14 - # 707, Chicago, IL 60611. E-mail: pmichelson@nwu.edu
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Summary |
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Epithelial cells attach to the basement membrane through adhesive contacts between the basal cells of the epithelium and the proteins of the extracellular matrix (ECM). The hemidesmosome (HD) is a specialized cellECM contact, that mediates the attachment of the epithelial cell basal surface to the ECM. In bronchial epithelial cells, the protein components that constitute the HD have not been demonstrated. Using immunohistochemical techniques, we determined that normal human bronchial epithelial (NHBE) cells express the HD cell surface integrin 6ß4 and produce laminin 5, the ECM protein associated with HDs. Furthermore, expression of the HD-associated structural proteins, bullous pemphigoid antigens 1 (BPAG 1) and 2 (BPAG 2), was demonstrated in NHBE cells by immunofluorescence microscopy and immunoblot analyses. In addition, we confirmed the presence of laminin 5 in the basement membrane (BM) of bronchial epithelial biopsy specimens and of BP230, BP180, and the
6ß4 integrin heterodimer at the site of bronchial epithelial cellECM interaction in vivo. Finally, using electron microscopy, we were able to demonstrate intact HDs in a glutaraldehyde-fixed NHBE cell monolayer. These findings suggest that bronchial epithelium forms HDs and that the laminin 5
6ß4 integrin interaction may be important in stabilizing epithelial cell adhesion to the BM in the lung. (J Histochem Cytochem 48:535544, 2000)
Key Words: hemidesmosome (HD), integrin, extracellular matrix (ECM)
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Introduction |
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Epithelial cell adhesion to the underlying basement membrane (BM) is essential for normal lung growth and development and for preservation of barrier function and host defense (
Integrins are heterodimeric transmembrane receptors that are composed of an -regulatory and a ß-signal-transducing subunit (
6ß4 integrin heterodimer with laminin 5, a heterotrimeric ECM glycoprotein composed of
3-, ß3-, and
2-subunits. Each HD also possesses three structural proteins, including the bullous pemphigoid antigens I (BPAG 1 or BP230) and II (BPAG 2 or BP180) and plectin (IFAP300) (
6ß4, and plectin (
A number of integrins that facilitate adhesion and attachment have been described in normal human bronchial epithelial (NHBE) cells, but the integrins and other structural proteins that are associated with the formation of bronchial cell HDs have not been identified (6ß4 and that the bronchial epithelium produces laminin 5, the ECM protein that co-localizes with the integrin heterodimer to form HDs (
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Materials and Methods |
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Cell Culture and Tissue Preparation
Normal human bronchial epithelial (NHBE) cells were obtained from Clonetics (Walkersville, MD). Cells were cultured at 37C in humidified 5% CO2 in air. NHBE cells were grown in bronchial epithelial growth medium (BEGM; Clonetics). BEGM consists of bronchial epithelial basal medium (BEBM) supplemented with recombinant human epidermal growth factor (rhEGF), insulin, hydrocortisone, transferrin, tri-iodothyronine, epinephrine, pituitary extract (PE), and retinoic acid. Cells were plated onto glass coverslips for 48 hr or until 75% confluence before fixation and immunofluorescent staining. SCC12 cells, a transformed keratinocyte cell line known to express most if not all hemidesmosomal antigens (
Normal bronchial tissues, surplus pathological specimens removed during staging pneumonectomy, were snap-frozen in liquid N2. Sections of frozen tissue material were prepared on a cryomicrotome and mounted on glass slides in the immunohistochemistry facility at Northwestern Memorial Hospital (Chicago, IL). Some sections were stained with hematoxylin and eosin for specimen orientation purposes, and others were prepared for immunofluorescence analyses (see below).
Antibodies
A description of antibodies is provided in Table 1. In brief, mouse monoclonal antibodies (MAbs) 3E1 and P1B5 (Gibco) and rat MAb GOH3 (Immunotech; Westbrook, ME) against cell surface integrins ß4, 3, and
6, respectively, were used in these studies. Integrin ß4 antibody AB1922 was purchased from Chemicon (Temecula, CA) for use in immunoblotting studies. For bullous pemphigoid antigens, we used 5E and 10C5A, MAbs against BP230 (BPAG I), whose preparation has been described previously (
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GB3 antibodies (Harlan Bioproducts for Science; Indianapolis, IN), which recognizes the -chain of laminin 5, was used for immunofluorescence microscopy. In addition, KS12 (Desmos; San Diego, CA), an
3-laminin chain-specific mouse MAb, and B1K (clone 17) (Transduction Laboratories; Lexington, KY), a ß3-laminin chain-specific antibody, were utilized for immunofluorescence studies. MAb 10B5, an
3-chain-specific antibody (
2-laminin chain of the laminin 5 molecule, were used for Western immunoblotting (Jones and Goldfinger, unpublished data). Antibody dilutions used to perform these studies are indicated in Table 1.
Secondary fluorescein-conjugated goat anti-mouse, goat anti-rat, goat anti-rabbit, and goat anti-human antibodies were purchased from Jackson Immunoresearch Laboratories (West Grove, PA). Horseradish peroxidase-conjugated goat anti-mouse, goat anti-rabbit, and goat anti-human secondary antibodies were obtained from the same supplier.
Immunofluorescence Microscopy
Cells on coverslips were rinsed in PBS. If the cells were to be probed with antibodies against the BP antigens or the matrix antibodies, the specimens were placed in acetone for 2 min at -20C and subsequently air-dried. Alternatively, those cells to be processed with integrin antibodies were fixed in 3.7% formaldehyde for 5 min at room temperature, extracted in -20C acetone, and then air-dried. Primary antibodies were overlaid on the cells plated on coverslips that were incubated for 1 hr at 37C. After a 5-min wash in PBS, appropriate secondary antibodies were overlaid on the cells for a second 1-hr incubation. After washes in PBS, the coverslips were mounted on slides in mounting medium and dried in the dark before examination (
Cryosections of tissue samples were fixed in acetone at -20C for 5 min. After incubation in primary antibody, the slides were gently washed in PBS to prevent disruption of the tissue specimens. After a 1-hr incubation with the secondary antibody, the sections were washed again in PBS, mounted, and dried as described above.
Protein Preparations, Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis, and Western Immunoblotting
HD protein and integrin expression were assessed in whole-cell extracts of cultured cells, whereas laminin 5 expression was determined in ECM preparations. The latter were prepared using a modification of a technique described by
Electron Microscopy
Cells maintained on tissue culture plastic until confluent were fixed for 30 min in 1% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2. After fixation, cells were washed three times in 0.1 M sodium cacodylate buffer and postfixed in 1% OsO4 containing 0.8% potassium ferricyanide. Preparations were then stained with uranyl acetate, dehydrated in ethanol, and embedded in EponAraldite resin (Tousimis; Rockville, MD). Thin sections of the embedded material were stained with lead nitrate and sodium citrate and viewed at 60 kV in a JEOL 100CX electron microscope (JEOL USA; Peabody, MA).
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Results |
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Immunofluorescence of HD Proteins in Human Bronchial Epithelial Cells In Vitro
To ascertain whether human bronchial epithelial cells express the HD integrins 6 and ß4 and the bullous pemphigoid proteins, we performed immunofluorescence staining, examining NHBE cells grown on glass coverslips. Antibodies 3E1 and GOH3 against ß4 and
6, respectively, stained NHBE cells in a "wavy" configuration with basilar localization, as determined by carefully examining the cells at different focal planes from the apical to basal surface (see arrows, Fig 1A and Fig 1C). This pattern was similar to that observed in keratinocytes and was not seen with negative controls, using appropriate secondary antibody alone (not shown) (
3 integrin, localized to the cellcell contacts (Fig 1E). For each image in which antibody localization is shown, a matched phase-contrast image is presented to demonstrate the cell morphology and provide spatial orientation (Fig 1B and Fig 1D).
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Staining for the bullous pemphigoid antigens BP180 and BP230 using antibodies J17 and 5E, respectively, also generated a similar wavy basal staining pattern as seen in keratinocytes (Fig 2A and Fig 2C) (
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Production of Laminin 5 in Human Bronchial Epithelial Cells In Vitro
We next examined laminin 5 production by NHBE cells. Laminin 5 is a ligand for the 6ß4 integrin and is essential for the assembly of HDs in epithelial cells (
3, ß3, and
2. For these studies, the MAbs KS-12 (
3), B1K (ß3), and GB3 (
2) were used, and all demonstrated significant ECM staining, as well as some intracellular staining (Fig 3A, Fig 3C, and Fig 3E). Phase-contrast images are presented for each fluorescence localization (Fig 3B, Fig 3D, and Fig 3F). It should be noted that the ß3 antibody probe generated more intense cytoplasmic staining of NHBE cells compared to the other laminin 5-subunit antibodies (compare Fig 3C with Fig 3A and Fig 3E). This may reflect differences in the ability of our three laminin 5-subunit antibodies to stain cytoplasmic pools of their respective antigens or, possibly, differences in the levels of the laminin 5-subunits in the cytosol.
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Immunoblotting of HD Proteins and HD ECM Elements in NHBE Cells In Vitro
To establish the nature of the HD proteins expressed by NHBE cells and to characterize the ECM produced and deposited by this cell type, we performed Western blot analyses. Using antibodies AB1922, J17, and 5E, we determined whether NHBE cells express integrin ß4 and the BP antigens (BP180 and BP230) and compared the electrophoretic gel mobility of the NHBE cell antigens with those in the squamous cell carcinoma cell line SCC12 (Fig 4). As demonstrated, both the ß4 integrin protein and the two BP proteins were readily expressed by NHBE cells and appeared to migrate identically to their keratinocyte counterparts.
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Analysis of the ECM deposited by the NHBE cells was then performed by Western immunoblotting. Antibodies 10B5, B1K, and 3D5, specific for the 3-, ß3-, and
2-subunits, respectively, recognize proteins of 190/160 kD, 145 kD, and 155 kD, respectively, in ECM preparations derived from NHBE cells (Fig 5). Species of similar molecular weight were recognized in SCC12 ECM preparations by the same set of antibodies (Fig 5). Whereas the predominant polypeptide recognized by the 10B5 antibody in the ECM of SCC12 cells was 190 kD, the same antibody showed more intense staining of the 160 kD species in NHBE ECM (
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Electron Microscopy of HDs in Bronchial Epithelial Cells In Vitro
Monolayers of NHBE cells grown under routine cell culture conditions were fixed in glutaraldehyde and prepared for EM as described. Mature HDs complete with dense plates were seen (Fig 6A). At high magnification, each mature HD plaque has a tripartite architecture that is associated with the keratin intermediate filament network (Fig 6B). In Fig 6B, one immature hemidesmosome was observed. This has a less well-defined cytoplasmic plaque than the mature hemidesmosome that lies nearby.
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Expression of Hemidesmosomal Integrins and Laminin 5 in Human Bronchial Biopsy Specimens
To relate the immunocytochemical results we obtained in cultured cells with expression of HD proteins in lung tissue, we next analyzed bronchial tissue specimens by immunofluorescence microscopy. As shown in Fig 7, bronchial biopsy specimens contained a characteristic pseudostratified columnar epithelium, and as seen in the phase-contrast images (Fig 7B, Fig 7D, Fig 7F, Fig 7H, and Fig 7J), the airway epithelium, lumen, and BM were clearly visible. In Fig 7A and Fig 7C, antibodies GOH3 and 3E1 showed strong immunofluorescence staining at the cellBM interface, confirming the localization of the integrins 6 and ß4 to the basal surface of the epithelial cell, with no significant staining at the cellcell contacts. Similarly, laminin 5 staining, using the GB3 antibody (Fig 7E), was confined to the BM. Like
6 and ß4 integrin, the hemidesmosomal proteins BP180 and BP230 were also localized to the epithelial cellBM interface (Fig 7G and Fig 7I).
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Discussion |
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In previous studies, laminin 1 production by bronchial epithelial cells has been reported in the developing airway preceding the presence of the epithelium (6ß4 and
3ß1 in the distal airways of patients undergoing biopsy for diagnosis of pulmonary carcinoma. However, the expression of laminin isoforms in the BM of airway epithelium has not been studied extensively. Using a panel of antibodies specific for the
3-, ß3-, and
2 laminin-subunits, we have shown by immunoblot analyses that primary human bronchial epithelial cells produce laminin 5 in vitro. Furthermore, utilizing immunofluorescence microscopy on human bronchial sections, we have been able to provide evidence that laminin 5 is a component of the bronchial BM.
In NHBE cells, we see two forms of the 3-subunit of laminin 5 by Western immunoblotting. The larger of the two migrates at 190 kD on SDS-PAGE, whereas the smaller shows a molecular weight of approximately 160 kD. Previous studies have shown that the
3-subunit is first secreted as a 190-kD species and then undergoes cleavage within its G-domain, resulting in the production of a 160-kD
3 isoform (
3-subunit is required for the nucleation of HD assembly (
3 laminin-subunit isoform and these cells assemble bona fide HDs.
In epithelial cells, the two major integrin receptors for laminin 5 are 6ß4 and
3ß1 (
3ß1 occurs primarily at sites of cellcell contact, whereas the integrin pair
6ß4, a component of HDs, occurs basally (
6 and ß4 on the basal surface of NHBE cells is similar to that observed for HD proteins, particularly the bullous pemphigoid antigens BP180 and BP230. Therefore, the HDs of NHBE cells contain all of the components of the classical or Type I HD seen in keratinocytes. This, taken together with the similarity between the appearance of NHBE HDs and epidermal HDs, leads us to propose that airway epithelial cells form the classical type of HD.
Demonstrating bona fide HDs in the airways has a multitude of implications. HDs are considered to be sites of stable cellECM anchorage that are involved in the maintenance of tissue integrity (
In epithelial cells, HDs have additional roles besides adhesion and morphogenesis (6ß4 (
Received for publication July 2, 1999; accepted November 24, 1999.
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