Pan-cadherin expression is along the cell membranes, CCSP in the cytoplasm and cell membranes, mostly on outer surface. B—E Immunofluorescence staining of sorted cell fractions. E Few cells in either fraction expressed pro-SPC red fluorescence.
Recovered cells were trypsinized in 0. Rabbit IgG staining was used as an isotype-matched negative control and CCSP staining with permeabilization of dissociated cells was used as a positive control.
Single and dual labeling of cells and tissue sections by immunofluorescence IF or immunohistochemistry IHC was performed according to previously described methods [16] — [17]. After 1 hour of incubation at room temperature, slides were washed in PBS three times. Secondary antibodies were added. For dual-labeling IF, additional primary antibodies were added after the third PBS wash, followed by incubation with secondary antibodies conjugated with Alexa fluor or Invitrogen.
Control slides were included in each analysis in which non-immune serum was substituted for primary antibodies and secondary antibodies individually. B The diameter of all spheroid colonies in the wells which cells were seeded. C Average spheroid colony number per well in serial diluted cells after one week sphere cell culture. Amplification was confirmed by ethidium bromide staining of the PCR products on an agarose gel.
After 1 week, cell spheroid colony numbers were counted and colony size was measured under a Zeiss Axio Observer Z1 Inverted Microscope Zeiss. Secondary sphere culture was performed after digestion of first sphere colonies by 0. Alzet pumps were implanted in mice and removed after one week. Mice were sacrificed 4 weeks after removal of the Alzet pumps.
Label-retaining cells were identified by BrdU immunofluorescence. BrdU and CCSP double staining was performed and cells exhibiting a nucleus and attachment to basement membrane were counted.
Bronchiolioles BLs were defined as intrapulmonary airways in which smooth muscle, but neither cartilage nor glands, could be seen. A total 49 TBs and 26 BL structures were analyzed in lung sections of five mice. Double positive cells were represented in TB Left and middle panel, white arrow.
CCSP which is widely used as a Clara cell marker is a cytoplasmic secretory protein [3]. Figure 2A revealed intense immunorectivity along the lining of mouse TB. Recently, Wong AP et al. Therefore, we postulated that CCSP may be expressed not only in the cytoplasm, but also in the cell membrane of Clara cells.
To obtain evidence for this, we used the well-known cell membrane marker pan-Cadherin [18]. Indeed, in high magnification photomicrographs we were able to demonstrate co-expression with pan-Cadherin in the cell membrane using confocal microscope Figure 2B.
These data suggested a possibility for isolating living Clara cells by flow cytometry sorting and lead us to develop the protocol outlined in this study. Sorted cells were plated into mm cell culture dish for overnight in an incubator prior to further studies.
Unattached dead cells were removed with the media. These data demonstrated that sorting by flow cytometry is a useful and simple method for harvesting purified CCSP-containing Clara cells. Spheroid culture is a common method to detect stem cell features in vitro [19]. Spheroid colony formation was tested by serial dilution technique. Dissociation of spheroid colonies into single cells resulted in reformation of the spheroid colonies, indicating that this phenotype was stable data not shown.
Quiescent or slow-cycling stem cells in adult tissues can retain BrdU over long periods by either segregating chromosomes asymmetrically or dividing slowly. Label-retaining cells can be used to identify populations that contain stem cells [20]. In fact, many such studies have been used to determine putative stem cell locations in mammalian tissues [21] , [22].
Our results showed that 1. This provides further evidence for the progenitor role that Clara cells may have in the mouse lung. In this study, we isolated and characterized significantly purified CCSP-expressing cell populations from mouse lung by using high concentrations of collagenase and a flow cytometric sorting method.
Accordingly, the novel method described herein is a significant step in the progress of isolating and characterizing highly purified Clara cells in primary cultures. Based on previous publications, the distribution of CCSP expression in non-ciliated Clara cells is described as cytoplasmic [23] , [24] , [25]. We made the surprising and novel discovery of CCSP immunoreactivity along cellular membranes of bronchiolar Clara cells.
Using pan-cadherin as a cell membrane marker in normal airway epithelium [18] , [26] , [27] we found CCSP was expressed not only in the cytoplasm, but also in the membrane. These findings gave rise to the possibility that living Clara cells can be isolated by flow cytometry using fluorescing tags. One explanation is that bronchiolar Clara cells secrete such large quantities of CCSP that part of it remains stuck to the outer surfaces of cell membranes, allowing sorting of CCSP-containing cells from suspension.
Clara cell isolation from rabbit was first reported in the early s by Devereux et al. They serve the function of progenitor cells, participate in xenobiotic metabolism, and regulate the pulmonary immune system. They are destroyed as early as 24 hours after the action of a harmful factor and are rebuilt after approximately 30 days [ 25 ]. As previously demonstrated, CCs secrete various proteins into the bronchial tree, blood, and urine; labeling these proteins has found its application in the diagnosis of some conditions.
Their concentration has been observed to increase in both pulmonary fluid and blood serum of patients with sarcoidosis or pulmonary fibrosis and in patients undergoing mechanical ventilation with high positive end-expiratory pressure PEEP values.
The authors of this report suggested that the concentration of the CC16 protein is a good non-invasive indicator for the assessment of the respiratory system's integrity [ 12 , 26 ].
These observations were confirmed by Wutzler et al. The authors concluded that the protein is a good indicator of secondary pulmonary insufficiency in patients with multiple injuries [ 27 ].
Elevated concentration of the CC16 protein in bronchiolar secretion is considered to be a good marker of increased lung-blood barrier permeability [ 12 ]. Still other authors have demonstrated that the serum concentration of the CC16 protein and surfactant is a useful marker for detecting the early form of silicosis [ 13 ]. Labeling this protein in blood and urine as a good marker for fibrotic changes in pneumoconiosis patients has also been proposed [ 28 ].
In turn, a large-scale study conducted on approximately patients with advanced lung cancer demonstrated that the serum concentration of the CC16 protein can be used to predict a mortality risk [ 29 ].
For many years now, the proteins secreted by CCs have been the subject of searching molecular and clinical studies. The reasons behind this interest include their anti-inflammatory and immunomodulatory properties; furthermore, studies on cell lines of human pulmonary adenocarcinoma have suggested that they also possess antineoplastic properties. It is to be hoped that further studies on CCs and their secretory proteins will give rise to their application in therapy for many lung conditions.
National Center for Biotechnology Information , U. Journal List Kardiochir Torakochirurgia Pol v. Kardiochir Torakochirurgia Pol. Published online Mar Author information Article notes Copyright and License information Disclaimer. Corresponding author. Address for correspondence: Prof. This article has been cited by other articles in PMC.
Abstract The report presents the cellular structure of the respiratory system as well as the history of club cells Clara cells , their ultrastructure, and location in the airways and human organs. Keywords: club cells, Clara cells, xenobiotic detoxification, progenitor cells. The epithelium lining the primary airways includes the following types of cells: ciliated cells with approximately cilia on the apical surface, goblet or beaker cells which produce and secrete mucus, brush cells with numerous microvilli on the surface, undifferentiated basal cells with high mitotic potential, small granule cells belonging to the group of APUD cells which regulate the secretory function of goblet cells and glands in the mucous membrane.
In the respiratory bronchioles, forming the margin between the conducting and respiratory zones, the following types of cells can be distinguished: ciliated cells the most common , microvillar cells few , small granule cells few , bronchiolar cells known as club cells or Clara cells CCs. In the pulmonary alveolar epithelium, the following cell types can be distinguished: type I pneumocytes, type II pneumocytes, type III pneumocytes rare , numerous pulmonary macrophages [ 1 ].
In physiological conditions, they are believed to have the following fundamental functions: they secrete the primary components of the extracellular substance lining the respiratory bronchioles, they are progenitor cells for both themselves and ciliated cells, they regulate the contents of secretion fluid in the distal segments of the respiratory tract, in the lungs, they play a key role in the biotransformation of inhaled xenobiotics together with cytochrome P and mixed-function monooxygenases.
However, it is believed that progenitor CCs participating in the repair of airway epithelial damage constitute causative factors behind the development of the nosological entities listed below probably by stimulating the development of mesenchymal fibroblasts : mucoviscidosis cystic fibrosis , ARDS, COPD, pulmonary fibrosis, pulmonary emphysema, pulmonary hypertension, subglottic tracheal stenosis, tracheomalacia [ 24 ].
Disclosure Authors report no conflict of interest. Open in a separate window. Regional differences in the cellular composition and function of pulmonary epithelium. References 1. Ostrowski K. Warszawa: PZWL; Kaiser S. Tradition on charge? J Anat. Singh G, Katyal SL. Clara cell proteins. Ann N Y Acad Sci. Ultrastructure of Clara cell stimulated by isoproterenol.
J Med Dent Sci. Clara cell: progenitor for the bronchiolar epithelium. Int J Biochem Cell Biol. Number and proliferation of clara cells in normal human airway epithelium.
A two-receptor pathway for catabolism of Clara cell secretory protein in the kidney. J Biol Chem. Clara cell secretory protein-expressing cells of the airway neuroepithelial body microenvironment include a label-retaining subset and are critical for epithelial renewal after progenitor cell depletion. Multiple secretoglobin 1A1 genes are differentially expressed in horses.
BMC Genomics. Generation of multiciliated cells in functional airway epithelia from human pluripotent stem cells. The secretion may contain enzymes. Its function is presumably to determine the chemical and physical properties of the lining of small airways, and it could behave as a kind of bronchiolar surfactant, limiting lung collapse. The Clara cells also contain much cytochrome P dependent mixed-function oxidases, which presumably play a detoxifying role.
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