ARTICLE |
Correspondence to: Liliana Luciano, Dept. of Cell Biology, Center of Anatomy, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. E-mail: luciano.liliana@mh-hannover.de
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Summary |
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It has been suggested that brush cells (BCs), a distinct type of cell occurring in various epithelia of the respiratory and gastrointestinal tracts, may function as receptor cells. The major characteristics of BCs are a prominent brush border and an unusually highly ordered arrangement of cytoskeletal elements (F-actin, microtubules, and intermediate filaments). In this study we aimed to characterize the nature of the intermediate filaments in BCs by light and electron microscopic immunostaining. Gallbladder and stomach specimens from mice and rats, respectively, were fixed in various solutions, embedded either in paraffin or epoxy resin, and processed for immunodetection. Commercially available, well-characterized antibodies against neurofilaments, peripherin, and cytokeratin peptide 18 were used. The polyclonal antiserum cocktail to neurofilaments was applied as a supplement in a double-labeling procedure with anti-actin and anti-cytokeratin 18 antibodies. The results demonstrate that the BCs of both organs express two types of intermediate filaments, i.e., neurofilaments and cytokeratin 18 filaments, and that these have a compartmentalized distribution in the cytoplasm. BCs do not express peripherin. The immunodetection of intermediate filaments distinctive for mature neurons in BCs supports their putative receptor function. The co-expression of neurofilaments and cytokeratins is shown for the first time in healthy tissues. (J Histochem Cytochem 51:187198, 2003)
Key Words: F-actin, microtubules, intermediate filaments, peripherin, cytokeratin 18, Western blot, immunoelectron microscopy, double labeling, epoxy resin
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Introduction |
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Brush cells (BCs) are distinctly structured cells that were first detected in the tracheal epithelium of the rat (
In this study we demonstrate by light (LM) and electron microscopy (EM) immunostaining that, in addition to CK 18-containing filaments, the BCs express IF type IV: the neurofilaments (NFs) which, as generally acknowledged, characterize the mature neurons. A combination of optimal morphological preservation and efficent immunolabeling of all cytoskeletal elements enabled us to recognize that NF and CK 18 IFs each have a distinct compartmentalized distribution in the cytoplasm.
The presence of neuron-specific IFs in BCs is a new characteristic that adds support to their putative function as receptor cells.
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Materials and Methods |
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Animals and Tissue Preparation
The gallbladders of six adult mice (male, NMRI breed; Hannover) and the stomachs of six male adult Wistar rats (200250 g bw) were used. The animals were decapitated and the organs were quickly removed in toto and immersed in the fixative. Tissue strips were then excised from both organs in the regions known to be rich in BCs (
The formaldehyde-fixed specimens (a) were washed in PBS, dehydrated in ascending concentrations of ethanol, and embedded either in the paraffin equivalent Histocomp (Vogel; Giessen, Germany) or in epoxy resin (Serva; Heidelberg, Germany). The specimens fixed in glutaraldehyde (b) and (c) were washed in cacodylate buffer, dehydrated in ethanol, and embedded in epoxy resin.
For LM, immunostaining was applied on both paraffin and epoxy (semithin) sections. Once the presence of BCs and their successful immunolabeling had been ascertained on semithin sections, parallel thin sections were prepared from the same tissue blocks and processed for immunogold EM.
Primary Antibodies
The rabbit polyclonal antiserum cocktail to neurofilaments (cat. no. NA 1297; batch Z02787; Biotrend Chemikalien, Köln, Germany), the mouse monoclonal anti-peripherin (clone PJM50; Novocastra Laboratories, Newcastle, UK), anti-cytokeratin peptide 18 (clone Ks 18.04; Progen, Heidelberg, Germany and clone CY-90; Sigma, Deisenhofen, Germany), anti-actin (clone C4; Chemicon International, Hofheim, Germany) were used. The working dilutions of primary antibodies had been assessed in preliminary tests and are indicated in Table 1.
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Immunostaining Procedures
Paraffin sections (about 4 µm thick) from gallbladder and stomach specimens were collected on glass slides pretreated with 3-(triethoxysilyl)propylamide (silane; Merck), dewaxed in xylene, and rehydrated in descending concentrations of ethanol. Endogenous peroxidase activity was blocked by treating the sections with 0.6% H2O2 in 96% ethanol for 20 min at RT. Later these sections were processed for antigen retrieval and immunostaining in the same way as the epoxy semithin sections (see below).
Epoxy semithin sections (about 1 µm thick) were collected on silane-treated slides, etched for 15 min in sodium ethoxide diluted to 50% with absolute ethanol, and rehydrated.
For both paraffin and epoxy sections, antigen retrieval was performed in a microwave oven (Sharp R-2S67; Sharp, Hamburg, Germany) by heating them in 0.01 M citrate buffer, pH 6.0 (
Thin sections (about 70 nm thick) were collected on nickel grids. Etching and antigen retrieval were performed as described elsewhere (
For all sections, after washing in PBS the appropriate secondary antibodies, goat anti-mouse and goat anti-rabbit, both conjugated with 10-nm gold particles (British BioCell; Cardiff, UK), were applied for 60 min at RT.
The immunoreaction was stabilized with 2.5% glutaraldehyde in PBS for 10 min (
Double labeling was performed on thin sections to detect NFs and F-actin as well as NFs and CK 18. For this purpose, the sections on nickel grids were processed in the same way as described previously except that both primary and both secondary antibodies were present simultaneously in the incubation solutions and that the binding of anti-actin and anti-CK 18 antibodies was detected with a goat anti-mouse IgG conjugated with 5-nm gold particles (British BioCell).
Controls
Negative Controls.
The primary antibodies were either omitted from the incubation solution or substituted by normal mouse or preimmune rabbit sera (Jackson Immunoresearch Laboratories) at the same concentrations as the primary antibodies.
Positive Controls. The specificity of the NF immunoreaction was tested on thin sections of the rat cerebellum from tissue blocks fixed according to procedure c and processed in parallel with thin sections from gallbladder and stomach specimens.
Immunoblotting
The antibodies detecting IFs in BCs by immunohistochemistry were also tested for specificity and species crossreactivity in Western blotting analysis. HeLa cells, which are known to contain a high amount of CK 18 and from which the immunogen for antibody Ks 18.04 had been derived, served as positive control.
Procedure.
HeLa cells, whole gallbladders, continuous strips of the stomach severed circumferentially along the stomach ridge (at the transition zone between pre- and glandular stomach; see
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Results |
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Immunodetection of NFs
In both paraffin sections and epoxy resin semithin sections, the BCs of the mouse gallbladder (Fig 1a1d) and rat stomach (Fig 1e1g) showed distinctive specific staining after incubation with antibody against NFs. In the gallbladder, the staining was prominent in the supranuclear region of the BCs where, in longitudinal sections, it appeared in the form of dark, parallel rows directed apicobasally (Fig 1a1c). This localization clearly indicates that the NFs were intermingled and ordered between the F-actin and microtubule bundles that characterize this cytoplasmic region of BCs. Serial semithin sections revealed that the immunostaining further delineated the periphery of the cell (Fig 1d and Fig 1d1), but this was only just visible in its basal cytoplasmatic processes and, moreover, only when BCs had lost contact with the lumen (Fig 1d1). In contrast, in the stomach of the rat, the many BCs present in the epithelium lining the distal slope of the ridge (
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In EM, the highly specific immunogold localization of F-actin was a major criterion that helped to identify, at least initially, the IF bundles intermixed with the actin and microtubule assemblies in the apical cytoplasm (Fig 2a). In fact, F-actin immunolocalization, by leaving the IF bundles unlabeled, made the differences in diameter between the two filament types immediately evident (Fig 2b) and even allowed their detection, otherwise difficult, in transverse sections of BC apical cytoplasm (Fig 2c). When thin sections, parallel to those used to demonstrate immunostaining of F-actin, were treated with the rabbit polyclonal antiserum cocktail to NFs, the IF bundles became positively labeled whereas the F-actin bundles remained negative (compare Fig 2b with Fig 2d).
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These results were confirmed in double-labeling experiments (Fig 2e), which further revealed the complex interlacing between F-actin and NFs with microtubules in some areas of the BC apical cytoplasm (Fig 2e). At the cell margins, around the nucleus, and below it, a large number of IFs organized in bundles of different thickness and orientation were present. However, in these regions they remained mostly negative after application of the antibody against NFs (see below).
Negative Control. After incubation in the absence of the primary antibody, all LM sections (paraffin and epoxy semithin) as well as thin sections (Fig 3a) remained unlabeled.
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Positive Control. The first indication of the specificity of the reaction was already given by LM sections showing a positive stain of both BCs and small nerve fibers in the gallbladder wall (Fig 1a) or BCs and small assemblies of ganglionic cells in the submucosa of the rat stomach (not shown). Nevertheless, final proof was provided by EM immunolabeling in sections of rat cerebellum, showing highly specific staining of axon NFs (Fig 3b).
Immunodetection of Peripherin
The use of the mouse monoclonal antibody against peripherin protein failed to detect BCs (Fig 4a and Fig 4b). The epithelium covering the stomach-limiting ridge in which many BCs occur was always negative, and thus were all epithelial cells lining the stomach glands (Fig 4b). In contrast, peripherin antibody labeled small assemblies of ganglion cells present in the stomach submucosa in the same section (Fig 4a and Fig 4c) as well as the ganglia lining the muscle sheets in both the gallbladder and stomach (not shown).
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Immunodetection of CK 18-containing Filaments
The two antibodies against CK peptide 18 applied in this study (Table 1) showed differences in species crossreactivity. The mouse monoclonal anti-CK 18 (clone CY 90) stained BCs only in the rat stomach and not in the mouse gallbladder (results not shown). In contrast, independent of the procedure with which the specimens were fixed and embedded, the mouse monoclonal anti-CK 18 clone Ks 18.04 labeled BCs in both mouse gallbladder and rat stomach (Fig 5a and Fig 5b). The immunoreaction in these organs was highly specific so that the BC shape was brought up among neighboring epithelial cells (Fig 5a and Fig 5b). The staining especially delineated the cell borders and the basal processes (Fig 5 b). The application of this same antibody on thin sections revealed that CK 18-positive bundles had a distinctive distribution in the cytoplasm. They were excluded in the region where F-actin, microtubules, and NFs dominate. In contrast, favorable section planes grazing BCs along a median plane enabled recognition that CK 18-positive bundles almost exclusively formed the prominent network that surrounds the nucleus (Fig 6a). However, and in agreement with LM observations, the double-labeling procedure demonstrated that near the lateral cell borders CK 18-positive bundles co-localized with NFs (Fig 6c and Fig 6d). The efficiency of the double labeling was highly dependent on the thin section plane, transverse or oblique to the filament bundles, there being more epitopes exposed in an oblique plane (
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Immunoblotting
In Western blotting analysis, the rabbit polyclonal antiserum cocktail to NFs detected bands at about 6870, 150, and 200210 kD in both mouse and rat brain extracts (Fig 7a). These bands corresponded to the low, medium, and heavy molecular weight of NF triplet proteins, thus demonstrating the species crossreactivity of the antibody for all three NF protein isoforms. The specificity and species crossreactivity of the two antibodies directed against CK 18 peptide were confirmed in Western blotting experiments. The monoclonal anti-CK 18 clone Ks 18.04 labeled a band of molecular weight slightly over 45 kD in HeLa cells, in mouse gallbladder, and in rat stomach (Fig 7b). In contrast, the anti-CK 18 clone CY 90 detected a corresponding band only in HeLa cells and in rat stomach but not in mouse gallbladder (Fig 7c), indicating, in parallel with the immunohistochemical results, differences in species crossreactivity.
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Discussion |
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The Cytoskeletal Network of BCs
It is rare for all three basic components of the cytoskeleton of eukaryotic cellsmicrotubules, F-actin, and IFto be so abundantly present and with a so highly ordered distribution in cytoplasm as in BCs. Many F-actin assemblies extend from the apex of the microvilli deep into the supranuclear region, where they become flanked by microtubule assemblies. This alternating arrangement of F-actin and microtubules, particularly evident in longitudinal sections of the cell, entails all organelles in between assuming the same apicobasal orientation and bestows the BC with its typical stiff aspect (
The cytoskeletal elements are known to be, in general, very delicate structures, subjected both in vivo and in vitro to alterations such as depolymerization, disassembly, and/or unraveling, being greatly influenced by chemical, thermal, and mechanical stress (reviewed by
Compartmentalized Distribution of IFs in the Cytoplasm of BCs
Immunodetection of NFs.
This study clearly demonstrates the presence of NFs in BCs. Western blotting analysis and immunostaining of thin sections of rat cerebellum (positive control) confirm the specificity of the rabbit antiserum cocktail to NFs used.
Immunogold labeling reveals that bundles of NFs are mainly intermingled with actin and microtubules in the apical cytoplasm, where they assume the same orientation. This location and arrangement, already predictable from the row-like labeling observed in LM, was at first surprising. In fact, the presence of IFs at this location had escaped notice in previous morphological studies. Conceivably, attention was distracted by the prominent number of F-actin and microtubule assemblies occurring here.
The presence of NFs in the BC cytoskeleton is of biological significance. It implies that the cell is able to synthesize this type of IF, a property generally considered to be restricted to mature neurons (
With the detection of NFs the BCs acquire a characteristic property of neurons (
In neurons the NFs are synthesized in the cell body and transported to the axon, but the mechanism of transport is controversial (
Further studies are needed to determine whether there are functional relatives beyond the structural analogies in cytoskeletal organization between BCs, axons, and hair cells.
Immunodetection of Peripherin.
Peripherin is an IF protein first described in neuroblastoma cells and subsequently found to be expressed mainly in neurons of the peripheral nervous system (
Immunodetection of CK 18.
CK 18 belongs to the group of acidic keratins type I IF (
EM immunogold labeling reveals that CK 18-positive IFs were absent in the apical cytoplasm where NFs, microtubules, and F-actin assemblies occur, although, from LM sections one would be induced to think so, the stain being mainly distributed over the whole cell. Assemblies of CK 18-positive filaments form, in contrast, the bulk of cytoskeletal elements in the perinuclear region, and in places they show close juxtaposition and even linkage with the nuclear envelope. CK 18-containing filaments and NFs are regularly co-expressed at the lateral cell margins and in the BC basal processes.
In recent years, growing evidence from studies using the green fluorescent protein-tagged IF fusion proteins, had led to the interpretation that these polymers are highly dynamic structures, able to assemble and disassemble, and to convert into protofilamentous aggregates. Their constituent proteins can move rapidly throughout the cytoplasm during various cell activities (
On the other hand, among IF types, cytokeratins are considered as major promoters of a specific epithelial cytoarchitecture and protectors against mechanical stress (
Co-expression of NF- and CK 18-containing Filaments in BCs
IFs are subdivided into six distinct types, which differ in the number of the genes that regulate their function, in sequence type, assembly group, molecular weight, and distribution in various tissues (
Under normal physiological conditions and in adult animals, co-expression of NFs and IF type III vimentin has thus far been immunohistochemically demonstrated in the horizontal cells of the mouse retina (
Under pathological conditions however, a co-expression of NFs and cytokeratins has been repeatedly reported. It occurs, for example, in tissue malignancies such as endocrine carcinomas of the respiratory and gastrointestinal tracts (
In the present study, in contrast, we demonstrated the co-existence of NF- and CK 18-containing filaments in normal and fully differentiated cells residing in common epithelia. This unexpected co-existence in BCs could reflect a functional requirement for tasks that are divergent enough to be fulfilled by only one type of IF. This possibility may, in turn, imply that some cells, including BCs, can retain the ability to synthesize IFs in different and more convenient molecular configurations, according to their functional needs. Conceivably, the co-expression of NF- and CK 18-containing filaments goes beyond the context of the BC function, assuming much more general biological interest.
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Conclusions |
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The results of the present study seem to provide a clear indication that the architecture of the BC cytoskeleton holds the secret to the way in which the cell functions. The presence of NFs in the BC cytoskeleton may add a piece to the puzzle.
New knowledge about the dynamics of cytoskeletal elements has steadily grown in recent years, and the numbers of associated proteins involved as crosslinkers and/or as motor proteins acting in transport mechanisms have progressively expanded (
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Acknowledgments |
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We wish to thank Prof Dr E. Ungewickell for critical reading of the manuscript, Mr F. Hurkuck for excellent technical assistance, Ms A. Hundt for photographic work, and Ms S. Fryk for linguistic corrections. The generous gift of anti-peripherin antibody by Ms A. Michalski (Loxo; Dossenheim, Germany) is greatly appreciated.
Received for publication May 6, 2002; accepted August 23, 2002.
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