Vimentin-positive Cells in the Epithelium of Rabbit Ileal Villi Represent Cup Cells but not M-cells
Institute of Anatomy, University of Lübeck, Lübeck, Germany (CR,AG), and Faculty of Biology, National University of Colombia, Bogota, Colombia (CR)
Correspondence to: Prof. Dr. A. Gebert, Inst. of Anatomy, University of Lübeck, 23538 Lübeck, Germany. E-mail: gebert{at}anat.uni-luebeck.de
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Summary |
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Key Words: M-cell cup cell vimentin lectin histochemistry gut rabbit
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Introduction |
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Immunohistochemistry (IHC) for the intermediate filament protein vimentin can be used to detect M-cells in the rabbit (Gebert et al. 1992; Jepson et al. 1992
). Many functional studies have confirmed that vimentin expression and tracer transport are closely related (Jepson et al. 1993b
,d
; Meynell et al. 1999
; Mantis et al. 2000
). Recently, individual vimentin-positive cells in the villi of the rabbit ileum, i.e., outside the domes of the Peyer's patches, were described (Fujimura and Iida 2001
; Iwatsuki et al. 2002
). Because these cells possessed morphological characteristics of (immature) M-cells, e.g., short microvilli and an electron-lucent cytoplasm (Bye et al. 1984
), they were considered to represent villous M-cells (Iwatsuki et al. 2002
). The presence of M-cells in ordinary villi would imply that antigenic matter can be transported from the lumen into the epithelium and lamina propria and that the potential pathogens come into contact with immunocompetent cells outside the organized gut-associated lymphoid tissue (GALT). Because the composition of lymphocyte subtypes and antigen-presenting cells largely differs between the organized lymphoid tissue of the domes and the diffusely distributed lymphoid cells of ordinary villi (Ermak and Owen 1986
; Bjerke et al. 1988
; Farstad et al. 1994
; Brandtzaeg et al. 1999
), it is unclear what kind of immune response could be initiated by the assumed M-cell-mediated antigen transport in the villi.
The aim of the present study was to test the hypothesis that the vimentin-positive cells in the villi of the rabbit ileum possess structural, histochemical, and functional characteristics of M-cells. Therefore, we tested potential histochemical markers to identify similarities and differences between the two vimentin-positive cell populations. Because lectins are useful tools to differentiate epithelial cell types, we used a panel of 26 lectins, including all typical saccharide specificities, that are typically expressed on intestinal epithelial cells. Using confocal fluorescence microscopy and lectingold-labeled ultrathin sections, we identified specific markers for the suspicious vimentin-positive cells in the villi and characterized their ultrastructure. In addition, uptake studies were performed using fluorescent latex microbeads as particulate tracers to investigate whether transepithelial transport similar to that of dome epithelial M-cells is performed by the vimentin-positive cells of the villi.
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Materials and Methods |
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Lectin Labeling
Cryosections (5-µm, 12-µm, 30-µm, and 60-µm thick) were fixed in a mixture of methanol and acetone for 10 min at -20C and transferred to PBS. The sections were incubated with a solution containing an anti-vimentin monoclonal antibody (MAb) (dilution 1:800 or 1:1600; clone V9; Sigma, Deisenhofen, Germany) conjugated to the red fluorescent dye Cy3 and one of the lectins (520 µg/ml) for 2 hr.
The following lectins conjugated with fluorescein (FITC) or biotin were tested in preliminary experiments to identify specific binding to the vimentin-positive cells: Abrus precatorius (APA), Arachis hypogaea (PNA), Bandeiraea simplicifolia I, isolectin B4 (BS-I-B4), Bandeiraea simplicifolia II (BS-II), Bauhinia purpurea (BPA), Canavalia ensiformis (ConA and succinylated ConA), Datura stramonium (DSA), Glycine max (SBA), Helix aspera (HAA), Helix pomatia (HPA), Jacalin (JAC), Lotus tetragonolobus (LTA), Lycopersicum esculentum (LEA), Maackia amurensis (MAA), Phaseolus vulgaris (PHA-L and PHA-E), Phytolacca americana (PWA), Psophocarpus tetragonolobus (WBA), Ricinus communis I (RCA-I), Sambucus nigra (SNA), Solanum tuberosum (STA), Triticum vulgaris (WGA), Ulex europaeus I (UEA-I), Vicia villosa (VVA), and Wisteria floribunda (WFA).
Biotinylated lectins (BPA, DSA, LTA, STA, WBA) were detected using streptavidin conjugated to the green fluorescent dye AlexaFluor 488 (Molecular Probes; Leiden, The Netherlands). The lectins MAA and SNA were purchased from EY Labs (San Mateo, CA), the others from Sigma. Nuclei were stained using the blue fluorescent dye bisbenzimide Hoechst 33258 (0.1 µg/ml in PBS for 1 hr; Sigma). Controls were performed by replacing the primary vimentin antibody with buffer and by preincubation of the lectins with their corresponding mono-, di-, or oligosaccharides overnight.
For lectin double labeling, cryosections were incubated with a mixture of LEAFITC (20 µg/ml), STAbiotin (10 µg/ml), and unconjugated anti-vimentin antibody V9 (1:800). After rinsing in PBS the sections were incubated in a mixture of streptavidin conjugated to the red fluorescent dye AlexaFluor 555 (2.5 µg/ml; Molecular Probes) and a goat anti-mouse antibody conjugated with the infrared-emitting fluorescence dye AlexaFluor 647 (1:100; Molecular Probes). Nuclei were counterstained with bisbenzimide as described above.
Fluorescence Microscopy
Cryosections of lectin labelings and microbead uptake experiments were examined using a conventional fluorescence microscope equipped with separate filter sets for blue, green, and red fluorescence, and a digital CCD camera (Zeiss; Jena, Germany). To resolve cellular details in thick sections (30-µm thick or more), individual sections were examined using a Zeiss LSM 510UV Meta confocal laser scanning microscope, equipped with lasers for 364-nm, 488-nm, 543-nm, and 633-nm excitation. In addition to fluorescence channels, a differential interference contrast (DIC) image was recorded simultaneously from each focal plane that allowed morphological details to be identified. Digital 3D multichannel data sets were processed using LSM 5 software (Zeiss), Photoshop software (version 6.0; Adobe, Edinburgh, UK), and the software package Erdas Imagine for geographical imaging (version 8.4; Erdas, Atlanta, GA).
Ultrathin Sections and LectinGold Labeling
Small tissue samples were fixed in a solution of 2% formaldehyde and 0.05% glutaraldehyde (grade I; Sigma) in 0.1 M phosphate buffer, pH 7.3, for 4 hr at 4C. They were then rinsed in PBS containing 1% L-lysine (Sigma) for 16 hr to block free aldehyde groups. The specimens were partially dehydrated in graded ethanol series (30%, 50%, 70%, 90%) and transferred to LR White resin, medium grade (Plano; Marburg, Germany). The tissue blocks were enclosed in gelatin capsules and allowed to polymerize at 50C for 48 hr. Ultrathin sections were mounted on formvar-coated nickel grids.
The lectingold labeling was performed in PBS containing 1.5% bovine serum albumin-c (BSA-c; Biotrend, Cologne, Germany). Free aldehyde groups were blocked in a drop of this buffer (PBSBSA) containing 0.7% L-lysine. After rinsing in PBS containing 5% BSA (Serva; Heidelberg, Germany), 0.1% coldwater fish skin gelatin (Biotrend), 1% normal goat serum (Sigma), and 0.05% Tween-20 (Serva), the sections were incubated with PBSBSA containing the respective lectins conjugated to biotin (20 µg/ml, 10 µg/ml, and 5 µg/ml) at 4C overnight. After rinsing the grids were incubated for 4 hr with a goat anti-biotin antibody conjugated to 10-nm or 15-nm colloidal gold (Biocell; Cardiff, U.K.). Finally, the grids were treated with 2% glutaraldehyde in PBS, washed in distilled water, and stained with uranium acetate and lead citrate. The sections were examined in a Philips EM400 electron microscope. Controls were carried out by omitting the lectin and by preincubating the lectins with their corresponding oligosaccharide (N,N',N''-triacetylchitotriose; Sigma) overnight.
Freeze-fracture Replicas
Small pieces of ileal mucosa, some of them containing a dome of a Peyer's patch, were briefly rinsed in Ringer's solution and immediately fixed in a solution of 2% formaldehyde, 2.5% glutaraldehyde, and 2 mg/ml CaCl2 in 0.1 M sodium cacodylate-HCl buffer, pH 7.3, at 4C. The freeze-fracture and replication of the fracture face were performed in a BA 360 M freeze-fracture device equipped with a QSG 201 quartz crystal thin film monitor and an EVM 052 electron beam gun (all from Balzers; Liechtenstein) at 2.7 x 10-4 Pa and -100C. The replicas were cleaned in commercial bleach, chromic acid, and distilled water, collected on formvar-coated copper grids, and examined in a Philips EM400T electron microscope.
Microbead Uptake Experiments
Adult New Zealand rabbits (n=10) were anesthetized with 100 mg ketamine (Alprecht; Aulendorf, Germany) IM, 5 mg midazolam (Curamed; Karlsruhe, Germany) IV, 10 mg PropofolLipuro 1% (Braun; Melsungen, Germany), and 150 mg Temgesic (Essex; Munich, Germany) IV. After intubation, the rabbits inhaled a mixture of oxygen and isoflurane (Abbot; Wiesbaden, Germany) for 1 hr or longer.
The abdominal cavity was opened and isolated loops of 2030-mm length, some containing a Peyer's patch, were prepared in situ from the ileum without disturbing the blood supply. They were then filled with a suspension of green fluorescent latex microbeads (diameter 0.5 µm; Polysciences, Eppelheim, Germany) in a dilution of 1:20 in PBS. The loops were removed after 45 min, briefly rinsed in saline, and frozen in liquid nitrogen. The animals were sacrificed by IV injection of T61. Vimentin labeling and microscopy were performed as described above.
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Results |
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In cryosections that contained both ileal villi and Peyer's patch domes, the M-cells were identified by their vimentin content, their cell shape, and their typical association with clusters of lymphocytes. Neither immature M-cells at the base of the domes nor mature M-cells on the upper flanks bound the lectins STA and LEA (Figure 2b). LEA and STA binding epithelial cells were even completely absent from the more than 50 domes screened in total. The remaining 24 lectins tested displayed different labeling patterns as described previously (Gebert and Hach 1993), but none of them specifically bound to both populations of V9+ cells (i.e., dome epithelial M-cells and V9+ villous epithelial cells).
Lectin double labeling of LEA with STA revealed that the two lectins bound to cells with identical subcellular labeling patterns (Figure 3). After preincubation of the lectins LEA and STA with N,N',N''-triacetylchitotriose, their binding intensity was reduced although not completely abolished.
LectinGold Labeling on Ultrathin Sections
Ultrathin sections gold-labeled with LEA or STA showed weak overall binding of the lectins to most tissue components and moderate binding to the brush border of enterocytes. A small number of epithelial cells (up to two per cross-sectioned villus) intensely bound the two lectins on their brush border (Figures 5 and 6)
. These individual lectin-positive epithelial cells were found in all of the five rabbits examined by lectingold labeling. The cells were columnar in shape and possessed microvilli that were shorter and slightly thicker than those of the neighboring enterocytes (Figure 5a). The brush border of many of these cells was indented so that the luminal surface was concave or cup-like (Figures 5a and 5b). The cytoplasm was less electron-dense than that of adjacent enterocytes, and small electron-lucent vesicles were present in its apical parts; most of them were lectin-positive (Figures 5a and 6). Some of the LEA+ and STA+ cells bound the lectin along the basolateral membrane (Figure 6b). The binding patterns of the lectins LEA and STA were almost identical. Other structures moderately labeled by the two lectins in the villous epithelium were the cell surface of intraepithelial lymphocytes and mucous granules in the goblet cells. The apical cell pole of a few enteroendocrine cells, identified by their typical electron-dense granules, was labeled by the LEA lectin, but such lectin-positive enteroendocrine cells were extremely rare and were found in only 3/85 grids examined in total.
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Freeze-fracture Electron Microscopy
Freeze-fracture replicas of the rabbit ileum contained the epithelial layer of the villi, consisting of enterocytes, goblet cells, and a few enteroendocrine cells, but brush cells, often seen in rat and mouse ileum, were not observed. Individual epithelial cells with the morphological characteristics of the V9+/LEA+/STA+ cells described for ultrathin sections could be identified in the freeze-fracture replicas as well. Compared to adjacent enterocytes, these cells possessed an often concave, cup-like brush border (Figure 7a)
as well as shorter and thicker microvilli (length about 1.2 µm; diameter 123 ± 7 nm, n=41; enterocytes 107 ± 7 nm, n=23). Whereas intramembranous particles (IMPs) present on P-faces were randomly distributed over the microvilli of enterocytes, the V9+/LEA+/STA+ cells contained linear aggregates of IMP on their P-faces, and corresponding grooves on the E-face of the microvillous membrane (Figure 7b). These arrays consisted of globular and rod-shaped particles and short fibrils that were orientated along an axis tilted 45° or less to the longitudinal axis of the microvillus. The present investigation and a previous detailed freeze-fracture study on the rabbit Peyer's patch dome epithelium (Gebert and Bartels 1991) revealed that epithelial cells with such typical arrays of IMPs were absent from the dome epithelium and that both mature and immature M-cells did not possess these unique membrane structures.
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Some fluorescent microbeads were attached to the brush border of the villous enterocytes, but no particles were seen in the epithelium and the lamina propria of the villi (Figure 4), except for a few regions where the epithelial layer was damaged and disrupted (probably because of mechanical alterations during ligation of the gut or dissection of the tissue). More than 300 V9+/LEA+ cells of the ileal villi were examined for microbeads lying in their cytoplasm or in close vicinity to them in the epithelium. None of these V9+/LEA+ cells contained fluorescent particles in the cytoplasm, and no particles were found close to them in the epithelium or lamina propria. In addition, adherence of microbeads to the brush borders of these cells was extremely rare and no more frequent than that of villous enterocytes.
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Discussion |
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The presence of individual vimentin-containing epithelial cells in ordinary villi of the rabbit ileum outside the Peyer's patches has recently been reported, and it was hypothesized that these columnar cells might represent cup cells (Fujimura and Iida 2001) or M-cells similar to those in the domes of GALT (Iwatsuki et al. 2002
). In the present study we reproduced these experiments using different antibodies, detection techniques, and microscopic methods, and found that 12% of the villous epithelial cells in normal rabbit ileum contain vimentin filaments. However, our results show that these vimentin-containing cells represent a population distinct from M-cells, because they differ considerably from mature and immature M-cells in the glycosylation pattern of their apical and cytoplasmic membranes and do not transcytose an experimentally applied tracer. In addition, the vimentin-containing cells in the villi bear linear arrays of particles in their apical membrane, which are known to be characteristic for the so-called "cup cells" described for various species (Neutra 1979
; Madara 1982
; Madara and Carlson 1985
).
The vimentin-positive epithelial cells in the villi are columnar in shape, contain electron-lucent vesicles in their apical cytoplasm and possess relatively short microvilli. They therefore resemble the immature M-cells found near the base of the Peyer's patch domes (Bye et al. 1984; see also Iwatsuki et al. 2002
). Immature M-cells are typically associated with only a few or even no lymphocytes and do not possess an elaborate pocket-like invagination of the basolateral membrane (Bye et al. 1984
). Although such morphological characteristics were likewise observed for the vimentin-containing cells in the villous epithelium (present study; and Iwatsuki et al. 2002
), lectin histochemistry revealed that the two cell types differ in the expression of N-acetyl-glucosamine oligomers. The lectins LEA and STA, which are both specific for di-, tri-, and tetramers of this saccharide (Allen and Neuberger 1973
), intensely bound to the vimentin-containing cells in the villi but not to mature and immature M-cells of the domes. Interestingly, the lectin DSA, which has a similar although not identical saccharide specificity (Crowley et al. 1984
), did not bind preferentially to these cells, indicating that cup cells express a constant and highly defined saccharide sequence in their brush borders. This interpretation is further supported by our lectin double labeling, demonstrating a constant binding of LEA and STA to all vimentin-containing epithelial cells in the villi.
Lectins represent excellent markers for the distinction of epithelial cell types and differentiation pathways (Falk et al. 1994), and many studies have revealed that the M-cells of mice and rabbits express specific glycoconjugates on their apical membrane that were sucessfully used as M-cell markers (Clark et al. 1993
; Gebert and Hach 1993
; Jepson et al. 1993a
; Gebert et al. 1996
,1999
). We therefore conclude that the differences in lectin binding between vimentin-containing cells in villi and domes reflect distinct glycosylation pathways in two different cell types. This view is further confirmed by studies in which other histochemical markers for rabbit M-cells were established, mostly unknown epitopes detected by monoclonal antibodies (Roy et al. 1987
; Pappo 1989
; Lelouard et al. 1999
). Although these markers detected most if not all M-cells in the domes, marker-positive cells were described to be absent from the villi, indicating that these markers also might have discriminated M-cells and cup cells. Although M-cells of different GALT sites may display divergent glycoconjugates in their apical membranes (Gebert and Hach 1993
; Jepson et al. 1993a
), no such variations have yet been described for different Peyer's patches in the same gut segment, e.g., the ileum, as investigated in the present study. Therefore, differences in the lectin binding properties among epithelial cells of the ileum most probably reflect distinct cell types.
In the present study, no uptake of microbeads by vimentin-containing cells was observed in the villi, whereas the M-cells in the domes actively transcytosed the tracer and thus served as positive controls in this well-established antigen sampling model (Pappo and Ermak 1989; Jepson et al. 1993c
; Meynell et al. 1999
). It could be objected that the vimentin-containing cells in the villi are immature M-cells, as hypothesized by Iwatsuki et al. (2002)
. Immature M-cells in the domes, however, do transport tracers (Bye et al. 1984
) and, within hours, develop into mature M-cells (Bhalla and Owen 1982
; Bye et al. 1984
) that acquire the typical morphological features of M-cells (e.g., a large pocket that contains clustered lymphocytes). The absence of such aggregates further supports our view that M-cells do not occur in ordinary villi and that the vimentin-containing cells in the villous epithelium represent a cell type distinct from M-cells.
Our lectin labeling at the electron microscopic level allowed the V9+/LEA+/STA+ cells of the villous epithelium to be identified ultrastructurally and revealed that they possess several characteristics of the so-called "cup cells" (Madara 1982). Because the unique feature of cup cells and of closely related cells described by Neutra (1979)
in primate large intestine consists of linear arrays of particles in the microvillus membrane, freeze-fracture replicas were examined and they confirmed that the V9+/LEA+/STA+ cells possess such typical arrays and thus represent cup cells. Previous freeze-fracture studies revealed that the apical membrane of M-cells differs from that of enterocytes in the density of intramembranous particles (Madara et al. 1984
; Gebert and Bartels 1991
), but that arrays of particles characteristic for cup cells are absent from M-cell membranes. In cup cells, arrays of intramembranous particles are restricted to the microvilli (present study; and Madara 1982
), while lectin binding was also found along the basolateral membrane and along that of cytoplasmic vesicles. It therefore appears not very likely that the intramembranous particles that form arrays in cup cell membranes are identical to the glycoconjugate that incorporates the binding sites for LEA and STA.
Cup cells comprise 16% of the epithelial cells in the ileum (present study; and Madara 1982) and therefore by far surpass the thoroughly studied enteroendocrine cells in frequency (the latter comprise 0.4% or less; Cheng and Leblond 1974
). Nevertheless, the function of cup cells is still obscure, but it has been proposed that they are attachment sites for certain bacteria (Madara and Carlson 1985
). In this context, the cup cell-specific glycoconjugates identified in the present study could serve as anchoring sites, because it is known that many pathogenic and commensal bacteria bind to oligosaccharide epitopes on epithelial surfaces via lectin-like substances on their outer membrane or pili (Wennerås et al. 1995
). The presence of unique glycoconjugates might reflect cup cell-specific glycoproteins that are involved in specific functions. The nature of such membrane-bound molecules in cup cells is unknown, but they might represent receptors associated with sensory or regulatory functions. Interestingly, it has been reported that individual villous cells in the rat and human intestine express the cystic fibrosis transmembrane conductance regulator (CFTR; Ameen et al. 1995
; Hayden and Carey 1996
). Because these cells resemble cup cells in both morphology and frequency (about 2% of the villous epithelial cells), it is tempting to speculate that the two cell types, and possibly a further cell type described by Heath et al. (1996)
, are identical. The present study provides for the first time histochemical markers for cup cells that might be employed in further attempts to elucidate the functions of these cell populations.
We conclude that the vimentin-containing epithelial cells in ordinary villi of the rabbit ileum represent cup cells but not M-cells. Although many ultrastructural, histochemical, and functional studies have been performed using various tracers and M-cell markers (Neutra 1998), no reports are available that can sustain the presence of M-cells outside the organised mucosal lymphoid tissues. The development of M-cells appears to be restricted to specialized crypts located at the periphery of the domes (Gebert et al. 1999
; Lelouard et al. 2001
), in which a unique microenvironment induces the formation of the M-cell lineage as a very early step of differentiation from stem cells (Neutra 1998
). In addition, this milieu suppresses the development of goblet cells and that of cup cells, as shown in this study. It must be assumed that both induction and the immunological function of M-cells are closely related to a special GALT microenviroment that has recently been shown to be critically influenced by B lymphocytes (Golovkina et al. 1999
; Debard et al. 2001
).
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Acknowledgments |
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The technical assistance of J. Endler, H. Manfeldt, C. Örün, G. Preiss, and D. Stöckmann is gratefully acknowledged.
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Footnotes |
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