ARTICLE |
Correspondence to: Andreas Gebert, Abt. Anatomie 2, Medizinische Hochschule Hannover, 30623 Hannover, Germany.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Intestinal M-cells are specialized epithelial cells located in the domes of the gut-associated lymphoid tissues, which transport antigens from the lumen to the underlying lymphoid tissue, thereby initiating immune reactions. It is assumed that M-cells arise from stem cells in the crypts, from which they migrate to the top of the domes. To study the differentiation pathway of M-cells, we used the rabbit cecal lymphoid patch in which the M-cells express high levels of 1-2-linked fucose and N-acetyl-galactosamine residues in their apical membrane. Dome areas were labeled with fluorescein- and rhodamine-conjugated lectins specific for
1-2-linked fucose and N-acetyl-galactosamine in vivo and in vitro, and were observed with confocal laser scanning microscopy. Ultrathin sections were double labeled with lectin-gold conjugates and the labeling density was quantified by computer-based image analysis. All cecal patch M-cells expressed
1-2-linked fucose and N-acetyl-galactosamine, but the amount of the two saccharides varied considerably depending on the position of the M-cells at the base, flank, or top of the dome. In eight of 18 rabbits studied, radial strips of M-cells with common glycosylation patterns were observed, each strip associated with an individual crypt. Confocal microscopy revealed that lectin-labeled M-cells were not restricted to the dome epithelium but were also detected in the upper third of crypts surrounding the domes. The results show that M-cells are heterogeneous concerning the glycosylation pattern of membrane glycoconjugates. This pattern is modified as the M-cells differentiate and migrate from the base to the top of the dome. Radial strips of M-cells with a common proclivity of glycoconjugate expression suggest that those M-cells that derive from the same crypt have a clonal origin. The presence of (pre-) M-cells in the crypts surrounding the domes indicates that M-cells derive directly from undifferentiated crypt cells and do not develop from differentiated enterocytes.(J Histochem Cytochem 45:1341-1350, 1997)
Key Words: M-cell, gut-associated lymphoid tissue, differentiation, lectins, glycoconjugates, lymphoid tissue, cecum, rabbit
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
M-cells are highly specialized epithelial cells that are present in the domes of the mucosa-associated lymphoid tissues (1-2-linked fucose and N-acetyl-galactosamine (GalNAc) on their apical membrane, a specialization that can be employed as a sensitive marker for M-cells at this location and in their differentiation pathway. In the present study, the cecal lymphoid patch of the rabbit was used as a model to study changes in the glycosylation patterns of the dome epithelial cells during their lifespan and their migration from the dome-associated crypts to the top of the domes. For this purpose, the distribution of M-cells with different lectin binding properties was investigated by two-channel confocal laser scanning microscopy using lectins conjugated to fluorescent dyes. To determine and quantify the amount and the ratio of the two saccharides expressed by individual M-cells, lectin-gold double labelings were performed and analyzed by electron microscopy and computer-based image processing.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adult male rabbits (n = 18, weight 2.0-3.3 kg, strains Chinchilla and New Zealand) were fed standard laboratory diet and had free access to water. The rabbits were kept under pathogen-free conditions (SPF) for at least 4 weeks before the experiments. After anesthetizing the animals with Ketanest and Nembutal (WDT; Garbsen, Germany), the abdominal cavity was opened and the cecal lymphoid patch removed. The rabbits were sacrificed by an intracardial or intravenous injection of T61 (Hoechst; Frankfurt, Germany). The excised samples were rinsed for 5-10 sec in Ringer's solution and immediately minced with razor blades. Small pieces were immersed in pure ethanol, Bouins's fluid, or in a solution of 2% formaldehyde and 0.1% glutaraldehyde in PBS, pH 7.3, for 4-16 hr.
Confocal Microscopy
Small pieces of ethanol- or Bouin-fixed tissue, each containing a single dome and adjacent nondome mucosa, were rinsed overnight in PBS and incubated in a solution containing 50 µg/ml Ulex europaeus agglutinin conjugated to rhodamine isothiocyanate (UEA-I-TRITC; Sigma, Deisenhofen, Germany) and 50 µg/ml Vicia villosa agglutinin conjugated to fluorescein isothiocyanate (VVA-FITC; Sigma) in PBS for 2 or 24 hr. After rinsing in PBS for at least 4 hr, the domes were observed with a BioRad MRC600 confocal laser scanning microscope equipped with an argon-krypton mixed gas laser using Comos software (version 6.0; BioRad, Munich, Germany). Maximal projections of serial optical sections were calculated and viewed using Lasersharp software (version 1.02; BioRad) and Photoshop software (version 3.05; Adobe Systems, Edinburgh, UK). In each rabbit, at least three domes were examined; a total of 117 confocal images taken from 76 domes were studied. Controls were carried out by omitting one of the lectins or both, and by preincubation of the lectin mixture with 0.06 M L-fucose or 0.06 M GalNAc, or both, overnight. In additional controls, a lectin (ConA; Sigma) was used that is known not to be specific for any of the epithelial cell types of the dome.
Lectin-Gold Labelings
Single domes, fixed in formaldehyde/glutaraldehyde for 4 hr (see above), were rinsed in PBS containing 1% L-lysine (Sigma) for 16 hr to block free aldehyde groups. The specimens were partially dehydrated in a graded series of ethanol dilutions (30%, 50%, 70%) and transferred to LR White resin, medium grade (Plano; Marburg, Germany). The tissue blocks were enclosed in gelatin capsules and were allowed to polymerize at 50C for 24 hr. Ultrathin sections 60-80 nm thick were cut with a diamond knife and mounted on 200-mesh nickel grids.
The lectin-gold labeling procedure was performed with PBS containing 0.15% bovine serum albumin-c (BSA-c; Biotrend, Cologne, Germany) and 0.1% sodium azide. Free aldehyde groups were blocked in a drop of this buffer (PBS-BSA) containing 0.7% L-lysine for 15 min. After rinsing in PBS containing 5% BSA (Serva; Heidelberg, Germany), 0.1% coldwater fish skin gelatin (Biotrend), 5% normal goat serum (Sigma), and 0.05% Tween-20 (Serva), the sections were incubated with a solution containing UEA-I-FITC (6.25 µg/ml; Sigma) and VVA-biotin (25 µg/ml; Sigma) in PBS-BSA at 4C overnight. The grids were rinsed in PBS-BSA for 30 min and incubated with a monoclonal anti-FITC antibody (clone B13-DE1;
Lectin Labeling of Living Tissue
To exclude possible alterations of the carbohydrate structure due to chemical fixation and to determine the accessibility of M-cell glycoconjugates in living tissue, the cecal patch of an additional rabbit was excised and immediately incubated in a solution containing 12.5 µg/ml UEA-I-TRITC and 12.5 µg/ml VVA-FITC in PBS for 15 min. After rinsing in PBS for 1 min and fixation in formaldehyde/glutaraldehyde (see above), single domes were dissected. Without further lectin labeling, some of the domes were examined by confocal microscopy and some were embedded in LR White resin. Unstained 1-µm sections were examined by conventional fluorescence microscopy using the appropriate filter sets for FITC and TRITC. Ultrathin sections were pretreated as described above and incubated with a monoclonal anti-FITC antibody (clone B13-DE1;
Image Analysis of Lectin-Gold Labeling
Photomicrographic negatives of the apical cell poles of M-cells taken at an electron optical magnification of 20,000:1 were digitalized with a scanner (PS2400X; Umax Data Systems, Taipei, Taiwan, ROC), and gray scale data sets were transferred to an IBAS image analysis system (version 2.0; Zeiss-Kontron, Munich, Germany). Regions of interest, including the brush border of M-cells, were selected interactively and the numbers of 10-nm and 20-nm gold particles per area were determined using algorithms supplied with the IBAS system. To exclude variation in the labeling procedure, photomicrographs of M-cells taken from single ultrathin sections were analyzed. A representative data set that includes 101 regions of interest is shown in the Results.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Confocal Microscopy
Maximal projections of serial optical sections showed that lectin-labeled cells, representing the M-cells, were most numerous at the flanks of the domes (Figure 1A). The M-cells were interspersed among the enterocytes and at the flanks comprised about 30% of all dome epithelial cells (Figure 1B and Figure 2). Corresponding pairs of single-channel black-and-white images of domes double labeled with UEA-I-TRITC and VVA-FITC revealed that the vast majority of M-cells were reactive for both lectins. However, the ratio of the two labeling intensities varied widely (Figure 1B and Figure 2). The prevailing specificity was visualized by two-channel color images. Cells located at the outer periphery of the domes were labeled in different shades of red, with only a small portion of green fluorescence (Figure 2), indicating that these cells predominantly bound UEA-I. Both the number of VVA-labeled cells and the VVA labeling intensity increased towards the middle of the flanks, whereas those of UEA-I-labeled cells decreased. At the transition from the flanks to the top of the domes, the labeling intensity for both lectins decreased and only a few mostly UEA-I-positive M-cells were present (Figure 1A and Figure 2).
|
In eight of 18 rabbits studied, the dome areas were characterized by radial strips differing in the prevailing fluorescent color (Figure 3). Although each strip was composed of M-cells with both saccharide specificities, one of the colorsred, green, or yellow
predominated. The strips varied in width between 30 µm and 200 µm and were irregularly arranged on the domes. Some of the strips, most of them UEA-I-positive, were tapered and reached the center of the dome (Figure 3). High-power views showed that each strip was associated with a single crypt opening into the cleft between the dome and the opposite nondome region. When radial strips were seen, all domes of the individual rabbit possessed such strips. The presence or absence of radial strips apparently did not correlate with the strain (New Zealand or Chinchilla), litter, or age of the rabbits, or with the fixation method.
|
To investigate the region of the dome-associated crypts, lectin-labeled domes were viewed from the side using high-power oil immersion lenses. At the base of the domes near the mouths of these crypts, an irregular pattern of mostly fucose-bearing M-cells and unlabeled enterocytes was seen. Image stacks that contained complete crypts showed that this pattern was also present in the upper third of the dome-associated crypts (Figure 4). Within each of these crypts and at their mouths, the labeled cells were oriented to all sides, i.e., dome and nondome. The proportion of labeled cells remained constant in the adjacent epithelium at the base of the dome. However, in the nondome epithelium opposite, the pattern of fucose-labeled and unlabeled cells disappeared within a distance of 30-70 µm from the crypt mouths. No patterns of labeled and unlabeled cells were found in crypts that were not associated with domes.
Controls omitting one of the lectin conjugates resulted in a drastically reduced signal in the corresponding channel, i.e., background noise in the red channel and some autofluorescence in the green channel. Preincubation of UEA-I-TRITC with fucose drastically reduced the binding to very few, weakly labeled cells per dome. Preincubation of VVA-FITC with GalNAc prevented any binding at all. Preincubation with GalNAc and with fucose did not influence the binding of UEA-I and of VVA, respectively. Controls with lectins not specific for cecal patch M-cells, e.g., ConA, showed a weak but uniform labeling of all dome epithelial cells.
Lectin-Gold Labelings
Results obtained with confocal microscopy gave the impression that the M-cells of the rabbit cecal patch could be divided into three distinct subtypes, defined by red, yellow, or green fluorescence. To prove this hypothesis, lectin-gold labelings were performed and quantified. On ultrathin sections, lectin-gold labeling was almost restricted to the apical membranes of M-cells and to the membranes of vesicles lying in the apical cytoplasm of the M-cells (Figure 5A). The labeling of enterocyte and lymphocyte membranes was negligible. The overall labeling density of UEA-I by far exceeded that of VVA because the 20-nm gold particles used for GalNAc labeling bind less efficiently than the 10-nm particles used for UEA-I. The labeling density of 10-nm particles, representing UEA-I binding sites, and 20-nm particles, representing VVA binding sites, varied considerably from cell to cell (Figure 5B-D). Image analysis revealed that both lectins showed a broad but continuous spectrum of labeling intensities, which did not allow the definition of three distinct subtypes at the electron microscopic level (Figure 6). Preincubation of the lectins with their corresponding saccharides drastically reduced or prevented the labeling.
|
|
Lectin Labeling of Living Tissue
Cecal patch domes that had been lectin-labeled in vivo showed a relatively low labeling intensity compared to fixed tissue. This is probably due to the reduced incubation time (15 min vs several hours) and the higher dilution of the lectins (12.5 µg/ml vs 50 µg/ml). Despite this low intensity, the labeling patterns found with confocal microscopy in in vivo lectin-labeled domes closely resembled those found in fixed tissue. Immunogold labelings on ultrathin sections, detecting the FITC label of VVA bound to cell surfaces, showed some binding of gold particles to the brush border of M-cells but no binding to the remaining cytoplasm of the M-cells nor to enterocytes and other cells. The labeling intensity of M-cell membranes varied from cell to cell in a similar manner to that described for on-section lectin labelings.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous lectin histochemical studies on the rabbit gut-associated lymphoid tissue demonstrated that M-cells of the rabbit cecal lymphoid patch express high levels of 1-2-linked fucose and GalNAc residues in their apical membrane (
Confocal light and lectin-gold electron microscopy reveal that the M-cells of an individual dome of the cecal patch are heterogeneous with respect to the expression of 1-2-linked fucose and GalNAc residues in the glycocalyx of their apical membrane, and that the M-cells form an irregular patchwork with various amounts of the two terminal saccharides. Both techniques applied in this study detected these variations and are necessary to give the full picture. Confocal laser scanning microscopy easily exhibits the distribution of labeled cells on the domes and in the crypts, but quantitations of the labeling intensities are unreliable because of bleaching effects and summation effects due to the maximal projection of the image stack performed during digital processing. Lectin-gold electron microscopy overcomes these problems because it allows the labeling densities to be quantified, the lectin binding sites in the glycocalyx to be localized, and the ultrastructural morphology of the labeled cells to be determined. Although some of the confocal images gave the impression that three subtypes of M-cells could exist according to the expression of
1-2-linked fucose or GalNAc, at the electron microscopic level no distinct subtypes were found, but rather a continuous spectrum of labeling intensities. Heterogeneity among the M-cells of individual domes has likewise been reported for the binding of the lectins UEA-I, AAA, and EEA to M-cells of murine Peyer's and cecal patches (
In addition to local variations of glycosylation among the M-cells, the base, flank, and top of the domes largely differ in the prevailing terminal saccharides on M-cell surfaces. These variations correlate with the crypt-villous axis of epithelial differentiation in general and the crypt-dome axis in the gut-associated lymphoid tissue. The M-cells and enterocytes of the dome epithelium are generated in the dome-associated crypts, subsequently migrate to the flanks, and are finally sloughed off near the center of the dome (
The radial strips of M-cells with similar lectin binding patterns were relatively clearly separated from each other and met at the center of the dome with tapered ends. Therefore, the population of M-cells deriving from an individual crypt migrates directly to the center of the dome and only a small portion blends with the M-cells from neighboring crypts. The tapering of the radial strips to the center of the domes suggests that a number of dome epithelial cells are sloughed off before reaching the center of the dome. This view is supported by findings in a mouse chimeric system of similar tapered and disrupted strips of enterocytes at the endings of duodenal villi (
In conclusion, this model implies that intestinal M-cells are derived from a single or only a few progenitor cells per crypt and are determined as M-cells even before they reach the dome epithelium. The heterogeneity of glycosylation patterns suggests that M-cells have a clonal origin in the dome-associated crypts and modify their glycosylation pattern as they migrate up the dome.
![]() |
Acknowledgments |
---|
Supported by the Deutsche Forschungsgemeinschaft (SFB280/C14).
We are grateful to Prof E. Reale, Department of Cell Biology and Electron Microscopy, for providing the EM facilities, Prof W.G. Forssmann, Lower Saxony Institute for Peptide Research, for providing the CLSM facilities, Prof D. Grube and Dr G. Bargsten for providing the image analysis system, and Prof B. Micheel for providing the anti-FITC antibody. We gratefully acknowledge the critical and helpful discussions with Prof H. Bartels, Prof R. Pabst, and Prof J. Westermann. The technical assistance of A. Beck, S. Fassbender, G. Preiss, and K. Werner, and the correction of the English text by S. Fryk, are gratefully acknowledged.
Received for publication February 26, 1997; accepted May 23, 1997.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amerongen HM, Weltzin R, Mack JA, Winner LS, Michetti P, Apter FM, Kraehenbuhl JP, Neutra MR (1992) M cell-mediated antigen transport and monoclonal IgA antibodies for mucosal immune protection. Ann NY Acad Sci 664:18-26 [Medline]
Bhalla DK, Owen RL (1982) Cell renewal and migration in lymphoid follicles of Peyer's patches and cecuman autoradiographic study in mice. Gastroenterology 82:232-242
[Medline]
Borghesi C, Regoli M, Bertelli E, Nicoletti C (1996) Modifications of the follicle-associated epithelium by short-term exposure to a non-intestinal bacterium. J Pathol 180:326-332 [Medline]
Bye WA, Allan CH, Trier JS (1984) Structure, distribution, and origin of M cells in Peyer's patches of mouse ileum. Gastroenterology 86:789-801 [Medline]
Cheng H, Bjerknes M (1996) Patterns of gene expression along the crypt-villus axis in mouse jejunal epithelium. Anat Rec 244:78-94 [Medline]
Cheng H, Leblond CP (1974) Origin, differentiation and renewal of the four main epithelial cells types in the mouse small intestine. V. Unitarian theory of the origin of the four epithelial cells types. Am J Anat 141:537-562 [Medline]
Clark MA, Jepson MA, Hirst BH (1995) Lectin binding defines and differentiates M-cells in mouse small intestine and caecum. Histochem Cell Biol 104:161-168 [Medline]
Clark MA, Jepson MA, Simmons NL, Booth TA, Hirst BH (1993) Differential expression of lectin-binding sites defines mouse intestinal M-cells. J Histochem Cytochem 41:1679-1687
Clark MA, Jepson MA, Simmons NL, Hirst BH (1994) Preferential interaction of Salmonella typhimurium with mouse Peyer's patch M cells. Res Microbiol 145:543-552 [Medline]
Etzler ME, Branstrator ML (1974) Differential localization of cell surface and secretory components in rat intestinal epithelium by use of lectins. J Cell Biol 62:329-343
Falk P, Roth KA, Gordon JI (1994) Lectins are sensitive tools for defining the differentiation programs of mouse gut epithelial cell lineages. Am J Physiol 29:G987-G1003
Gebert A (1996) M-cells in the rabbit tonsil exhibit distinctive glycoconjugates in their apical membrane. J Histochem Cytochem 44:1033-1042
Gebert A, Hach G (1993) Differential binding of lectins to M cells and enterocytes in the rabbit cecum. Gastroenterology 105:1350-1361 [Medline]
Gebert A, Rothkötter HJ, Pabst R (1996) M cells in Peyer's patches of the intestine. Int Rev Cytol 167:91-159 [Medline]
Giannasca PJ, Giannasca KT, Falk P, Gordon JI, Neutra MR (1994) Regional differences in glycoconjugates of intestinal M cells in mice: potential targets for mucosal vaccines. Am J Physiol 267:G1108-G1121
Jepson MA, Clark MA, Simmons NL, Hirst BH (1993) Epithelial M cells in the rabbit caecal lymphoid patch display distinctive surface characteristics. Histochemistry 100:441-447 [Medline]
Jepson MA, Simmons NL, Hirst GL, Hirst BH (1993) Identification of M cells and their distribution in rabbit intestinal Peyer's patches and appendix. Cell Tissue Res 273:127-136 [Medline]
Leblond CP (1981) The life history of cells in renewing systems. Am J Anat 160:113-158
Maiuri L, Raia V, Fiocca R, Solcia E, Cornaggia M, Norèn O, Sjostrom H, Swallow D, Auricchio S, Dabelsteen E (1993) Mosaic differentiation of human villus enterocytes: patchy expression of blood group A antigen in A nonsecretors. Gastroenterology 104:21-30 [Medline]
Maiuri L, Raia V, Potter J, Swallow D, Ho MW, Fiocca R, Finzi G, Cornaggia M, Capella C, Quaroni A, Auricchio S (1991) Mosaic pattern of lactase expression by villous enterocytes in human adult-type hypolactasia. Gastroenterology 100:359-369 [Medline]
Maury J, Bernadac A, Rigal A, Maroux S (1995) Expression and glycosylation of the filamentous brush border glycocalyx (FBBG) during rabbit enterocyte differentiation along the crypt-villus axis. J Cell Sci 108:2705-2713
Micheel B, Jantscheff P, Böttger V, Scharte G, Kaiser G, Stolley P, Karawajew L (1988) The production and radioimmunoassay application of monoclonal antibodies to fluorescein isothiocyanate (FITC). J Immunol Methods 111:89-94 [Medline]
Neutra MR, Frey A, Kraehenbuhl JP (1996) Epithelial M cells: gateways for mucosal infection and immunization. Cell 86:345-348 [Medline]
Nordström C, Dahlquist A, Josefsson L (1967) Quantitative determination of enzymes in different parts of the villi and crypts of rat small intestine. Comparison of alkaline phosphatase, disaccharidases and dipeptidases. J Histochem Cytochem 15:713-721 [Medline]
Owen RL (1977) Sequential uptake of horseradish peroxidase by lymphoid follicle epithelium of Peyer's patches in the normal unobstructed mouse intestine: an ultrastructural study. Gastroenterology 72:440-451 [Medline]
Owen RL, Jones AL (1974) Epithelial cell specialization within human Peyer's patches: an ultrastructural study of intestinal lymphoid follicles. Gastroenterology 66:189-203 [Medline]
Ponder BAJ, Schmidt GH, Wilkinson MM, Wood MJ, Monk M, Reid A (1985) Derivation of mouse intestinal crypts from single progenitor cells. Nature 313:689-691 [Medline]
Rubin DC, Ong D, Gordon JI (1989) Cellular differentiation in the emerging fetal rat small intestinal epithelium: mosaic patterns of gene expression. Proc Natl Acad Sci USA 86:1278-1282 [Abstract]
Savidge TC (1996) The life and times of an intestinal M cell. Trends Microbiol 4:301-306 [Medline]
Savidge TC, Smith MW, James PS, Aldred P (1991) Salmonella-induced M cell formation in germ-free mouse Peyer's patch tissue. Am J Pathol 139:177-184 [Abstract]
Schmedtje JF (1980) Lymphocyte positions in the dome epithelium of the rabbit appendix. J Morphol 166:179-195 [Medline]
Schmidt GH, Wilkinson MM, Ponder BAJ (1985) Cell migration pathway in the intestinal epithelium: an in situ marker system using mouse aggregation chimeras. Cell 40:425-429 [Medline]
Sicinski P, Rowinski J, Warchol JB, Bem W (1986) Morphometric evidence against lymphocyte-induced differentiation of M cells from absorptive cells in mouse Peyer's patches. Gastroenterology 90:609-616 [Medline]
Smith MW, Jarvis LG, King IS (1980) Cell proliferation in follicle-associated epithelium of mouse Peyer's patch. Am J Anat 159:157-166 [Medline]
Smith MW, Peacock MA (1980) "M" cell distribution in follicle-associated epithelium of mouse Peyer's patch. Am J Anat 159:167-175 [Medline]
Smith MW, Peacock MA (1982) Lymphocyte induced formation of antigen transporting "M" cells from fully differentiated mouse enterocytes. In Robinson JWL, Dowling RH, Riecken EO, eds. Mechanisms of Intestinal Adaption. Lancaster, MTP, 573-583
Winton DJ, Blount MA, Ponder BAJ (1988) A clonal marker induced by mutation in mouse intestinal epithelium. Nature 333:463-466 [Medline]
Wolf JL, Rubin DH, Finberg R, Kauffman RS, Sharpe AH, Trier JS, Fields BN (1981) Intestinal M-cells: a pathway for entry of reovirus into the host. Science 212:471-472 [Medline]