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Further characterization of M cells in gut-associated lymphoid tissues of the chicken
IMMUNOLOGY Further Characterization of M Cells in Gut-Associated Lymphoid Tissues of the Chicken SUZAN H. M. JEURISSEN,*,1 FRANS WAGENAAR,† and E. MARGA JANSE* *Department of Immunology, Pathobiology, and Epidemiology and †Department of Avian Virology, ID-DLO, Lelystad, The Netherlands of the FAE. The numbers of intra-epithelial leucocytes (IEL) increased rapidly after hatch, reaching innumerable at 6 wk of age. Most IEL were T lymphocytes expressing CD8 and only about 30% of them were B lymphocytes. Nevertheless, double staining of M cells (WGA/SBA) and IEL showed that M cells were much fewer than IEL. These results indicate that M cells are not solely induced by the intra-epithelial localization of leucocytes. Because the phenotype of IEL reflected the content of the adjacent underlying lamina propria, IEL immigrate the FAE locally and do not migrate along with the epithelial cells from the crypts. In conclusion, M cells exist in the chicken, but their phenotype and function are less well demarcated from neighbor epithelial cells than is seen in mammals.
(Key words: intestine, follicle-associated epithelium, leucocytes, M cell, antigen absorption) 1999 Poultry Science 78:965–972
are considered to be of epithelial origin. The numbers of M cells in the FAE vary considerably between species, being about 50% in the rabbit (Pappo et al., 1988) and about 10 to 15% in mice (Smith and Peacock, 1980). Originally, M cells were characterized by their typical ultrastructural features, including reduction in number and height of microvilli and the extracellular pocket that encloses lymphocytes (Owen and Jones, 1974). These M cell-related lymphocytes possibly induce, directly or indirectly, the development of M cells (Smith and Peacock, 1980). Furthermore, M cells were functionally characterized by the efficient uptake of an array of pathogens such as reovirus (Wolf et al., 1983), Cryptosporidium (Marcial and Madara, 1986), Vibrio cholerae (Owen et al., 1986), and Salmonella typhimurium (Clark et al., 1994), and of soluble and particulate antigens, such as horseradish peroxidase (Owen, 1977), ferritin (Bockman and Cooper, 1973), and latex particles (Pappo and
INTRODUCTION Since the first description of a highly absorptive cell in the follicle-associated epithelium of the bursa of Fabricius in 1973 by Bockman and Cooper, M cells have been considered to be the most effective cells for the transport of specific antigens from the intestinal lumen into the underlying lymphoid tissue. Since 1973, they have been described in many different species, including humans (Owen and Jones, 1974), mice (Smith and Peacock, 1980), rats (Bockman and Cooper, 1973), guinea pigs (Rosner and Keren, 1984), rabbits (Fujimura, 1986), pigs (Gebert et al., 1994), and ruminants (Momotyani et al., 1988). M cells are located in the follicle-associated epithelium (FAE) covering the B cell follicles in mucosaassociated lymphoid tissues in the intestines. They are connected to adjacent epithelial cells with tight junctions and desmosomes and they derive directly from undifferentiated crypt cells (Bye et al., 1984); therefore, they
Received for publication May 14, 1998. Accepted for publication February 2, 1999. 1To whom correspondence should [email protected]
be
Abbreviation Key: AAA = Anguilla anguilla agglutinin; FAE = follicle-associated epithelium; GS-II = Griffonia simplicifolia type II agglutinin; IEL = intra-epithelial leucocyte; LTA = Lotus tetragonolobus agglutinin; PNA = Arachis hypogaea (peanut) agglutinin; SBA = Glycine max agglutinin; UEA-I = Ulex europaeus type II agglutinin; WGA = Triticum vulgaris (wheatgerm) agglutinin.
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ABSTRACT M cells are considered to be the most effective cells for the transport of antigens from the intestinal lumen into the gut-associated lymphoid tissue. M cells are characterized by their ultrastructural appearance, the selective uptake of antigens, the binding of lectins, and the presence of underlying lymphocytes. Little attention has been paid to the interaction of intraepithelial leucocytes and M cells in chickens; therefore, we have investigated both cell types separately and using double immunocytochemical staining in cecal tonsils and Meckel’s diverticulum. In the follicleassociated epithelium (FAE), cells were present that differ from their neighbors by short, irregular microvilli. Ferritin was absorbed by these putative M cells, but also by other epithelial cells. The lectins of Triticum vulgaris (WGA) and Glycine max (SBA) showed a patchy staining
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MATERIALS AND METHODS
Animals White Leghorn chickens were hatched and kept under routine specific-pathogen-free conditions with free access to feed and water. In all experiments, groups of three chickens were used. To study the numbers of IEL in the FAE of cecal tonsils and Meckel’s diverticulum, chickens were used at 20 d of incubation, and at 5 d and 2, 6, and 12 wk after hatch. To study the phenotypes of IEL, cecal tonsils and Meckel’s diverticulum were isolated from chickens at 8 wk of age. To study the binding of various lectins and antibodies, cecal tonsils were isolated from 6-wk-old chickens. Meckel’s diverticulum and cecal tonsils were removed after exsanguination, snap-frozen in liquid nitrogen, and stored at –20 C. To investigate the uptake of antigens at an electronmicroscopic level, chickens (14 wk old) were anaesthe-
tized by intravenous injection of Nembutal (30 mg/kg body weight). A small incision was made in the abdomen and the small intestine partly lifted out. Meckel’s diverticulum was injected at the blunt end with 100 mL ferritin solution.2 After 2 h, Meckel’s diverticulum was collected, the blunt end was carefully removed, and the remaining part was treated for electronmicroscopy.
Immunocytochemistry Cryostat sections of 8 mm thickness were placed on slides, air-dried, and fixed in pure acetone for 10 min. Then sections were incubated with (monoclonal) antibodies in an appropriate dilution for 45 min at room temperature. Slides were rinsed three times in PBS. Sections were subsequently incubated with peroxidase labeled rabbit anti-mouse Ig.3 After 30 min, slides were rinsed again. Peroxidase activity was detected with a solution of 0.5 mg 3,3′-diaminobenzidine-tetrahydrochloride4 and 0.01% H2O2/mL TRIS-HCl buffer (0.05 M, pH 7.6).
(Monoclonal) Antibodies The following mouse monoclonal antibodies specific for chicken cell populations were used: HIS-C7, specific for CD45 on leucocytes (Jeurissen et al., 1988a); HIS-C1, specific for Bu1 on B lymphocytes (Jeurissen et al., 1988a); HIS-C12, specific for IgM (Jeurissen et al., 1988a); CVIChIgG-47.3, specific for IgG (Jeurissen et al., 1988a); antiCD3, specific for all T lymphocytes;5 anti-CD4, specific for T-helper lymphocytes;4 CVI-ChT-74.1, specific for CD8 on cytotoxic T lymphocytes (Jeurissen et al., 1994); anti-TCR1, specific for gd T lymphocytes;4 anti-TCR2, specific for ab T lymphocytes;4 CVI-ChNL-68.1, specific for mononuclear phagocytes (Jeurissen et al., 1988b); CVI-ChNL-74.2, specific for mature macrophages (Jeurissen et al., 1992). In addition rabbit anti-cytokeratin 18 antibody6 was used to visualize M cells.
Lectins The following lectins were used: Glycine max (SBA) specific for N-acetyl-D-galactosamine;5 Lotus tetragonolobus (LTA), Ulex europaeus type 1 (UEA-1), and Anguilla anguilla (AAA), specific for a-L-Fucose;5 Griffonia simplicifolia type II (GS-II), and Triticum vulgaris (WGA), specific for N-acetyl-D-Glucosamine;5 Arachis hypogaea (PNA) specific for a/b-D-Galactose.5 All lectins were used at a concentration of 2 to 5 mg/mL in PBS.
Electronmicroscopy 2Serva, Heidelberg, Germany. 3DAKO, Glostrup, Denmark. 4Sigma Chemical Co., St. Louis, MO 63178-9916. 5Southern Biotechnology Associates, Birmingham, 6E-Y Labs, San Mateo, CA 94401.
AL 35226.
Meckel’s diverticulums were cut in blocks of about 1 mm3. Blocks were immediately fixed in a mixture of 0.8% glutaraldehyde and 0.8% osmium tetroxide in a veronal
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Ermak, 1989). M cells are thought to sample antigens from the intestinal lumen and to transport them to the associated lymphocytes (Trier, 1991). In a single species, the rabbit, monoclonal antibodies have been developed specific for M cells (Pappo, 1989). M cells can be distinguished by antibodies against cytokeratin 18 in pigs (Gebert et al., 1994) and by antibodies against vimentin in rabbits (Gebert, 1995). In addition, M cells can also be recognized by their selective binding of lectins. The type of lectin, however, is dependent on the species. M cells in the mouse can be specifically recognized by Ulex europaeus agglutinin type I (UEA-I) and by Psophocarpus tetragonolobus (WBA; Clark et al., 1993). Rabbit M cells specifically bind UEA-I and Lotus tetragonolobus (LTA) among others (Gebert, 1996). In contrast, no lectin of a group of 27 lectins was found to react specifically with human M cells (Sharma et al., 1996). Since their first description in the bursa of Fabricius, however, little attention has been paid to M cells in birds. Only two reports on the ultrastructural determination of M cells in the Peyer’s patch of the chicken small intestine are known (Befus et al., 1980; Burns and Maxwell, 1986) and one report on M cells in cecal tonsils (Kato et al., 1992). Especially, the link between M cells and intra-epithelial leucocytes (IEL) has not been addressed. In this report, our previous work on the structure and function of the Meckel’s diverticulum and cecal tonsils (Jeurissen et al., 1989, 1991, 1994) is extended to the phenotypes and functioning of M cells and IEL, and to the link between both cell populations.
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acetate buffer (0.15 M, pH 7.4), washed, and stained in 1% uranyl acetate with microwave stimulation (Wagenaar et al., 1993). Further processing for electronmicroscopy was performed according to standard procedures. Ultrathin sections, counterstained with lead citrate, were examined in a Philips CM10 transmission electron microscope at 80 kV.
RESULTS
Ultrastructural Appearance
Uptake of Ferritin The uptake of antigens from the lumen was examined at the ultrastructural level using ferritin that was injected in vivo into Meckel’s diverticulum. Ferritin could be detected as a dark homogenous substance in the lumen and attached to the apical side of epithelial cells. Occasionally, M cells with their short, irregular microvilli were seen to absorb ferritin (Figure 2a). In the direct neighborhood of these cells, ferritin was found as well in the extracellular space and around IEL situated deeper in the epithelium. In addition at several sites, ferritin was detected in the extracellular space in between regular epithelial cells (Figure 2b). Ferritin was only found at the basal side of the tight junctions that looked to be intact. At very high magnification, ferritin could be seen in epithelial cells as small droplets in the microvilli and cytoplasm (Figure 2c), indicating a physiological transport route. Ferritin could also be detected more basally in the extracellular space, where IEL were located, and it was found surrounding these cells (Figure 2d).
Binding of Lectins and Antibodies against the Cytoskeleton Sections that were not incubated with any lectin showed no staining at all.
FIGURE 1. Ultrastructural appearance of a M cell in the Meckel’s diverticulum of the chicken. The short and irregular microvilli (arrows) discriminate the M cell from its neighboring epithelial cells (E). The M cell is connected to neighboring epithelial cells by desmosomes (double arrow). M = microvilli. Bar = 1 mm.
WGA. This lectin showed a faint, but extensive background staining of the connective tissue in the lamina propria (Figure 3a). Crypt epithelium was negative, but the tips of the villi were stained. In the FAE a dark patchy staining was seen. SBA. This lectin bound to the inner walls of large blood vessels in the lamina propria and showed a faint background staining in the lamina propria and muscle layers (Figure 3b). Crypt epithelium was negative. In contrast, the tips of the villi and the epithelium covering the lymphoid tissue were weakly stained. Only in the FAE some small patches of intense staining were seen. AAA. In the lamina propria of the lymphoid tissue, AAA selectively stained germinal centers and some round clusters. Epithelium of the crypts, the villi, and the lymphoid tissue was all positively stained, but the staining in the FAE was patchy. GS-II. (Figure 3c) In the subepithelial area of the lymphoid tissue, GS-II bound to stromal cells. Crypt epithelium was negative. The epithelium of villi and lymphoid tissue showed a strong overall staining of the apical side of the cells and the brush border. The staining was interrupted where goblet cells were present. LTA. This lectin bound to clusters of stromal cells in the lamina propria. Furthermore, it stained all epithelium of crypts, villi, and lymphoid tissue, including the basal membrane. The LTA bound to epithelial cells at the basal and the apical sides. PNA. In the lamina propria of the lymphoid tissue and the villi, just beneath the basal membrane, 10 to 20 mm
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Using electronmicroscopy, the ultrastructural appearance of the epithelial cells covering the lymphoid tissue of cecal tonsils and Meckel’s diverticulum was examined. Among regular epithelial cells, some distinct epithelial cells were seen (Figure 1). These cells were characterized by the presence of short and irregular microvilli on the apical side. Furthermore, the cytoplasm of these cells was darker than that of surrounding epithelial cells. These cells were connected with tight junctions and desmosomes to the neighboring epithelial cells, confirming their epithelial nature. Based on these observations, these distinct epithelial cells were considered to represent M cells. In addition, many leucocytes were detected in the epithelium. These leucocytes were located between and under regular columnar epithelial cells with regular microvilli and near M cells, thus showing no strict relation to M cells.
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round structures were stained. At this site, many capillaries and macrophages are located. The crypt epithelium was negative. Epithelium of the villi and lymphoid tissue showed a faintly stained brush border.
FIGURE 2. Absorption of ferritin on ultrastructural level. a) A M cell has absorbed ferritin (arrow) from the lumen (L). b) In addition, ferritin (arrows) is also abundantly found in the intracellular space of regular epithelial cells (E). c) Ferritin (arrows) was detected in the microvilli and apical part of the epithelial cells as small grains. d) Ferritin (arrow) was collected around intra-epithelial lymphoid cells (IEL). M = microvilli. Bar = 2.5 mm (a, b, d), 0.25 mm (c).
UEA-I. The lectin UEA-1 showed no staining in the epithelium, or in the lamina propria, although it reacted positively with simultaneously stained porcine tissue. Anti-Cytokeratin 18. This antibody showed no staining in the lamina propria. In addition, crypt epithelium and lymphoid tissue epithelium were both negative. Faint staining was seen in the villi epithelium at the basal side of the cells near the basal membrane. Anti-Vimentin. With anti-vimentin, all leucocytes were stained, which resulted in an extensive staining of the lamina propria. The IEL in crypt and villus epithelium were stained as well. In the epithelium covering the lymphoid tissue, all intra-epithelial leucocytes were stained. In contrast, the regular epithelial cells were not
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FIGURE 3. Binding of various lectins to the follicle-associated epithelium of cecal tonsils and Meckel’s diverticulum. The localization of the basal membrane, which separates the follicle associated epithelium from the lamina propria (LP), is indicated with a dotted line. a) WGA and b) SBA showed a patchy staining (arrows) of the follicle-associated epithelium of cecal tonsils. c) GS-II stained epithelial cells of the villi and the lymphoid tissue. G = germinal center; V = villi. Bar = 50 mm.
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stained. No indications were found that chicken M cells were recognized by anti-vimentin.
Age-Dependent Development of Intra-Epithelial Lymphocytes
distributed over the FAE and more numerous when they were located on top of a B cell area. Of these B lymphocytes, only some expressed IgM or IgG. No IgM or IgG plasma cells were detected in the epithelium. No IgA B lymphocytes or IgA plasma cells were detected in the epithelium. In contrast, the FAE contained soluble IgA antibodies, when located on top of IgA plasma cells in the lamina propria. Nonlymphoid Cells. Monocytes and macrophages were sporadically found among IEL and were always situated apically of epithelial nuclei close to the lumen. Mature macrophages were not present in the epithelium. Downloaded from http://ps.oxfordjournals.org/ at University of Illinois at Urbana-Champaign on March 10, 2015
At all ages, the epithelium covering lymphoid tissues was characterized by the absence of goblet cells. The agedependent development of IEL was examined in Meckel’s diverticulum and cecal tonsils using the chicken CD45 determinant. Day 20 of Incubation. In Meckel’s diverticulum and cecal tonsils, small undifferentiated aggregates of CD45positive lymphocytes were present. The epithelium covering these infiltrates sporadically contained IEL; in Meckel’s diverticulum about 6 IEL/mm epithelium and in cecal tonsils about 20 IEL/mm epithelium were found. Day 5. Both lymphoid tissues consisted of larger, but still undifferentiated aggregates of lymphocytes (Figure 4a). The epithelium contained an increasing number of IEL; in Meckel’s diverticulum about 30 IEL/mm epithelium and in cecal tonsils about 50 IEL/mm epithelium were found. These IEL were located basally and apically of the row of epithelial nuclei. Day 14. By Day 14, Meckel’s diverticulum and cecal tonsils comprised separate T and B cell areas, although only in cecal tonsils germinal centers were present (Figure 4b). In Meckel’s diverticulum about 70 IEL/mm epithelium were found. In cecal tonsils, the epithelium contained countless numbers of IEL. Basally of epithelial nuclei, IEL formed more or less a closed row of cells, whereas apically of the epithelial nuclei, many single IEL were found. Six and 12 Weeks. Both lymphoid tissues were fully developed with many germinal centers (Figure 4c). The numbers of IEL in both lymphoid tissues were innumerable. Epithelial cells were hardly detectable anymore, and IEL were located at all levels in the epithelium. Especially at 12 wk, the epithelium appeared to be pseudostratified.
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Phenotype of IEL The phenotypes of IEL were investigated in the follicleassociated epithelium of Meckel’s diverticulum and cecal tonsils of 8-wk-old chickens. As no differences were found between these organs, the results are presented simultaneously for both tissues. T Lymphocytes. Between 60 and 80% of IEL expressed CD3. The distribution in the epithelium was uneven, because intraepithelial T lymphocytes were even more numerous when they were located on top of a T cell area in the lamina propria. Most of these T lymphocytes expressed CD8 (Figure 5a), whereas only some T lymphocytes expressed CD4. Within the intra-epithelial T lymphocyte population, cells expressing TCR1 (Figure 5b) and TCR2 (Figure 5c) were equally distributed. B Lymphocytes. About 30% of IEL expressed Bu-1. Like the T lymphocytes, B lymphocytes were unevenly
FIGURE 4. Posthatching development of CD45-positive leucocytes in the follicle-associated epithelium. The localization of the basal membrane, which separates the follicle associated epithelium from the lamina propria (LP), is indicated with a dotted line. a) At Day 5 after hatch, only few leucocytes were detected in cecal tonsils. b) At 2 wk after hatch, numerous leucocytes were located in the follicle-associated epithelium of Meckel’s diverticulum. c) At the age of 12 wk, leucocytes had completely occupied the follicle-associated epithelium. G = germinal center; L = lumen; M = Muscularis externa. Bar = 50 mm.
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Simultaneous Detection of M Cells and Intra-Epithelial Leucocytes Double immunocytochemical staining using the lectins WGA, SBA, or AAA to visualize M cells and anti-CD45 or anti-CD3 to visualize IEL demonstrated that the IEL by far outnumbered the M cells. Even in younger chickens with low numbers of IEL, only few M cells were stained. This result coincided with that of the separate single stainings of M cells and IEL.
DISCUSSION
FIGURE 5. Phenotype of leucocytes in the follicle-associated epithelium.The localization of the basal membrane, which separates the follicle associated epithelium from the lamina propria (LP), is indicated with a dotted line. a) Most leucocytes in the follicle-associated epithelium of cecal tonsils were CD8-positive. b) Of these CD8-positive cells the majority bore the TCR1. c) The rest of the cells was TCR2-positive. G = germinal center; P = lamina propria (LP). Bar = 50 mm.
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At an ultrastructural level, we recognized specific epithelial cells in the FAE that most probably represent M cells in the chicken. Nevertheless, we were unable to find selective cell surface markers for these cells and did not find indications of increased antigen absorption. The capacity to absorb particles and soluble antigens from the gut lumen is thought to differ between regular cuboidal epithelium cells and M cells. Although both types of epithelial cells can absorb soluble antigens, the efficiency of M cells is supposed to be much higher. In our study, however, no difference was observed in the uptake of ferritin between putative M cells and regular epithelial cells of the Meckel’s diverticulum. Using the soluble antigen horseradish peroxidase, the results were similar in cecal tonsils, because uptake was observed throughout the FAE and not specifically in M cells (Kato et al., 1992). It can be argued that the preferential uptake of antigens by M cells is more distinct for particles. The ferritin solution used, however, can be considered as a mixture of soluble antigen and particles, as ferritin is known for the formation of protein complexes. The FAE of chicken gut-associated lymphoid tissues therefore seem to lack specialized epithelial cells dedicated to antigen absorption. As lectins can be divided into subgroups depending on the type of sugar they specifically adhere to (Giannasca et al., 1994), we have chosen one or more lectins from each subgroup. Three lectins, WGA, SBA, and AAA, were found to show a patchy staining of the FAE that might be indicative of M cells. The M cell staining by WGA has been described previously (Kato et al., 1992). With all three lectins, this staining was not specific for M cells, because WGA and SBA also stained mature epithelial cells of the villi and AAA even all epithelial cells. Surprisingly, the three lectins are specific for three different glycoconjugates and they even recognize different epitopes on these molecules (Giannasca et al., 1994).The results with the lectins WGA and SBA coincided with the observations on villi and crypt epithelium of Alroy et al (1989). In contrast, PNA and UEA-I were found negative in this study, whereas they stained villi epithelium in the study of Alroy et al (1989). The staining was, however, not consistently found in 100% of the chickens, indicating that genetic heterogeneity may influence the results (Alroy et al., 1989).
Using immunocytochemical staining on paraffin embedded cecal tonsils, Kato and coworkers (1992) detected some staining with PNA, but no staining with UEA-1. This finding indicates that the processing of the intestinal tissue also can influence the staining results. The lectins widely used to recognize murine and porcine M cells, UEA-1 and anti-cytokeratin 18, did not react with cells of chicken follicle-associated epithelium at all. Next to the phenotypical and functional characterization of chicken M cells, this study was undertaken to investigate the link between M cells and IEL. From a few days before hatch until several weeks after hatch, an increasing number of IEL were found in the epithelium covering the lymphoid tissue of Meckel’s diverticulum
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ACKNOWLEDGMENTS The authors thank Gerard Kok and Thys van der Wal for expert technical support of the electronmicroscopical work and lectin-binding work, respectively.
REFERENCES Alroy, J., V. Goyal, M. W. Lukacs, R. L. Taylor, R. G. Strout, H. D. Ward, and M.E.A. Pereira, 1989. Glycoconjugates of the intestinal epithelium of the domestic fowl (Gallus domesticus): a lectin histochemistry study. Histochem. J. 21: 187–193. Befus, A. D., N. Johnston, G. A. Leslie, and J. Bienenstock, 1980. Gut-associated lymphoid tissue in the chicken. 1. Morphology, ontogeny, and some functional characteristics of Peyer’s patches. J. Immunol. 125:2626–2632. Bockman, D. E., and M. D. Cooper, 1973. Pinocytosis by epithelium associated with lymphoid follicles in the bursa of Fabricius, appendix, and Peyer’s patches. An electron microscopic study. Am. J. Anat. 136:455–478. Burns, R. B., and M. H. Maxwell, 1986. Ultrastructure of Peyer’s patches in the domestic fowl and turkey. J. Anat. 147:235–243.
Bye, W. A., C. H. Allan, and J. S. Trier, 1984. Structure, distribution, and origin of M cells in Peyer’s patches of mouse ileum. Gastroenterology 86:789–801. Clark, M. A., M. A. Jepson, N. L. Simmons, T. A. Booth, and B. H. Hirst, 1993. Differential expression of lectin-binding sites defines mouse intestinal M cells. J. Histochem. Cytochem. 41:1679–1687. Clark, M. A., M. A. Jepson, N. L. Simmons, and B. H. Hirst, 1994. Preferential interaction of Salmonella typhimurium with mouse Peyer’s patch M cells. Res. Microbiol. 145: 543–552. Fujimura, Y., 1986. Functional morphology of microfold cells (M cells) in Peyer’s patches—Phagocytosis and transport of BCG by M cells into rabbit Peyer’s patches. Gastroenterol. Jpn. 21:325–335. Gebert, A., 1995. Identification of M cells in the rabbit tonsil by vimentin immunohistochemistry and in vivo protein transport. Histochem. Cell Biol. 104:211–220. Gebert, A., 1996. M cells in the rabbit tonsil exhibit distinctive glycoconjugates in their apical membranes. J. Histochem. Cytochem. 44:1033–1042. Gebert, A., H. J. Rothkotter, and R. Pabst, 1994. Cytokeratin 18 is an M cell marker in porcine Peyer’s patches. Cell Tissue Res. 276:213–221. Giannasca, P. J., K. T. Giannasca, P. Falk, J. I. Gordon, and M. R. Neutra, 1994. Regional differences in glycoconjugates of intestinal M cells in mice: potential targets for mucosal vaccines. Am. J. Physiol. 267:G1108–G1121. Jeurissen, S.H.M., E. Claassen, and E. M. Janse, 1992. Histological and functional differentiation of nonlymphoid cells in the chicken spleen. Immunology 77:75–80. Jeurissen, S.H.M., E. M. Janse, S. Ekino, P. Nieuwenhuis, G. Koch, and G. F. de Boer, 1988a. Monoclonal antibodies as probes for defining cellular subsets in the bone marrow, thymus, bursa of Fabricius, and spleen of the chicken. Vet. Immunol. Immunopathol. 19:225–238. Jeurissen, S.H.M., E. M. Janse, G. Koch, and G. F. de Boer, 1988b. The monoclonal antibody CVI-ChNL-68.1 recognizes cells of the monocyte-macrophage lineage in chickens. Dev. Comp. Immunol. 12:855–864. Jeurissen, S.H.M., E. M. Janse, G. Koch, and G. F. de Boer, 1989. Postnatal development of mucosa-associated lymphoid tissues in chickens. Cell Tissue Res. 258:119–124. Jeurissen, S.H.M., D. van Roozelaar, and E. M. Janse, 1991. Absorption of carbon from the yolk into gut-associated lymphoid tissues of chickens. Dev. Comp. Immunol. 15: 437–442. Jeurissen, S.H.M., L. Vervelde, and E. M. Janse, 1994. Structure and function of lymphoid tissues of the chicken. Poult. Sci. Rev. 5:183–207. Kato, A., Y. Hashimoto, Y. Kon, and M. Sugimura, 1992. Are there M cells in the cecal tonsil of chickens? J. Vet. Med. Sci. 54:999–1006. Kerneis, S., A. Bogdanova, J. P. Kraehenbuhl, and E. Pringault, 1997. Conversion by Peyer’s patch lymphocytes of human enterocytes into M cells that transport bacteria. Science 277:949–952. Marcial, M. A., and J. L. Madara, 1986. Cryptosporidium: Cellular localisation, structural analysis of absorptive cell parasite membrane-membrane interactions in guinea pigs, and suggestion of protozoan transport by M cells. Gastroenterol. 90:583–594. Momotyani, E., D. L. Whipple, A. B. Thiermann, and N. F. Cheville, 1988. Role of M cells and macrophages in the entrance of Mycobacterium paratuberculosis into domes of ileal Peyer’s patches in calves. Vet. Pathol. 25:131–137.
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and cecal tonsils. These leucocytes comprised about 70% T cells and about 30% B cells, and only few other cell types. On a light microscopic level using double immunocytochemistry and on an ultramicroscopic level, the localization of leucocytes in the epithelium was not related, however, to the occurrence of M cells. In fact, the phenotype of IEL was strongly related to the phenotype of the underlying lamina propria leucocytes, indicating that IEL migrate to the FAE locally. The fact that no correlation exists between M cells and IEL in the chicken contradicts recent results obtained with in vitro culture of human epithelial cells, where, in particular, human B cells were found to induce the development of M cells (Kerneis et al., 1997). The most probable explanation for this difference is the lesser degree of specialization of epithelial cells in birds than in mammals. The high number of IEL vs the low number of M cells also indicates that IEL are more important than M cells in the defense mechanisms of the gut-associated lymphoid tissues. In summary, the epithelium covering lymphoid tissue in Meckel’s diverticulum and cecal tonsils contains epithelial cells that differ from their neighbors in that they have a dark stained cytoplasm and short, irregular microvilli. These cells can be visualized with the lectins WGA, SBA, and AAA. Nevertheless, their number is much smaller than the number of IEL, indicating that the localization of IEL is not determined by M cells and that M cells are not induced by the intra-epithelial localization of leucocytes. Uptake of ferritin by these M cells has been seen, but uptake also occurs by other epithelial cells. We conclude that their phenotype and function are less well demarcated from regular epithelial cells than is seen in mammals.
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JEURISSEN ET AL. Rosner, A. J., and D. F. Keren, 1984. Demonstration of M cells in the specialized follicle-epithelium overlying isolated lymphoid follicles in the gut. J. Leucocyte Biol. 35: 397–404. Sharma, R., E.J.M. van Damme, W. J. Peumans, P. Sarsfield, and U. Schumacher, 1996. Lectin binding reveals divergent carbohydrate expression in human and mouse Peyer’s patches. Histochem. Cell Biol. 105:459–465. Smith, M. W., and M. A. Peacock, 1980. “M” cell distribution in follicle-associated epithelium of mouse Peyer’s patch. Am. J. Anat. 159:167–175. Trier, J. S., 1991. Structure and function of intestinal M cells. Gastroenterol. Clin. North Am. 20:531–547. Wagenaar, F., G. L. Kok, J. M. Broekhuijsen-Davies, and J.M.A. Pol, 1993. Rapid cold fixation of tissue samples by microwave irradiation for use in electron microscopy. Histochem. J. 25:719–725. Wolf, J. L., R. S. Kauffman, R. Finberg, R. Dambrauskas, B. N. Fields, and J. S. Trier, 1983. Determinants of reovirus interaction with the intestinal M cells and absorptive cells of murine intestine. Gastroenterology 85:291–300.
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Owen, R. L., 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. Owen, R. L., and A. L. Jones, 1974. Epithelial cell specialisation with human Peyer’s patches: An ultrastructural study of intestinal lymphoid follicles. Gastroenterology 66:189–203. Owen, R. L., N. F. Pierce, R. T. Apple, and W. C. Cray, 1986. M cell transport of Vibrio cholerae from the intestinal lumen into Peyer’s patches: A mechanism for antigen sampling and for microbial transepithelial migration. J. Infect. Dis. 153:1108–1118. Pappo, J., 1989. Generation and characterisation of monoclonal antibodies recognising follicle epithelial M cells in rabbit gut-associated lymphoid tissues. Cell. Immunol. 120:31–41. Pappo, J., and T. H. Ermak, 1989. Uptake and translocation of fluorescent latex particles by rabbit Peyer’s patch follicle epithelium: A quantitative model for M cell uptake. Clin. Exp. Immunol. 76:144–148. Pappo, J., H. J. Steger, and R. L. Owen, 1988. Differential adherence of epithelium overlying gut-associated lymphoid tissue. Lab. Invest. 58:692–697.
(Key words: intestine, follicle-associated epithelium, leucocytes, M cell, antigen absorption) 1999 Poultry Science 78:965–972
are considered to be of epithelial origin. The numbers of M cells in the FAE vary considerably between species, being about 50% in the rabbit (Pappo et al., 1988) and about 10 to 15% in mice (Smith and Peacock, 1980). Originally, M cells were characterized by their typical ultrastructural features, including reduction in number and height of microvilli and the extracellular pocket that encloses lymphocytes (Owen and Jones, 1974). These M cell-related lymphocytes possibly induce, directly or indirectly, the development of M cells (Smith and Peacock, 1980). Furthermore, M cells were functionally characterized by the efficient uptake of an array of pathogens such as reovirus (Wolf et al., 1983), Cryptosporidium (Marcial and Madara, 1986), Vibrio cholerae (Owen et al., 1986), and Salmonella typhimurium (Clark et al., 1994), and of soluble and particulate antigens, such as horseradish peroxidase (Owen, 1977), ferritin (Bockman and Cooper, 1973), and latex particles (Pappo and
INTRODUCTION Since the first description of a highly absorptive cell in the follicle-associated epithelium of the bursa of Fabricius in 1973 by Bockman and Cooper, M cells have been considered to be the most effective cells for the transport of specific antigens from the intestinal lumen into the underlying lymphoid tissue. Since 1973, they have been described in many different species, including humans (Owen and Jones, 1974), mice (Smith and Peacock, 1980), rats (Bockman and Cooper, 1973), guinea pigs (Rosner and Keren, 1984), rabbits (Fujimura, 1986), pigs (Gebert et al., 1994), and ruminants (Momotyani et al., 1988). M cells are located in the follicle-associated epithelium (FAE) covering the B cell follicles in mucosaassociated lymphoid tissues in the intestines. They are connected to adjacent epithelial cells with tight junctions and desmosomes and they derive directly from undifferentiated crypt cells (Bye et al., 1984); therefore, they
Received for publication May 14, 1998. Accepted for publication February 2, 1999. 1To whom correspondence should [email protected]
be
Abbreviation Key: AAA = Anguilla anguilla agglutinin; FAE = follicle-associated epithelium; GS-II = Griffonia simplicifolia type II agglutinin; IEL = intra-epithelial leucocyte; LTA = Lotus tetragonolobus agglutinin; PNA = Arachis hypogaea (peanut) agglutinin; SBA = Glycine max agglutinin; UEA-I = Ulex europaeus type II agglutinin; WGA = Triticum vulgaris (wheatgerm) agglutinin.
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ABSTRACT M cells are considered to be the most effective cells for the transport of antigens from the intestinal lumen into the gut-associated lymphoid tissue. M cells are characterized by their ultrastructural appearance, the selective uptake of antigens, the binding of lectins, and the presence of underlying lymphocytes. Little attention has been paid to the interaction of intraepithelial leucocytes and M cells in chickens; therefore, we have investigated both cell types separately and using double immunocytochemical staining in cecal tonsils and Meckel’s diverticulum. In the follicleassociated epithelium (FAE), cells were present that differ from their neighbors by short, irregular microvilli. Ferritin was absorbed by these putative M cells, but also by other epithelial cells. The lectins of Triticum vulgaris (WGA) and Glycine max (SBA) showed a patchy staining
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MATERIALS AND METHODS
Animals White Leghorn chickens were hatched and kept under routine specific-pathogen-free conditions with free access to feed and water. In all experiments, groups of three chickens were used. To study the numbers of IEL in the FAE of cecal tonsils and Meckel’s diverticulum, chickens were used at 20 d of incubation, and at 5 d and 2, 6, and 12 wk after hatch. To study the phenotypes of IEL, cecal tonsils and Meckel’s diverticulum were isolated from chickens at 8 wk of age. To study the binding of various lectins and antibodies, cecal tonsils were isolated from 6-wk-old chickens. Meckel’s diverticulum and cecal tonsils were removed after exsanguination, snap-frozen in liquid nitrogen, and stored at –20 C. To investigate the uptake of antigens at an electronmicroscopic level, chickens (14 wk old) were anaesthe-
tized by intravenous injection of Nembutal (30 mg/kg body weight). A small incision was made in the abdomen and the small intestine partly lifted out. Meckel’s diverticulum was injected at the blunt end with 100 mL ferritin solution.2 After 2 h, Meckel’s diverticulum was collected, the blunt end was carefully removed, and the remaining part was treated for electronmicroscopy.
Immunocytochemistry Cryostat sections of 8 mm thickness were placed on slides, air-dried, and fixed in pure acetone for 10 min. Then sections were incubated with (monoclonal) antibodies in an appropriate dilution for 45 min at room temperature. Slides were rinsed three times in PBS. Sections were subsequently incubated with peroxidase labeled rabbit anti-mouse Ig.3 After 30 min, slides were rinsed again. Peroxidase activity was detected with a solution of 0.5 mg 3,3′-diaminobenzidine-tetrahydrochloride4 and 0.01% H2O2/mL TRIS-HCl buffer (0.05 M, pH 7.6).
(Monoclonal) Antibodies The following mouse monoclonal antibodies specific for chicken cell populations were used: HIS-C7, specific for CD45 on leucocytes (Jeurissen et al., 1988a); HIS-C1, specific for Bu1 on B lymphocytes (Jeurissen et al., 1988a); HIS-C12, specific for IgM (Jeurissen et al., 1988a); CVIChIgG-47.3, specific for IgG (Jeurissen et al., 1988a); antiCD3, specific for all T lymphocytes;5 anti-CD4, specific for T-helper lymphocytes;4 CVI-ChT-74.1, specific for CD8 on cytotoxic T lymphocytes (Jeurissen et al., 1994); anti-TCR1, specific for gd T lymphocytes;4 anti-TCR2, specific for ab T lymphocytes;4 CVI-ChNL-68.1, specific for mononuclear phagocytes (Jeurissen et al., 1988b); CVI-ChNL-74.2, specific for mature macrophages (Jeurissen et al., 1992). In addition rabbit anti-cytokeratin 18 antibody6 was used to visualize M cells.
Lectins The following lectins were used: Glycine max (SBA) specific for N-acetyl-D-galactosamine;5 Lotus tetragonolobus (LTA), Ulex europaeus type 1 (UEA-1), and Anguilla anguilla (AAA), specific for a-L-Fucose;5 Griffonia simplicifolia type II (GS-II), and Triticum vulgaris (WGA), specific for N-acetyl-D-Glucosamine;5 Arachis hypogaea (PNA) specific for a/b-D-Galactose.5 All lectins were used at a concentration of 2 to 5 mg/mL in PBS.
Electronmicroscopy 2Serva, Heidelberg, Germany. 3DAKO, Glostrup, Denmark. 4Sigma Chemical Co., St. Louis, MO 63178-9916. 5Southern Biotechnology Associates, Birmingham, 6E-Y Labs, San Mateo, CA 94401.
AL 35226.
Meckel’s diverticulums were cut in blocks of about 1 mm3. Blocks were immediately fixed in a mixture of 0.8% glutaraldehyde and 0.8% osmium tetroxide in a veronal
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Ermak, 1989). M cells are thought to sample antigens from the intestinal lumen and to transport them to the associated lymphocytes (Trier, 1991). In a single species, the rabbit, monoclonal antibodies have been developed specific for M cells (Pappo, 1989). M cells can be distinguished by antibodies against cytokeratin 18 in pigs (Gebert et al., 1994) and by antibodies against vimentin in rabbits (Gebert, 1995). In addition, M cells can also be recognized by their selective binding of lectins. The type of lectin, however, is dependent on the species. M cells in the mouse can be specifically recognized by Ulex europaeus agglutinin type I (UEA-I) and by Psophocarpus tetragonolobus (WBA; Clark et al., 1993). Rabbit M cells specifically bind UEA-I and Lotus tetragonolobus (LTA) among others (Gebert, 1996). In contrast, no lectin of a group of 27 lectins was found to react specifically with human M cells (Sharma et al., 1996). Since their first description in the bursa of Fabricius, however, little attention has been paid to M cells in birds. Only two reports on the ultrastructural determination of M cells in the Peyer’s patch of the chicken small intestine are known (Befus et al., 1980; Burns and Maxwell, 1986) and one report on M cells in cecal tonsils (Kato et al., 1992). Especially, the link between M cells and intra-epithelial leucocytes (IEL) has not been addressed. In this report, our previous work on the structure and function of the Meckel’s diverticulum and cecal tonsils (Jeurissen et al., 1989, 1991, 1994) is extended to the phenotypes and functioning of M cells and IEL, and to the link between both cell populations.
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acetate buffer (0.15 M, pH 7.4), washed, and stained in 1% uranyl acetate with microwave stimulation (Wagenaar et al., 1993). Further processing for electronmicroscopy was performed according to standard procedures. Ultrathin sections, counterstained with lead citrate, were examined in a Philips CM10 transmission electron microscope at 80 kV.
RESULTS
Ultrastructural Appearance
Uptake of Ferritin The uptake of antigens from the lumen was examined at the ultrastructural level using ferritin that was injected in vivo into Meckel’s diverticulum. Ferritin could be detected as a dark homogenous substance in the lumen and attached to the apical side of epithelial cells. Occasionally, M cells with their short, irregular microvilli were seen to absorb ferritin (Figure 2a). In the direct neighborhood of these cells, ferritin was found as well in the extracellular space and around IEL situated deeper in the epithelium. In addition at several sites, ferritin was detected in the extracellular space in between regular epithelial cells (Figure 2b). Ferritin was only found at the basal side of the tight junctions that looked to be intact. At very high magnification, ferritin could be seen in epithelial cells as small droplets in the microvilli and cytoplasm (Figure 2c), indicating a physiological transport route. Ferritin could also be detected more basally in the extracellular space, where IEL were located, and it was found surrounding these cells (Figure 2d).
Binding of Lectins and Antibodies against the Cytoskeleton Sections that were not incubated with any lectin showed no staining at all.
FIGURE 1. Ultrastructural appearance of a M cell in the Meckel’s diverticulum of the chicken. The short and irregular microvilli (arrows) discriminate the M cell from its neighboring epithelial cells (E). The M cell is connected to neighboring epithelial cells by desmosomes (double arrow). M = microvilli. Bar = 1 mm.
WGA. This lectin showed a faint, but extensive background staining of the connective tissue in the lamina propria (Figure 3a). Crypt epithelium was negative, but the tips of the villi were stained. In the FAE a dark patchy staining was seen. SBA. This lectin bound to the inner walls of large blood vessels in the lamina propria and showed a faint background staining in the lamina propria and muscle layers (Figure 3b). Crypt epithelium was negative. In contrast, the tips of the villi and the epithelium covering the lymphoid tissue were weakly stained. Only in the FAE some small patches of intense staining were seen. AAA. In the lamina propria of the lymphoid tissue, AAA selectively stained germinal centers and some round clusters. Epithelium of the crypts, the villi, and the lymphoid tissue was all positively stained, but the staining in the FAE was patchy. GS-II. (Figure 3c) In the subepithelial area of the lymphoid tissue, GS-II bound to stromal cells. Crypt epithelium was negative. The epithelium of villi and lymphoid tissue showed a strong overall staining of the apical side of the cells and the brush border. The staining was interrupted where goblet cells were present. LTA. This lectin bound to clusters of stromal cells in the lamina propria. Furthermore, it stained all epithelium of crypts, villi, and lymphoid tissue, including the basal membrane. The LTA bound to epithelial cells at the basal and the apical sides. PNA. In the lamina propria of the lymphoid tissue and the villi, just beneath the basal membrane, 10 to 20 mm
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Using electronmicroscopy, the ultrastructural appearance of the epithelial cells covering the lymphoid tissue of cecal tonsils and Meckel’s diverticulum was examined. Among regular epithelial cells, some distinct epithelial cells were seen (Figure 1). These cells were characterized by the presence of short and irregular microvilli on the apical side. Furthermore, the cytoplasm of these cells was darker than that of surrounding epithelial cells. These cells were connected with tight junctions and desmosomes to the neighboring epithelial cells, confirming their epithelial nature. Based on these observations, these distinct epithelial cells were considered to represent M cells. In addition, many leucocytes were detected in the epithelium. These leucocytes were located between and under regular columnar epithelial cells with regular microvilli and near M cells, thus showing no strict relation to M cells.
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round structures were stained. At this site, many capillaries and macrophages are located. The crypt epithelium was negative. Epithelium of the villi and lymphoid tissue showed a faintly stained brush border.
FIGURE 2. Absorption of ferritin on ultrastructural level. a) A M cell has absorbed ferritin (arrow) from the lumen (L). b) In addition, ferritin (arrows) is also abundantly found in the intracellular space of regular epithelial cells (E). c) Ferritin (arrows) was detected in the microvilli and apical part of the epithelial cells as small grains. d) Ferritin (arrow) was collected around intra-epithelial lymphoid cells (IEL). M = microvilli. Bar = 2.5 mm (a, b, d), 0.25 mm (c).
UEA-I. The lectin UEA-1 showed no staining in the epithelium, or in the lamina propria, although it reacted positively with simultaneously stained porcine tissue. Anti-Cytokeratin 18. This antibody showed no staining in the lamina propria. In addition, crypt epithelium and lymphoid tissue epithelium were both negative. Faint staining was seen in the villi epithelium at the basal side of the cells near the basal membrane. Anti-Vimentin. With anti-vimentin, all leucocytes were stained, which resulted in an extensive staining of the lamina propria. The IEL in crypt and villus epithelium were stained as well. In the epithelium covering the lymphoid tissue, all intra-epithelial leucocytes were stained. In contrast, the regular epithelial cells were not
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FIGURE 3. Binding of various lectins to the follicle-associated epithelium of cecal tonsils and Meckel’s diverticulum. The localization of the basal membrane, which separates the follicle associated epithelium from the lamina propria (LP), is indicated with a dotted line. a) WGA and b) SBA showed a patchy staining (arrows) of the follicle-associated epithelium of cecal tonsils. c) GS-II stained epithelial cells of the villi and the lymphoid tissue. G = germinal center; V = villi. Bar = 50 mm.
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stained. No indications were found that chicken M cells were recognized by anti-vimentin.
Age-Dependent Development of Intra-Epithelial Lymphocytes
distributed over the FAE and more numerous when they were located on top of a B cell area. Of these B lymphocytes, only some expressed IgM or IgG. No IgM or IgG plasma cells were detected in the epithelium. No IgA B lymphocytes or IgA plasma cells were detected in the epithelium. In contrast, the FAE contained soluble IgA antibodies, when located on top of IgA plasma cells in the lamina propria. Nonlymphoid Cells. Monocytes and macrophages were sporadically found among IEL and were always situated apically of epithelial nuclei close to the lumen. Mature macrophages were not present in the epithelium. Downloaded from http://ps.oxfordjournals.org/ at University of Illinois at Urbana-Champaign on March 10, 2015
At all ages, the epithelium covering lymphoid tissues was characterized by the absence of goblet cells. The agedependent development of IEL was examined in Meckel’s diverticulum and cecal tonsils using the chicken CD45 determinant. Day 20 of Incubation. In Meckel’s diverticulum and cecal tonsils, small undifferentiated aggregates of CD45positive lymphocytes were present. The epithelium covering these infiltrates sporadically contained IEL; in Meckel’s diverticulum about 6 IEL/mm epithelium and in cecal tonsils about 20 IEL/mm epithelium were found. Day 5. Both lymphoid tissues consisted of larger, but still undifferentiated aggregates of lymphocytes (Figure 4a). The epithelium contained an increasing number of IEL; in Meckel’s diverticulum about 30 IEL/mm epithelium and in cecal tonsils about 50 IEL/mm epithelium were found. These IEL were located basally and apically of the row of epithelial nuclei. Day 14. By Day 14, Meckel’s diverticulum and cecal tonsils comprised separate T and B cell areas, although only in cecal tonsils germinal centers were present (Figure 4b). In Meckel’s diverticulum about 70 IEL/mm epithelium were found. In cecal tonsils, the epithelium contained countless numbers of IEL. Basally of epithelial nuclei, IEL formed more or less a closed row of cells, whereas apically of the epithelial nuclei, many single IEL were found. Six and 12 Weeks. Both lymphoid tissues were fully developed with many germinal centers (Figure 4c). The numbers of IEL in both lymphoid tissues were innumerable. Epithelial cells were hardly detectable anymore, and IEL were located at all levels in the epithelium. Especially at 12 wk, the epithelium appeared to be pseudostratified.
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Phenotype of IEL The phenotypes of IEL were investigated in the follicleassociated epithelium of Meckel’s diverticulum and cecal tonsils of 8-wk-old chickens. As no differences were found between these organs, the results are presented simultaneously for both tissues. T Lymphocytes. Between 60 and 80% of IEL expressed CD3. The distribution in the epithelium was uneven, because intraepithelial T lymphocytes were even more numerous when they were located on top of a T cell area in the lamina propria. Most of these T lymphocytes expressed CD8 (Figure 5a), whereas only some T lymphocytes expressed CD4. Within the intra-epithelial T lymphocyte population, cells expressing TCR1 (Figure 5b) and TCR2 (Figure 5c) were equally distributed. B Lymphocytes. About 30% of IEL expressed Bu-1. Like the T lymphocytes, B lymphocytes were unevenly
FIGURE 4. Posthatching development of CD45-positive leucocytes in the follicle-associated epithelium. The localization of the basal membrane, which separates the follicle associated epithelium from the lamina propria (LP), is indicated with a dotted line. a) At Day 5 after hatch, only few leucocytes were detected in cecal tonsils. b) At 2 wk after hatch, numerous leucocytes were located in the follicle-associated epithelium of Meckel’s diverticulum. c) At the age of 12 wk, leucocytes had completely occupied the follicle-associated epithelium. G = germinal center; L = lumen; M = Muscularis externa. Bar = 50 mm.
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Simultaneous Detection of M Cells and Intra-Epithelial Leucocytes Double immunocytochemical staining using the lectins WGA, SBA, or AAA to visualize M cells and anti-CD45 or anti-CD3 to visualize IEL demonstrated that the IEL by far outnumbered the M cells. Even in younger chickens with low numbers of IEL, only few M cells were stained. This result coincided with that of the separate single stainings of M cells and IEL.
DISCUSSION
FIGURE 5. Phenotype of leucocytes in the follicle-associated epithelium.The localization of the basal membrane, which separates the follicle associated epithelium from the lamina propria (LP), is indicated with a dotted line. a) Most leucocytes in the follicle-associated epithelium of cecal tonsils were CD8-positive. b) Of these CD8-positive cells the majority bore the TCR1. c) The rest of the cells was TCR2-positive. G = germinal center; P = lamina propria (LP). Bar = 50 mm.
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At an ultrastructural level, we recognized specific epithelial cells in the FAE that most probably represent M cells in the chicken. Nevertheless, we were unable to find selective cell surface markers for these cells and did not find indications of increased antigen absorption. The capacity to absorb particles and soluble antigens from the gut lumen is thought to differ between regular cuboidal epithelium cells and M cells. Although both types of epithelial cells can absorb soluble antigens, the efficiency of M cells is supposed to be much higher. In our study, however, no difference was observed in the uptake of ferritin between putative M cells and regular epithelial cells of the Meckel’s diverticulum. Using the soluble antigen horseradish peroxidase, the results were similar in cecal tonsils, because uptake was observed throughout the FAE and not specifically in M cells (Kato et al., 1992). It can be argued that the preferential uptake of antigens by M cells is more distinct for particles. The ferritin solution used, however, can be considered as a mixture of soluble antigen and particles, as ferritin is known for the formation of protein complexes. The FAE of chicken gut-associated lymphoid tissues therefore seem to lack specialized epithelial cells dedicated to antigen absorption. As lectins can be divided into subgroups depending on the type of sugar they specifically adhere to (Giannasca et al., 1994), we have chosen one or more lectins from each subgroup. Three lectins, WGA, SBA, and AAA, were found to show a patchy staining of the FAE that might be indicative of M cells. The M cell staining by WGA has been described previously (Kato et al., 1992). With all three lectins, this staining was not specific for M cells, because WGA and SBA also stained mature epithelial cells of the villi and AAA even all epithelial cells. Surprisingly, the three lectins are specific for three different glycoconjugates and they even recognize different epitopes on these molecules (Giannasca et al., 1994).The results with the lectins WGA and SBA coincided with the observations on villi and crypt epithelium of Alroy et al (1989). In contrast, PNA and UEA-I were found negative in this study, whereas they stained villi epithelium in the study of Alroy et al (1989). The staining was, however, not consistently found in 100% of the chickens, indicating that genetic heterogeneity may influence the results (Alroy et al., 1989).
Using immunocytochemical staining on paraffin embedded cecal tonsils, Kato and coworkers (1992) detected some staining with PNA, but no staining with UEA-1. This finding indicates that the processing of the intestinal tissue also can influence the staining results. The lectins widely used to recognize murine and porcine M cells, UEA-1 and anti-cytokeratin 18, did not react with cells of chicken follicle-associated epithelium at all. Next to the phenotypical and functional characterization of chicken M cells, this study was undertaken to investigate the link between M cells and IEL. From a few days before hatch until several weeks after hatch, an increasing number of IEL were found in the epithelium covering the lymphoid tissue of Meckel’s diverticulum
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ACKNOWLEDGMENTS The authors thank Gerard Kok and Thys van der Wal for expert technical support of the electronmicroscopical work and lectin-binding work, respectively.
REFERENCES Alroy, J., V. Goyal, M. W. Lukacs, R. L. Taylor, R. G. Strout, H. D. Ward, and M.E.A. Pereira, 1989. Glycoconjugates of the intestinal epithelium of the domestic fowl (Gallus domesticus): a lectin histochemistry study. Histochem. J. 21: 187–193. Befus, A. D., N. Johnston, G. A. Leslie, and J. Bienenstock, 1980. Gut-associated lymphoid tissue in the chicken. 1. Morphology, ontogeny, and some functional characteristics of Peyer’s patches. J. Immunol. 125:2626–2632. Bockman, D. E., and M. D. Cooper, 1973. Pinocytosis by epithelium associated with lymphoid follicles in the bursa of Fabricius, appendix, and Peyer’s patches. An electron microscopic study. Am. J. Anat. 136:455–478. Burns, R. B., and M. H. Maxwell, 1986. Ultrastructure of Peyer’s patches in the domestic fowl and turkey. J. Anat. 147:235–243.
Bye, W. A., C. H. Allan, and J. S. Trier, 1984. Structure, distribution, and origin of M cells in Peyer’s patches of mouse ileum. Gastroenterology 86:789–801. Clark, M. A., M. A. Jepson, N. L. Simmons, T. A. Booth, and B. H. Hirst, 1993. Differential expression of lectin-binding sites defines mouse intestinal M cells. J. Histochem. Cytochem. 41:1679–1687. Clark, M. A., M. A. Jepson, N. L. Simmons, and B. H. Hirst, 1994. Preferential interaction of Salmonella typhimurium with mouse Peyer’s patch M cells. Res. Microbiol. 145: 543–552. Fujimura, Y., 1986. Functional morphology of microfold cells (M cells) in Peyer’s patches—Phagocytosis and transport of BCG by M cells into rabbit Peyer’s patches. Gastroenterol. Jpn. 21:325–335. Gebert, A., 1995. Identification of M cells in the rabbit tonsil by vimentin immunohistochemistry and in vivo protein transport. Histochem. Cell Biol. 104:211–220. Gebert, A., 1996. M cells in the rabbit tonsil exhibit distinctive glycoconjugates in their apical membranes. J. Histochem. Cytochem. 44:1033–1042. Gebert, A., H. J. Rothkotter, and R. Pabst, 1994. Cytokeratin 18 is an M cell marker in porcine Peyer’s patches. Cell Tissue Res. 276:213–221. Giannasca, P. J., K. T. Giannasca, P. Falk, J. I. Gordon, and M. R. Neutra, 1994. Regional differences in glycoconjugates of intestinal M cells in mice: potential targets for mucosal vaccines. Am. J. Physiol. 267:G1108–G1121. Jeurissen, S.H.M., E. Claassen, and E. M. Janse, 1992. Histological and functional differentiation of nonlymphoid cells in the chicken spleen. Immunology 77:75–80. Jeurissen, S.H.M., E. M. Janse, S. Ekino, P. Nieuwenhuis, G. Koch, and G. F. de Boer, 1988a. Monoclonal antibodies as probes for defining cellular subsets in the bone marrow, thymus, bursa of Fabricius, and spleen of the chicken. Vet. Immunol. Immunopathol. 19:225–238. Jeurissen, S.H.M., E. M. Janse, G. Koch, and G. F. de Boer, 1988b. The monoclonal antibody CVI-ChNL-68.1 recognizes cells of the monocyte-macrophage lineage in chickens. Dev. Comp. Immunol. 12:855–864. Jeurissen, S.H.M., E. M. Janse, G. Koch, and G. F. de Boer, 1989. Postnatal development of mucosa-associated lymphoid tissues in chickens. Cell Tissue Res. 258:119–124. Jeurissen, S.H.M., D. van Roozelaar, and E. M. Janse, 1991. Absorption of carbon from the yolk into gut-associated lymphoid tissues of chickens. Dev. Comp. Immunol. 15: 437–442. Jeurissen, S.H.M., L. Vervelde, and E. M. Janse, 1994. Structure and function of lymphoid tissues of the chicken. Poult. Sci. Rev. 5:183–207. Kato, A., Y. Hashimoto, Y. Kon, and M. Sugimura, 1992. Are there M cells in the cecal tonsil of chickens? J. Vet. Med. Sci. 54:999–1006. Kerneis, S., A. Bogdanova, J. P. Kraehenbuhl, and E. Pringault, 1997. Conversion by Peyer’s patch lymphocytes of human enterocytes into M cells that transport bacteria. Science 277:949–952. Marcial, M. A., and J. L. Madara, 1986. Cryptosporidium: Cellular localisation, structural analysis of absorptive cell parasite membrane-membrane interactions in guinea pigs, and suggestion of protozoan transport by M cells. Gastroenterol. 90:583–594. Momotyani, E., D. L. Whipple, A. B. Thiermann, and N. F. Cheville, 1988. Role of M cells and macrophages in the entrance of Mycobacterium paratuberculosis into domes of ileal Peyer’s patches in calves. Vet. Pathol. 25:131–137.
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and cecal tonsils. These leucocytes comprised about 70% T cells and about 30% B cells, and only few other cell types. On a light microscopic level using double immunocytochemistry and on an ultramicroscopic level, the localization of leucocytes in the epithelium was not related, however, to the occurrence of M cells. In fact, the phenotype of IEL was strongly related to the phenotype of the underlying lamina propria leucocytes, indicating that IEL migrate to the FAE locally. The fact that no correlation exists between M cells and IEL in the chicken contradicts recent results obtained with in vitro culture of human epithelial cells, where, in particular, human B cells were found to induce the development of M cells (Kerneis et al., 1997). The most probable explanation for this difference is the lesser degree of specialization of epithelial cells in birds than in mammals. The high number of IEL vs the low number of M cells also indicates that IEL are more important than M cells in the defense mechanisms of the gut-associated lymphoid tissues. In summary, the epithelium covering lymphoid tissue in Meckel’s diverticulum and cecal tonsils contains epithelial cells that differ from their neighbors in that they have a dark stained cytoplasm and short, irregular microvilli. These cells can be visualized with the lectins WGA, SBA, and AAA. Nevertheless, their number is much smaller than the number of IEL, indicating that the localization of IEL is not determined by M cells and that M cells are not induced by the intra-epithelial localization of leucocytes. Uptake of ferritin by these M cells has been seen, but uptake also occurs by other epithelial cells. We conclude that their phenotype and function are less well demarcated from regular epithelial cells than is seen in mammals.
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JEURISSEN ET AL. Rosner, A. J., and D. F. Keren, 1984. Demonstration of M cells in the specialized follicle-epithelium overlying isolated lymphoid follicles in the gut. J. Leucocyte Biol. 35: 397–404. Sharma, R., E.J.M. van Damme, W. J. Peumans, P. Sarsfield, and U. Schumacher, 1996. Lectin binding reveals divergent carbohydrate expression in human and mouse Peyer’s patches. Histochem. Cell Biol. 105:459–465. Smith, M. W., and M. A. Peacock, 1980. “M” cell distribution in follicle-associated epithelium of mouse Peyer’s patch. Am. J. Anat. 159:167–175. Trier, J. S., 1991. Structure and function of intestinal M cells. Gastroenterol. Clin. North Am. 20:531–547. Wagenaar, F., G. L. Kok, J. M. Broekhuijsen-Davies, and J.M.A. Pol, 1993. Rapid cold fixation of tissue samples by microwave irradiation for use in electron microscopy. Histochem. J. 25:719–725. Wolf, J. L., R. S. Kauffman, R. Finberg, R. Dambrauskas, B. N. Fields, and J. S. Trier, 1983. Determinants of reovirus interaction with the intestinal M cells and absorptive cells of murine intestine. Gastroenterology 85:291–300.
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Owen, R. L., 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. Owen, R. L., and A. L. Jones, 1974. Epithelial cell specialisation with human Peyer’s patches: An ultrastructural study of intestinal lymphoid follicles. Gastroenterology 66:189–203. Owen, R. L., N. F. Pierce, R. T. Apple, and W. C. Cray, 1986. M cell transport of Vibrio cholerae from the intestinal lumen into Peyer’s patches: A mechanism for antigen sampling and for microbial transepithelial migration. J. Infect. Dis. 153:1108–1118. Pappo, J., 1989. Generation and characterisation of monoclonal antibodies recognising follicle epithelial M cells in rabbit gut-associated lymphoid tissues. Cell. Immunol. 120:31–41. Pappo, J., and T. H. Ermak, 1989. Uptake and translocation of fluorescent latex particles by rabbit Peyer’s patch follicle epithelium: A quantitative model for M cell uptake. Clin. Exp. Immunol. 76:144–148. Pappo, J., H. J. Steger, and R. L. Owen, 1988. Differential adherence of epithelium overlying gut-associated lymphoid tissue. Lab. Invest. 58:692–697.