Rabbit Tonsil-associated M-cells Express Cytokeratin 20 and Take Up Particulate Antigen
Department of Pharmacology "Giorgio Segre," Section of Morphology, University of Siena, Siena, Italy (AC,MR,LF,EB); Laboratory of Gut Immunology, Institute of Food Research, Norwich, United Kingdom (CN); and Department of Evolutionary Biology, University of Siena, Siena, Italy (LE)
Correspondence to: Eugenio Bertelli, Dept. of Pharmacology "Giorgio Segre," Section of Morphology, Via Aldo Moro 4, University of Siena, Siena, Italy. E-mail: bertelli5{at}unisi.it
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: M-cells cytokeratin 20 tonsils MALT antigen transport
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals and Tissues
Five male New Zealand rabbits (1.52 kg bw) (Charles River Italia Laboratories; Rome, Italy) were used for this study. Animals were treated according to current Italian law. Palatine tonsils were sampled from animals previously anesthetized with an IV injection of pentobarbital (30 mg/kg) and sacrificed with an overdose of the same anesthetic. Some animals, anesthetized as above, were tracheotomized and, to expose palatine tonsils, the anterior wall of the pharynx was opened along the median plane. Forty µl of a PBS solution containing 2.5 x 109/ml FITC-conjugated Fluoresbrite polystyrene microparticles (Polysciences; Eppelheim, Germany) were placed with a micropipette into the tonsil crypts for 15 min. At this point, tonsils were quickly removed, briefly rinsed in PBS to wash away the excess microparticles, and processed as all other samples. Specimens were mounted on Tissue-Tek OCT (Sakura Finetek Europe; Zoeterwoude, The Netherlands) and immediately frozen in isopentane prechilled in liquid nitrogen. Samples were stored at 80C or directly transferred to a cryostat (Frigocut N 2800; Reichert-Jung, Wetzlar, Germany) in which consecutive sections (710 µm thick) were cut at 20C and mounted on SuperFrost Plus slides (Menzel-Gläser; Mannheim, Germany). Sections were air-dried, fixed with cold acetone for 10 min at 20C, and stored at 80C. Sections from tonsils incubated with Fluoresbrite microparticles were directly stored without the fixation step with acetone.
Immunohistochemistry, Lectin Histochemistry, and Confocal Microscopy
Sections for single-labeling experiments were placed in PBS for 10 min and then blocked with 1% BSA for 10 min at RT. Then the slides were incubated overnight at 4C with anti-CK20 at a dilution of 1:100 and for 90 min with 1:100 FITC-conjugated anti-mouse IgG antibody. Almost the same protocol was followed with unfixed sections from tonsils incubated with 0.5-µm-diameter fluorescein Fluoresbrite microparticles. The only change was that the secondary antibody employed was a TRITC-conjugated anti-mouse IgG antibody.
Sections for vimentin/CK20 double-labeling experiments were incubated overnight at 4C with goat anti-vimentin PAb at a 1:50 dilution. After washing, sections were incubated for 3 hr at RT with TRITC-conjugated anti-goat IgG at a dilution of 1:100. Slides were then incubated for 18 hr at 4C with MAb anti-CK20 diluted 1:100 and, after washing, sections were finally incubated for 3 hr at RT with FITC-conjugated anti-mouse IgG. Controls for all IHC experiments were performed by omission of the primary antibodies or by their replacement with the appropriate non-immune serum.
Sections for CK20/rabbit IgG double-labeling experiments were incubated for 18 hr at 4C with MAb anti-CK20 diluted 1:100. After washing, sections were incubated for 3 hr at RT with FITC-conjugated anti-mouse IgG. Slides were rinsed and incubated with TRITC-conjugated anti-rabbit IgG.
Some sections were double labeled using UEA-I agglutinin and the MAb anti-CK20. For this purpose, slides were processed as follows. After washing in PBS, sections were blocked with 10% goat serum for 10 min at RT and then incubated overnight at 4C with MAb anti-CK20 (diluted 1:100). After rinsing in PBS, sections were blocked as above and incubated for 4 hr at RT with TRITC-conjugated anti-mouse IgG (diluted 1:50). Slides were thoroughly rinsed and then fixed with a solution of 1% glutaraldehyde and 2% paraformaldehyde in PBS for 10 min at RT. After washing in PBS, sections were dehydrated in increasing concentrations of ethanol and left in 100% ethanol for 10 min at RT. Slides were then rehydrated in a series of decreasing concentrations of ethanol and washed in PBS. Then the sections were blocked with 2.5% BSA for 20 min at RT and incubated overnight at 4C with UEA-I at a concentration of 5 µg/ml in 1% PBS-BSA. Controls were performed by preincubation of UEA-I with fucose or mannose separately.
Unfixed sections (1025-µm thick) of tonsils incubated with fluorescent polystyrene microparticles were washed with PBS, blocked with 10% goat serum for 10 min at RT, and then incubated overnight at 4C with MAb anti-CK20 (diluted 1:100). After rinsing in PBS, sections were incubated for 3 hr at RT with TRITC-conjugated anti-mouse IgG (diluted 1:100) and thoroughly washed.
Fluorescent staining was observed with a Zeiss Axioplan microscope or a TCS 4D Leica laser scanning confocal microscope.
SDS-PAGE and Western Blotting of IF-enriched Cytoskeletal Fractions
To confirm the IHC results, Western blotting analysis with anti-CK20 antibody was carried out on proteins of the IF-enriched cytoskeletal fraction of palatine tonsil epithelium separated by SDS-PAGE. As a positive control, proteins of the IF-enriched cytoskeletal fraction of the intestinal epithelium were separated and blotted in parallel with tonsil samples. IF-enriched cytoskeletal fractions were prepared as previously reported (Achtstaetter et al. 1986), with minor modifications. Briefly, tonsils were removed and their epithelia were isolated by microdissecting away the submucosa, including lymphoid follicles. Then the epithelia were minced and placed in 1 ml of homogenization buffer (96 mM NaCl, 8 mM KH2PO4, 5.6 mM Na2PO4·2H2O, 1.5 mM KCl, 10 mM EDTA, 0.1 mM dithiothretol (DTT), 2.5 mg/ml aprotinin, 100 mM PMSF, pH 6.8). Intestinal epithelium, isolated by gentle scraping of the intestinal surface with a scalpel, was placed in 1 ml of the above homogenization buffer. Both samples (tonsil and intestinal epithelia) were homogenized with Dounce homogenizers and then filtered through four layers of gauze. Three ml of very high salt buffer (2 M KCl, 200 mM NaCl, 10 mM Tris-HCl, 0.1 mM DTT, 2.5 mg/ml aprotinin, 100 mM PMSF, pH 7.4) was added to each homogenate and the obtained suspensions were stirred for 30 min on ice. Samples were next homogenized once more in Dounce homogenizers and centrifuged at 10,000 x g for 20 min at 4C. After removal of the supernatants, each pellet was resuspended in 3 ml of high salt buffer (10 mM Tris-HCl, 140 mM NaCl, 1.5 M KCl, 5 mM EDTA, 0.5% w/v Triton X-100, 2.5 mg/ml A, 100 mM PMSF, pH 7.6) with Dounce homogenizers. Samples were gently agitated for 30 min on ice, centrifuged at 10,000 x g for 20 min at 4C, and resuspended in 3 ml of high salt buffer. These steps were repeated once more but the obtained pellets, instead of being resuspended in high salt buffer, were washed in PBS, pH 7.4, with 0.1 mM DTT. After a centrifugation at 10,000 x g for 20 min at 4C, the supernatants were discarded and the final pellets were stored at 20C until use.
IF-enriched cytoskeletal fractions were separated by electrophoresis through a 10% polyacrylamide gel according to Laemmli (1970) and transferred to nitrocellulose in a Bio-Rad Transblot apparatus (Bio-Rad; Hercules, CA). A 5% (w/v) solution of skimmed milk in TBS was used to quench nonspecific protein adherence of the antibodies. Membranes were incubated overnight at RT with 1:500 anti-CK20 antibody in 5% (w/v) solution of skimmed milk in TBS. Specific bands were detected using an electrochemiluminescence kit (Roche Diagnostics; Milan, Italy).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Specificity of CK20 staining is confirmed by the following elements: control experiments (see Materials and Methods) result in labeling suppression; confocal microscopy demonstrates that CK20 immunoreactivity is localized, as expected, to a complex of cytoskeletal elements that differ from the vimentin network; and Western blotting of the IF-enriched cytoskeletal fraction displays only a single band of the appropriate molecular weight co-migrating with intestinal CK20-immunoreactive proteins.
Vimentin/CK20 double-labeling experiments show that CK20-immunoreactive cells also express vimentin. The converse, however, does not apply. Clearly, many vimentin+/CK20 cells should be classified as cells that massively infiltrate the epithelium (i.e., macrophages and other immunocompetent cells) but, for the time being, we cannot rule out the possibility that some of these cells may correspond to a minor subset of tonsil M-cells. However, because most of these cells are located in the lower layers of the epithelium, an alternative possibility is that they may represent immature M-cells. Unfortunately, the uncertainty of assigning vimentin+/CK20 cells to the population of M-cells or to the infiltrating immunocompetent cells prevents us from producing any quantitative or semiquantitative data that can reliably determine the ratio between vimentin+/CK20 M-cells and the entire M-cell population.
Even though we have identified CK20+ cells provided with fucose residues that label with specific lectins, the use of UEA-I in our hands did not prove useful to confirm that CK20+ cells corresponded to the entire population of M-cells. Indeed, probably due to the peculiar protocol that we had to follow to obtain an acceptable double-staining of both CK20 and fucose residues, the labeling with UEA-I appeared largely unspecific, involving a population of epithelial cells much broader than was expected and that was previously reported (Gebert 1997). Clearly, CKs cannot be a useful tool for M-cell identification when M-cells are embedded in an epithelium with the same profile of CK expression (Kucharzik et al. 1998
). However, when M-cells are located in epithelia with a different CK profile (i.e., composed epithelia), the presence of M-cell-specific CKs can be used as a valuable marker. This is the case for M-cells from palatine tonsils, in which we have demonstrated specific CK20 staining. Even though with the limitations due to the possible existence of a subpopulation of vimentin+/CK20 M cells, in palatine tonsils CK20 can be added to the very short list of documented M-cell markers.
From a functional point of view, we demonstrated that CK20+ M-cells have the ability to form wide pockets harboring lymphoid cells and to take up fluorescent polystyrene microparticles from the pharyngeal lumen. These features are considered distinctive of functionally competent M-cells (Bye et al. 1984; Kernéis et al. 1997
; Gebert et al. 1999
). Indeed, not all tonsil CK20+ M-cells display the characteristic pockets, this aspect being mainly confined to the M-cells located close to the pharyngeal lumen. This observation could be in accordance with a progressive maturation of M-cells moving from the basal layers of the tonsil epithelium towards the surface, somewhat resembling what occurs elsewhere. In the intestine, for example, M-cells originate from dome-surrounding intestinal crypts and, during their migration along the flanks of the dome, progressively acquire morphological and functional maturity (Bye et al. 1984
; Gebert et al. 1999
).
We have reported the presence of IgG+ cells in M-cell pockets. This observation confirms that the cells harbored within CK20+ M-cell hollows are actually immunocompetent cells, corroborating the acquisition by M-cells of a functionally mature phenotype. However, several cells contained in M-cell pockets do not stain with anti-IgG antibodies. This was expected because of the heterogeneous population of lymphoid cells infiltrating M-cells. Indeed, in this population, which accounts mostly for T and B memory cells (Farstad et al. 1994), IgG+ B-cells correspond only to a subset of cells, even though they are well represented at least in human tonsils (Liu et al. 1995
).
A very important function of M-cells, and perhaps the most important, is the ability to take up a large set of microorganisms and even inert particles that are steered into the pockets where they meet immunocompetent cells (Jepson and Clark 1998; Meynell et al. 1999
; Nicoletti 2000
). Exactly what happens in the pockets is not yet clear, but it is believed that important interactions may occur between antigens and memory B-cells that might substantially contribute to the initiation of the immune response (Liu et al. 1995
; Yamanaka et al. 2001
). Therefore, the demonstration that tonsil CK20+ M-cells are able to take up fluorescent microparticles is particularly important to demonstrate their full operational capabilities. The kinetics of microparticle uptake appear to be extremely rapid because even after 15 min some of the particles can be found in M-cells situated even in middle layers of the epithelium. However, probably owing to the short time of exposure to the fluorescent beads, the number of beads that could be spotted in the middle layers of epithelium was low. Therefore, to evaluate the actual impact of this phenomenon on the generation of the immune response, longer incubation time points must be investigated. The ability of M-cells to sample material from the pharyngeal lumen was first observed by Gebert (1995)
. In that case, horseradish peroxidase was used as tracer. To our knowledge, this is the first time that active transport of particulate antigen has been documented. The ability of tonsil-associated M-cells to carry out antigen-sampling activity might have important implications for the development of new strategies for the delivery of certain types of vaccine. It has been recently shown that significant levels of specific IgA antibody to Streptococcus mutans were detected in the saliva of human volunteers after tonsillar administration of a glucosyltransferase-enriched preparation (Childers et al. 2002
). In addition, it is noteworthy that high levels of a specific salivary antibody to other pathogens, such as S. sobrinus, can be induced via tonsillar immunization and that the immunological function of palatine tonsils can be modulated by the use of regulatory cytokines, such as IL-1 (Kokuryo et al. 2002
). These findings point to tonsils as a possible site for antigen delivery to obtain a local immune response against oral pathogens. In this context, our observation that tonsil-associated M-cells can take up particles opens the way to the development of a particle-based vaccine that can be delivered in a simple and effective way.
![]() |
Acknowledgments |
---|
We are indebted to Prof. F. Rosati (Dept of Evolutionary Biology, University of Siena, Siena) for supplying us with UEA-I lectin.
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Achtstaetter T, Hatzfeld M, Quinlan RA, Parmelee DC, Franke WW (1986) Separation of cytokeratin polypeptides by gel electrophoretic and chromatographic techniques and their identification by immunoblotting. Methods Enzymol 134:355371[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 180:326332[CrossRef][Medline]
Brandtzaeg P (1996) History of oral tolerance and mucosal immunity. Ann NY Acad Sci. 778:127[Abstract]
Brandtzaeg P, Baekkevold ES, Farstad IN, Jahnsen FL, Johansen F, Nilsen EM, Yamanaka T (1999) Regional specialization in the mucosal immune system: what happens in the microcompartments? Immunol Today 20:141151[CrossRef][Medline]
Bye WA, Allan CH, Trier JS (1984) Structure, distribution, and origin of M cells in Peyer's patches of mouse ileum. Gastroenterology 86:789801[Medline]
Childers NK, Tong G, Li F, Desanayaka AP, Kirk K, Michalek SM (2002) Humans immunized with Streptococcus mutans antigens by mucosal routes. J Dent Res 81:4852
Farstad IN, Halstensen TS, Fausa O, Brandtzaeg P (1994) Heterogeneity of M cell-associated B and T cells in human Peyer's patches. Immunology 83:457464[Medline]
Gebert A (1995) Identification of M-cells in the rabbit tonsil by vimentin immunohistochemistry and in vivo protein transport. Histochem Cell Biol 104:211220[Medline]
Gebert A (1996) M-cells in the rabbit tonsil exhibit distinctive glycoconjugates in their apical membranes. J Histochem Cytochem 44:10331042
Gebert A (1997) M cells in the rabbit palatine tonsil: the distribution, spatial arrangement and membrane subdomains as defined by confocal lectin histochemistry. Anat Embryol 195:353358[CrossRef][Medline]
Gebert A, Fassbender S, Werner K, Weissferdt A (1999) The development of M cells in Peyer's patches is restricted to specialized dome-associated crypts. Am J Pathol. 154:15731582
Gebert A, Rothkotter HJ, Pabst R (1994) Cytokeratin 18 is an M-cell marker in porcine Peyer's patches. Cell Tissue Res 276:213221[CrossRef][Medline]
Gebert A, Rothkotter HJ, Pabst R (1996) M cells in Peyer's patches of the intestine. Int Rev Cytol 167:91159[Medline]
Hartig R, Huang Y, Janetzko A, Shoeman R, Grub S, Traub P (1997) Binding of fluorescence- and gold-labeled oligodeoxyribonucleotides to cytoplasmic intermediate filaments in epithelial and fibroblast cells. Exp Cell Res 233:169197[CrossRef][Medline]
Jepson MA, Clark MA (1998) Studying M cells and their role in infection. Trends Microbiol 6:359365[CrossRef][Medline]
Jepson MA, Mason CM, Bennett MK, Simmons NL, Hirst BH (1992) Co-expression of vimentin and cytokeratins in M cells of rabbit intestinal lymphoid follicle-associated epithelium. Histochem J 24:3339[Medline]
Kernéis S, Bogdanova A, Kraehenbuhl J-P, Pringault E (1997) Conversion by Peyer's patch lymphocytes of human enterocytes into M cells that transport bacteria. Science. 277:949951
Kokuryo S, Inoue H, Fukuizumi T, Tsujisawa T, Tominaga K, Fukuda J (2002) Evaluation of IL-1 as a mucosal adjuvant in immunization with S. sobrinus cells by tonsilar application in rabbits. Oral Microbiol Immunol 17:163171[CrossRef][Medline]
Kucharzik T, Lugering N, Schmid KW, Schmidt MA, Stoll R, Domschke W (1998) Human intestinal M cells exhibit enterocyte-like intermediate filaments. Gut 42:5462
Laemmli UK (1970) Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature. 227:680685[Medline]
Liu Y-J, Barthélémy C, de Bouteiller O, Arpin C, Durand I, Banchereau J (1995) Memory B cells from human tonsils colonize mucosal epithelium and directly present antigen to T cells by rapid up-regulation of B71 and B72. Immunity 2:239248[Medline]
Meynell HM, Thomas NW, James PS, Holland J, Taussig MJ, Nicoletti C (1999) Up-regulation of microsphere transport across the follicle-associated epithelium of Peyer's patch by exposure to Streptococcus pneumoniae R36a. FASEB J 13:611619
Moll R, Franke WW, Schiller DL, Geiger B, Krepler R (1982) The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31:1124[Medline]
Moll R, Löwe A, Laufer J (1992) Cytokeratin 20 in human carcinomas. A new histodiagnostic marker detected by monocolonal antibodies. Am J Pathol. 140:427447[Abstract]
Moll R, Schiller DL, Franke WW (1990) Identification of protein IT of the intestinal cytoskeleton as a novel type I cytokeratin with unusual properties and expression patterns. J Cell Biol 111:567580[Abstract]
Nicoletti C (2000) Unsolved mysteries of intestinal M cells. Gut 47:735739
Rautenberg K, Cichon C, Heyer G, Demel M, Schmidt MA (1996) Immunocytochemical characterization of the follicle-associated epithelium of Peyer's patches: anti-cytokeratin 8 antibody (clone 4.1.18) as a molecular marker for rat M cells. Eur J Cell Biol 71:363370[Medline]
Regoli M, Bertelli E, Borghesi C, Nicoletti C (1995a) Three-dimensional (3D-) reconstruction of M cells in rabbit Peyer's patches: definition of the intraepithelial compartment of the follicle-associated epithelium. Anat Rec 243:1926[Medline]
Regoli M, Borghesi C, Bertelli E, Nicoletti C (1994) A morphological study of the lymphocyte traffic in Peyer's patches after an in vivo antigenic stimulation. Anat Rec 239:4754[Medline]
Regoli M, Borghesi C, Bertelli E, Nicoletti C (1995b) Uptake of a gram-positive bacterium (Streptococcus pneumoniae R36a) by the M cells of rabbit Peyer's patches. Anat Anz 177:119124[Medline]
Shalaby WS (1995) Development of oral vaccines to stimulate mucosal and systemic immunity: barriers and novel strategies. Clin Immunol Immunopathol 74:127134[CrossRef][Medline]
Yamanaka T, Straumfors A, Morton H, Fausa O, Brandtzaeg P, Farstad I (2001) M cells pockets of human Peyer's patches are specialized extension of germinal centers. Eur J Immunol 31:107117[CrossRef][Medline]