54 Gut 1998;42:54–62 Human intestinal M cells exhibit enterocyte-like intermediate filaments Gut: first published as 10.1136/gut.42.1.54 on 1 January 1998. Downloaded from T Kucharzik, N Lügering, K W Schmid, M A Schmidt, R Stoll, W Domschke Abstract antigens.5612 As M cells have a high capacity Background—The derivation and ul- for transcytosis of a wide range of microorgan- trastructural composition of M cells cov- isms and macromolecules, they are believed to ering the lymphoid follicles of Peyer’s act as an antigen sampling system.34 M cells patches is still unknown. Results from dif- can be characterised electron microscopically ferent animal models have shown that by their characteristic morphology, notably there are species specific diVerences in their atypical microvilli and the presence of an the composition of intermediate filaments invagination of the basolateral membrane har- between M cells and neighbouring entero- bouring leucocytes.47Since the first report on cytes. Little is known, however, about M cells in the human ileum1 and appendix,2 intermediate filaments of human M cells. numerous studies have been done to elucidate Aims—To compare components of the their morphology and to investigate functional cytoskeleton of human M cells with those aspects of M cells in diVerent animal species of adjacent absorptive enterocytes. (for review see Trier7). Little is known, Methods—The expression and localisation however, about the function and morphology of diVerent cytokeratins, vimentin, and of M cells in humans. Concerning their desmin in M cells was determined on folli- morphology, human M cells reveal an anasto- cle associated epithelia of human appen- mosing, short, ridgelike network of folds and dix using immunohistochemistry and occasionally short microvilli in strong contrast immunogold electron microscopy. to the regular appearance of microvilli of adja- Results—Cytokeratins specific for human cent absorptive cells.113 intestinal epithelial cells such as cytok- DiVerent enzymatic markers for the recogni- eratins 8, 18, 19, and 20 were expressed in tion of M cells have been established, such as both absorptive enterocytes and M cells alkaline phosphatase in the brush border.14 with no diVerences in intensity and cellu- Other investigators have determined immuno- lar distribution between both cell types. histochemical markers for M cells, but all are http://gut.bmj.com/ Vimentin and desmin, tissue specific restricted to single animal species. Gebert et al markers of either mesenchymal or myo- have shown that cytokeratin 18 (CK-18) is a genic cells, as well as other cytokeratins sensitive marker for porcine M cells15 while were not detectable in enterocytes or M vimentin is selectively expressed in rabbit M cells. cells.16 M cells in rats are detected by Conclusion—This is the first study on the monoclonal antibodies (clone 4.1.18) raised structure of intermediate filaments in 17 against cytokeratin 8. Allthough all markers on September 28, 2021 by guest. Protected copyright. human intestinal M cells. Our results identified thus far seem to be species specific, show that in contrast to several animal these studies did show that M cells of various models, human M cells apparently do not animals diVer in their composition of cytoskel- diVer from adjacent enterocytes in the eton proteins from adjacent enterocytes. These Department of composition of their intermediate fila- observations suggest that diVerent intermedi- Medicine B ment cytoskeleton. The presence of ente- ate filaments serve variable functions15 16 which T Kucharzik rocyte like cytokeratins and the absence of N Lügering could explain the diVerent properties of M cells W Domschke other cytokeratins as well as of vimentin compared with enterocytes. R Stoll and desmin supports the hypothesis of an Within the system of intermediate filaments, epithelial origin of human intestinal M the subgroup of cytokeratins might serve as a Department of cells and suggests that M cells may derive system of especially potent diVerentiation Pathology from diVerentiated enterocytes. K W Schmid markers, because the diverse patterns of cyto- (Gut 1998;42:54–62) keratin polypeptides are correlated with diVer- Centre for Molecular 18 Keywords: human intestinal M cells; appendix; ent pathways of epithelial diVerentiation. To Biology of cytokeratin; intermediate filaments; follicle associated our knowledge, no data are available concern- Inflammation, epithelium ing the components of the cytoskeleton of University of Münster, Münster, Germany human M cells. There is controversy as to M A Schmidt whether M cells are of epithelial–intestinal or M cells are known as specialised antigen mesenchymal origin. Whether M cells derive Correspondence to: sampling cells that are interspersed between from intestinal crypt cells or from fully Dr T Kucharzik, di erentiated enterocytes and whether this Department of Medicine B, epithelial cells of the follicle associated epithe- V University of Münster, lia (FAE) of the gastrointestinal and respiratory stage in diVerentiation is induced by lymphoid Albert-Schweitzer-Str. 33, tracts.1–7 They diVer morphologically and tissues is also not known. D-48129 Münster, Germany. enzymatically from the adjacent enterocytes, As the properties of specialised M cells in the Accepted for publication show preferential aYnity for certain FAE are diVerent from those of common ente- 16 July 1997 microorganisms,8–11 and take up and transport rocytes, the aim of our study was to investigate Intermediate filaments in human intestinal M cells 55 Table 1 Antibodies Antibody Clone Supplier Dilution Gut: first published as 10.1136/gut.42.1.54 on 1 January 1998. Downloaded from Anticytokeratin 1/2, 10/11 AE2 Progen 1/50 Anticytokeratin 3 AE5 Progen 1/50 Anticytokeratin 4 215B8 Boehringer 1/200 Anticytokeratin 5/6 D5/16B4 Boehringer 1/1000 Anticytokeratin 6 Ks 6KA12 Progen 5 µg/ml Anticytokeratin 7 Ks 7.18 Progen 5 µg/ml Anticytokeratin 8 4.1.18 Boehringer 1/40 Ks 8.7 Progen 5 µg/ml Ks 8.10 Progen 5 µg/ml Anticytokeratin 10/11, 1/2 K8.60 Progen 1/50 Anticytokeratin 13 KS13.1 Progen 1/20 Anticytokeratin 14 LL002 Medac 1/20 Anticytokeratin 16 LL025 Medac 1/40 Anticytokeratin 17 Ks 17.E3 Progen 5 µg/ml Anticytokeratin 18 CY-90 Sigma IH 1/800 EM 1/1000 Ks 18.04 Progen 5 µg/ml Anticytokeratin 19 A53-B/A2 Sigma IH 1/10 EM 1/2 Anticytokeratin 20 Ks 20.10 Progen 1/20 Ks 20.8 Progen 5 µg/ml Anticytokeratin8+18 CAM5.2 Becton-Dickinson 1/2 Antivimentin V9 Immunotech 1/50 Antidesmin DE-R-11 Medac 1/300 IH, immunohistochemistry; EM, immunoelectron microscopy. whether this functional diVerence might be reflected in an unusual range of cytoskeletal proteins. We therefore examined diVerent components of the cytoskeleton in M cells of the human appendix in comparison with adja- cent absorptive enterocytes by immunohisto- chemistry and immunogold electron micros- copy. We determined the expression of tissue specific cytoskeletal proteins such as diVerent cytokeratins, vimentin, an intermediate fila- Figure 2 Photomicrograph of frozen sections of FAE from human appendix. The AP reaction is prominent over the ment protein present in mesenchymal cells, FAE of human appendix but is intermittently broken by and desmin, a marker for myogenic cells in gaps (arrows) (a). Because of the lack of staining for AP, human intestinal M cells. these cells are characterised as M cells. Labelling of the same area with a monoclonal antibody against CK-20 http://gut.bmj.com/ (green fluorescence) shows that there is no diVerence in the staining pattern of M cells and adjacent enterocytes (b). Materials and methods The arrowhead in (a) indicates a goblet cell. Original TISSUE magnification ×400. F,follicle. To examine human intestinal M cells, we used females, nine males; mean age 52 years, range surgical specimens of human appendix. Ap- 27–81) undergoing cystectomy because of car- pendices were obtained from 13 patients (four cinoma of the urinary bladder. All patients gave written informed consent to participate in the on September 28, 2021 by guest. Protected copyright. study. The study protocol was approved by the Human Studies Committee of the University of Münster. Macroscopically and microscopi- cally the appendix was not inflamed in any of the patients investigated. Tissue was taken immediately after resection of the appendix and rinsed with cold Ringer’s solution. Small pieces were fixed overnight at 4°C in 2.5% glutaraldehyde (Serva, Heidel- berg, Germany). Samples that were taken for cryosections were embedded in OCT (Miles, Elkhart, Indiana, USA), snap frozen, and stored in liquid nitrogen. Cryosections were serially cut at –25°C with a Jung Frigocut N2800 (Leica, Nussloch, Germany) (5 µm), fixed in acetone (–20°C) for 10 minutes, and stored at –70°C until use. For paraYn wax sec- tions, the material was fixed in 10% formalde- hyde for 20 hours at room temperature and embedded in paraYn wax. Sections of 2 µm were dewaxed, rehydrated in a series of alcohol, Figure 1 Typical M cell overlying a lymphoid follicle of human appendix embedded in Epon. The surface of the M and transferred to phosphate buVered saline cell (M) shows microfolds and fewer, shorter microvilli than (PBS) (pH 7.2). For morphological evaluation, the adjacent absorptive enterocytes (E). A mononuclear cell the material was stained with haematoxylin in the central hollow (MN) is surrounded by M cell cytoplasm. The cell nucleus (N) is located at the base of the (Merck, Darmstadt, Germany). Sections with M cell (Bar = 0.5 µm). activated lymphoid follicles and corresponding 56 Kucharzik, Lügering, Schmid, et al of alcohol and transferred to PBS (pH 7.2). The sections were blocked with 50% goat serum containing 0.1% bovine serum albumin (BSA) in PBS for one hour and then incubated Gut: first published as 10.1136/gut.42.1.54 on 1 January 1998.