Cyclophilin A is a new M cell marker of bovine intestinal epithelium.

Microfold (M) cells in the follicle-associated epithelium (FAE) of Peyer's patches contribute to the mucosal immune response by the transcytosis of microorganisms. The mechanism by which M cells take up microorganisms, and the functional proteins by which they do this, are not clear. In order to explore one such protein, we developed a 2H5-F3 monoclonal antibody (2H5-F3 mAb) through its binding to bovine M cells, and identified the antibody reactive molecule as cyclophilin A (Cyp-A). The localization patterns of Cyp-A were very similar to the localization pattern of cytokeratin (CK) 18-positive M cells. Cyp-A was identified at the luminal surface of CK18-positive M cells in bovine jejunal and ileal FAE. The membranous localization of Cyp-A in the bovine intestinal cell line (BIE cells) increased as cells differentiated toward M cells, as determined by flow cytometry analysis. Additionally, BIE cells released Cyp-A to the extracellular space and the differentiation of BIE cells to M cells increased the secretion of Cyp-A, as determined by western blotting. Accordingly, Cyp-A may be localized in M cells in the small intestinal epithelium of cattle. The rise of the membranous localization and secretion of Cyp-A by differentiation toward M cells indicates that Cyp-A has an important role in the function of M cells. While Cyp-A of the M cell membrane may contribute to the uptake of viruses with peptidyl-prolyl cis-trans isomerase activity, in the extracellular space Cyp-A may work as a chemokine and contribute to the distribution of immuno-competent cells.

Effect of myostatin on chemokine expression in regenerating skeletal muscle cells.

Transforming growth factor-ß (TGF-ß) is implicated in the regulatory expression of chemokines that control multiple steps in myogenesis. However, it remains to be established whether myostatin, a member of the TGF-ß superfamily, affects chemokine expression in skeletal muscle. We investigated the effects of myostatin on the expression of mRNAs and proteins for 4 chemokines (CXCL1, CXCL2, CXCL6, CCL2) in intact and regenerating musculus longissimus thoracis from normal-muscled (NM) and double-muscled (DM) cattle. These chemokines were expressed in regenerating muscle, and their expression was always lower in DM than in NM cattle. Immunohistochemistry revealed that CXCL1 and CXCL6 were detected in the regenerating areas of myoblasts and myotubes in both NM and DM cattle. In cultures of myoblasts isolated from the regenerating muscles, significantly less CXCL1, CXCL2 and CCL2 mRNA was expressed in DM myoblasts than in NM myoblasts during the proliferating stage (P-stage). The expression of CXCL1, CXCL2 and CCL2 mRNAs in NM myoblasts and CXCL1, CXCL2 and CXCL6 mRNAs in DM myoblasts decreased upon switching from P-stage to fusion stage (F-stage). Also, the expression of CXCL1, CXCL2 and CXCL6 mRNAs was significantly lower in DM than in NM myoblasts during the F-stage. The addition of 100 ng/ml myostatin during the F-stage attenuated the expression of CXCL1 and CXCL2 mRNAs and augmented that of CCL2. These results show for the first time that myostatin regulates the differential expression of chemokines in skeletal muscle cells.

Cytokeratin 18 is a specific marker of bovine intestinal M cell.

Microfold (M) cells in the follicle-associated epithelium (FAE) of Peyer's patches have an important role in mucosal immune responses. A primary difficulty for investigations of bovine M cells is the lack of a specific molecular marker. To identify such a marker, we investigated the expression of several kinds of intermediate filament proteins using calf Peyer's patches. The expression patterns of cytokeratin (CK) 18 in jejunal and ileal FAE were very similar to the localization pattern of M cells recognized by scanning electron microscopy. Mirror sections revealed that jejunal CK18-positive cells had irregular and sparse microvilli, as well as pocket-like structures containing lymphocytes, typical morphological characteristic of M cells. However, CK18-negative cells had regular and dense microvilli on their surface, typical of the morphology of enterocytes. In contrast, CK20 immunoreactivity was detected in almost all villous epithelial cells and CK18-negative cells in the FAE. CK18-positive proliferating transit-amplifying cells in the crypt exchanged CK18 for CK20 above the mouth of the crypt and after moving to the villi; however, CK18-positive M cells in the crypt continued their expression of CK18 during movement to the FAE region. Terminal deoxynucleotidyl-transferase-mediated deoxyuridine-triphosphate-biotin nick-end labeling-positive apoptotic cells were specifically detected at the apical region of villi and FAE in the jejunum and ileum, and all were also stained for CK20. These data indicate that CK18 may be a molecular marker for bovine M cells in FAE and that M cells may transdifferentiate to CK20-positive enterocytes and die by apoptosis in the apex of the FAE.