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Intestinal M-cells are specialized epithelial cells located in the domes of the gut-associated lymphoid tissues, which transport antigens from the lumen to the underlying lymphoid tissue, thereby initiating immune reactions. It is assumed that M-cells arise from stem cells in the crypts, from which they migrate to the top of the domes. To study the differentiation pathway of M-cells, we used the rabbit cecal lymphoid patch in which the M-cells express high levels of α1–2-linked fucose and N-acetyl-galactosamine residues in their apical membrane. Dome areas were labeled with fluorescein- and rhodamine-conjugated lectins specific for α1–2-linked fucose and N-acetyl-galactosamine in vivo and in vitro, and were observed with confocal laser scanning microscopy. Ultrathin sections were double labeled with lectin–gold conjugates and the labeling density was quantified by computer-based image analysis. All cecal patch M-cells expressed α1–2-linked fucose and N-acetyl-galactosamine, but the amount of the two saccharides varied considerably depending on the position of the M-cells at the base, flank, or top of the dome. In eight of 18 rabbits studied, radial strips of M-cells with common glycosylation patterns were observed, each strip associated with an individual crypt. Confocal microscopy revealed that lectin-labeled M-cells were not restricted to the dome epithelium but were also detected in the upper third of crypts surrounding the domes. The results show that M-cells are heterogeneous concerning the glycosylation pattern of membrane glycoconjugates. This pattern is modified as the M-cells differentiate and migrate from the base to the top of the dome. Radial strips of M-cells with a common proclivity of glycoconjugate expression suggest that those M-cells that derive from the same crypt have a clonal origin. The presence of (pre-) M-cells in the crypts surrounding the domes indicates that M-cells derive directly from undifferentiated crypt cells and do not develop from differentiated enterocytes.

Glycoconjugate Expression Defines the Origin and Differentiation Pathway of Intestinal M-cells