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Intestinal villous M cells: an antigen entry site in the mucosal epithelium - PubMed

  • ️Thu Jan 01 2004

Intestinal villous M cells: an antigen entry site in the mucosal epithelium

Myoung Ho Jang et al. Proc Natl Acad Sci U S A. 2004.

Abstract

M cells located in the follicle-associated epithelium of Peyer's patches (PP) are shown to be the principal sites for the sampling of gut luminal antigens. Thus, PP have long been considered the gatekeepers of the mucosal immune system. Here, we report a distinct gateway for the uptake of gut bacteria: clusters of non-follicle-associated epithelium-associated Ulex europaeus agglutinin (UEA)-1(+) cells, which we have designated intestinal villous M cells. Interestingly, villous M cells are developed in various PP [or gut-associated lymphoid tissue (GALT)]-null mice, such as in utero lymphotoxin beta receptor (LTbetaR)-Ig-treated, lymphotoxin alpha (LTalpha)(-/-), tumor necrosis factor/LTalpha(-/-), and inhibition of differentiation 2 (Id2)(-/-) mice. Intestinal villous M cells have been observed to take up GFP-expressing Salmonella, Yersinia, and Escherichia coli-expressing invasin, as well as gut bacterial antigen for subsequent induction of antigen-specific immune responses. Thus, the identified villous M cells could be an alternative and PP-independent gateway for the induction of antigen-specific immune responses by means of the mucosal compartment.

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Figures

Fig. 1.
Fig. 1.

Confocal view of UEA-1+ cells in villous epithelium (a–i) and FAE of PP (j–m) isolated from naive BALB/c mice. M cell- and columnar epithelial cell-specific UEA-1-TRITC and WGA-FITC, respectively, were applied to the whole-mount preparation of the small intestine (a–f, j, and k). M cells were stained by UEA-1 (red, arrow), enterocytes by WGA (green), and goblet cells by UEA-1 and WGA (yellow, arrowhead). Villous M cells were found as two different distribution forms, dense (a and b) and diffuse (d and e) types. In contrast to the epithelial and goblet cells, M cells in the villous epithelium were completely negative to the WGA staining (c and f). Frozen sections were prepared and stained with UEA-1-TRITC alone (h and l) or with UEA-1-TRITC and B220 mAb-FITC (i and m) and the M cells were shown to have a pocket membrane and pocket lymphocytes (arrow) whereas the goblet cells do not (arrowhead). M cells were doubly negative cells for alkaline phosphatase activity demonstrated by red/pink color substrate, and alcian blue staining (white; g). The scale bar for a, d, and j is 50 μm; for b, c, e, f, h, and l is 20 μm; and for g, i, k, and m is 10 μm.

Fig. 2.
Fig. 2.

The presence of villous M cells in PP-null mice, such as in utero LTβR-Ig-treated C57BL/6 mice (Upper Left), LTα-/- mice of C57BL/6 background (Upper Right), TNF/LTα-/- mice of 129 × C57BL/6 background (Lower Left), and Id2-/- mice of 129×Sv background (Lower Right). The scale bar for all pictures is 10 μm. The whole-mount preparations of small intestine were stained with FITC-WGA and TRITC-UEA-1.

Fig. 3.
Fig. 3.

(A) Immunohistochemistry for antigen uptake by UEA-1+ villous M cells. Each panel shows histological features for sampling of GFP-expressing Salmonella (a, b and e, f) and Yersinia (c, d and g, h)byUEA+ cells in the small intestine of wild-type (a–d) and PP-null TNF/LTα-/- mice (e–j). Whole mount (a–d and e–h) and frozen sections of small intestine after exposure of GFP-expressing Salmonella were prepared and stained with UEA-1-TRITC (i and j). The scale bars are as follows: for a, c, and g,50 μm; for b, e, and i,20 μm; and for d, f, h, and j,10 μm. (B) Localization of GFP-expressing Salmonella in the intracellular region of UEA-1+ villous M cells. An ileal loop infection experiment using rSalmonella-GFP was performed for 30 min, and whole-mount tissues and UEA-1+ IEC cells were analyzed by sequential confocal planar microscopy. Sequential Z plans of whole-mount staining revealed the localization of rSalmonella-GFP in the intracellular region of villous UEA-1+ cells (a, b, and c). Further, the cytospin analysis revealed that rSalmonella-GFP also existed in the intracellular region of villous UEA-1+ cells (d). (C) Antigen uptake by UEA-1+ villous M cells. Cells (5 × 108) of GFP-expressing S. typhimurium PhoPc (Salmonella), Y. pseudotuberculosis (Yersinia), E. coli-invasin (E. coli-Inv), and E.-coli were administered into a 10-cm loop of the small intestine of naive wild-type mice (Top and Middle) or TNF/LTα-/- mice (Bottom). After 10 min of incubation in situ, IECs were isolated from PP and villous epithelium. After being fixed with 4% paraformaldehyde, IECs were stained by UEA-1-TRITC, and uptake efficiency was analyzed by fluorescence-activated cell sorter (FACS). Data demonstrate the frequency of GFP+ cells in the UEA+ (filled bar) and UEA- (open bar) cells isolated from PP and villous epithelium (VE). The results represent the mean values ± SD from three separate experiments (three mice per group). *, P < 0.05 vs. the UEA- IEC group.

Fig. 4.
Fig. 4.

Scanning and transmission electron microscopy of M cells in villous epithelium and FAE. Scanning electronic microscopy demonstrates that the M cells (arrow) in villous epithelium (a–c) and PP (d and e) are distinguished from enterocytes and goblet cells (arrowhead) by their relatively depressed and dark brush border. A transmission electron microscopy view of villous M cells shows short stub-like microvilli (f, g, and h) and the presence of infiltrating mononuclear cells in the pocket of villous M-cells (h; arrowhead). (i–l) The presence of villous M cells and the uptake of bacteria in the villous epithelium (arrowhead) after intestinal exposure of Salmonella (see Fig. 3 legend) in PP-deficient TNF/LTα-/- mice. The scale bars are as follows: for a, d, and i,50 μm; for b, e, f, and j,5 μm; for g and h, 1.0 μm; and for c, k, and l, 0.5 μm.

Fig. 5.
Fig. 5.

Induction of Ag-specific immune responses in PP-deficient mice. Shown are PP-deficient (TNF/LTα-/-) and wild-type mice, which were orally immunized with rSalmonella-ToxC (filled bar) or rSalmonella alone (open bar). Serum samples were obtained 21 days after oral immunization for the assessment of tetanus toxoid (TT)-specific antibody responses by ELISA. The results represent the mean values ± SD from three separate experiments (three mice per group). There is no statistically significant difference between TNF/LTα-/- and wild-type mice analyzed by unpaired Mann–Whitney U test.

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References

    1. Madara, J. L. (1998) Annu. Rev. Physiol. 60, 143-159. - PubMed
    1. Frey, A., Giannasca, K. T., Weltzin, R., Giannasca, P. J., Reggio, H., Lencer, W. I. & Neutra, M. R. (1996) J. Exp. Med. 184, 1045-1059. - PMC - PubMed
    1. Kraehenbuhl, J. P. & Neutra, M. R. (2000) Annu. Rev. Cell Dev. Biol. 16, 301-332. - PubMed
    1. Neutra, M. R., Frey, A. & Kraehenbuhl, J. P. (1996) Cell 86, 345-348. - PubMed
    1. Neutra, M. R., Mantis, N. J., Frey, A. & Giannasca, P. J. (1999) Semin. Immunol. 11, 171-181. - PubMed

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