Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Apr;23(4):261-9.
doi: 10.1093/intimm/dxq478. Epub 2011 Mar 21.

CCR6hiCD11c(int) B cells promote M-cell differentiation in Peyer's patch

Masashi Ebisawa  1 , Koji Hase, Daisuke Takahashi, Hiroshi Kitamura, Kathryn A Knoop, Ifor R Williams, Hiroshi Ohno
Affiliations

CCR6hiCD11c(int) B cells promote M-cell differentiation in Peyer's patch

Masashi Ebisawa et al. Int Immunol. 2011 Apr.

Abstract

M cells are responsible for uptake of mucosal antigens in Peyer's patches (PPs). Differentiation of M cells is thought to be induced by interactions between follicle-associated epithelium and PP cells; however, it remains elusive what types of immune cells function as M-cell inducers. Here, we attempted to identify the cells that serve as an M-cell inducer in PP. We found that a unique B-cell subset characterized by CCR6(hi)CD11c(int) resided in the subepithelial dome (SED) in mouse PP. CCR6(hi)CD11c(int) B cells showed chemotactic migration in response to CCL20. Furthermore, this unique B-cell subset substantially decreased in PP of CCR6-deficient mice, indicating that the SED localization of CCR6(hi)CD11c(int) B cells is most likely regulated by the CCL20-CCR6 system. Concomitantly, CCR6 deficiency caused remarkable decrement of M cells. Moreover, adoptive transfer of CCR6(hi)CD11c(int) B cells from wild-type mice restored the M-cell decrement in CCR6-deficient mice. Collectively, the spatial regulation of CCR6(hi)CD11c(int) B cells via the CCL20-CCR6 system may play a vital role in M-cell differentiation in mice.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
An important role of the CCL20–CCR6 system in differentiation of M cells. (A) Sections of the mouse small intestine including PP were stained with anti-CCL20 antibody (green) and DAPI (blue) for a nuclei counter stain. Lower pictures are higher magnification of the FAE dome region indicated by a square in upper pictures. Scale bars represent 100 μm. (B) Whole-mount specimens of PP from WT and CCR6-deficient mice were stained with anti-GP2 mAb and phalloidin to visualize M cells and F-actin, respectively. Scale bars represent 40 μm. (C) Number of M cells in WT and CCR6-deficient mice were quantified. The number was normalized to the area of observation field. Values are mean ± SD (n = 5). *P < 0.05 (Student's t-test). Results are representative of three separate experiments.
Fig. 2.
Fig. 2.
Identification of CCR6hiCD11cint B cells. (A) The dot plot shows expression of CD19 and CD11c of PP cells. The histograms show CCR6 expression of the cells in R1–R4 gates. Filled and blank areas show staining with anti-CCR6 mAb and isotype control, respectively. A representative result of three independent experiments is shown. (B) PP cells from WT and CCR6-deficient mice were monitored for surface expression of CD19 and CD11c. Percentage of CD11cintCD19+ cells in total cell population is shown. The result is a representative of four independent experiments.
Fig. 3.
Fig. 3.
Expression profiles of surface marker on CD11cintCD19+ cells. (A) PP cells from WT mice were stained with indicated specific antibodies (gray area) or isotype controls (blank area). The cells in the CD11cintCD19+ gate were shown. Three independent experiments gave similar results and the representative data are shown. (B) PCR was performed with cDNAs derived from CD11cintCD19+, CD11c−CD19+ or CD11c+CD19− cell fraction and primer sets specific for the rearranged V region of Igμ (30 cycles) or glyceraldehyde 3-phosphate dehydrogenase (25 cycles) as an internal control.
Fig. 4.
Fig. 4.
Detection of CD11cint B cells in PP, MLN and the spleen (SPL). (A) Cells from PP, MLN and the SPL of WT mice were analyzed for surface expression of CD11c and CD19. (B) CD11cint B cells were gated and analyzed for surface expression of CCR6. Filled and blank histograms show staining with anti-CCR6 mAb and isotype control, respectively. Three independent experiments gave similar results and the representative data are shown.
Fig. 5.
Fig. 5.
Distribution of CD11cint B cells in PP. (A) Chemotactic activity of CD11cint B cells was examined with various concentrations of CCL20. Values are mean ± SD (n = 3). *P < 0.05, **P < 0.01 (one-way analysis of variance followed by Tukey's post hoc test). A representative result of three independent experiments is shown. (B) CD11cint B cells prepared from PP of WT mice were labeled with PKH26 red fluorescent dye and intravenously injected into recipient WT mice. PP tissue sections were prepared 48 h after the injection. The sections were counterstained with DAPI. Scale bar represents 100 μm. (C) Whole-mount specimens of PP were stained with an anti-CD11c (green) and CD19 (red) mAbs and phalloidin (blue). Arrows and Arrowheads represent CD11cint B cells. The X–Z image of FAE at the position indicated by a solid line in the X–Y image is shown. Scale bars represent 40 μm.
Fig. 6.
Fig. 6.
Involvement of CD11cint B cells in differentiation of M cells. (A) CD11cint and CD11c− B cells from WT mice were adoptively transferred to CCR6-deficient mice. Three days later, whole-mount specimens of PP were stained with an anti-GP2 (green) mAb and phalloidin (blue) to visualize M cells and F-actin, respectively. Scale bars represent 40 μm. (B) Quantification of M cells in FAE of CCR6-deficient mice after the adoptive transfer of CD11cint and CD11c− B cells. The number of M cells was counted in FAE region, and the value was normalized to the area of observation field. Values are mean ± SD of 15 observation fields in three mice per group. *P < 0.05 (Mann–Whitney U-test). (C) The number of nanoparticles internalized into PP was quantified in CCR6-deficient mice receiving CD11cint B cells or CD11c− B cells. Values are mean ± SD of three mice per group. At least 10 observation fields were examined per mice. *P < 0.05 (Student's t-test).
Fig. 7.
Fig. 7.
RANKL is normally expressed in PP in the absence of CD11cint B cells. (A) Frozen sections of PP from WT and CCR6-deficient mice were stained with anti-RANKL antibody (red) and DAPI (blue) for a nuclei counter stain. Scale bars represent 100 μm. (B) Total RNA samples were isolated from PP of the same mice as described in A and were subjected to real-time PCR analysis. The mRNA expression level of Tnfsf11 encoding RANKL was normalized to the value of GAPDH. Data are mean ± SD (n = 3).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kraehenbuhl JP, Neutra MR. Epithelial M cells: differentiation and function. Annu. Rev. Cell Dev. Biol. 2000;16:301. - PubMed
    1. Gebert A, Fassbender S, Werner K, Weissferdt A. The development of M cells in Peyer's patches is restricted to specialized dome-associated crypts. Am. J. Pathol. 1999;154:1573. - PMC - PubMed
    1. Knoop KA, Kumar N, Butler BR, et al. RANKL is necessary and sufficient to initiate development of antigen-sampling M cells in the intestinal epithelium. J. Immunol. 2009;183:5738. - PMC - PubMed
    1. Kerneis S, Pringault E. Plasticity of the gastrointestinal epithelium: the M cell paradigm and opportunism of pathogenic microorganisms. Semin. Immunol. 1999;11:205. - PubMed
    1. Kanaya T, Miyazawa K, Takakura I, et al. Differentiation of a murine intestinal epithelial cell line (MIE) toward the M cell lineage. Am. J. Physiol. Gastrointest. Liver Physiol. 2008;295:G273. - PubMed

Publication types

MeSH terms