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. 2010 Aug;177(2):666-76.
doi: 10.2353/ajpath.2010.090811. Epub 2010 Jul 8.

CD137 is required for M cell functional maturation but not lineage commitment

En Hui Hsieh  1 , Xiomara Fernandez, Jing Wang, Mary Hamer, Stephanie Calvillo, Michael Croft, Byoung S Kwon, David D Lo
Affiliations

CD137 is required for M cell functional maturation but not lineage commitment

En Hui Hsieh et al. Am J Pathol. 2010 Aug.

Abstract

Mucosal immune surveillance depends on M cells that reside in the epithelium overlying Peyer's patch and nasopharyngeal associated lymphoid tissue to transport particles to underlying lymphocytes. M cell development is associated with B lymphocytes in a basolateral pocket, but the interactions between these cells are poorly understood. In a cell culture model of M cell differentiation, we found lymphotoxin/tumor necrosis factor alpha induction of CD137 (TNFRSF9) protein on intestinal epithelial cell lines, raising the possibility that CD137 on M cells in vivo might interact with CD137L expressed by B cells. Accordingly, while CD137-deficient mice produced UEA-1+ M cell progenitors in nasopharyngeal associated lymphoid tissue and Peyer's patch epithelium, they showed an abnormal morphology, including the absence of basolateral B cell pockets. More important, CD137-deficient nasopharyngeal associated lymphoid tissue M cells were defective in microparticle transcytosis. Bone marrow irradiation chimeras confirmed that while induction of UEA-1+ putative M cell precursors was not CD137-dependent, full M cell transcytosis function required expression of CD137 by radioresistant stromal cells as well as by bone marrow-derived cells. These results are consistent with a two-step model of M cell differentiation, with initial CD137-independent commitment to the M cell lineage followed by a CD137-CD137L interaction of M cells with CD137-activated B lymphocytes or dendritic cells for functional maturation.

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Figures

Figure 1
Figure 1
Induction of CD137 expression in cytokine treated Caco-2BBe cells. Caco-2BBe cells were cultured alone or with a LTβR agonist antibody plus TNFα and stained for CD137 (green) and fibronectin (red). The inset shows co-localization of cytoplasmic CD137 with fibronectin (arrows).
Figure 2
Figure 2
Electron microscopy analysis of PP and NALT. PP and NALT from wild-type (BALB/c) and CD137-deficient mice were prepared for SEM and TEM to assess the morphology of M cells in mucosal lymphoid tissues. A: SEM of wild-type PP M cells. B: SEM of CD137-deficient PP M cells, showing abnormally narrowed apical surfaces of M cells (arrows). C: SEM of wild-type NALT. D: SEM of CD137-deficient NALT. E: TEM of wild-type NALT, showing M cell (M) lacking apical cilia (asterisk), but with basolateral membranes (arrows) extending around basolateral pocket lymphocyte (L). F: TEM of CD137-deficient NALT, showing narrow cross-section and absence of apical cilia (asterisk) of M cells (M). Most M cells lack basolateral lymphocytes; the M cell at upper left may be associated with a lymphocyte, although membrane extensions contacting the lymphocyte are not evident.
Figure 3
Figure 3
Confocal analysis of Peyer’s patch M cells. PP whole mounts were prepared for confocal analysis; serial z-sections are projected so that a single follicle could be visualized in a single image at low magnification. A: UEA-1 (green) staining of wild-type PP follicle, with radial pattern of M cells evident, and at higher magnifications the broad polygonal apical surface is evident. B: UEA-1 staining of CD137-deficient PP, showing a similar radial pattern, but with a clearly narrowed apical morphology. C: UEA-1 staining of wild-type adjacent crypts, showing pattern consistent with migration of M cells from crypts (asterisk, with arrows indicating apparent course of migration). D: UEA-1 staining of CD137-deficient adjacent crypts (asterisks and arrows as in C). E: B220 (red) versus UEA-1 (green) in wild-type PP, showing weaker expression of B220 by B cells in and near the follicle epithelium (arrows). F: staining MHC class II (red) staining in wild-type PP, showing broad apical surface of UEA-1-positive M cells (green) and basolateral pocket B lymphocyte (arrows). G: MHC class II staining in CD137-deficient PP, where epithelial MHC II+ cells are evident (red, arrows), but UEA-1-positive M cells (green) are narrower, with less clearly established basolateral pockets. The axis is for scale and orientation.
Figure 4
Figure 4
Confocal analysis of NALT M cells. NALT whole mounts were prepared for confocal analysis as described for PP. A: UEA-1 staining of wild-type NALT, showing distribution and broadly rounded apical surface of UEA-1-positive M cells. B: UEA-1 staining of CD137-deficient NALT, showing less uniform morphology of M cells. C: Three-dimensional rendering of wild-type NALT M cell, viewed from below so that the basolateral pocket (arrows) can be seen. D: Three-dimensional rendering of CD137-deficient NALT M cell also viewed from below, showing narrowed morphology. The axis is to show 3-dimensional orientation.
Figure 5
Figure 5
Microparticle uptake in NALT. Fluorescent streptavidin-biotinylated peptide-coated styrene (A–C) or uncoated latex (D) microparticles were administered to nasal passages to enable uptake into NALT. A: Uptake of CPE30-coated styrene beads (red) in wild-type NALT, showing that after 10 minutes the beads have already passed the epithelial layer (UEA-1-positive M cells stained green) and entered the follicle. B: Uptake of CPE30-coated beads in CD137-deficient NALT, showing far fewer beads within the follicle. C: Statistical analysis of CPE30-coated bead uptake, showing poor uptake of styrene beads coated with control peptide but significant uptake of CPE30-coated styrene beads by wild-type NALT, but little uptake of either type of particle in CD137-deficient NALT. D: Statistical analysis of uncoated latex bead uptake in wild-type and CD137-deficient NALT, showing uptake of beads by wild-type NALT but minimal uptake by CD137-deficient NALT. Note that uncoated latex beads are taken up slightly less efficiently than CPE30-coated styrene beads. The axis is for scale and orientation.
Figure 6
Figure 6
M cell development and function in irradiation chimeras. Irradiation chimeras were analyzed for development of NALT UEA-1+ M cells (A–D), latex particle uptake, and density of M cells (E). Confocal images of representative areas of NALT epithelium showed similar size and distribution of M cells in all four chimeras: wild-type to wild-type (A); wild-type to CD137KO (B); CD137KO to wild-type (C); CD137KO to CD137KO (D). Among these chimeras, only the wild-type→wild-type chimeras showed significant latex particle uptake (E). The density of UEA-1+ M cells across the NALT epithelium was similar for all chimeras (F), suggesting that the defect in uptake was due to functional deficits rather than absence of committed M cell progenitors.
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