Separate populations of M cells have been detected in the follicle-associated epithelium of Peyer’s patches (PPs) and the villous epithelium of the small intestine, but the traits shared by or distinguishing the two populations have not been characterized. Our separate study has demonstrated that a potent mucosal modulator cholera toxin (CT) can induce lectin Ulex europaeus agglutinin-1 and our newly developed M cell-specific mAb NKM 16-2-4-positive M-like cells in the duodenal villous epithelium. In this study, we determined the gene expression of PP M cells, CT-induced villous M-like cells, and intestinal epithelial cells isolated by a novel approach using FACS. Additional mRNA and protein analyses confirmed the specific expression of glycoprotein 2 and myristoylated alanine-rich C kinase substrate (MARCKS)-like protein by PP M cells but not CT-induced villous M-like cells. Comprehensive gene profiling also suggested that CT-induced villous M-like cells share traits of both PP M cells and intestinal epithelial cells, a finding that is supported by their unique expression of specific chemokines. The genome-wide assessment of gene expression facilitates discovery of M cell-specific molecules and enhances the molecular understanding of M cell immunobiology.

As a unique epithelial cell type specializing in Ag sampling, microfold or membranous cells (M cells) are present in the follicle-associated epithelium (FAE)4 of both GALT and nasopharynx-associated lymphoid tissue, which act as a major inductive site for Ag-specific mucosal immune responses (1, 2). Recently, we also identified M cells in the small intestinal villous epithelium, at effector sites far from the FAE, suggesting that Ag sampling via villous M cells may be responsible for induction of systemic Ag-specific immune responses, such as IgG production via the oral route (3). Still missing, however, were a characterization of the shared and distinctive traits of Peyer’s patches (PPs) and villous M cells and a better understanding of the immunological nature of each.

Recent comprehensive gene expression analyses using microdissected FAE or whole cells dissociated from the FAE identified genes specifically expressed by PP M cells (4, 5, 6). Similar data, however, have not been available for villous M cells, in part because sufficient numbers of M cells are difficult to isolate from the surrounding intestinal epithelial cells (IECs). In mice, lectin Ulex europaeus agglutinin-1 (UEA-1) possessing affinity for α (1, 2) fucose has been routinely used for the detection of such M cells (3, 7). UEA-1, however, does not alone suffice to identify M cells because it also reacts to goblet cells (3). Our laboratory has recently succeeded in distinguishing M cells from goblet cells by developing a mAb (NKM 16-2-4 mAb) that specifically reacts to murine PP and villous M cells but not goblet cells and IECs (8). Furthermore, our recent separate studies have demonstrated that oral administration of cholera toxin (CT) as mucosal adjuvant resulted in the induction of NKM 16-2-4 mAb+ and UEA-1+ M-like cells, which have pocket structure and Ag uptake ability, in the duodenal villous epithelium (Terahara et al., submitted for publication). These recent advances in our understanding of M cells allowed us to define gene expression profiles capable of distinguishing PP M cells, CT-induced villous M-like cells, and IECs.

BALB/c mice were purchased from Japan SLC. These mice were maintained under specific pathogen-free conditions in horizontal flow cabinets in our experimental animal facility at the University of Tokyo. Following a previously established protocol (9, 10), CT (List Biologic Laboratories) was dissolved in PBS (20 μg/mouse) and then orally administered to BALB/c mice. Two days after CT administration, mice were used for experiments. All animal experiments were approved by the Animal Care and Use Committee of University of Tokyo.

The following fluorescence-conjugated lectins and Abs were used for the identification of PP and villous M cells by FACS and histochemistry: PE-conjugated UEA-1 (Biogenesis), rhodamine-conjugated UEA-1 (Vector Laboratories), biotin-conjugated UEA-1 (Vector Laboratories), FITC-conjugated wheat germ agglutinin (WGA) (Vector Laboratories), FITC-conjugated or biotin-conjugated M cell-specific NKM 16-2-4 mAb (8), and allophycocyanin-Cy7-conjugated anti-mouse CD45 mAb (30-F11; BD Biosciences).

PPs from the naive duodenum and PP-free segments from the duodenum of naive or CT-administered mice were washed with cold PBS. Cells were dissociated from the small intestinal epithelium using a previously described mechanical procedure with some modifications (11). In brief, the tissues were incubated in PBS containing 0.5 mM EDTA with a stirrer for 10 min at 37°C. More than 90% of the dissociated cells survived as confirmed by a trypan blue exclusion test. The cells were stained with 1 μg/ml FITC-conjugated NKM 16-2-4 mAb, 5 μg/ml PE-conjugated UEA-1, and 1 μg/ml allophycocyanin-Cy7-conjugated anti-mouse CD45 mAb for 40 min before being reacted with 7-amino actinomycin (7-AAD; BD Biosciences) diluted 1/5 in DMEM containing 10% FCS for 10 min on ice. After washing with DMEM containing 10% FCS, the stained cells were analyzed using a flow cytometer FACSAria (BD Biosciences), and suitable cell populations gated on CD45 and 7-AAD cells were sorted.

Total RNA was extracted from the freshly isolated PP M cells, CT-induced villous M-like cells, and IECs of BALB/c mice using a High Pure RNA Tissue kit (Roche). Biotinylated cRNA was prepared using a two-cycle target-labeling assay in accordance with the protocol of the manufacturer (Affymetrix). The cRNA was hybridized with DNA probes on a GeneChip Mouse Genome 430 2.0 array (Affymetrix), washed, and fluorescence-labeled in accordance with the standard amplification protocol for eukaryotic targets developed by Affymetrix. The arrays were scanned with a GeneChip Scanner 3000 7G (Affymetrix). The fluorescence intensity of each probe was taken to represent the raw expression level and was quantified using GeneChip Operating software (Affymetrix). Data obtained from three independent experiments for PP M cells, CT-induced villous M-like cells, and IECs were normalized and statistically analyzed by Welch’s ANOVA using GeneSpring 7.3.1 software (Silicon Genetics). In addition, both qualitative indices (“Present Call,” “Marginal Call,” and “Absent Call”) based on p-value and a quantitative index (raw value) were also determined using GeneSpring 7.3.1 software. All microarray data described in this study have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geo/) with the accession no. GSE7838.

DNA fragments encoding GP2 (GenBank: NM_025989) and myristoylated alanine-rich C kinase substrate (MARCKS)-like protein (MLP; GenBank: NM_010807) were amplified by PCR from PP FAE-derived cDNA. The following sets of primers were used: GP2, sense, 5′-GGGTGATGGAGGAGTGAAGA-3′, anti-sense, 5′-CTCCAGGATGTTCCCACAGT-3′; and MLP, sense, 5′-AATTAACCCTCACTAAAGGGGAAGGCCAACGGACAGGAGA-3′, anti-sense, 5′-TAATACGACTCACTATAGGGCTTCTTGGGGGTCTCCTTGG-3′ (T3 and T7 promoter sequences are shown by italics). The PCR products for GP2 were subcloned into a pCR4-TOPO vector (Invitrogen). After sequencing, digoxigenin-labeled sense and anti-sense RNA probes were transcribed in vitro from the subcloned plasmids or from T3 and T7 promoter-conjugated PCR products with DIG RNA labeling mix (Roche). Paraffin-embedded sections of small intestinal tissues (6 μm) from naive BALB/c mice were obtained from Genostaff. ISH was performed as previously described (12). The bound probes were detected with BM purple AP substrate (Roche), before being counterstained with Kernechtrot stain solution (Muto Pure Chemicals) or reacted with 0.25 μg/ml biotin-conjugated UEA-1 at 4°C overnight after treatment with 3% H2O2. The sections labeled with biotin-conjugated UEA-1 were further reacted with HRP-conjugated streptavidin, followed by staining with 3,3′ diaminobenzidine (Vector Laboratories).

For the generation of GP2- and MLP-specific Abs, the open reading frames of GP2 and MLP genes were amplified by PCR from PP FAE-derived cDNA. The following sets of primers were used: GP2, sense, 5′-GACATGCTAGCATGAAAAGGATGGTGGGTTGTGAC-3′, anti-sense, 5′-GTATCGAATTCTCAGAACAGTAGAGCCAGGAAGAC-3′; and MLP, sense, 5′-TGACTGAATTCATGGGCAGCCAGAGCTCTAAGGCT-3′, anti-sense, 5′-TACATGTCGACCTACTCATTCTGCTCAGCACTGGC-3′, [NheI and EcoRI (GP2), and EcoRI and SalI (MLP) restriction enzyme sites are shown by italics]. For generation of GP2-specific mAbs, amplified GP2 gene was subcloned into pIRES2-EGFP vector (BD Biosciences) and the plasmid (pIRES2-GP2-EGFP) was then introduced in rat IEC line IEC-6 (ATCC, CRL-1592). After 2 days of transformation, EGFP-positive cells were purified by FACSAria and injected into the footpads of SD rats (1 × 106 cells/rat) five times at 2-wk intervals with TiterMax Gold (TiterMax) as an adjuvant. Four days after the final immunization, lymphocytes isolated from inguinal lymph node of the immunized rats were fused with P3×63-AG8.653 myeloma cells (ATCC, CRL-1580) in the presence of 50% (w/v) polyethylene glycol 1500 (Roche). Established hybridomas were injected into Crlj; CD1-Foxn1nu mice and mAbs were purified from ascites by using Protein G-Sepharose (GE Healthcare). For generation of MLP-specific polyclonal Abs, the amplified MLP gene was subcloned into pGEX-4T-1 (GE Healthcare) and the plasmid (pMLP-GEX-4T-1) was then introduced in Escherichia coli DH5α. After induction of MLP expression with 0.1 mM isopropyl β-D-thiogalactoside, the GST-fused MLP was purified on a Glutathione-Sepharose 4B (GE Healthcare) and subsequently removed the GST-tag with thrombin (GE Healthcare). The purified rMLP was then immunized into New Zealand white rabbits and anti-MLP pAbs were purified from the antiserum by using rMLP-conjugated TOYOPEARL AF-Tresyl-650M (Tosoh).

The histochemical analyses were performed with whole-mount tissues and frozen-section specimens prepared from mucus-free tissues fixed with 4% paraformaldehyde in PBS as previously described (3). For GP2 staining, the specimens were incubated with 1 μg/ml rat anti-GP2 mAb (10F5-9-2) or the isotype control Ab (rat IgG2a; BD Biosciences) at 4°C overnight. For MLP staining, tissue sections were incubated with 10 μg/ml anti-MLP pAb or normal rabbit IgG at 4°C overnight. The specimens were then treated with 3 μg/ml Cy5-conjugated donkey anti-rat IgG or 3 μg/ml Cy5-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories) together with 10 μg/ml tetramethylrhodamine isothiocyanate-conjugated UEA-1 (Vector Laboratories) and/or 5 μg/ml FITC-conjugated WGA (Vector Laboratories) for 1 h at room temperature. Finally, the section specimens were reacted with 400 ng/ml 4′-6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) and the signal was observed under a confocal laser-scanning microscope (TCS SP2; Leica). For counterstaining with our recently established M cell-specific mAb (NKM 16-2-4; rat IgG2c), the same section specimens were incubated with 5 μg/ml biotin-conjugated NKM 16-2-4 at 4°C overnight followed by 1.25 μg/ml HRP-conjugated streptavidin (Pierce) for 1 h at room temperature. The signal was then developed with 3,3′ diaminobenzidine and the nucleus was finally stained with hematoxylin.

FACS analysis demonstrated that the large forward-scatter CD45 cell population could be divided into three subpopulations by NKM 16-2-4 mAb and UEA-1 staining (Fig. 1). Because of the known specificities of NKM 16-2-4 mAb and UEA-1, cells positive for NKM 16-2-4 and UEA-1 were identified as M cells (or M-like cells) in this study. In the CD45 epithelial cell population isolated from the duodenum of naive BALB/c mice, the frequency of PP M cells averaged 7%. Perhaps due to the nature of the isolation technique used, the harvest of PP M cells far outstripped that of villous M cells, the frequency of the latter being so low (1.7 ± 1.5%) as to render harvest extremely difficult. We recently found that the number of villous M-like cells could be increased by oral administration of CT (64.5 ± 15.7% and Terahara et al., submitted for publication), and so decided to use the villous M-like cells induced by CT treatment for this DNA microarray analysis. A FACS with a purity of 90–99% was used to isolate PP M cells (NKM 16-2-4+/UEA-1+), CT-induced villous M-like cells (NKM 16-2-4+/UEA-1+), and IECs (NKM 16-2-4/UEA-1).

FIGURE 1.

Frequency of PP M cells (PP-M) and CT-induced villous M-like cells (Vi-M) in the duodenal epithelium. Dot plots are shown by NKM 16-2-4-FITC and UEA-1-PE staining gated on CD45 and 7-AAD duodenal epithelial cell populations from naive BALB/c mice or from those that had been orally treated with CT. PP-M (NKM 16-2-4+ and UEA-1+), Vi-M (NKM 16-2-4+ and UEA-1+), and IEC (NKM 16-2-4 and UEA-1) were isolated by FACS. Numbers are the mean percentage ± SD in CD45 and 7-AAD epithelial cell populations from three independent experiments.

FIGURE 1.

Frequency of PP M cells (PP-M) and CT-induced villous M-like cells (Vi-M) in the duodenal epithelium. Dot plots are shown by NKM 16-2-4-FITC and UEA-1-PE staining gated on CD45 and 7-AAD duodenal epithelial cell populations from naive BALB/c mice or from those that had been orally treated with CT. PP-M (NKM 16-2-4+ and UEA-1+), Vi-M (NKM 16-2-4+ and UEA-1+), and IEC (NKM 16-2-4 and UEA-1) were isolated by FACS. Numbers are the mean percentage ± SD in CD45 and 7-AAD epithelial cell populations from three independent experiments.

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DNA microarrays containing 45,101 probes were used to determine the comprehensive gene expression of PP M cells, CT-induced villous M-like cells, and IECs. Comparison of these profiles revealed correlation coefficients of 0.285 for PP M cells and IECs, of 0.402 for PP M cells and CT-induced villous M-like cells, and of 0.410 for CT-induced villous M-like cells and IECs (Fig. 2,A). Based on the constructed gene profiling, we categorized probes showing significant expression into seven groups (Groups A-G) using our own criteria (Fig. 2,B). The 1272, 4, and 7 probes were regarded as significant for PP M cells (Group A), CT-induced villous M-like cells (Group B), and IECs (Group C), respectively (Fig. 2 C). The relative expression levels and gene names of the significant probes are provided in Supplementary Table I.5 Our gene-profiling database allowed us to confirm previous findings that Group A includes the transcripts of peptidoglycan recognition protein-S, secretory granule neuroendocrine protein 1, and annexin V that are specifically expressed by PP M cells (4, 5, 6).

FIGURE 2.

Gene expression profiles for PP M cells (PP-M), CT-induced villous M-like cells (Vi-M), and IECs. A, Scatter plots of the normalized expression level on each DNA microarray probe for PP-M and IEC, PP-M and Vi-M, and Vi-M and IEC with correlation coefficients (r2). B, Grouping of probes based on their significance for PP-M, Vi-M, and IEC using the criteria outlined in the qualitative (“Present Call” or “Marginal Call”) and quantitative (raw value) indices as assessed by GeneSpring 7.3.1 software. C, Venn diagram showing the categorization of significant probes into seven groups (Group A–G).

FIGURE 2.

Gene expression profiles for PP M cells (PP-M), CT-induced villous M-like cells (Vi-M), and IECs. A, Scatter plots of the normalized expression level on each DNA microarray probe for PP-M and IEC, PP-M and Vi-M, and Vi-M and IEC with correlation coefficients (r2). B, Grouping of probes based on their significance for PP-M, Vi-M, and IEC using the criteria outlined in the qualitative (“Present Call” or “Marginal Call”) and quantitative (raw value) indices as assessed by GeneSpring 7.3.1 software. C, Venn diagram showing the categorization of significant probes into seven groups (Group A–G).

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In an effort to identify molecules that could be expressed on the apical surface of PP M cells, we looked for genes showing a higher expression level in Group A. During the ISH analysis, we found that GP2 mRNA was specifically expressed in the FAE of PPs throughout the small intestine (Fig. 3,A, 1) and that its expression was distinctively colocalized with UEA-1+ M cells (Fig. 3,A, 3 and 4). A negative control using sense cRNA probes did not show any positive signals (Fig. 3,A, 2). Immunohistochemical analysis with newly established anti-GP2-specific mAb (10F5-9-2) revealed that the GP2 protein was highly expressed in UEA-1+ PP M cells (Fig. 3,B). A negative control using isotype rat IgG2a did not show any positive signals in the dome epithelium of PPs (Fig. 3,B). The expression of GP2 in M cells was further confirmed by counterstaining with our recently established M cell-specific mAb NKM 16-2-4 (Fig. 3,B). Supporting the histochemical analyses, whole-mount staining analysis also demonstrated GP2 was expressed on the apical surface of UEA-1+ PP M cells, which were not recognized by enterocyte-reactive lectin WGA (Fig. 3 C). Supporting the gene profiling data (Supplementary Table I), GP2 protein was not detected in CT-induced villous M-like cells (data not shown).

FIGURE 3.

GP2 was specifically expressed by PP M cells in the small intestine. A, ISH for GP2 mRNA with positive signals (blue) of hybridized anti-sense or sense cRNA probes on duodenal PPs and adjacent villi of naive BALB/c mice. Tissues were also counterstained with Kernechtrot (pink, 1 and 2) or labeled with UEA-1-HRP before being stained with 3,3′ diaminobenzidine (brown, 4). 3 and 4, High magnification of a PP FAE before (3) and after (4) labeling with UEA-1. Scale bar = 200 μm (1 and 2) and 40 μm (3 and 4). B, Confocal images of frozen sections of PPs stained with anti-GP2-specific mAb (10F5-9-2) or isotype control (rat IgG2a). The specific expression of GP2 in M cells was confirmed by counterstaining with our recently established M cell-specific mAb (NKM 16-2-4). Arrows and arrowheads show M cells and goblet cells, respectively. Scale bar = 30 μm. C, Confocal images of whole-mount duodenal PP domes stained with anti-GP2-specific mAb (10F5-9-2), UEA-1, and WGA. Scale bar = 30 μm.

FIGURE 3.

GP2 was specifically expressed by PP M cells in the small intestine. A, ISH for GP2 mRNA with positive signals (blue) of hybridized anti-sense or sense cRNA probes on duodenal PPs and adjacent villi of naive BALB/c mice. Tissues were also counterstained with Kernechtrot (pink, 1 and 2) or labeled with UEA-1-HRP before being stained with 3,3′ diaminobenzidine (brown, 4). 3 and 4, High magnification of a PP FAE before (3) and after (4) labeling with UEA-1. Scale bar = 200 μm (1 and 2) and 40 μm (3 and 4). B, Confocal images of frozen sections of PPs stained with anti-GP2-specific mAb (10F5-9-2) or isotype control (rat IgG2a). The specific expression of GP2 in M cells was confirmed by counterstaining with our recently established M cell-specific mAb (NKM 16-2-4). Arrows and arrowheads show M cells and goblet cells, respectively. Scale bar = 30 μm. C, Confocal images of whole-mount duodenal PP domes stained with anti-GP2-specific mAb (10F5-9-2), UEA-1, and WGA. Scale bar = 30 μm.

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Candidates for FAE-specific genes including MLP (also known as MacMARCKS or MRP) have been previously proposed (5, 6). Most of these genes together with MLP could be identified as PP M cell-significant genes by the DNA microarray analysis (Supplementary Table I). The subsequent ISH analysis demonstrated a unique expression pattern of MLP mRNA in the small intestine, i.e., MLP mRNA was detected in the FAE and B cell zones of PPs throughout the small intestine (Fig. 4,A, 1). A negative control using sense cRNA probes did not show any positive signals (Fig. 4,A, 2). In the FAE, the expression of MLP mRNA was exclusively colocalized with UEA-1+ M cells (Fig. 4,A, 3 and 4). Immunohistochemical analysis further elucidated the complicated expression pattern of MLP, revealing that the MLP protein was also found in B cell zones and the cytoplasm of M cells in PPs throughout the small intestine (Fig. 4,B), but not in CT-induced villous M-like cells (data not shown). A negative control using normal rabbit IgG did not show any positive signals (Fig. 4 B).

FIGURE 4.

MLP was expressed by M cells and by the B cell zones of PPs but not by villi. A, ISH for MLP mRNA with positive signals (blue) of hybridized anti-sense or sense cRNA probes on duodenal PPs and adjacent villi of naive BALB/c mice. Tissues were also counterstained with Kernechtrot (pink, 1 and 2) or labeled with UEA-1-HRP before being stained with 3,3′ diaminobenzidine (brown, 4). 3 and 4, High magnification of a PP FAE before (3) and after (4) labeling with UEA-1. Scale bar = 200 μm (1 and 2) and 40 μm (3 and 4). B, Confocal images of frozen sections of duodenal PPs stained with anti-MLP-specific pAb or control rabbit IgG. Confocal images of frozen sections of duodenal PPs from naive BALB/c mice labeled with UEA-1-rhodamine, DAPI, and immune complexes of FITC-labeled anti-rabbit IgG secondary Ab with anti-MLP polyclonal Ab or normal rabbit IgG. 1 and 5, Merged images of DAPI (blue) and MLP (green) (1) or normal rabbit IgG (green) (5). 2–4, High magnification of a PP FAE. 2, Merged image of DAPI- (blue) and MLP (green)-staining. 3, Single image of UEA-1-staining (red). 4, Merged image of (2) and (3). Arrows show PP M cells expressing MLP. Scale bar = 100 μm (1), 50 μm (5), and 10 μm (2–4).

FIGURE 4.

MLP was expressed by M cells and by the B cell zones of PPs but not by villi. A, ISH for MLP mRNA with positive signals (blue) of hybridized anti-sense or sense cRNA probes on duodenal PPs and adjacent villi of naive BALB/c mice. Tissues were also counterstained with Kernechtrot (pink, 1 and 2) or labeled with UEA-1-HRP before being stained with 3,3′ diaminobenzidine (brown, 4). 3 and 4, High magnification of a PP FAE before (3) and after (4) labeling with UEA-1. Scale bar = 200 μm (1 and 2) and 40 μm (3 and 4). B, Confocal images of frozen sections of duodenal PPs stained with anti-MLP-specific pAb or control rabbit IgG. Confocal images of frozen sections of duodenal PPs from naive BALB/c mice labeled with UEA-1-rhodamine, DAPI, and immune complexes of FITC-labeled anti-rabbit IgG secondary Ab with anti-MLP polyclonal Ab or normal rabbit IgG. 1 and 5, Merged images of DAPI (blue) and MLP (green) (1) or normal rabbit IgG (green) (5). 2–4, High magnification of a PP FAE. 2, Merged image of DAPI- (blue) and MLP (green)-staining. 3, Single image of UEA-1-staining (red). 4, Merged image of (2) and (3). Arrows show PP M cells expressing MLP. Scale bar = 100 μm (1), 50 μm (5), and 10 μm (2–4).

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Focusing on chemokines whose presence was statistically identified regardless of the raw value, we found seven chemokines to be expressed by PP M cells and/or CT-induced villous M-like cells (Table I). In addition to the previously reported CCL9 and CCL20 that are expressed by the M cell-containing PP FAE (13, 14, 15), we found significant expression of CXCL13 and chemokine-like factor (CKLF) in PP M cells. Although CT-induced villous M-like cells and IECs constitutively expressed CCL6 and CCL28, the expression level of these chemokines was highest in PP M cells (thereby categorized in Group G; Fig. 2, B and C). Next, we also examined the expression pattern of chemokines in CT-induced villous M-like cells, finding, as in PP M cells, an up-regulation of CCL9, CKLF, and CCL6 mRNAs. Their raw values and expression levels were higher than those seen in IECs (Table I). Furthermore, CT-induced villous M-like cells showed the highest expression of CXCL16 mRNA, with expression levels 1.9–2.5-fold higher than in PP M cells and 2.1–2.3-fold higher than in IECs (Table I).

Table I.

Chemokines expressed by PP M cells and/or CT-induced villous M-like cellsa

NameGenBankAffymetrix Probe No.Group (see Fig. 2 C)Relative Expression Level against IEC
PP-MVi-M
CCL9 AF128196 1417936_at 124.8 7.0 
CXCL13 AF030636 1417851_at 64.4 – 
CKLF BE852312 1436242_a_at 52.2 8.3 
CCL6 AV084904 1420249_s_at 30.4 5.1 
 BC002073 1417266_at 12.5 4.6 
CCL20 AF099052 1422029_at 10.9 
CCL28 BE196980 1455577_at 2.8 0.7 
CXCL16 BC019961 1449195_s_at 1.1 2.1 
  1418718_at 0.9 2.3 
NameGenBankAffymetrix Probe No.Group (see Fig. 2 C)Relative Expression Level against IEC
PP-MVi-M
CCL9 AF128196 1417936_at 124.8 7.0 
CXCL13 AF030636 1417851_at 64.4 – 
CKLF BE852312 1436242_a_at 52.2 8.3 
CCL6 AV084904 1420249_s_at 30.4 5.1 
 BC002073 1417266_at 12.5 4.6 
CCL20 AF099052 1422029_at 10.9 
CCL28 BE196980 1455577_at 2.8 0.7 
CXCL16 BC019961 1449195_s_at 1.1 2.1 
  1418718_at 0.9 2.3 
a

Expression levels on probes identified as “Present Call” in PP M cells (PP-M) or CT-induced villous M-like cells (Vi-M) were compared with those in IECs. Minus indicated no expression in Vi-M and IECs. CKLF, Chemokine-like factor.

In this study, we combined the advanced techniques of M cell purification and DNA microarrays to construct a gene-profiling database for PP M cells, CT-induced villous M-like cells, and IECs. The lack of M cell-specific markers has long presented an obstacle to the isolation of M cells. We overcame this barrier by using M cell-specific NKM 16-2-4 mAb. Our knowledge that villous M (or M-like) cells, usually low frequency in the duodenum, could be increased by CT allowed us to separately isolate PP M cells, CT-induced villous M-like cells, and IECs using FACS. Of course, we cannot yet exclude the possibility that individual cell-sorted fractions were mildly contaminated by other cell types; however, we regard the database as reliable because most of the previously reported PP M cell-specific genes encoding peptidoglycan recognition protein-S, secretory granule neuroendocrine protein 1, and annexin V (4, 5, 6) were found in the PP M cell-significant group (Group A). Thus, the gene-profiling database presented here has several advantages: it appears reliable because it was capable of confirming already established findings; it includes villous M (or M-like) cells as well as PP M cells and IECs; and it is based on a purified cell population. These advantages could make this gene-profiling database a reliable and useful tool for identifying new molecules expressed by M cells and for deepening our understanding of M cell immunobiology.

A mucosal vaccine delivery system targeting PP M cells would be more effective at generating not only efficient mucosal but also systemic immunity. When UEA-1 was used as an Ag delivery vehicle, the administration of PP M cell-targeted Ags induced Ag-specific mucosal and systemic immune responses (16) despite the cospecificity of the lectin for M cells and goblet cells (3). The gene-profiling database was, therefore, used to look for candidate target molecules in the vaccine delivery system. We focused on GP2, which is a GPI-anchored protein expressed at a higher level in Group A. GP2 is associated with lipid rafts, is sorted to the apical plasma membrane (17), and is likely to possess a similar distribution in PP M cells. Additional mRNA and protein analyses by ISH and immunohistochemistry identified the specific expression of GP2 in the apical plasma membrane of PP M cells. Although the role played by GP2 in a unique Ag-sampling system of PP M cells remains obscure, GP2 is not required for PP M cell development, as evidenced by the presence of M cells in the FAE of PPs from GP2−/− mice (data not shown). Taken together, these findings support the candidacy of GP2 as an M cell-targeting molecule. If it is in fact confirmed to be so, it could greatly contribute to the development of a mucosal vaccine delivery system.

In addition to the identification of GP2, reliance of the gene-profiling database is further supported by the identification of specific expression of MLP by PP M cells. Not only MLP but also other genes have been previously reported as FAE-specific genes (5, 6). Using DNA microarray analysis, we were able to identify most such genes, including MLP, as PP M cell-significant genes. This study demonstrated for the first time the histological distribution of expressed MLP mRNA and protein in the small intestine. MLP, a member of protein kinase C substrates, binds calcium/calmodulin and actin (18, 19), and has been implicated in integrin-dependent phagocytosis by macrophages (20, 21). However, that contention was challenged in another study by Underhill and coworkers (22) using MLP−/− macrophages. Interestingly, β1 integrin, which is expressed by the apical membrane of PP M cells but not of IECs in the murine small intestine, is involved in the uptake of Yersinia by PP M cells via integrin-invasin binding (23). Thus, MLP may also account for the Ag uptake/sampling process, including integrin-dependent Ag uptake, of M cells located within the FAE of PPs.

Our constructed gene profiling also provides additional information for M cell immunobiology. Both PP and villous M cells contain in their basolateral region immunocompetent cells characterized by a pocket formation (3, 24), the contents of which are influenced by the repertoire of chemokines expressed by M cells. So far, three chemokines, CCL9, CCL20, and CXCL16, have been reported to be specifically expressed by the M cell-containing FAE of PPs and to contribute to the spatial distribution of dendritic cells or T cells in the subepithelial dome as well as in the basolateral pocket regions of M cells (13, 14, 15, 25). Our examination for the gene expression pattern of chemokines using the gene-profiling database showed that CXCL13, CKLF, CCL6, and CCL28, in addition to CCL9 and CCL20, are specifically or highly expressed by PP M cells, suggesting that CXCL13, CKLF, CCL6, and CCL28 may also play a role in regulating the recruitment of various immunocompetent cells into the pocket region of PP M cells.

Noticeably, CT-induced villous M-like cells share with PP M cells the expression of certain chemokines, including CCL6, CCL9, and CKLF. Furthermore, the highest expression of CXCL16 mRNA was observed in CT-induced villous M-like cells, although CXCL16 has previously been shown to be specifically expressed in the FAE of PPs (25). This discrepancy may result from our exclusive use of duodenal tissues for the analysis of M cell gene profiling. CXCL16 is a chemoattractant for activated CD8+ T cells and, to a lesser extent, for activated CD4+ T cells (25, 26); the CXCL16 receptor is expressed by intraepithelial lymphocytes (IELs) (26). In the small intestine, the distribution patterns for CD4+ and CD8+ T cells are distinct, with CD4+ T cells primarily located in the lamina propria and CD8+ T cells residing along the epithelium (27). When CT was orally administered, CD8+ IELs were rapidly and transiently depleted (28). Interestingly, we observed that CD8+ IEL numbers recovered following the generation of CT-induced villous M-like cells (data not shown). Therefore, our current finding that CT-induced villous M-like cells express a higher level of CXCL16 makes it plausible that CD8+ T cells are retained in the intestinal epithelium, mainly into the pocket of villous M-like cells. Furthermore, up-regulation of CCL9, CKLF, CCL6, and CXCL16 in CT-induced villous M-like cells could account for the CT-induced recruitment of immunocompetent cells to the site of Ag sampling from the intestinal lumen via CT-induced villous M-like cells.

Although the development mechanism of villous M cells remains unclear, we hypothesize that villous M cells are differentiated from IECs by exogenous stimuli because oral CT administration resulted in the induction of villous M-like cells in the middle to upper regions of villi (Terahara et al., submitted for publication), i.e., where IECs normally migrate from the crypts to the villus (29). Our hypothesis is also informed by the suggestions offered by other groups that IECs in the FAE of PPs could be converted to M cells by bacterial infection and inflammation (30, 31). We propose that CT-induced villous M-like cells have a gene expression pattern that is intermediate between PP M cells and IECs, as evidenced by the very similar correlation coefficient values obtained when the comprehensive gene profile of CT-induced villous M-like cells was compared with that of PP M cells (r2 = 0.402) and IECs (r2 = 0.410). The intermediate nature of CT-induced villous M-like cells between PP M cells and IECs is further confirmed by chemokine expression profiles. In this study, we have attempted to use gene profiling to elucidate the development mechanism of villous M cells.

In conclusion, our gene-profiling database should prove a valuable tool in identifying suitable M cell-targeting molecules, thereby speeding the development of a mucosal vaccine delivery system as well as allowing for a better understanding of M cell immunobiology.

We thank the members of our laboratory for technical advice and helpful discussions. We also extend our thanks to Dr. K. McGhee for editorial help.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported in part by grants from Core Research for Evolutional Science and Technology of the Japan Science and Technology Corporation, the Ministry of Education, Science, Sports, and Culture, and the Ministry of Health and Welfare in Japan.

4

Abbreviations used in this paper: FAE, follicle-associated epithelium; 7-AAD, 7-amino actinomycin; CKLF, chemokine-like factor; CT, cholera toxin; DAPI, 4′-6-diamidino-2-phenylindole; IEC, intestinal epithelial cell; IEL, intraepithelial lymphocyte; ISH, in situ hybridization; MLP, myristoylated alanine-rich C kinase substrate (MARCKS)-like protein; PP, Peyer’s patch; UEA-1, Ulex europaeus agglutinin-1; WGA, wheat germ agglutinin.

5

The online version of this article contains supplemental material.

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