ARTICLE |
Correspondence to: Ram Sharma, Human Morphology, U. of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK.
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Summary |
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Peyer's patches in the intestinal mucosa are characterized by the presence of several lymphatic follicles and interfollicular T-cell regions. Luminal antigens are transported across the intestinal epithelium to stimulate the Peyer's patch pre-B-cells in the follicles that proliferate and migrate to distant sites. Evidence suggests that antigen priming of B-lymphocytes is responsible for the number and location of Peyer's patches during postnatal life, but little is known about the histogenesis of Peyer's patches and their overlying membranous (M) cells. To examine whether T- and B-lymphocytes in Peyer's patches have an influence on M-cell generation, we studied the development of Peyer's patches and M-cells in severe combined immunodeficient (SCID) mice reconstituted with bone marrow cells from normal syngeneic mice. Our experiments demonstrate that the donor bone marrow cells in the host SCID mice repopulate to form single (primary) follicles and aggregates of lymphoid nodules, the Peyer's patches. Use of the lectins Anguilla anguilla (AAA) and Ulex europaeus I (UEA-I) as positive markers of mouse Peyer's patch M-cells revealed that M-cells develop in the dome epithelium overlying the single primary follicles and Peyer's patches of reconstituted SCID mice. This experimental system therefore allows the study of the histogenesis of Peyer's patches and M-cells. (J Histochem Cytochem 46:143148, 1998)
Key Words: bone marrow, follicle-associated epithelium, lectins, lymphocytes, M-cells, Peyer's patches, SCID mice
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Introduction |
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The gut-associated lymphoid tissue (GALT), which is present in all vertebrates as ileal Peyer's patches, consists of tightly packed follicles that are separated by small T-cell areas and contain 95% surface IgM-positive B-cells (
The severe combined immunodeficiency (SCID) disease impairs lymphopoiesis, and consequently the individuals with this defect are deficient in immune functions mediated by T- and B-lymphocytes. Mice homozygous for this mutation (SCID mice) are devoid of pre-B-, B-, and T-lymphocytes (
The aim of this study was to assess the potential of bone marrow progenitor cells to repopulate the host's GALT and, in particular, to determine whether the repopulating lymphocytes induce Peyer's patch and M-cell formation. We used a mouse model of SCID disease and made bone marrow transfers from normal Balb/c mice which are syngeneic to SCID mice. To define the developmental pattern of mucosal follicles in reconstituted SCID mice, we distinguished the primary follicles from Peyer's patches. To verify the extent of reconstitution of Peyer's patches by donor bone marrow cells, we used fucose-specific lectins Anguilla anguilla agglutinin (AAA) and Ulex europaeus agglutinin I (UEA-I) to identify the M-cells in the repopulated mice. Both lectins have been shown to be markers for M-cells in mice (
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Materials and Methods |
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Animals and Bone Marrow Transfer
Twenty-four 34-month-old pathogen-free Balb/c C57BL/Kalgh-I male scid/scid (SCID) mice were sampled from the breeding program of the University of Southampton Biomedical Sciences Building animal house. A laminar flow isolator was used during manipulations of the animals, which were housed up to five in a filter-top cage on a sterile bedding and were provided with a sterile food and water ad libitum. Ileal specimens from 12 normal male Balb/c mice fed a standard rodent diet and maintained under conventional laboratory conditions from the same animal facilities were examined as controls.
For bone marrow transplantation, bone marrow was removed from the femora of adult male Balb/c mice. Each end of the femur was cut off and Hank's balanced salt solution (HBSS) was flushed through the bone using a 23-gauge syringe needle on a 10-ml syringe. The harvested cells were centrifuged after washing in HBSS and counted. They were resuspended in HBSS and 0.1 ml containing 6.3 x 106 cells was injected IV into the tail vein of each SCID mouse. Fifteen SCID mice were divided into groups of five and were sacrificed 2, 4, or 6 weeks after transplantation. In addition, nine transplanted SCID mice were left for 10 weeks. The mice were sacrificed by cervical dislocation and three or four pieces of the terminal part of the ileum containing lymphoid follicles were dissected under a binocular microscope. The tissues were swiss-rolled, fixed in a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 18 hr at 4C, and processed routinely for wax histology.
Sample Processing and Statistics
One hundred serial transverse 56-µm sections were cut from swiss-rolled ileal samples of each of 12 Balb/c control mice and 24 reconstituted SCID mice. Every tenth equidistant section was stained with hematoxylin and eosin (H&E) and the numbers of single primary follicles and Peyer's patches were counted on 10 H&E sections from each animal. Statistical comparisons between the groups were analyzed using one-way analysis of variance (ANOVA; Tukey's multiple comparison test) and differences were considered significant at p<0.05.
Lectin Histochemistry
Paraffin sections were deparaffinized in xylene, hydrated through a series of graded alcohols, and brought to 0.05 M Tris-buffered saline (TBS) at pH 7.7 containing 0.1% calcium chloride. Sections were then trypsinized with 0.1% trypsin (Sigma; St Louis, MO) in 0.05 M TBS for 30 min at 37C. After a wash in TBS, the endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol. Sections were washed again in TBS and incubated for 30 min at room temperature in a Shandon Sequenza immuno-staining center with the biotinylated lectins at a concentration of 10 µg ml-1 in TBS. Biotinylated (1-2)-fucose-specific lectins from Anguilla anguilla (AAA; EY Laboratories, San Mateo, CA) and Ulex europaeus Type I (UEA-I; Sigma) were used to characterize M-cells. The names, sugar specificities, and inhibitors of the lectins used in this study are listed in Table 1. After a further wash in TBS, sections were treated with ABC complex (peroxidase standard PK-4000; Vector Laboratories, Peterborough, UK) for 30 min, washed again in TBS, and then incubated in diaminobenzidine tetrahydrochloride (DAB) in Tris-HCl buffer, pH 7.3, with 0.3% hydrogen peroxide for 510 min. The control experiments were carried out by omission of the lectin and by incubation of the sections with the lectins and their appropriate inhibitory sugar at a concentration of 0.3 M, except for MAA, in which a neuraminidase predigestion was used (
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Results |
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In all the SCID animals reconstituted with bone marrow progenitor cells from normal syngeneic mice, GALT was identified as single primary follicles and aggregated Peyer's patches in the terminal ileum. The numbers of single follicles and Peyer's patches in Balb/c control mice and in SCID mice sacrificed 2, 4, 6, or 10 weeks after bone marrow cell transfer are shown in Table 2. Although no significant differences in the number of Peyer's patches were found between the Balb/c and reconstituted SCID mice at 2, 4, or 6 weeks after the bone marrow reconstitution, the number of Peyer's patches at 10 weeks after the bone marrow transfer were significantly higher in reconstituted SCID mice compared with the Balb/c control mice. In reconstituted SCID mice, the number of single follicles was significantly higher at 2, 4, 6, or 10 weeks compared with the Balb/c control mice.
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The FAE covering the domes of the Peyer's patches of SCID mice was composed of enterocytes, goblet cells, and M-cells, whereas the FAE overlying the single follicles lacked goblet cells. In the present study, AAA (Figure 1 Figure 2 Figure 3) and UEA-I (Figure 4 and Figure 5) selectively stained the cell membranes and cytoplasmic contents of M-cells in the FAE of the single primary follicles and Peyer's patches of bone marrow-reconstituted SCID mice and Balb/c controls. The adjacent enterocytes and goblet cells on FAE were not stained with these lectins. Other lectins, including those with specificities for mannose (ConA), N-acetylgalactosamine (DBA), or sialic acid (MAA) did not bind to M-cells. In all sections examined, ConA (Figure 6) and MAA (Figure 7) were bound to the subepithelial connective tissue, whereas DBA binding was seen in the goblet cells (Figure 8). Preincubations of UEA-I and AAA with fucose resulted in complete inhibition of their binding to M-cells (Figure 9). In other sections incubated with biotinylated ConA, DBA, and MAA in the presence of specific monosaccharides, complete loss of their binding to connective tissue and goblet cells was found.
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Discussion |
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The development of GALT depends on the presence of T- and B-lymphocytes, and therefore SCID mice are deficient in Peyer's patches (
In this study, when the percentage distribution and the relative proportion of primary follicles and Peyer's patches in reconstituted SCID mice were compared with the Balb/c controls, there was an increase in the percentage of primary follicles and a decrease in the percentage of Peyer's patches between 2 and 6 weeks after the bone marrow transfer. However, after 10 weeks there was no difference between the reconstituted SCID mice and controls. These data suggest that after transfer of syngeneic bone marrow into SCID mice, lymphocytes initially infiltrate the gut lamina propria to home on the single primary follicles, and the reconstitution of mucosal tissue by the donor population is well established in Peyer's patches by 10 weeks after the cell transfer.
The M-cells characteristically display short, irregular microvilli and basal intraepithelial pockets containing lymphocytes and macrophages (-L-fucose-specific lectins bind selectively to cell membranes and cytoplasmic contents of M-cells (
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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Bosma GC, Custer RP, Bosma MJ (1983) A severe combined immunodeficiency mutation in the mouse. Nature 301:527-530[Medline]
Bye WA, Allan CH, Trier JS (1984) Structure, distribution, and origin of M cells in Peyer's patches of mouse ileum. Gastroenterology 86:789-801[Medline]
Clark MA, Jepson MA, Simmons NL, Booth TA, Hirst BH (1993) Differential expression of lectin-binding sites defines mouse intestinal M-cells. J Histochem Cytochem 11:1679-1687
Danguy A, Akif F, Pajak B, Gabius HJ (1994) Contribution of carbohydrate histochemistry to glycobiology. Histol Histopathol 9:155-171[Medline]
Dorschkind K, Keller GM, Phillips RA, Miller RG, Bosma GC, O'Toole M, Bosma GC (1984) Functional status of cells from lymphoid and myeloid tissues in mice and severe combined immunodeficient disease. J Immunol 132:1804-1808
Ermak TH, Owen RL (1987) Phenotype and distribution of T lymphocytes in Peyer's patches of athymic mice. Histochemistry 87:321-325[Medline]
Falk P, Roth KA, Gordon GI (1994) Lectins are sensitive tools for defining the differentiation programs of mouse gut epithelial cells lineages. Am J Physiol 266:G987-1003
Gebert A, Hach G (1993) Differential binding of lectins to M cells and enterocytes in the rabbit caecum. Gastroenterology 105:1350-1361[Medline]
Giannasca PJ, Giannasca KT, Falk P, Gordon GI, Neutra MR (1994) Regional differences in glycoconjugates of intestinal M cells in mice:potential targets for mucosal vaccines. Am J Physiol 267:G1108-1121
Giannasca PJ, Neutra MR (1994) Interactions of microorganisms with intestinal M cells: mucosal invasion and induction of secretory immunity. Infect Agent Dis 2:242-248
Hilbert DM, Anderson AO, Holmes KL, Rudikoff S (1994) Long-term reconstitution of SCID mice suggests self-renewing B and T cell populations in peripheral and mucosal tissues. Transplantation 58:466-475[Medline]
Kirchgessner CU, Patil CK, Evans JW, Cuomo CA, Fried LM, Carter T, Oettinger MA, Brown JM (1995) DNA-dependent kinase (P350) as a candidate gene for the murine SCID defect. Science 267:1178-1183[Medline]
Kraehenbuhl J-P, Neutra MR (1992) Molecular and cellular basis of immune protection of mucosal surfaces. Physiol Rev 72:853-879
Neutra MR, Frey A, Kraehenbuhl J-P (1996a) Epithelial M cells: gateways for mucosal infection and immunization. Cell 86:345-348[Medline]
Neutra MR, Pringault E, Kraehenbuhl J-P (1996b) Antigen sampling across epithelial barriers and induction of mucosal immune responses. Annu Rev Immunol 14:275-300[Medline]
Plendl J, Schönleber B, Schmahl W, Schumacher U (1989) Comparison of the unmasking of lectin receptors by neuraminidase and by enzyme free buffer alone. J Histochem Cytochem 37:1743-1744[Medline]
Reichman FM, Bosma MJ, Hardy RR (1993) B-lineage cells in mu-transgenic SCID mice proliferate in response to IL-7 but fail to show evidence of immunoglobulin light chain gene arrangement. Int Immunol 5:303-310[Abstract]
Reynaud C-A, Mackay CR, Müller RG, Weill J-C (1991) Somaticgeneration of diversity in a mammalian primary lymphoid organ: the sheep ileal Peyer's patches. Cell 64:995-1005[Medline]
Savidge TC (1996) The life and times of an intestinal M cell. Trends Microbiol 4:301-306[Medline]
Sharma R, van Damme Els JM, Peumans WJ, Sarsfield P, Schumacher U (1996) Lectin binding reveals divergent carbohydrate expression in human and mouse Peyer's patches. Histochem Cell Biol 105:459-465[Medline]