Secondary Logo

Journal Logo

Original articles

Histological, immunohistochemical, and morphometric study on the postnatal development of Peyer's patches in albino rat

Hassan, Mahmoud M.; El-Aleem, Somaya Abd; Hammouda, Gehad A.; Shaban, Ibrahim

Author Information
The Egyptian Journal of Histology: June 2011 - Volume 34 - Issue 2 - p 198-207
doi: 10.1097/01.EHX.0000396502.60108.fa
  • Free

Abstract

Introduction

Peyer's patches (PPs) are large masses of confluent lymphatic nodules situated in the wall of the small intestine, particularly the ileum. Such permanent aggregates of confluent uncapsulated lymphatic nodules, together with solitary nodules of the temporary type, make up a large part of the gut-associated lymphoid tissue [1].

In humans, there are up to 200 oval lumps of lymphoid tissue along the length of the small intestine. Each is an accumulation of up to 60 lymphoid follicles composed of B lymphocytes with T lymphocytes in the interfollicular areas [2].

These patches are situated just beneath the gut epithelium and gut antigens can readily pass into the lymphoid tissue. The epithelium covering PPs is modified. A new cell type, called M cells, appeared and acted as antigen sampling cells allowing the passage of gut antigens through them [3]. Such antigen sampling is thought to be an important stage in the process of generating immunoglobulin A-producing plasma cells. Secretion of immunoglobulin A is an effective defense mechanism that guards against infection by way of the mucosal membranes. Moreover, they are able to decrease the adherence of microbes to the epithelial surface and they can neutralize viruses and intraluminal bacterial toxins [4].

PPs are involved in defense against pathogens that may be colonizing the gut, but they are also involved in oral tolerance. Food antigens are foreign and the immune system should recognize food as an antigenic, potentially threatening challenge to the body's survival. However, humans are clearly able to absorb food antigens through the gut into the bloodstream with no obvious ill effects [5]. The underlying detailed mechanisms are still not well understood, but it would seem that PPs are involved in monitoring food antigens. The PPs decide what are the dangerous gut pathogens to which the body should respond and what are the food antigens that the body should accept. The lymphoid tissue is somehow capable of telling the lymphocytes that they should not respond to food antigens. The lymphocytes are tolerized and become nonresponsive to these antigens [5]. Therefore, this study was designed to shed the light on structural and correlated functional modifications that occur during the postnatal development of PPs.

Materials and methods

Fifty male albino rats were used in this study. Rats were kept under good hygienic conditions with free access to food and water ad libitum. The study was conducted according to the guidelines of care and use of laboratory animals. The animals were divided into five equal groups according to age. Group 1 (1 day old), group 2 (1 week old), group 3 (2 weeks old), group 4 (4 weeks old), and adult group 5 (8 weeks old). The unweaned rats (below the age of 3 weeks) were not separated from their nursing mothers till they were killed. At the specified age of each group, the animals were killed; the terminal part of the ileum was excised and processed for light and electron microscopic examinations. The specimens were fixed in 10% formol saline, and processed for paraffin sectioning (5 μm thick). The sections were stained with:

  • (1) Hematoxylin and eosin.
  • (2) Periodic acid-Schiff stain (PAS).
  • (3) Immunohistochemical staining for detection of CD3 and CD20 (markers of T and B lymphocytes, respectively) [6].

Immunohistochemical staining was carried out using the avidin–biotin peroxidase system. The primary antibody used was a mouse monoclonal antibody (Sigma, St.Louis, USA) and avidin–biotin peroxidase universal kits were used (Nova Castra Laboratories Ltd, London, UK). Negative control specimens were prepared without the primary antibody to test for its specificity. Lymph node sections were used as a positive control. Immunohistochemical staining was performed in the pathology unit in Ain Shams Specialized Hospital.

For scanning electron microscopic (SEM) examination, small pieces of the terminal ileum were immediately fixed in 2% paraformaldehyde/2.5% glutaraldehyde in 0.1 m of phosphate buffer for 3 h. Specimens were examined with an XL30 SEM (PHILIPS, Amsterdam, the Netherlands) in the anatomy Department, Faculty of Medicine, Ain Shams University.

For transmission electron microscopic (TEM) examination, small pieces were immediately fixed in buffered formol glutaraldehyde and postfixed in 1% osmium tetroxide. Ultrathin sections were stained with uranyl acetate and lead citrate [7]. The specimens were examined using TEM (PHILIPS, Amsterdam, the Netherlands) in Electron Microscopic unit in Ain Shams specialized Hospital.

Quantitative morphometric measurements

Quantitative morphometry was performed using Digimizer image analysis software (Version 3.0.4.0, 2007, MedCalc Software, Mariakerke, Belgium). Equally magnified images of selected areas were captured by Photography Microscope (Carl Zeiss, Jena, Germany) and areas of PPs were encircled by line path.

  • (1) Lymphocytes were counted, and consequently cellular density was estimated in hematoxylin and eosin-stained sections.
  • (2) B lymphocytes were counted in anti-CD20-stained sections.
  • (3) T lymphocytes were counted in anti-CD3-stained sections.
  • (4) The number of goblet cells was counted per unit length of epithelial surface in PAS-stained sections.

Statistical analysis

Measured variables were collected, tabulated, and statistically analyzed in comparison with the preceding age group and/or within the same age group. SPSS (SPSS Inc., Illinois, USA) was used for data entry and analysis [8]. The parameters were tested using Student's t-test, and the results were significant when P value was less than 0.05.

Results

Light microscopic examination

Results of this study revealed that in group 1 rats, the primitive PPs were identified as small dome-shaped epithelial elevations; each overlies a small aggregate of lymphocytes (Fig. 1). The primitive PPs were populated mainly with T lymphocytes (Fig. 2), whereas B lymphocytes were occasionally found and represented a minority (Fig. 3).

Figure 1
Figure 1:
Showing the structure of a primitive Peyer's patch of a 1-day-old rat. Small aggregates of lymphocytes observed in its core of connective tissue (↑). The overlying epithelial surface lacks villi and crypts (↑↑). H&E×250.
Figure 2
Figure 2:
Showing positive reaction to CD3 in the majority of lymphocytes (↑) in the primitive Peyer's patch of a 1-day-old rat. Avidin–biotin peroxidase and hematoxylin counterstain ×6400.
Figure 3
Figure 3:
Showing positive reaction to CD20 in few lymphocytes (↑) in the primitive Peyer's patch of a 1-day-old rat. Avidin–biotin peroxidase and hematoxylin counterstain ×640.

PPs in rats of group 2 showed an increase in the patch size accompanied by a statistically significant increase in the density of lymphocytic infiltration (Fig. 4). B lymphocytes increased markedly compared with group 1; however, T lymphocytes were still the major type that was found. No separate zones of B and T lymphocytes were found (Figs 5 and 6).

Figure 4
Figure 4:
Showing the structure of Peyer's patch of a 1-week-old rat, with dense lymphocytic infiltration (↑). Notice ordinary villi (V), crypts (C). H&E×250.
Figure 5
Figure 5:
Showing positive reaction to CD3 in the majority of lymphocytes (↑) in a 1-week-old rat Peyer's patch. Avidin–biotin peroxidase and hematoxylin counterstain ×640.
Figure 6
Figure 6:
Showing positive reaction to CD20 in few lymphocytes in a 1-week-old rat Peyer's patch (↑). Avidin–biotin peroxidase and hematoxylin counterstain ×640.

The PPs in rats of group 3 showed further marked increase in patch size, with significant increase in the density of lymphocytic population in mucosa and extending to the submucosa when compared with group 2 (Fig. 7). Moreover, PPs at this age showed prominent primary B-lymphocytic follicles separated by the diffuse T-lymphocytic follicles (Figs 8 and 9).

Figure 7
Figure 7:
Showing the Peyer's patch of a 2-week-old rat, with densely packed lymphocytes extending to the submucosa. The overlying epithelial surface (FAE ↑) lacks villi and crypts. H&E×250.
Figure 8
Figure 8:
Showing positive reaction to CD3 in the interfollicular area (IFA) of the Peyer's patch of a 2-week-old rat whereas the follicular area (FA) negatively stained. Avidin–biotin peroxidase and hematoxylin counterstain ×640.
Figure 9
Figure 9:
Showing positive reaction to CD20 B lymphocytes in the follicular area (FA) of Peyer's patch of a 2-week-old rat. Avidin–biotin peroxidase and hematoxylin counterstain ×640.

In group 4 rats, an increase in the patch size and the number of lymphoid follicles were noticed, with significant increase in the density of lymphocytic infiltration especially in follicular areas. Moreover, some of the lymphoid follicles showed germinal centers (Fig. 10). Lymphoid follicles were also primarily populated with densely packed B lymphocytes, whereas T lymphocytes were found in between the follicles (Figs 11 and 12).

Figure 10
Figure 10:
Showing the Peyer's patch of a 4-week-old rat. Follicular area (FA) is filled with densely packed lymphocytes. Notice the absence of villi and crypts, presence of pale germinal center in lymphoid follicles (GC). H&E×250.
Figure 11
Figure 11:
Showing CD3-positive T lymphocytes (↑) in the interfollicular area (IFA) of the Peyer's patch of a 4-week-old rat whereas the follicular area (FA) is negatively stained. Avidin–biotin peroxidase and hematoxylin counterstain ×640.
Figure 12
Figure 12:
Showing densely packed B lymphocytes with positive reaction to CD20 (↑) in the follicular area (FA) of the Peyer's patch of a 4-week-old rat. Avidin–biotin peroxidase and hematoxylin counterstain ×640.

In group 5 rats, no remarkable changes were noticed apart from increase in the size of the PP and significant increase in the density of the lymphocytic population. Densely packed B lymphocytes were populating the follicular area, whereas T lymphocytes were present in the interfollicular area as in group 4.

The mucosa at the site of PPs was lacking the presence of villi and crypts in all studied age groups; instead dome-shaped elevations were found. Moreover, the covering epithelium showed a significantly reduced number of PAS positive goblet cells when compared with neighboring villi and crypts (Figs 13 and 14).

Figure 13
Figure 13:
Showing the presence of periodic acid-Schiff (PAS) positive goblet cells (↑) in the primitive Peyer's patch (PP) of a 1-day-old rat, in both epithelial surfaces overlying the PPs and also the epithelial covering of villi. PAS ×250.
Figure 14
Figure 14:
Showing the follicle-associated epithelium (FAE) of Peyer's patch (PP) of a 8-week-old rat, lacking the presence of goblet cells. Note the numerous goblet cells (↑) in the nearby villi and crypts. Periodic acid-Schiff ×250.

Ultrastructurally, scanning electron microscopic examination of groups 1 and 2 showed the dome-shaped epithelium overlying the PPs without M cells and also the villi with their overlying epithelium (Fig. 15). However, M cells were detected from the age of 2 weeks onward and were characterized by the lower apical surface that lacks the microvillous carpet (Fig. 16).

Figure 15
Figure 15:
Scanning electron micrograph of the ileum of a 1-day-old rat, showing the epithelial surface overlying the Peyer's patch. The surface is covered with absorptive columnar cells and goblet cells (↑). ×4250.
Figure 16
Figure 16:
Scanning electron micrograph of the ileum of a 8-week-old rat, showing the surface of a dome overlying the Peyer's patch. Notice absorptive columnar cells with carpet of microvilli and M cell without this microvillus carpet (↑). ×2250.

TEM examination of group 5 specimens showed that the M cell was shorter with fewer microvilli and their cytoplasm appeared paler with fewer organelles. The basal region of the M cell showed presence of two nuclei; one of which appeared kidney shaped mostly belonged to the macrophage within the basolateral pocket. The basal lamina underneath the M cell was incomplete (Fig. 17).

Figure 17
Figure 17:
Transmission electron micrograph of the ileum of a 8-week-old rat, showing a goblet cell (G) with mucous granules (mv), absorptive columnar cell (AC) with many microvilli (mv), and an M cell (M) with fewer microvilli on the free border. Note the incomplete basal lamina underlying the M cell (↑). M cells exhibit an oval dark nucleus (N1), another kidney-shaped nucleus (N2) in the basolateral pocket. ×2950.

Morphometric results

Total lymphocytic cellular density

All lymphocytes were counted per unit area of PPs in different age groups, and total lymphocytic cellular density was expressed in n/1000 μ2. Statistically, the total lymphocytic cell count increased significantly (P<0.05) in all age groups compared with the preceding age except for the adult age Histogram 1.

Histogram 1
Histogram 1:
Histogram 1. Mean count of all lymphocytes per unit area of Peyer's patches in different age groups (n/1000 μ2).
Histogram 2
Histogram 2:
Histogram 2. Mean count of B and T lymphocytes per unit area of Peyer's patches in different age groups (n/1000 μ2).
Differential lymphocytic cellular density

The count of B lymphocytes increased progressively in all age groups and it was significant (P<0.05) in 2 and 4 week age groups compared with the preceding ages. T lymphocytes were also increasing progressively in all age groups and it was significant in 1, 2, and 4 week age groups (P<0.05). Comparing B-lymphocytic and T-lymphocytic cell counts within the same group revealed statistical significance (P<0.05) in young age groups (1 day and 1 week). Meanwhile, the B-lymphocyte count was significantly higher in the last two groups (P<0.05) Histogram 2.

Count of goblet cells

The count of goblet cells decreased significantly in the epithelium covering PPs compared with those covering the villi which was increasing (P<0.05) Histogram 3.

Histogram 3
Histogram 3:
Histogram 3. Mean count of goblet cells per unit length of epithelium on Peyer's patches and villi in different age groups (n/100 μ).

Discussion

This study was conducted to outline the postnatal development of PPs from birth to adult life. In 1-day-old rats, the primitive PPs appeared as small dome-shaped epithelial elevations overlying small aggregates of lymphocytes with no special arrangement. This result was in accordance with those of some researchers [9] who demonstrated the presence of small clusters of lymphocytes in developing PPs in newly born rats. Moreover, these primitive PPs were detected earlier in prenatal life using monoclonal antibodies specific for detection of T lymphocytes and dendritic cells in rat and bovine fetuses [10–12]. In 1-day-old rats, PPs were populated mainly with T lymphocytes (CD3), whereas B lymphocytes (CD20) were occasionally found. T cells in early life are supposed to express a distinct pattern of homing molecules, allowing them to interact with and transmigrate through the postcapillary high endothelial venules [13]. Such high endothelial venules were detected in postnatal life from birth to adult age in this study. In other researchers' study [14–16], dendritic cells were proved to be the first nonlymphoid cells to appear in the PPs during its prenatal development. These dendritic cells were supposed to be the initiating factor for development of PPs through their interaction with T lymphocytes, facilitating their homing and localization in PPs [13,17].

PPs of 1-week-old rats showed an increase in the patch size with significant increase in density of lymphocytic infilteration. Furthermore, B lymphocytes increased markedly but were still less than T lymphocytes. In addition, no separate zones of B and T lymphocytes were found. B cells were found in small groups in between the T cells. These results were in agreement with those of some researchers [9] who found the same results. On the contrary, other researchers [16] found that the primary follicles appeared at day 3 of life in rats. This controversy might be due to difference in the monoclonal antibody used.

PPs of 2-week-old rats showed further increase in patch size and lymphocytic density in addition to the appearance of primary lymphoid follicles. Lymphoid nodules were primarily populated with B lymphocytes whereas T lymphocytes were present between the follicles. These findings were in accordance with those of some researchers [16] who found similar results at ages between 9 and 14 days after birth. The appearance of dense B-lymphocytic aggregates was proved to be due to dendritic cells, which secretes chemokines and attracting molecules, attracting B lymphocytes to the developing PPs [13].

PPs of 4-week-old rats revealed an increase in patch size and significant increase in density of lymphocytic infiltration with appearance of germinal centers. These results were in agreement with those of some researchers [9] who found similar results. On the contrary, other researchers [17] found that maturity of PPs was reached at 21 days after birth. B lymphocytes were found mainly in lymphoid nodules, whereas T lymphocytes were predominant in the interfollicular areas. The development of secondary B lymphocytic nodules was supposed to be due to antigenic challenge initiated by the change in intestinal flora accompanying weaning [18].

Apart from the increase in size of PPs, the results of the adult age group were entirely identical to those of 4-week-old rats.

In this study, the epithelium overlying PPs showed marked decrease in the number of goblet cells till adult age. This result was in accordance with those of some researchers [19] who found such decrease from 3 weeks onward.

SEM study revealed that M cells were detected from the age of 2 weeks onward. TEM examination confirmed the appearance of M cells in the adult age group by their specific structural criteria. None of the available literature reported the age at which M cell first appears. Two conflicting theories were hypothesized for the origin of M cell. One theory assumes that M cell originates from absorptive enterocytes by a series of morphofunctional changes induced by lymphocytes in the PPs. This theory is supported by a number of in-vivo and in-vitro studies. PPs lymphoid cells were injected under epithelium of nonpatch area, in intestine of mice and rabbit, which caused the development of PP-like structure with appearance of epithelial cell similar in structure and function to M cell. Moreover, depletion of B and T lymphocytes (by irradiation) did not permit the development of typical follicle-associated epithelium including M cells [20–23]. The other theory postulates that M cells arise from unique cell lineage different from that of absorptive enterocytes [24–27]. The results of this study supposed that the coincidence of lymphoid follicles and M cells might have been induced by antigenic stimulation, which occurs after weaning.

Conclusion

It could be concluded that many changes occurring during the development of PPs follow a plan of structure–function adaptation. This might explain the gradual decrease of goblet cells, with concomitant appearance and increase of M cells to adapt for the immunological function of PPs. Similarly, the absence of villi and crypts in areas of PPs indicates its weak absorptive and secretory functions when compared with other areas of the small intestinal mucosa.

Table
Table:
No title available.

References

1. Cormack DH Ham's histology. 19879th ed Philadelphia Lippincott Co.
2. Debard N, Sierro F, Kraehenbuhl JP. Development of Peyer's patches, follicle-associated epithelium and M cell: lessons from immunodeficient and knockout mice. Semin Immunol. 1999;11:183–191
3. Mebius RE. Organogenesis of lymphoid tissues. Nat Rev Immunol. 2003;3:292–303
4. Befus AD, Bienenstock JHay J. The mucosa associated immune system of the rabbit. Animal models of immunological processes. 1982 London, UK Academic Press:167
5. Mowat AM, Viney JL. The anatomical basis of intestinal immunity. Immunol Rev. 1997;156:145–166
6. Kiernan JA Histological and histochemical methods: theory and practice. 20003rd ed Oxford, Boston Butterworth Heinemann
7. Glauert AM, Lewis PR Biological specimen preparation for transmission electron microscopy. 19981st ed London, UK Princeton University Press
8. Armitage P, Berry GD Statistical methods in medical research. 19943rd ed Oxford, UK Blackwell Science
9. Sminia T, Janse EM, Plesch BE. Ontogeny of Peyer's patches of the rat. Anat Rec. 1983;207:309–316
10. Recher S, Raccurt M, Lambert A, Lobie PE, Mertani HC, Morel G. Prenatal and adult growth hormone gene expression in rat lymphoid organs. J Histochem Cytochem. 2001;49:347–354
11. Campbell DJ, Butcher EC. Rapid acquisition of tissue-specific homing phenotypes by CD4(+) T cells activated in cutaneous or mucosal lymphoid tissues. J Exp Med. 2002;195:135–141
12. Yasuda M, Ogawa D, Nasu T, Yamaguchi T, Murakami T. Kinetics and distribution of bovine gammadelta T-lymphocyte in the intestine: gammadelta T cells accumulate in the dome region of Peyer's patch during prenatal development. Dev Comp Immunol. 2005;29:555–564
13. Johansson Lindbom B, Agace WW. Generation of gut-homing T cells and their localization to the small intestinal mucosa. Immunol Rev. 2007;215:226–242
14. Johansson C, Kelsall BL. Phenotype and function of intestinal dendritic cells. Semin Immunol. 2005;17:284–294
15. Stenstad H, Ericsson A, Johansson Lindbom B, Svensson M, Marsal J, Mack M, et al. Gut-associated lymphoid tissue-primed CD4+ T cells display CCR9-dependent and -independent homing to the small intestine. Blood. 2006;107:3447–3454
16. Chen D, Hoshi H, Tanaka K, Murakami G. Postnatal development of lymphoid follicles in rat Peyer's patches, with special reference to increased follicle number. Arch Histol Cytol. 1995;58:335–343
17. Mora JR, Bono MR, Manjunath N, Weninger W, Cavanagh LL, Rosemblatt M, Von Andrian UH. Selective imprinting of gut-homing T cells by Peyer's patch dendritic cells. Nature. 2003;424:88–93
18. Pabst O, Ohl L, Wendland M, Wurbel MA, Kremmer E, Malissen B, Forster R. Chemokine receptor CCR9 contributes to the localization of plasma cells to the small intestine. J Exp Med. 2004;199:411–416
19. Onori P, Franchitto A, Sferra R, Vetuschi A, Gaudio E. Peyer's patches epithelium in the rat: a morphological, immunohistochemical and morphometrical study. Dig Dis Sci. 2001;46:1095–1104
20. Fujimura Y, Kihara T, Hosobe M, Ohtani K, Kamoi R, Kato T, et al. Measurement of microvilli of microfold cells (M-cells) and absorptive cells in follicle-associated epithelium of mouse Peyer's patches. Gastroenterol Jpn. 1990;25:508
21. Savidge TC, Smith MW. Evidence that membranous (M) cell genesis is immuno-regulated. Adv Exp Med Biol. 1995;371A:239–241
22. Kerneis S, Bogdanova A, Kraehenbuhl JP, Pringault E. Conversion by Peyer's patch lymphocytes of human enterocytes into M cells that transport bacteria. Science. 1997;277:949–952
23. Yamanaka T, Helgeland L, Farstad IN, Fukushima H, Midtvedt T, Brandtzaeg P. Microbial colonization drives lymphocyte accumulation and differentiation in the follicle-associated epithelium of Peyer's patches. J Immunol. 2003;170:816–822
24. Rautenberg K, Cichon C, Heyer G, Demel M, Schmidt MA. Immunocytochemical characterization of the follicle-associated epithelium of Peyer's patches: anti-cytokeratin 8 antibody (clone 4.1.18) as a molecular marker for rat M cells. Eur J Cell Biol. 1996;71:363–370
25. Gebert A, Posselt W. Glycoconjugate expression defines the origin and differentiation pathway of intestinal M-cells. J Histochem Cytochem. 1997;45:1341–1350
26. 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;154:1573–1582
27. Debard N, Sierro F, Browning J, Kraehenbuhl JP. Effect of mature lymphocytes and lymphotoxin on the development of the follicle-associated epithelium and M cells in mouse Peyer's patches. Gastroenterology. 2001;120:1173–1182
Keywords:

development; gut-associated lymphatic tissue; Peyer's patches; postnatal

© 2011 The Egyptian Journal of Histology