Unique Microanatomy of Ileal Peyer's Patches of the One Humped Camel (Camelus dromedarius) is not Age-Dependent.код для вставкиСкачать
THE ANATOMICAL RECORD 291:1023–1028 (2008) Unique Microanatomy of Ileal Peyer’s Patches of the One Humped Camel (Camelus dromedarius) Is Not Age-Dependent MOHAMED ZIDAN1 AND REINHARD PABST2* Department of Histology and Cytology, Faculty of Veterinary Medicine, Alexandria University, Egypt 2 Institute of Functional and Applied Anatomy, Hannover Medical School, Germany 1 ABSTRACT The Peyer’s patches (PP) have been intensely investigated in several species because this is an important entry site for antigens and infectious agents. There are many PP in the jejunum, and in some species such as ruminants, carnivores, and omnivores, a different continuous PP is found in the terminal ileum. This PP disappears with age in these species studied. So far the ileal PP (IPP) has only been examined in the camel by light microscopy. Therefore, the localization of ileal Peyer’s patches in the dromedary camel at different ages, as well as the histology and ultrastructures were now investigated. The IPP were characteristically seen as dark rose-colored isolated structures in the shape of a cup, arranged in three irregular rows. The central row was antimesenteric. Each patch was formed by several mainly elongated dome regions ﬂanked by intestinal villi. In cross-sections these domes appeared as short, wide villi. The domes were formed from lymphoid follicles covered with a typical domeassociated epithelium of enterocytes and M cells without any goblet cells. The M cells showed variable appearance depending on the functional status. The lymphoid follicles expressed clear germinal centers. High endothelial venules were localized in the interfollicular region. In contrast to other species the IPP were still present with a comparable macroscopic and histological structure in camels of 25 years of age. Anat Rec, 291:1023–1028, 2008. Ó 2008 Wiley-Liss, Inc. Key words: ileal Peyer’s patches; dome area; age-dependence; camel; electron microscopy Peyer’s patches (PP) are the most frequently studied structures in the gut-associated lymphoid tissues forming a central part of the inductive site of the mucosal immune system (Brandtzaeg and Pabst, 2004). PP are subepithelial aggregations of lymphoid tissue located along the antimesenteric side of the small intestine (Owen and Jones, 1974; Owen, 1977). The number, size, and distribution of PP vary according to species (LieblerTenorio and Pabst, 2006). The PP in the jejunum have been studied in rodents and many larger species including humans in much detail. These PP are composed of germinal center, corona, dome, and interfollicular region (Abe and Ito, 1977). The follicle-associated epithelium (FAE) covering the domes of these lymphoid structures Ó 2008 WILEY-LISS, INC. Abbreviations used: FAE 5 follicle-associated epithelium; IPP 5 ileal Peyer’s patch; PP 5 Peyer’s patches; SEM 5 scanning electron microscopy. Grant sponsor: Deutsche Forschungsgemeinschaft; Grant number: SFB 621, A10. *Correspondence to: Reinhard Pabst, Anatomie II 4120, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Fax: 49-511-532-2948. E-mail: firstname.lastname@example.org Received 4 December 2007; Accepted 25 February 2008 DOI 10.1002/ar.20697 Published online 30 April 2008 in Wiley InterScience (www. interscience.wiley.com). 1024 ZIDAN AND PABST contains specialized M (membrane) cells that constitute a very thin epithelial barrier (Bockman and Cooper, 1973; Owen and Jones, 1974). These cells have characteristic ultrastructural features with short, irregular microvilli and basal pockets harboring lymphocytes and macrophages (Bye et al., 1984; Jarry et al., 1989; Gebert et al., 1996). However, M cells show different morphological characteristics in various species (Owen, 1977; Bye et al., 1984; Madara et al., 1984; Sicinski et al., 1986; Gebert and Hach, 1993; Gebert and Bartels, 1995). There are different morphologies of M cells in calves (Liebler et al., 1988, 1991) and in rats (Onishi et al., 2007). M cells play an important role in transporting particulate, microbial and soluble antigenic substances from the lumen to the subepithelial immunocompetent cells, thereby initiating mucosal immune responses (Gebert et al., 1996). In several species, in the terminal part of the ileum a different PP has been described. It was documented in sheep that this ileal or continuous PP is the site of B lymphocytopoiesis and it regresses with age (for review, see Pabst and Rothkötter, 2006). In pigs the ileal PP (IPP) disappears much later (at 4.5 years only scattered lymphoid aggregations are seen) but the PP in the jejunum remain present into older age (reviewed in Binns and Pabst, 1994). Alluwaimi et al. (1998) had described the anatomy and histology of IPP of six camels. This study showed the basic histology and revealed that the IPP of the camel were cup-shaped structures elevated approximately 1 cm above the lumen and distributed in the antimesenteric side of the ileum. Each patch consisted of an aggregation of secondary lymphatic nodules occupying the submucosa. The FAE consisted of simple columnar cells and M cells but without goblet cells. This epithelium was inﬁltrated by lymphocytes. However, no details of the age were given. Fath El-Bab et al. (2000) described the alkaline and acid phosphatase enzymatic reactivity of the IPP of the camel and showed that the M cells appeared as unstained sites in the FAE which expressed stained luminal borders. As these studies of the camel IPP were limited to light microscopic investigations, the ultrastructure has now been studied by transmission electron microscopy and scanning electron microscopy (SEM) in addition to light microscopy. These ﬁndings might be used to study the antigen uptake in oral vaccination protocols in this species, which is economically important in various parts of the world. MATERIALS AND METHODS Specimens The ilea of 10 clinically healthy male camels (5 camels aged 3–5 years and 5 camels aged 20–25 years) were obtained directly after slaughtering in Koom Hamada slaughterhouse, Behera, Egypt. IPP were identiﬁed macroscopically and dissected for the following studies. Light Microscopy. IPP were ﬁxed in 10% phosphate buffered formalin. Specimens were processed for parafﬁn embedding. Sections (5 mm) were prepared and stained with hematoxylin and eosin, Gomori reticulin, and Crossman trichrome stains. Scanning Electron Microscopy. Specimens were ﬁxed in 10% phosphate buffered formalin, rinsed in phosphate buffered saline (PBS) pH 7.3 and dehydrated in a series of acetone dilutions. Critical-point drying was done using a Balzers CPD 030 (Balzers, Liechtenstein) and sputter-coating using a Polaron E5400 (Polaron, Watford, UK) and a gold-palladium target. The specimens were examined in a PSEM 500 scanning electron microscope (Philips, Eindhoven, The Netherlands) equipped with a PC-based device for recording digital images at a high resolution (Gebert and Preiss, 1998). Transmission Electron Microscopy. Fresh specimens, approximately 1 mm3 in size, were obtained from the IPP and ﬁxed in 4% phosphate buffered glutaraldehyde pH 7.4 for 2 hr at 48C (McDowell and Trump, 1976). After post-ﬁxation in 1% solution of phosphate buffered osmium tetroxide at 48C, specimens were dehydrated in ascending grades of ethanol and embedded in Epon (Serva, Heidelberg, Germany) according to standard protocols. Semithin sections (1 mm) were prepared, stained with toluidine blue and examined by light microscopy. Suitable areas for electron microscopic examination were selected. From these areas ultrathin sections (50–70 nm) were cut by a diamond knife and stained with urinyl acetate followed by lead citrate. The sections were observed with a Zeiss EM10 electron microscope (Zeiss, Oberkochen, Germany) at 80 kV. RESULTS The IPP were identiﬁed macroscopically on the lateral surface of the terminal 20 cm of the ileum as dark rosecolored, isolated patches distributed in the wall. On the mucosal surface, the IPP were arranged in three irregular rows, the central row facing the mesentery. The other two rows extended at both sides of the central one. The IPP appeared as elevated, dark rose-colored cupshaped structures with central 1–3 nodules (Fig. 1A). No age-related macroscopic variations in the shape or the arrangement of the IPP were observed despite the increased number of intestinal plicae in the old camels (Fig. 1B). The IPP consisted of groups of lymphoid follicles with clear germinal centers arranged in more than one layer located in the submucosa of the ileum (Fig. 1C). These lymphoid follicles were formed by lymphocytes supported by a network of reticular ﬁbers and follicular dendritic cells (DC). The interfollicular zone consisted of diffusely arranged lymphocytes with obvious high endothelial venules. Lymphoid tissue above the apical follicles bulged toward the lumen as a dome. The dome looked like a short wide intestinal villus. It was covered with FAE. This epithelium consisted of columnar cells with microvilli as typical for enterocytes. No goblet cells were observed in this region (Fig. 1D). SEM investigation revealed that each camel IPP consisted of several dome regions which were mainly of elongated shape. A few domes were hemispherical. Intestinal villi encircled each dome (Fig. 2A,B). It was difﬁcult to determine any M cells during SEM investigation. In the interfollicular area high endothelial venules ILEAL PEYER’S PATCHES OF THE ONE HUMPED CAMEL 1025 were obvious with lymphocytes migrating through the wall (Fig. 2C). DC were seen scattered in the dome area (Fig. 2D). Seen by transmission EM several M cells were distributed among enterocytes (Fig. 3A). In general, M cells were rich in mitochondria with their cytoplasm elevated above that of neighboring enterocytes. Depending on the number of apical processes, M cells were variable in appearance, ranging from M cells without any processes (Fig. 3B), and M cells with a few short processes (Fig. 3C) to M cells with several short processes (Fig. 3D). Several cytoplasmic pockets contained lymphocytes or macrophages. The M cells were tightly connected to the neighboring enterocytes by junctional complexes of tight junctions, desmosomes, and interdigitations (Fig. 3E). The histology revealed no age-dependence of the size of the dome follicular or interfollicular area with similar HEV. The more round or oval-shaped follicles and domes sometimes observed depended on the direction of the sections but were not age-correlated. DISCUSSION The current work supports the ﬁndings of Alluwaimi et al. (1998) that the camel IPP were distributed in the terminal 20 cm of the ileum. The present study extends these observations by detailed scanning and transmission EM data and documents that the IPP has a characteristic arrangement and shape different from that of other species (Liebler-Tenorio and Pabst, 2006). It is arranged in three rows of cup-shaped structures. The central one is antimesenteric, while cattle, sheep, pigs and dogs have one continuous patch in the ileum (for review see Liebler-Tenorio and Pabst, 2006). Macroscopically no PP could be identiﬁed in the jejunum at any age. It cannot be excluded that isolated follicles could be found microscopically and these fulﬁll the function of jejunal PP in other species (Brandtzaeg and Pabst, 2004). In contrast to the ﬁndings of Alluwaimi et al. (1998), there is no age-related involution of IPP of the camel, as the present study showed the same structure in 25-yearold camels. In other species, IPP disappear at approximately 15 months of age in sheep and goat, and in old horses. Similarly, age-related involution occurs in IPP of cattle and sheep over 2 years of age and in the pig some time later at approximately 4 years of age (reviewed in Liebler-Tenorio and Pabst, 2006; Yasuda et al., 2006). This makes it very likely that the camel IPP plays a continuous role in immunity during the animal’s life. Alluwaimi et al. (1998) showed by routine histology that the mucosa above the IPP of the one humped camel Fig. 1. The IPP are arranged in three rows (1–3). A: Ileum of a 3year-old camel with a few intestinal plicae. B: Ileum of a 25-year-old camel with several plicae. C: The histological overview demonstrates villi (V), the dome (D) and lymphoid follicle and the interfollicular area (IFA). D: Higher magniﬁcation of a dome (D). It consists of lymphoid aggregation bulging into the lumen. The FAE (e) differs from the surrounding nondome epithelium of the villi (V) by the presence of numerous lymphocytes (arrowheads) and the absence of goblet cells (arrows). Hematoxylin and eosin stain. E: Semithin section of the dome area (D) showing the distribution of M cells (arrowheads) among the enterocytes. Toluidine blue stain. L, lymphocytes. 1026 ZIDAN AND PABST Fig. 2. A: Scanning electron micrograph showing that the camel IPP is formed from several dome areas (D) bulging toward the gut lumen and surrounded by villi (arrows). The majority of the dome areas are elongated. B: Scanning electron microscopy of IPP shows one hemispherical dome area (D) surrounded by intestinal villi (V). C: High endothelial venule with lymphocytes (arrows) on their migratory route into the T cell area of the PP. D: A dendritic cell (D) in the dome area engulfs a plasma cell (P). The arrows indicate the width of the plasma cell. A lymphocyte (L) is also marked. was devoid of intestinal villi while the present study documents the presence of a single row of intestinal villi encircling each dome area. Similar ﬁndings were described in other species (Kuhn and Kaup, 1996; Gebert, 1997; Beier and Gebert, 1998). The domes of the camel IPP appeared like a short but wide villus similar to that described in ruminants called pseudovilli (Liebler-Tenorio and Pabst, 2006). The domes were hemispherical in rhesus macaques (Kuhn and Kaup, 1996) and mice and gerbils (Komazawa et al., 1991). In agreement with Aleksandersen et al. (1991) and Alluwaimi et al. (1998), the FAE were devoid of goblet cells. The lymphoid follicles of the IPP of the camel were characteristically arranged in more than one row where the lymphoid tissue above the top of the follicles bulged toward the lumen as a dome. Similar to other species (Liebler-Tenorio and Pabst, 2006), follicular DC were observed in the follicles and also high endothelial venules were identiﬁed in the interfollicular region, which is the T lymphocyte region in other species. The DC are essential for antigen presentation and the high endothelial venules are the only entrance site for recirculating lymphocytes. The M cells of camel IPP showed variable morphological appearance. In contrast, in cattle IPP the M cells were uniform in appearance (Landsverk, 1981; TorresMedina, 1981; Landsverk et al., 1991; Parsons et al., 1991). In the pig M cells of the large intestine have a more variable appearance than those of the small intestine (for review, see Liebler-Tenorio and Pabst, 2006). In bovine PP, differences in the microvillus development on M cells, particularly obvious in the large intestine, were interpreted as different stages of cellular maturation (Liebler et al., 1988, 1991). The different morphologies of M cells in the rat might be reﬂected by the functional difference in uptake of luminal antigens between immature M cells and typical M cells (Onishi et al., 2007). Therefore, the variable appearance of M cells of the camel may be related to different stages of development or different functional stages. As there are no afferent lymphatics in the mucosal-associated lymphoid tissue, antigen sampling occurs only by means of the M cells (Liebler-Tenorio and Pabst, 2006). In agreement with previous observations (Faulk et al., 1971; Owen and Jones, 1974; Torres-Medina, 1981), the present study showed that the M cells were tightly con- ILEAL PEYER’S PATCHES OF THE ONE HUMPED CAMEL Fig. 3. A: Electron micrograph showing an M cell (M) with elevated cytoplasm rich in mitochondria and some apical processes. E, enterocytes; mac, macrophage. B: Electron micrograph showing an M cell (M) with elevated cytoplasm rich in microfolds and long microﬁlament bundles. E, enterocyte. C: Electron micrograph showing an M cell (M) containing a group of lymphocytes (L) within its cytoplasmic pockets. 1027 D: Electron micrograph showing an M cell (M) with elevated cytoplasm rich in mitochondria and absence of any apical processes. L, migrating lymphocytes; E, enterocytes. E: Electron micrograph showing the junctional complex (arrow) of tight junctions and desmosomes between an M cell (M) and enterocytes (E). 1028 ZIDAN AND PABST nected to the neighboring enterocytes by junctional complexes of tight junctions, desmosomes and interdigitations. These junctions act as a barrier against invading antigens and support the antigen sampling function of M cells. The IPP of the camel are well organized lymphoid structures that persist without age-related involution. Their M cells probably play an important role in antigen sampling. The macrophages and DC are essential for antigen presentation. The lymphoid follicles produce activated lymphocytes, which might later mature into plasma cells to produce speciﬁc IgA antibodies for mucosal immunity. The presence of lymphocytes in the wall of high endothelial venules indicates the participation of IPP in the route of recirculating lymphocytes. 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