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Unique Microanatomy of Ileal Peyer's Patches of the One Humped Camel (Camelus dromedarius) is not Age-Dependent.

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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: pabst.reinhard@mh-hannover.de
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.
In conclusion, the IPP in camels might be the site of
entry for antigens throughout life, and, therefore, might
be used for enteric vaccination procedures in this species.
ACKNOWLEDGMENTS
The technical assistance of K. Westermann, S. Fassbender, and G. Preiss, the expert help of M. Peter in preparing the figures and the revision of the English text
by S. Fryk are gratefully acknowledged.
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