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Linköping University Medical Dissertation No. 1006
Barrier Function of the Follicle‐Associated Epithelium in Stress and Crohn’s disease
Åsa Keita
Division of Surgery, Department of Biomedicine and Surgery,
Faculty of Health Sciences,
SE‐581 85, Linköping, Sweden
Linköping 2007
© Åsa Keita, 2007
Copyright Åsa Keita pages 1‐105, Paper III and IV. Paper I and II have been
reprinted with the permission from the respective journal. All drawings and
photos are made by the author.
The studies in this thesis were supported by The Swedish Research Council ‐
Medicine (Project 12618), The Swedish Society for Medical Research, The Broad
Medical Research Program of the Eli and Edythe L. Broad Foundation, The Åke
Wiberg Foundation and The ʺLions Forskingsfond mot folksjukdomarʺ.
Printed by LiU‐Tryck, Linköping, Sweden, 2007.
ISBN: 978‐91‐85831‐79‐1
ISSN: 0345‐0082
Även en tusenmilafärd börjar med ett steg
Kinesiskt ordspråk
Till Alpha och Mathilda
ABSTRACT The earliest observable signs of Crohn’s disease are microscopic erosions in the follicle‐associated epithelium (FAE) covering the Peyer’s patches. The FAE, which contains M cells, is specialised in sampling of luminal content and delivery to underlying immune cells. This sampling is crucial for induction of protective immune responses, but it also provides a route of entry for microorganisms into the mucosa. Crohn’s disease is associated with an increased immune response to bacteria, and the disease course can be altered by stress. The overall aim of this thesis was to study the effects of stress on the FAE and elucidate the role of FAE in the development of intestinal inflammation, specifically Crohn’s disease. Initially, rats were submitted to acute and chronic water avoidance stress to study the effects of psychological stress on the FAE. Stressed rats showed enhanced antigen and bacterial passage, and the passage was higher in FAE than in regular villus epithelium (VE). Further, stress gave rise to ultrastructural changes. Subsequent experiments revealed the stress‐induced increase in permeability to be regulated by corticotropin‐releasing hormone and mast cells. Furthermore, vasoactive intestinal peptide (VIP) mimicked the stress effects on permeability, and the VIP effects were inhibited by a mast cell stabiliser. Human studies of ileal mucosa from patients with non‐inflammatory disease and healthy controls showed a higher antigen and bacterial passage in FAE than in VE. In patients with Crohn’s disease, the bacterial passage across the FAE was significantly increased compared to non‐inflammatory and inflammatory controls (ulcerative colitis). Furthermore, there was an enhanced uptake of bacteria into dendritic cells, and augmented TNF‐α release in Crohn’s disease mucosa. Taken together this thesis shows that stress can modulate the uptake of luminal antigens and bacteria via the FAE, through mechanisms involving CRH and mast cells. It further shows that human ileal FAE is functionally distinct from VE, and that Crohn’s disease patients exhibit enhanced FAE permeability compared to inflammatory and non‐inflammatory controls. This thesis presents novel insights into regulation of the FAE barrier, as well as into the pathophysiology of Crohn’s disease by demonstrating a previously unrecognised defect of the FAE barrier function in ileal Crohn’s disease. Keywords: Corticotropin‐releasing hormone, Crohn’s disease, 51Cr‐ EDTA, Escherichia coli, follicle‐associated epithelium, horseradish peroxidase, human, ileum, inflammatory bowel disease, intestinal mucosa, mast cell, M cell, permeability, Peyer’s patches, rat, Ussing chamber, vasoactive intestinal peptide, villus epithelium
LIST OF PAPERS
This thesis is based on the following papers, which are referred to by
their Roman numerals.
I. Increased antigen and bacterial uptake in follicle‐
associated epithelium induced by chronic psychological stress in rats.
Åsa K Velin, Ann‐Charlott Ericson, Ylva Braaf, Conny
Wallon and Johan D Söderholm. Gut 2004; 53:494‐500.
II. Characterization of antigen and bacterial transport in the
follicle‐associated epithelium of human ileum.
Åsa V Keita, Elisabet Gullberg, Ann‐Charlott Ericson,
Sa’ad Y Salim, Conny Wallon, Anders Kald, Per Artursson
and Johan D Söderholm. Lab. Invest. 2006;86:504–516.
III: Increased uptake of non‐pathogenic E. coli via the
follicle‐associated epithelium in ileal Crohn’s disease.
Åsa V Keita, Sa’ad Y Salim, Tieshan Jiang, Ping‐Chang
Yang, Lennart Franzén, Peter Söderkvist, Karl‐Eric
Magnusson and Johan D Söderholm. Submitted
manuscript 2007.
IV. Stress‐induced barrier disruption of the follicle‐
associated epithelium involves corticotropin‐releasing
hormone, vasoactive intestinal peptide and mast cells.
Åsa V Keita, Johan D Söderholm and Ann‐Charlott
Ericson. Manuscript 2007.
CONTENTS_____________________________________
1. INTRODUCTION 9
1.1. Crohn’s disease 9
1.1.1. History 9
1.1.2. Epidemiology and symptoms 9
1.1.3. General treatment 11
1.1.4. Aetiology 11
2. BACKGROUND TO THE STUDY 18
2.1. Structure and function of the small intestine 18
2.1.1. The small intestinal wall covered by VE 19
2.1.2. The Peyer’s patches covered by FAE 21
2.3. Intestinal barrier function 27
2.3.1. Permeability 28
2.3.2. Uptake and transport 28
2.3.3. The junctional complex 30
2.3.4. Endocytosis 33
2.3.5. Transcytosis 35
2.3.6. Regulation of endocytosis and transcytosis 37
2.4. Studies of intestinal permeability 38
2.4.1. In vivo 38
2.4.2. In vitro 38
2.4.3. The Ussing chamber 39
2.5. Stress 41
2.5.1. The stress concept 41
2.5.2. Stress and intestinal disease 43
2.5.3. Animal stress models 44
3. AIMS OF THE THESIS 47
4. SUBJECTS AND METHODOLOGY 48
4.1. Animals 48
4.2. Patients 48
4.3. Stress protocol 49
4.4. Permeability studies 50
4.4.1. Tissue preparation 50
4.4.2. Ussing chamber experiments 53
4.4.3. Permeability markers 54
4.5. In vitro co‐culture model of FAE 56
4.6. Immunohistochemistry 57
4.7. Microscopy 58
5. RESULTS 60
6. DISCUSSION 68
7. CONCLUSIONS 77
8. TACK 79
9. SVENSK SAMMANFATTNING 81
10. REFERENCES 82
ABBREVIATIONS 51Cr‐EDTA 51chromium‐EDTA CRH corticotropin‐releasing hormone CRH‐R corticotropin‐releasing hormone receptor E. coli Esherichia. coli FAE follicle‐associated epithelium HRP horseradish peroxidase IBD inflammatory bowel disease Isc short circuit current M cell membranous or microfold cell NK‐1R neurokinin‐receptor 1 PD transepithelial potential difference SED subepithelial dome TER transepithelial electrical resistance VE villus epithelium VIP vasoactive intestinal peptide VIPR vasoactive intestinal peptide receptor WAS water avoidance stress
SUPERVISORS
Johan Dabrosin Söderholm, Associate Professor of Surgery, Division of Surgery, Department of Biomedicine and Surgery, Faculty of Health Sciences, SE‐581 85, Linköping, Sweden Ann‐Charlott Ericson, Associate Professor of Cellbiology, Division of Cellbiology, Department of Biomedicine and Surgery, Faculty of Health Sciences, SE‐581 85, Linköping, Sweden
1. INTRODUCTION
1.1. Crohn’s disease
1.1.1. History
Clinical descriptions of gastrointestinal disease resembling Crohn’s
disease go back to the 16th century 1, when G. F. Hildenus noted during
an autopsy of a boy suffering from abdominal pain and diarrhoea, that
the ulcerated cecum was contracted and ivaginated into the ileum.
Similar reports during the 16th‐19th centuries indicated the appearance of
a unique intestinal inflammatory disease that today would be identified
as Crohn’s disease. Although Dalziel already published a paper in 1913
of a series of patients with granulomatous small bowel inflammation 2, it
is the report by Crohn, Ginzburg and Oppenheimer at Mt Sinai Hospital
in New York that is considered as the original description 3. In this classic
paper, Crohn and his colleagues describe a condition of abdominal pain,
emaciation, diarrhoea and fever. Originally, Crohn himself named the
disease terminal ileitis since it was believed to be strictly localised to the
small bowel. However, criticism was raised that the disease could also
occur at other locations, and the name was changed to regional enteritis
in the publication 3. Today it is well established that Crohn’s disease is a
chronic episodic, inflammatory disease that can affect the entire
gastrointestinal tract, from the mouth to the anus, however, the ileocaecal
region of the bowel is most commonly affected.
1.1.2. Epidemiology and symptoms
Crohn’s disease is a Western world disease with the highest incidence
rates in Scandinavia, Great Britain and North America 4. The disease has
a slightly female predominance and onset at young adulthood with a
peak incidence between 15‐30 years. In 1991, the incidence in Sweden
9
was 6.1 in 100 000 per year and the prevalence was 146 in 100 000 5. Since
this date, no further epidemiological studies in Sweden in general have
been reported, however, a recent study showed that the incidence in
Stockholm between 1990 and 2001 was 8.3 in 100 000 per year and the
prevalence in the 1st of Jan 2002 was 213 in 100 000 6. Together with
ulcerative colitis, Crohn’s disease constitutes the main condition of
inflammatory bowel diseases (IBD).
The symptoms of Crohn’s disease are dependent on the location of the
inflammation but abdominal pain, diarrhoea, weight loss, fever and
vomiting are common features. The presence of abdominal and perianal
fistulae are typical for the disease.
It is not fully understood how Crohn’s disease is initiated, however,
studies have shown that the first observable signs of the disease are ileal
aphtoid lesions, well recognised by endoscopy 7. These lesions have
shown to progress over time to larger ulcerations and stricturing of the
lumen 8. Initially, it was observed that the lesions mainly occur at the
lymphoid follicles 7. It has been shown that they can vary in size from
barely visible to 3 mm in diameter. They are found in 70 % of the Crohn’s
disease patients 9, and most commonly in the clusters of lymphoid
follicles called the Peyer’s patches of the distal ileum 8‐10. Further,
magnifying endoscopy and scanning electron microscopy have been used
to demonstrate that the aphtoid lesions of Crohn’s disease are preceded
by 150‐200 μm sized ultra‐structural erosions of the epithelium covering
the Peyer’s patches, the so called follicle‐associated epithelium (FAE) 11.
The early inflammation in Crohn’s disease is often located at the distal
ileum 7, where Peyer’s patches are more frequent 12. Taken together, these
observations suggest that the lymphoid follicles are the sites of initial
inflammation in ileal Crohn’s disease, where the ulcerations originate
10
from small erosions over the FAE. The FAE and Peyer’s patches are
further discussed in paragraph 2.1.2.
1.1.3. General treatment
In the report from 1932, Crohn et al. proposed resection of the diseased
segment as a cure, and for a long time, radical bowel resection was the
only treatment. However, the development of anti‐inflammatory drugs in
the 1950’s, and increased knowledge about the disease as a panenteric
disorder has lead to a more restrictive surgical approach 13.
The treatment of Crohn’s disease is unsatisfactory, since none of the
existing treatments such as 5‐aminosalicylates, corticosteroids,
immunomodulators (e.g. azathioprine and methotrexate) or surgery, are
curative. Although these treatments have a positive effect on most
patients, the occurrence of relapse is high.
An example of a newer biological medication is infliximab (Remicade®)
that induce remission of the disease by antibodies against TNF‐α 14.
Infliximab and other new immunomodulators are utilised with the goal
of keeping the disease in remission and there is very little evidence that
these treatments alter the natural history and disease course 15.
1.1.4. Aetiology
The exact cause of Crohn’s disease is unknown but evidence shows that
genetic, immunological and environmental factors all contribute to the
pathogenesis of the disease 14;16 (Fig. 1).
11
Fig. 1. Pathogenesis of Crohn’s disease.
Genetics
Epidemiological studies have shown that ethnic background and family
history are of importance in the susceptibility of Crohn’s disease. First
degree relatives of patients with Crohn’s disease show a 10 to 30 times
increased risk of acquiring the disease 17. A number of studies have
reported that 10‐15 % of first degree relatives have increased intestinal
permeability in the absence of clinical symptoms 18‐20, and approximately
30 % have increased intestinal permeability compared to controls, after
ingestion of acetylsalicylic acid 21. Furthermore, twin studies have
demonstrated a higher pair concordance rate in monozygotic twins with
Crohn’s disease, than in dizygotic twins 22.
In 2001, the first susceptibility gene for Crohn’s disease, CARD15/NOD2,
was identified on chromosome 16 (IBD1) 23;24, and since then, the results
have been widely replicated 25. Whether the Crohnʹs‐associated CARD15
mutations lead to a loss or gain of function of the NOD2 receptor is
subject to controversy 25, and by which mechanisms this change in
function might increase the susceptibility to Crohn’s disease is still under
investigation 26. A recent study showed that high mucosal permeability in
12
healthy first degree relatives is associated with the presence of CARD15
3020insC mutation, indicating that genetic factors may be involved in
impairment of intestinal barrier function in families with IBD 27. Since
CARD15/NOD2 variants only seem to account for 10‐30 % of Crohn’s
disease patients 28, several groups have focused on finding other
candidate susceptibility genes associated with the disease. Additional
putative loci have been mapped to chromosome 5 (OCTN gene), 6 (IBD3),
10 (DLG5 gene), 12 (IBD2), 14 (IBD4), and 19 (IBD6) 25;26;29.
The barrier defect seen in IBD share several pathophysiological and
clinical characteristics with other barrier disorders and studies have
revealed many disease‐associated genes. For example, CARD15/NOD2
mutations have also been observed in Blau syndrome, and also in early
onset of sarcoidosis, suggesting a role for the NOD2 gene in the
development of granolomatous diseases, probably by inappropriate
activation of the immune system 30. The association of NOD2 mutations
has also been shown in allergic diseases and atopy, which might indicate
that NOD2 also plays a role in the Th2 pathway 30.
Aside from the genetic studies in humans, several animal models have
been developed to map the genes involved in the aetiology of IBD 31;32.
The overall lesson from animal studies is that genetics alone is not
enough to cause Crohn’s disease, but an interaction with alterations in
the microflora, epithelial barrier, immune response, enteric nerves and
other components of the intestine, can contribute to the development of
the disease.
Immunology
Under normal conditions the immune system acquires tolerance towards
luminal antigens. The tolerance is induced by regulatory T‐cells, and/or
anergy or deletion of antigen specific T‐cells. This phenomenon is called
oral tolerance. It has been found that T‐cells from patients with Crohn’s
13
disease, in contrast to the normal population, produce cytokines in
response to dietary antigens and the patients’ own microfloral antigens 33;34. This suggests that patients with Crohn’s disease have impaired oral
tolerance, possible caused by a defect barrier. Moreover, studies show a
disturbed IgA‐production, with a reduced in vivo secretion of IgA in
Crohn’s disease 35. Large numbers of immunoglobulin producing cells in
the lamina propria have been observed and in inflamed mucosa
increased numbers of T‐cells, mast cells, and macrophages are seen
together with inflammatory mediators like prostaglandin E2, leukotriene
B4, histamine, substance P and nitric oxide 36. Crohn’s disease is thought
to be of Th1 type inflammation, as shown by increased production of
cytokines important in cell‐mediated immunity such as IL‐2, IL‐6, IL‐1β,
IFN‐γ and TNF‐α, and, in inflamed mucosa, HLA class II molecules on
the epithelial cells 37‐39. Though, in early stages of Crohn’s disease,
increased levels of the Th2 cytokine IL‐4 have been found 40.
Environment
Microflora
Luminal enteric bacteria are the most important inflammation‐driving
environmental factor in Crohn’s disease 41;42. Patients with Crohn’s
disease have an increased number of adherent‐invasive Esherichia (E.) coli
in the mucosa 43;44, and the concentration of bacteria increases
progressively with the severity of the disease 42;45. Although no specific
pathogen has been proven as a causative factor, several bacteria have
been linked to Crohn’s disease, for example Mycobacterium
paratuberculosis 46 and Yersinia pseudotuberculosis 47, and the role of the
luminal microflora is evident. There are some animal models present
where inflammation is caused by microbal infection, such as models of
infectious murine colitis 48. Studies in these models have helped to define
the interactions between bacterial pathogens and host defence. For
14
instance it has been shown that rats susceptible to intestinal inflammation
do not develop IBD when bred under germfree conditions 49.
Aside from being inflammation‐driving, bacteria can also be protective,
and probiotics like bifidobacteria and lactobacilli are thought to protect
against IBD 50.
Diet
The influence of food in Crohn’s disease aetiology has been widely
investigated in the Western world. It has been shown that dietary factors
may have effect on disease activity 51, and an enhanced intake of fast
food, fat and refined sugar, and reduced intake of fresh fruit are
important risk factors in the development of Crohn’s disease 4. Moreover,
elemental diet can be used for inducing remission in the disease and for
nutrition before a bowel resection 52;53. An increasing incidence of Crohn’s
disease has been found in Asia 6;54, formerly a low‐incidence part of the
world. A probable cause could be the influence of Western style, such as
food.
Smoking
In several studies it has been shown that a higher degree of patients with
Crohn’s disease are smokers compared to controls 55. Although a few
studies have shown that smoking has no negative effect on the
development of Crohn’s disease 56‐58, it is generally believed that smoking
more than doubles the risk of acquiring the disease 59;60 and that patients
with Crohn’s disease that smoke are associated with greater disease
activity and higher surgical rates 61;62.
15
Stress
Environmental stress has been shown to alter the course of Crohn’s
disease 63;64. Stress and its effects on gastrointestinal disease are further
described in paragraph 2.5.2.
Barrier function
A disturbed intestinal barrier function has been suggested as a factor for
Crohn’s disease 65;66. Normally, only small amounts of protein antigens
cross the epithelium and interact with the immune system. However, in
Crohn’s disease there is an increased small bowel permeability to
medium sized probes and antigens 65;67;68, leading to increased antigen
exposure to immune cells, that in turn can contribute to inflammation
and gastrointestinal disease 37;69‐71. Several studies have shown an
increased permeability also in healthy relatives of patients with Crohn’s
disease 18;20;21, and both patients with Crohn’s disease, and their relatives,
have demonstrated an increased intestinal permeability when exposed to
NSAIDs 19;21;72. Moreover, studies have reported a high prevalence of
increased permeability in spouses of patients with Crohn’s disease 20;21;73,
suggesting that environmental factors are of importance in the
development of the disease. Though, recently a very extensive study
showed that a common environment with Crohn’s disease patients was
not associated with increased permeability in family members 27.
Crohn’s disease has been suggested as a tight junction disorder 74.
Structural changes 75 and leaky of the tight junctions in response to
luminal stimuli 76 has been demonstrated in Crohn’s disease mucosa.
Another factor in Crohn’s disease is mucus. It has been shown that
inflamed colon mucosa of Crohn’s disease patients has a thicker mucus
layer 77 and an altered structure of mucus glycoproteins 78;79. This might
result in less protective mucus layer, despite its increased thickness.
16
Since patients with Crohn’s disease have shown an enhanced
permeability, and the barrier function of FAE has not previously been
studied, studies regarding environment, barrier function and immune
system are of importance to elucidate the initial steps of the pathogenesis
of Crohn’s disease.
17
2. BACKGROUND TO THE STUDY
2.1. Structure and function of the small intestine The human small intestine is approximately 4‐5 m long and is divided
into duodenum, jejunum and ileum, where the ileum is the most distal
part. The total surface area of the intestinal epithelium is 3‐400 square
meters. From the stomach to the rectum, the inner surface is covered by a
single‐cell polarised epithelial layer that digests and absorbs nutrients at
the same time as it constitutes a barrier between the inner and outer
milieu.
The epithelium that lines the mucosal surfaces of the small bowel consists
of villus epithelium (VE) and FAE 80 (Fig. 2). The exact distribution of
FAE and VE in the human intestine is not known. The entire FAE
presents a biochemical face to the lumen that is distinct from the
surrounding VE 81. While the VE is specialised for digestion and
absorption of nutrients, the FAE is specialised in antigen sampling.
VE
FAE
Follicle
VE
FAE
Follicle
Fig. 2. Villus epithelium (VE) and follicle‐associated epithelium (FAE) of the
ileum.
18
2.1.1. The small intestinal wall covered by VE
The small intestinal wall lined by VE consists of three main tissue
structures; mucosa, submucosa and muscle layers (Fig. 3). The small bowel
has digestive, absorptive, secretory and immunological functions that
mainly take place in the mucosal layer.
Epithelium
Lamina propriaMuscularis mucosae
Mucosa
Submucosa
Muscularis propria
Serosa
Junctional complex
Basal lamina
Microvilli
Epithelium
Lamina propriaMuscularis mucosae
Mucosa
Submucosa
Muscularis propria
Serosa
Junctional complex
Basal lamina
Microvilli
Fig. 3. Schematic illustration of the small intestinal wall covered by
villus epithelium.
The mucosa can be further divided into epithelium, lamina propria and
muscularis mucosae. The epithelium is organised in fingerlike villi, which
project into the lumen, and crypts, that extend down into the basal layer
and often reach the muscularis mucosae. The VE surface area is increased
by submucosal foldings, so called plicae circulares, and the covering of
the villi by tightly packed microvilli. The epithelial cells are connected
laterally to each other by junctional complexes (see paragraph 2.3.3), and
are separated from the underlying lamina propria by a thin basement
19
membrane, basal lamina, which is mainly composed of collagens,
laminins, and proteoglycans (Fig. 3).
The lamina propria lies beneath the epithelium and consists of loose
connective tissue forming the core of the villi and surrounds the crypts.
The most abundant cell types in lamina propria are mononuclear
immunocompetent cells like plasma cells, lymphocytes, and
macrophages, but, eosinophils, mast cells, fibroblasts, myofribroblasts,
smooth muscle cells also occur. Underneath the lamina propria is the
muscularis mucosae which is a thin sheet of smooth muscle cells
bordering the underlying submucosa. The physiological role of
muscularis mucosae is unclear, but it is thought that it may contribute to
the movement of villi and emptying of luminal contents of the crypts.
The submocosa is a more densely collageneous, less cellular structure
than the mucosa. Major blood vessels, lymphatics, nerves, ganglia, and
occasionally lymphoid collections are located here.
The muscle layer consists of the muscularis propria, constituting of the
inner circular and the outer longitudinal muscle layer, and the serosa
consisting of loose, connective tissue with fat, collagen and elastic tissue.
Cell types in VE
As the major function of the small bowel epithelium is nutrient
absorption, enterocytes constitute 85 % of the cells lining the villi 82. The
absorptive capacity of the enterocytes is 20‐40 times increased by the
lining of a brush border membrane constituting of closely packed
microvilli on the enterocytes surface. Anchored to the brush border is the
glycocalyx which protects the epithelium and prevents uptake of
antigens and pathogens. It mainly constitutes of large carbohydrates, but
also enzymes and proteins essential in digestion and absorption.
Both the crypts and villi consist to 20 % of the mucin‐producing goblet
cells. The mucins are glycosylated proteins constituting the main part of
20
the mucus that protects the epithelial surface throughout the intestine.
The exact regulation of goblet cells is unclear.
About 3.5 % of the epithelial cells are found to be Paneth cells. Located at
the base of the crypts they prevent microorganism proliferation by a
variety of secretory granules enfolding for example lysozyme, TNF,
phospholipases, and antimicrobial peptides called defensins 83.
Enteroendocrine cells are spread throughout the epithelium, and in
response to changes in the external microenvironment, or signalling from
enteric nerves, they release gastrointestinal hormones like secretin,
neurotensin and somatostatin.
Remaining cells of the epithelium include the so called cup cells that are
thought to have an affinity for distinctive bacterial pathogens, and the
tuft cells whose role is unknown.
The ability of VE to face the outer environment is enhanced by the non
cellular defenses produced by epithelial cells. These are among others,
mucins, defensins, and secretory antibodies. The most important
antibody in mucosal surface protection is IgA, that is produced by
plasma cells in the lamina propria and then transported to the apical
surface and secreted into the gut lumen where they bind to the
pathogens, thus limiting their adherence and colonisation 84.
2.1.2. The Peyer’s patches covered by FAE
The small intestinal wall lined by FAE constitutes of clusters of organised
lymphoid structures that are spread through out the human intestine 85. It
was more than 300 years ago that J.K Peyer described these aggregations
of lymphoid cells in the small intestinal wall, and named them “folliculi
lymphatici aggregate”, consequently there has to be more than one
follicle to form a Peyer’s patch. Peyer’s patches are found in all parts of
the small bowel. In humans, they develop well before birth, though the
21
full development of the patches as inductive sites requires acquired
antigenic challenge 86. In adolescence more than 240 patches are found in
the small intestine, however, the number regresses with age and in 90‐
year olds, the number of observable patches is around 50 87. In 2002, Van
Kruiningen et al described the anatomic distribution of the Peyer’s
patches in humans 12. By using distal ileum, obtained at autopsy from 55
adults without intestinal disease, the number, location and size of patches
were recorded. The mean number of patches in the distal ileum was
evaluated to 24 (range 19‐30), however, the number of patches turned out
to vary between individuals. In addition, it was revealed that also the
size of a single patch varies between individuals, with the largest patches,
approximately 50 cm2, in 21‐30 years‐old compared to approximately 30
cm2 in younger and older individuals. The variation in distribution, size
and numbers of the human Peyer’s patches have been demonstrated
previously 87.
The Peyer’s patches consist of numerous follicles separated from each
other by interfollicular zones, characterised by high endothelial venules
(HEVs) surrounded by densely packed lymphocytes, mainly T cells, but
also dendritic cells 88 (Fig. 4.). The follicles consist of B‐cell germinal
centres and a marginal zone constituting of proliferating B lymphocytes
expressing IgM and IgG, and phagocytotic macrophages. Between the
FAE and the follicle, a specialised tissue called the dome area or the
subepithelial dome (SED) is located. The SED constitutes of T cells and B
cells, and is rich in phagocyting dendritic cells, macrophages and
monocytes. It has been proposed that luminal antigens and pathogens
sampled by the FAE are further captured by immature dendritic cells
within the SED and ferried to adjacent interfollicular T cells where
dendritic cell maturation and antigen presentation would occur 89.
22
BT
BB
BB BB
B
TT
T
TT
T
Follicle
IFRIFR
GC
MZ
B
TT
T
B
BT
T
B BBB
B
FAE
IFR
MZ
GC
SEDFAE
IFR
B
T
= B cell= T cell = macrophage
= dendritic cell
= high endothelialvenule (HEV)
SEDBT
BB
BB BB
B
TT
T
TT
T
Follicle
IFRIFR
GC
MZ
B
TT
T
B
BT
T
BB BBBBBB
B
FAE
IFR
MZ
GC
SEDFAE
IFRIFR
MZ
GC
SEDFAE
IFR
BB
TT
= B cell= T cell = macrophage
= dendritic cell
= high endothelialvenule (HEV)
SED
Fig. 4. Structure of the follicle‐associated epithelium (FAE) and underlying
Peyer’s patches. SED = subepithelial dome, IFR = interfollicular region, MZ =
marginal zone, GC = germinal centre.
After initiation of an immune reaction, primed B lymphocytes in Peyer’s
patches preferentially migrate as precursors of IgA secreting plasma cells
via the lymphatics, mesenteric lymph nodes and peripheral blood to the
lamina propria 85. Because of this homing of lymphocytes, the term “gut
associated lymphoid tissue” or GALT was introduced. In following
studies it was shown that the lymphoblasts also migrated into other
organs lined with mucosal membranes, which gave rise to the term
“mucosa‐associated lymphoid tissue” or MALT.
Cell types in FAE
The entire FAE presents a surface to the lumen that is different from the
surrounding VE 81. As in VE, enterocytes are the most common cell, and
as their counterparts in VE they have a complex network of glycocalyx,
but they are not identical to VE enterocytes. For example, they express
lower amounts of the membrane‐associated hydrolases involved in
digestive functions, and the glycosylation patterns differ from those in
23
VE enterocytes 80;90;91. These features together facilitate the recognition
and adherence of microorganisms to the FAE.
The number of Paneth cells in the FAE crypts is decreased and there are
less goblet and enteroendocrine cells. In addition, the FAE lacks the
subepithelial myofoibroblast sheath, and the basal lamina is more porous
compared with that in VE 92.
Moreover, the entire FAE lacks polymeric IgA receptors and therefore it
is unable to transport protective IgA to the lumen 80. These characteristics
together promote local contact of intact antigens and pathogens with the
FAE surface.
In contrast to VE, the FAE contains so called membranous or microfold (M)
cells, specialised in antigen sampling and transport 88 (Fig. 5).
EE EEMM
antigen, bacteria
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EE EEMM
antigen, bacteria
EE EEMM
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EE EEMM
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LL LL
MMEE
X10000X10000LL LL
MMEE
X10000X10000
Fig. 5. Photograph and schematic illustration of an M cell (M) between to
enterocytes (E) in the follicle‐associated epithelium. Internalised antigen
(arrows) and bacteria are transported in vesicles across the M cell and delivered
to immune cells (T and B lymphocytes, macrophages and dendritic cells) in the
M cell pocket and underlying subepithelial dome. L = lymphocyte.
24
In humans 93 and rats 88 it is known that approximately 10 % of the FAE
constitutes of M cells. M cells differ in morphology from the adjacent
FAE enterocytes. The apical membrane has microfolds, or ruffles, rather
than microvilli 85 and are further characterised by the lack of an organised
brush border, short and irregular microvilli, and reduced expression of
digestive enzymes, such as sucrase, isomaltase and alkaline phosphatase 80. They have a high endocytotic activity of adherent, as well as fluid‐
phase macromolecules and particles, and have very few lysosomes 80. The
basolateral plasma membrane is deeply invaginated and forms pockets
containing T and B lymphocytes in addition to professional antigen‐
presenting cells 94. These pockets decrease the travelling distance for the
endocytotic vesicles, from the apical to the basolateral side, ensuring
rapid and efficient transcytosis 80.
There are several different theories concerning the origin of M cells. The
first one is that they are generated from the same pluripotent stem cells
as enterocytes, Paneth cells and goblet cells 95, and this theory is
supported by a recent study 96 showing the presence of M cells in regular
VE. The second theory is that M cells are formed directly from the
enterocytes under the influence of B lymphocytes or bacteria in vivo 97‐101.
In contrast, a third group of scientists believe that M cells are formed in
specific FAE‐associated crypts when stimulated with lymphocyte‐
derived factors 102. Finally, the last theory is that M cells are of clonal
origin and represent a separate cell line 100;103.
Unfortunately, the histochemical properties of FAE and M cells vary
according to species and location 85. For example, markers have been
identified for pig, rabbit and mouse 104‐106, however, there is today no
reliable rat or human M cell marker 107, which hampers the possibility to
elucidate the actual role of M cells in barrier function of the FAE in rats
and humans.
25
Bacterial uptake via FAE and M cells
The key features of FAE that face the lumen is the facilition of the uptake
of antigens and various microorganisms such as bacteria, viruses and
protozoa 108. Several microorganisms, particularly bacteria, take
advantage of the transcytotic function of the M cells and use them to
cross the otherwise impermeable epithelial lining of the gut. Both in
experimental animal models, and when cultured in vitro together with
human intestinal biopsies, strains of E. coli, Yersinia, Salmonella and
Shigella exhibit specific adherence to FAE and M cells 109;110.
The adhesion of enterohaemorrhagic E. coli (EHEC) and
enteropathogenic E. coli (EPEC) to FAE is characterised by intimin
binding to the translocated intimin receptor, which is exported by the
bacteria and integrated into the host cell plasma membrane 109;110.
Studies have shown that Yersinia enterocolitica utilises the FAE and M
cells as entry sites via specific binding of invasin to β1‐integrin on the
surface 111;112. In mouse FAE, β1‐integrin is specifically expressed on M
cell surface 112, and in a human model FAE, we recently showed,
increased β1‐integrin expression in FAE compared to VE 113.
Salmonella typhimurium crosses the epithelial barrier by attaching to either
M cells or enterocytes 109. Subsequent events include M cell/enterocyte
destruction and subepithelial migration of macrophages.
Shigella flexneri uses the M cells for its initial entry and once across the
luminal surface, the bacteria may invade adjacent enterocytes 109. Shigella
flexneri has shown to direct its own uptake into rectal and colonic mucosa
through membrane ruffling and secretion of effector proteins that induce
endocytosis of Shigella by colonic M cells. After crossing the M cell, the
bacteria are engulfed by macrophages leading to the induction of
inflammatory responses.
26
2.3. Intestinal barrier function The intestinal mucosa is continuously exposed to a heavy load of
antigenic molecules from ingested food, resident/invading bacteria and
viruses. The ability to control invasion of harmful substances from lumen
is defined as intestinal barrier function. The exact mechanisms involved are
as yet not fully elucidated but barrier function can be divided into four
components. The importance of the barrier components described
depends on the chemical, physical and immunological nature of the
luminal contents.
The first component is the lumen itself, where bacteria and antigens are
degraded by gastric acid, pancreatic and biliary juices. The second
component is the microclimate constituting of the unstirred water layer,
glycocalyx, IgA, and the mucus layer, that prevents adhesion of
pathogenic bacteria to the epithelium by mucin‐binding sites that
compete with the epithelial binding sites, thus impeding bacterial‐
epithelial interaction. The third component is the epithelium with chloride
secretion, basal lamina and epithelial cells, connected to each other via
junctional complexes. Within the epithelium there are numerous
antimicrobial peptides whose function is to kill microorgansims, attract
monocytes, and potentiate macrophage opsonisation. One family of
antimicrobial peptides is the defensins, which can be found in the Paneth
cells of the crypts of the small bowel (α‐defensins) and throughout the
colonic epithelium (β‐defensins). Finally, lamina propria make up the last
barrier component and consists of immunoglobulins, cells of acquired
and innate immunity, the enteric nervous system, hormones and the
endothelium of the capillaries. In addition, intestinal propulsive motility,
rapid repair process and cell turnover also are involved in barrier
function 114. If the control of the barrier function is disturbed, it can lead
to enhanced antigen and bacterial passage which in turn may damage the
mucosa leading to pathological conditions 115.
27
2.3.1 Permeability
Intestinal permeability is defined as the non‐mediated intestinal passage
of medium‐sized hydrophilic molecules, i.e. passage of molecules down a
concentration gradient without the assistance of a carrier system 116;117.
When measuring intestinal permeability it is necessary to use
simplifications, since the definition permeability refers to the passage of a
solute through a simple membrane, whereas the intestinal membrane
consists of several layers and different cell types.
Several clinical disorders are believed to result from altered permeability,
induced by loss of mucosal integrity. The most common ones are IBD,
celiac disease, intestinal ischemia, food intolerance, rheumatoid arthritis,
allergy and malnutrition 116‐119. Moreover, mucosal integrity can also be
affected by treatment with acetylsalicylic acids 120;121.
In vivo, intestinal permeability is usually measured as the permeability to
paracellular markers that are taken up via the junctional complex. In in
vitro systems, intestinal permeability is often measured as the
transepithelial electrical resistance (TER) which correlates with the ability
for passive diffusion of ionic charge across the epithelia 122.
2.3.2 Uptake and transport
There are several ways for solutes to cross the intestinal epithelium (Fig.
6). Lipid soluble or small hydrophilic compounds pass through the cells
via passive diffusion into the lipid bilayers, while larger hydrophilic
molecules pass via the tight junctions and intercellular spaces in the
paracellular route. The paracellular route, via the junctional complexes, is
believed to be impermeable to protein‐sized molecules and thus, under
normal conditions, it constitutes an effective barrier to antigenic
macromolecules. A controlled protein uptake by the intestinal mucosa is
physiologic and essential for antigen surveillance in the gastrointestinal
28
tract 71. Small hydrophilic molecules can also pass the barrier
transcellularly via aqeous pores.
A B C D EA B C D E
Fig. 6. Schematic illustration of passage routes across the epithelium. (A)
Transcellular route (lipophilic and small hydrophilic compounds). (B)
Paracellular route (larger hydrophilic compounds). (C) Transcellular route via
aqueous pores (small hydrophilic compounds). (D) Active carrier‐mediated
absorption (nutrients). (E) Endocytosis, followed by transcytosis and exocytosis
(larger peptides, proteins and particles).
In addition to the non‐mediated permeation routes described, there are
two active ways to cross the epithelium. First, there are numerous carrier‐
mediated mechanisms for uptake, utilised for sugars, amino acids and
vitamins, and second, larger peptides, proteins and particles may be
endocytosed into endosomal vesicles and transported through the cells
via transcytosis.
29
2.3.3. The junctional complex
The junctional complex was first described in 1963 when Farquhar and
Palade 123 showed that enterocytes were attached to each other via tight
junctions, adherens junctions, desmosomes and gap junctions (Fig. 7).
Tight junction
Adherens junction
Desmosome
Actin and myosinfilaments
Intermediatefilaments
ConnexinGap junction
Tight junction
Adherens junction
Desmosome
Actin and myosinfilaments
Intermediatefilaments
ConnexinGap junction
Fig. 7. The junctional complex.
Tight junctions, also called zonula occludens (ZO) are located at the apical
part of the lateral membrane forming a network of linking strands (Fig.
8). They are important in epithelial transport towards and away from the
lumen (gate function), as well as in maintaining the polarity of lipid
substances by preventing diffusion from the apical to the basolateral
region in the outer cell membrane (fence function). These functions seem
to be regulated in different ways 124;125. The gate function is important in
the intestinal barrier regulation and has been shown to be affected by
many intracellular second messengers such as cAMP, Ca2+,
phospholipase C, protein kinase C, calmodulin and G‐proteins 126‐128.
There is a size and charge‐selectivity within the tight junction
permeability barrier, where positively charged molecules and ions pass
30
more easily. Tight junctions appear as focal contacts (“kisses”) on the
plasma membrane. These contacts correspond to continuous fibrils,
where fibrils on one cell interact with fibrils on the adjacent cell. The
fibrils are formed by at least two types of transmembrane proteins,
occludin and different variants of the claudin family 125.
ZO-1
ZO-2
p130
actin filaments
cingulin7H6symplekinZA-1TJ
N
C
Plasma membranecell 1 cell 2
Occludin
ZAK
N
CParacellular
space
Claudin
N
C
N
C
ZO-1
ZO-2
p130
actin filaments
cingulin7H6symplekinZA-1TJ
N
C
Plasma membranecell 1 cell 2
Occludin
ZAK
N
CParacellular
space
Claudin
N
C
N
C
Fig. 8. The tight junction and related cytoskeletal proteins.
The discovery of claudins in 1998 129 significantly advanced the
understanding of the tight junction barrier. The human claudin family
includes at least 24 members 130 and the distribution of them varies in
different tissues, which probably explains the variable permeability seen
in diverse tissues 125. Occludin and claudins are connected to protein
complexes called cytoplasmic plaques. These constitute of ZO‐1, ZO‐2,
31
ZO‐3, cingulin, 7H6 antigen, and several other proteins with unidentified
function including symplekin, ZA‐1TJ, p130 and ZAK 131. The
cytoplasmic plaques are further linked to F‐actin filaments of the
cytoskeleton 132;133. The exact way in which F‐actin is linked to the tight
junctions is unknown, but strong evidence show that the paracellular
permeability is influenced by the state of perijunctional actin 134.
Interestingly, a recent study showed that actin‐depolymerising drugs
caused disruption of the tight junctions by removing occludin via
endocytosis 135. Although this observation does not elucidate the
functional role of occludin, or the detailed mechanisms of actin
depolymerisation‐induced tight junction disruption, it does suggest an
important role for occludin endocytosis in the regulation of tight
junctions.
Alterations of tight junction structure and/or function have been found in
several disease states, and structural changes have, for example, been
found in inflamed mucosa of Crohn’s and celiac disease patients 75.
Several inflammatory mediators involved in inflammatory diseases have
been shown to affect tight junction permeability, such as nitric oxide 136,
and the inflammatory cytokines INF‐γ 137;138, TNF‐α 139;140 and IL‐4 141;142.
Some pathogens also have the ability to disrupt the intestinal barrier via
the tight junctions, for example zonula occludens toxine (ZOT) from
Vibrio cholera 143 and the toxin A derived from Clostridium difficile 144.
Moreover, abnormalities in ZO‐1 localisation have been found in
epithelia infected with Salmonella typhimurium 145.
Adherens junctions, located below the tight junctions, are actin‐ and
myosin‐associated membrane structures formed by adhesion molecules
of the cadherin family and their cytoplasmatic binding proteins α‐, β‐,
32
and γ‐catenin. It has been suggested that adherens junctions together
with tight junctions form one single functional unit 146.
Desmosomes form spot‐like dense adhesions between the epithelial cells
and are connected to the intermediate filaments of the cytoskeleton.
Desmosomes are dispersed all over the lateral cell surfaces, however,
they are frequently found to be concentrated below the adherens
junctions.
Gap junctions are arrangements of cylindrical tubuli consisting of proteins
called connexins. Gap junctions function as intercellular channels
allowing ions and small molecules to pass between cells, thus linking the
interior of adjacent cells.
2.3.4. Endocytosis
Endocytosis is defined as uptake of extracellular particles and molecules
into the cells by invagination of the plasma membrane and vesicle
formation. Endocytosis in epithelial cells can occur in different ways,
depending on the nature of the substance that is taken up (Fig. 9).
R
R R
Clathrin-mediatedendocytosis
Phagocytosis Macropinocytosis Lipid rafts / Caveolae
R
R R
Clathrin-mediatedendocytosis
Phagocytosis Macropinocytosis Lipid rafts / Caveolae
Fig. 9. Uptake of extracellular material via different types of endocytosis.
R = receptor.
33
Endocytosis occurs in enterocytes of both VE and FAE, however, it is well
known that endocytosis of bacteria and particles primarily occur via the
M cells in the FAE 147.
The first route, present in both enterocytes and M cells, is via clathrin‐
mediated endocytosis 148‐150, a highly specific receptor‐mediated process,
utilised mainly by immunoglobulins, viruses and growth factors from
breast milk. The clathrin‐coated vesicles seldom become larger than 150
nm in diameter 151. In this special type of endocytosis the cells synthesise
receptors and internalise molecules that have bound specifically to them 152.
Larger (up to several μm in size) bacteria, viruses, and particles are taken
up via an adsorptive endocytosis, or phagocytosis 153, involving binding
of molecules to the cell membrane via receptors. Phagocytosis is relevant
for the non‐specific uptake of luminal dietary and bacterial antigens, and
the process is triggered by secreted solubles from the invading bacterium 154. Phagocytosis is a more common process in M cells than in enterocytes 109;155‐157.
Both enterocytes and M cells are capable of actin‐dependent non‐specific
fluid‐phase endocytosis, or macropinocytosis, where substances in the
luminal fluid are internalised 153. The process resembles phagocytosis, but
is not receptor‐mediated 152. For example, the protein antigen horse‐
radish peroxidase (HRP) is known to be taken up via macropinocytosis,
preferentely via M cells 158;159, but the exact way how HRP is sorted and
transported after endocytosis is not fully elucidated.
In recent years, attention has been paid to a fourth mechanism, referred
to as lipid rafts / caveolae. This endocytotic event involves a flask‐shaped
34
invagination of cholesterol‐enriched microdomains within the plasma
membrane that may contain a coat protein, caveolin 160. Endocytosis via
lipid rafts / caveolae is most common in endothelial cells but occurs also
in enterocytes, although this type of endocytosis is rare in M cells as they
contain few to no caveolae 161. Studies have shown that for example
certain enterotoxins and viruses are endocytosed via rafts /caveolae. In
addition, the endocytosis of occludin discussed in paragraph 2.3.3 has
shown to occur via caveolae‐mediated endocytosis 135.
2.3.5. Transcytosis
Following endocytosis, uptaken molecules must be transported through
the cells via transcytosis. For this, enterocytes and M cells have different
systems.
Enterocytes
Enterocytes have apical and basolateral sorting compartments, so called
“apical early endosomes” and “basolateral early endosomes”, that share
a “common recycling compartment” 162. These compartments are used
during clathrin‐mediated endocytosis and phagocytosis. In enterocytes,
transcytosis can occur in three ways.
1) When vesicles bud off from the apical membrane, they can merge with
the apical early endosomes and then be recycled back to the apical
membrane, with or without cytoplasmic release of their content. The
content (protein, virus or particle) bound to the internalised receptor is
most often released into the cytoplasm upon acidification of the vesicles,
while the receptor is delivered back to the cell membrane. However,
large molecules like peptides and proteins may be degraded on their way
to the basolateral membrane since they diffuse rather slowly through the
cytoplasm.
35
2) The vesicle can join with the common recycling compartment and from
there, the content (protein, virus or particle) is often directed into
pathways leading to lysosomal degradation in lysosomes.
3) The vesicle can be transported from the apical to the basolateral side
for subsequent merging with vesicles from the basolateral early
endosomes, although this is a quite rare process compared to the others
described 152;162;163.
Transport of intact proteins or carrier‐mediated systems across
enterocytes is not easy to achieve, however, studies have demonstrated
that the endosomal sorting mechanisms can be modified in order to
decrease apical vesicle recycling, thereby increasing the transcytosis of
proteins across the epithelium 164.
It is known that enterocytes not only transport internalised antigen, in
addition they can, during chronic inflammatory diseases such as IBD and
celiac disease, act as non‐professional antigen‐presenting cells and
promote inflammation 165‐167.
M cells
Endocytosis via the FAE and M cells is well characterised, however, the
transcytosis and fate of internalised content have not been very well
studied 168. M cells only contain few lysosomes 156 and can not express
MHC‐II, consequently they can not function as true antigen‐presenting
cells 166. It is known that the apical part of the M cell cytoplasm contains
several endosomes and vesicles with lysosomal markers on the surface 149;156. Ultrastructural studies have demonstrated that soluble tracer
proteins infused into the lumen are incorporated into the membrane
vesicles of the M cells and rapidly transported across the narrow bridge
36
of the apical cytoplasm, and released by exocytosis into the sequestered
intraepithelial space 158.
2.3.6. Regulation of endocytosis and transcytosis
Both endocytosis and transcytosis can be influenced by numerous factors.
Although the mechanisms are not fully elucidated, bacterial exposure in
one way or another leads to enhanced uptake and transport across the
intestinal epithelium 169. Bacterial stimulation also leads to the production
of pro‐inflammatory cytokines, increasing endocytosis and transcytosis.
For example, TNF‐α has shown to induce HRP endocytosis in intestinal
epithelial cell culture 170;171, and increased transcytosis of HRP could be
correlated to TNF‐α mRNA levels in the underlying mucosal tissue 170.
Another factor affecting epithelial uptake is intestinal disease, in which
dysfunctional intestinal motility can prolong the exposure time to
luminal bacteria. Furthermore, studies have shown that antigen‐binding
speeds up the transcytosis. For example, when conjugating HRP to IgE,
the protein was carried across the epithelial membrane into the lamina
propria within three minutes compared with hours for unconjugated
HRP 165.
In FAE, the size and number of Peyer’s patches and M cells are of
importance for endocytosis and subsequent transcytosis. Smith et al
reported that the number of M cells increased after transfer of germ‐free
mice to normal housing conditions 172. Subsequently, several other
studies have shown an increased number of M cells and enhanced
particle uptake after bacterial stimulation 99;101;173;174. However, Gebert et al 169 recently found that the enhanced uptake seen after bacterial
stimulation depends on increased transport capacity of the M cells
already present in the FAE, and not an increase in numbers. In addition,
intestinal inflammation may increase the M cell numbers. For example,
indomethacin‐induced ileitis in rats increases the M cell number and
37
apoptosis, which may alter the intestinal barrier function 90. Moreover, an
increased number of M cells has been found in ileal mucosa of patients
with spondylarthropathy, and the observation of M cell cytoplasm
disruption, with lymphocytes entering the gut lumen at these sites,
suggest a possible mechanism for the development of aphtoid ulcers 175.
2.4. Studies of intestinal permeability
2.4.1. In vivo
Intestinal permeability in vivo in humans was first studied using
intestinal infusion of solutes 176. Today, intestinal barrier function is
mainly studied as the urinary or blood excretion of orally ingested
markers. Obviously, the characteristics of the permeability probes are of
high importance and the ideal probe should be water soluble, non‐toxic,
nondegradable, and not be metabolised. Moreover, the probe should
ensure complete urinary excretion and the analysis should be sensitive,
accurate and uncomplicated. There is, of course, no probe that fulfils all
these criteria, but some probes are close and consequently used for
permeability studies. The most commonly used small pore markers (5‐8
Å) are monosaccharides (mannitol, rhamnose), and polyethylene glycols
(PEG), with molecular weight around 400 Da. The most frequently used
large pore markers (9.5‐11 Å) are disaccharides (lactulose, cellobiose), 51chromium‐EDTA (51Cr‐EDTA), and PEG, with molecular weight around
1000 Da. Permeability is usually presented as the ratio between the large
pore and small pore marker.
2.4.2. In vitro
The current in vivo methods for permeability studies of the human
intestinal mucosa cannot elucidate passage routes and mechanisms
involved in barrier function in IBD. In addition, there are considerable
38
difficulties with in vivo studies of intestinal uptake of intact protein 177. In
vitro techniques offer possibilities to study processes in human tissue that
would be impossible to study in vivo, and much of the basic knowledge
of gastrointestinal physiology has been achieved through in vitro
techniques. Several techniques for in vitro studies of intestinal mucosa
have been developed, and one of them is mucosal sheets in Ussing
chambers 178;179. The good viability‐supporting possibilities with
oxygenation and effective circulation of the fluid on both sides of the
tissue, combined with the possibility to monitor membrane
electrophysiological parameters, provide the Ussing chamber technique
with important advantages compared to other in vitro techniques for
intestinal tissue experiments 180.
2.4.3. The Ussing chamber
The Ussing chamber was first described in 1951 by the Danish
physiologists Ussing and Zerhan 181. The Ussing chamber technique has
many applications, but mostly it is used to study ion transport, drug
absorption, protein absorption, and studies of several pathophysiological
processes in both animals and humans 179;180;182;183. The initial methodology
of the Ussing chambers, that was rather complicated, was modified and
simplified by Grass et al in 1988 184. The principle is that a flat sheet of
mucosa is mounted between two half‐chambers filled with continuously
oxygenated buffer (Fig. 10). The arrangement of the gas ports provide
buffer circulation, which gives efficient mixing of the fluid and reduces
the thickness of the unstirred water layer to physiological levels 185. The
chambers are kept at 37°C and two pairs of electrodes enable the
monitoring of electrophysiological parameters during the experiment.
The marker solution is added to the mucosal buffer, and at defined time
intervals samples are redrawn from the serosal buffer as a measurement
of passage.
39
A B C
D
IPt
PDAg/AgCl
Mucosa
Gasinlet
BuffersolutionMarker
solution
Fluidcirculation
Platinum-electrodes
Ag/AgCl-electrodes
Samplecollection
A B C
D
IPt
PDAg/AgCl
Mucosa
Gasinlet
BuffersolutionMarker
solution
Fluidcirculation
Platinum-electrodes
Ag/AgCl-electrodes
Samplecollection
Fig. 10. The Ussing chamber. (A‐B) Schematic drawing and photograph of the
Ussing chamber. Tissue is mounted between the two chamber halves and is
continuously oxygenated. One pair of Ag/AgCl‐electrodes enables measurement
of potential difference and one pair of platinum‐electrodes gives current to the
system. (C) Tissue is carefully mounted so it covers the entire surface area of the
opening that connects the two chamber halves. (D) After mounting, chambers
are filled with buffer and put in the 37°C Ussing chamber system.
The preparation techniques of intestinal tissue for the use in Ussing
chambers depend on the species and bowel segment being studied 180.
Generally, the intestinal specimens are dissected and immediately
transported to the laboratory, in oxygenated buffer without circulation.
Tissues are either unstripped, or stripped, which means dissection of the
muscle layers. In unstripped bowel, the whole bowel wall is intact, and it
is possible to evaluate effects of the enteric nerves on intestinal tissue.
However, unstripped segments are not that often used in permeability
studies because of the longer diffusion distance for the probe molecules.
In stripped tissues, the external muscle layers and myenteric plexus are
removed and the mucosa mounted thus consists of the epithelium, the
underlying lamina propria and the muscularis mucosae. After mounting
40
in the chambers, tissues are equilibrated for 20‐60 minutes to achieve
steady state conditions in transepithelial potential difference (PD).
Electrophysiology
A characteristic for all transporting epithelia is the ability to maintain a
PD. The ability depends on all the electrogenic ion pumps activity in the
epithelial cell membrane, mainly Na+ / K+‐ATPase, and on the epithelial
barrier function 186. Theoretically these two can be separated into the
short circuit current (Isc) and TER. The Isc represents the current needed
to nullify the PD and is a function of the ion pumps activity. The TER
reflects the electrical resistance of the paracellular routes, mainly via the
tight junctions. The electrodes enabling the monitoring of
electrophysiological parameters during the experiments consist of one
pair of Ag / AgCl‐electrodes with agar‐salt bridges and one pair of
current‐giving platinum electrodes. Since active ion transport requires
energy production, the basal PD or Isc can be used as a measure of tissue
viability. By passing the current (I) through the epithelium, the change in
PD can determine the TER by Ohm’s law, PD = I x R. However, this
calculation relies on a simplified epithelial model, viewing the epithelium
as a parallel circuit consisting of parcellular and transcellular pathways.
2.5. Stress
2.5.1. The stress concept
Stress is a normal part of life and has been defined in many different
ways 187, for example as “any threat to the homeostasis of an organism” 188 or “the range of tensions of modern life” 189. An adequate stress
response is essential for survival, but the ways of coping with stress is
highly individual 190. Under normal circumstances, physiological systems
are turned on and off in response to stress, matching the duration and
41
severity of the stressors, a so‐called adaptive response. However, in some
individuals stress may become harmful and cause damage to the
organism, a so‐called maladaptive response, also referred to as
pathologic stress. Maladaptive responses are often associated with
chronic daily life stressors such as losses, financial problems,
unemployment, and have been linked to exacerbations of irritable bowel
syndrome symptoms 191.
Regardless if the threat is real (physical), perceived (physiological) or
environmental, the principal stress responses triggered to maintain
homeostasis are quite similar 192. Normally, the stress response
constitutes of a behavioural response (e.g. anxiety), an autonomic
response (e.g. raised heart rate), and a hypothalamic‐pituitary‐adrenal
(HPA) ‐axis mediated response (e.g. cortisol release) 193 (Fig. 11).
STRESS
HypothalamusParaventricular nucleus
Periaqueductal gray
Pituitary gland
CRH
Adrenal gland
Visceral pathophysiology
Pontomedullary nuclei
ACTH
+
+
Cortisol-
+
Autonomic nervous system
+
+
CRHNEACh
-+
+ cytokines
STRESS
HypothalamusParaventricular nucleus
Periaqueductal gray
Pituitary gland
CRH
Adrenal gland
Visceral pathophysiology
Pontomedullary nuclei
ACTH
+
+
Cortisol-
+
Autonomic nervous system
+
+
CRHNEACh
-+
+ cytokines
Fig. 11. The hypothalamic‐pituitary‐adrenal (HPA)–axis. CRH = corticotropin‐
releasing hormone, ACTH = adrenocorticotropic hormone, NE = norepinephrine,
ACh = acetylcholine.
42
The stress response is generated through a network of brain structures,
mainly the paraventricular nucleus of the hypothalamus, amygdale, and
periaqueductal gray. These structures receive input from cortical
structures, and visceral and somatic afferents 194.
2.5.2. Stress and intestinal disease
The influence of stress on intestinal disorders has for long been described
by patients, but the actual effects of chronic stress on gastrointestinal
diseases are still a matter of debate. However, studies are providing
increasing evidence that chronic stress plays an important role in
intestinal physiology, including more importantly mucosal barrier
dysfunction 195.
There are several studies present confirming the importance of stress in
gastrointestinal disease in humans. Barclay and Turnberg showed in the
late 1980´s that psychological stress, induced by dichotomous listening 196
or cold‐induced hand pain 197, reduced mean net water absorption and
transformed net sodium and chloride absorption to secretion. These
effects were inhibited by atropine, suggesting the involvement of
cholinergic neurons (achetylcholine). Ten years later, Santos et al
extended this model and found that jejunal water secretion induced by
cold pain stress was associated with luminal release of histamine and
tryptase, typical mast cell mediators 198. These findings pointed to an
interaction between the intestinal mucosa, mast cells, and central nervous
system during stress in humans.
Since then, numerous studies have been performed in this field, and it is
now known that chronic stress can modulate the inflammatory activity in
IBD 194;199. Severe stress episodes are an important risk factor for the
development and reactivation of intestinal inflammation 191;200. Moreover,
a significant association has been found between acute daily stress and
43
bowel symptoms (e.g. pain, nausea, and diarrhea) in patients with
Crohn’s disease 201.
2.5.3. Animal stress models
Since it is hard to study the effects of psychological stress on mucosal
function in humans, several animal models have been developed. The
stress model can be applied as acute or chronic stress. In acute stress,
animals are exposed to stress at one session, and in chronic stress,
exposure is repeated daily. The models include animals (most often
rodents) which are exposed to restraint stress, water avoidance stress
(WAS), swimming stress, crowding stress, social defeat stress or maternal
deprivation stress 195. All these stress models have been developed with
the intention to mimic psychological stress in daily life of humans. In
WAS, animals are put on a small platform surrounded by water at room
temperature for 20‐60 min at one session (acute) or repeated daily
(chronic) (Fig. 12).
Fig. 12. Rat submitted to water avoidance stress.
44
Acute stress
Acute stress in rats has shown that stress‐induced mucosal dysfunction
mainly involves corticotropin‐releasing hormone (CRH), mast cells and
cholinergic neurons 202‐204, and that stress increases the intestinal
permeability to macromolecules without causing changes in gut
morphology 205‐207. In an early study by Saunders et al 205, rats submitted
to acute restraint stress showed increased conductance and enhanced
jejunal permeability to the paracellular markers mannitol and 51Cr‐EDTA.
In subsequent experiments the stress‐susceptible strain Wistar‐Kyoto rats
(rats with low cholinesterase activity) revealed an enhanced jejunal
permeability after stress compared to the parent Wistar strain 203. The fact
that atropine inhibited the stress response further confirms the
involvement of cholinergic neurons in stress. These experiments were
followed by studies on macromolecular uptake by Kiliaan et al 206 that
could show an increased uptake of HRP in jejunum after stress. Stress‐
induced increases in colonic permeability have been reported as well 207;208. These studies suggested a role for CRH in colonic epithelial
pathophysiology and in another study by Pfeiffer et al 209 increased
paracellular permeability was seen after cold restraint stress and seemed
to be induced by cholinergic neurons and mast cells.
Chronic stress
Several studies have shown that repetitive stress give more pronounced
gut changes than acute stress. Rats submitted to 5‐10 days of 1 hour WAS
reduced their food intake and lost weight 210. Further chronic stress
increased the Isc, conductance and macromolecular permeability and also
gave raise to bacterial attachment, mast cell hyperplasia, mitochondrial
swelling and initiation of inflammation 211;212. Mast cell‐deficient rats
exposed to chronic stress also lost weight but showed no signs of changes
45
in epithelial function and gut morphology 211, confirming an important
role for the mast cells in barrier function.
The involvement of mast cells and neuropeptides in stress
Several studies have confirmed the role of mast cells in stress‐related
changes of intestinal mucosal function. Castagliuolo et al 213 found
increased levels of the mast cell protease RMCPII during acute restraint
stress in rats and the stress‐induced increase in mucin was inhibited by
adding mast cell stabilisers 207. Mast cells are often found close to neurons
and are activated by neuropeptides, such as CRH, substance P and
acetylcholine 214. CRH is known to be involved in the increased intestinal
permeability seen after stress 211 and peripheral injection of CRH mimics
stress‐induced permeability changes in rats 207;215;216. There are very few
studies present regarding the role of vasoactive intestinal peptide (VIP)
in mucosal stress and barrier function. However, recent studies showed
increased VIP levels in mouse ileum after acute psychological stress 217,
and in a human cell‐culture model, a neural activated increase in
paracellular permeability was inhibited by blocking of the VIP receptor 218.
46
3. AIMS OF THE THESIS
As mentioned in the introduction, the earliest observable signs of Crohn’s
disease are aphtoid ulcers originating over the FAE. Crohn’s disease is
associated with an increased immune response to bacteria, and the
disease course can be altered by stress. With this in mind, together with
the knowledge that FAE is an entry site for antigens and bacteria, we set
up an overall aim to elucidate the effects of stress on the FAE and to
reveal the role of FAE in the development of Crohn’s disease.
The specific aims of the separate studies were to:
I. Elucidate if the FAE barrier function could be modulated by stress. For
this we set up an animal stress model and investigated the functional and
morphological effects of stress in rat FAE.
II. Establish a technique for the identification of FAE in human ileal
tissue and characterise the normal barrier properties of human FAE and
compare it with regular VE.
III. Investigate the FAE barrier in patients with Crohn’s disease, using
non inflammatory and inflammatory controls, respectively.
IV. Elucidate the mechanisms involved in the stress‐induced increase in
FAE permeability and to map the chemical coding of neuropeptides in
rat FAE and Peyer´s patches.
47
4. SUBJECTS AND METHODOLOGY
For information on the statistics used, and other details on subjects and
methodology, see the individual papers.
4.1. Animals Since we have prior experience from stress experiments in male Wistar
rats, we chose to work with this strain in the animal studies (Paper I and
IV). From an immunological view, mice would have been preferable,
however, from previous research we know that stress experiments in
mice are difficult to perform.
A total of 102 rats, weighing 150‐200 g at arrival, were acclimatised for
one week and then handled daily for two weeks before the experiments
started. The animals were kept in pathogen free conditions with a 12:12 h
light:dark cycle and fed ad libitum on a diet of standard rat pellets and
tap water. Studies were approved by the Animal Ethics Committee of the
Faculty of Health Sciences at Linköping University, Sweden.
4.2. Patients The study group in Paper II consisted of 19 patients undergoing surgery
for right‐sided colon cancer and 11 healthy persons undergoing
colonoscopy for the surveillance of colonic polyps.
The study population in Paper III included 14 of the patients undergoing
surgery for colonic cancer in Paper II, and 10 of the healthy persons
undergoing colonoscopy. In addition, the study group consisted of 25
patients with Crohn’s disease (19 surgery and 6 follow‐up colonoscopy)
and 6 patients with extensive ulcerative colitis (2 surgery and 4 follow‐up
colonoscopy). All specimens were taken from the terminal ileum next to
the ileocaecal valve or from the neo‐terminal ileum, during surgery or
48
colonoscopy at the University Hospital of Linköping from March 2003 to
May 2006. Both studies were approved by the Committee of Human
Ethics, Linköping, University Hospital, and all subjects had given their
informed consent. The colon cancer patients had no signs of generalised
disease and none had received preoperative chemo‐ or radiotherapy. The
Crohn’s disease patients had mild to moderate ileal inflammation and the
ulcerative colitis patients showed signs of mild to severe inflammation in
the colon.
The mean age for the Crohn’s disease patients was 48 (21‐73) years,
ulcerative colitis 61 (42‐73), healthy controls 59 (38‐75) and colon cancer
controls 74 (47‐85). Since intestinal permeability does not seem to be
affected by aging 116;219;220, the age differences between the study groups
are probably not important for the results.
4.3. Stress protocol As mentioned in the introduction, there are several ways to generate
stress in animals. In Paper I and IV, we used WAS, since it induces
minimal physical stress and is considered a good psychological stress
model of mimicking daily life stress in humans 195. In addition to studies
on intestinal function, it has also been widely used in psychiatric research
as a model of depression. Moreover, this model has previously been used
in studies to achieve pure psychological stress effects on intestinal
permeability 211. Prior to experimental start, rats were handled daily by
the researcher for two weeks to decrease the influence of stress due to
human contact, as we wanted the stress to be induced only by water
avoidance, and evade stress effects in the control groups.
Rats were submitted to acute and/or chronic WAS. Rats to be stressed
were placed on a platform (h=8 cm, d=6 cm) in a plastic container (h=56
cm, d=50 cm) with 25°C water, 1 cm below the platform. Control rats
were either placed on a similar platform in a waterless container (Paper I)
49
or left in their cages (Paper IV). After one hour, the number of faecal
pellets were counted as an indirect index of changes of colonic propulsive
activity 221. In chronic stress, this procedure was repeated for ten
consecutive days, one hour per day. To minimise the effect of circadian
rhythm, the procedures were started between 9 and 10 a.m. Before each
session, rats were weighed.
Rats were anaesthesised by isofluran inhalation and 15‐cm segments
from the distal ileum (starting 5 cm proximal to the ileocecal valve) were
taken out and immediately transported to the laboratory in ice‐cold
oxygenated Krebs buffer (115 mM NaCl, 1.25 mM CaCl2, 1.2 mM MgCl2,
2 mM KH2PO4 and 25 mM NaHCO3, pH 7.35).
Blood for corticosterone analysis (Paper I) was collected by cardiac
puncture, centrifuged, and stored at –70°C until further use for
corticosterone assay (IDS, Boldon, U.K).
In Paper IV, animals were intraperitoneally injected with neuropeptide
receptor antagonists or mast cell blocker, before being submitted to acute
WAS. We chose to inject rats with anti‐neurokinin‐receptor 1 (NK‐1R)
and anti‐CRH receptor (CRH‐R), since the neuropeptides substance P
and CRH have been shown to be involved in stress and permeability
changes in VE. We also planned to study the effects of blocking the VIP
receptor (VIPR), however, at the time of the study no such one was
available.
4.4. Permeability studies
4.4.1. Tissue preparation
In Paper I and IV, rat distal ileal specimens were, while immersed in
Krebs buffer, stripped of external muscle and myenteric plexus, and FAE
and VE were identified macroscopically.
50
In Paper II and III, human tissue from surgery and colonoscopy was
used. Surgical specimens of distal ileum were immediately, after division
of the ileocolic artery, put in Krebs, macroscopically reviewed by a
pathologist, and transported to the laboratory. While rinsed in Krebs,
specimens were stripped of external muscle and myenteric plexus. Tissue
was then put with the mucosa up in a Petri dish and carefully stretched
out with needles (Fig. 13). For identification of FAE, we modified a
technique previously described for post‐mortem ileal tissue 12. The dish
was placed on a horizontal X‐ray view box and by transillumination from
below, regions of VE and FAE could be identified in a dissection
microscope (Fig. 13).
VEVE
FAEFAE
VEVE
FAEFAE
Fig. 13. Identification of villus epithelium (VE) and follicle‐associated epithelium
(FAE) in surgical tissue. The border between VE and FAE is indicated by a
stitched line. Note the regular pattern of villi (dark dots) in close apposition in
the VE, compared to the more irregular pattern of multiple follicles surrounded
by sparse villi in FAE.
Biopsies, identified as VE or FAE with magnification endoscopy, were
taken at colonoscopy with a biopsy forceps without a central lance. They
were directly put in a dish with Krebs, and epithelial type was identified
with dissection microscopy as for surgical tissue.
51
Following the Ussing experiments, histological assessment verified
epithelial type in each chamber (Fig. 14).
LFLF
FAEFAEVEVE
X20X20X20X20
LFLF
FAEFAEVEVE
X20X20X20X20
Fig. 14. Histological assessment of VE (left panel) and an ileal lymphoid follicle
(LF) and the overlaying FAE, following Ussing chamber experiments.
Our intention in Paper I was to histologically identify M cells within the
FAE of stressed rats and controls. There is one paper present that
describes the identification of rat M cells by cytokeratine‐8, in
combination with the negative markers alkaline phosphatase and alcian
blue 222. However, earlier studies have shown conflicting results 90;223 and
we were not able to obtain a specific staining with these markers.
To date there is no reliable human M cell marker, and consequently, we
did not stain for M cells in the FAE specimens of Paper II and III.
However, a few markers have been suggested. One is Sialyl Lewis A
Antigen 224, but we (unpublished observations) and others 107, have not
been able to confirm this finding. Other suggested M cell markers are
Cathepsin E 225 and β‐1 integrin 226. In a recent paper 113, we could show
52
that β‐1 integrin, and also CD9, were several times higher expressed in
FAE compared to VE, however, if the expressions were associated
exclusively with the M cells and not to the other cells of the FAE could
not be determined.
4.4.2. Ussing chamber experiments
Sections of VE and FAE were cut in appropriate sizes and mounted in
modified Ussing chambers (Harvard apparatus Inc., Holliston, MA, USA) 227. When mounting FAE, the segments were carefully adjusted so that
the patches covered the entire exposed tissue surface area of 9.6 mm2 for
rat tissue, 4.9 mm2 for surgical specimens and 1.8 mm2 for biopsies.
Mucosal compartments were filled with 1.5 ml cold 10 mM mannitol in
Krebs buffer and the serosal compartments were filled with 10 mM
glucose in Krebs buffer. The chambers were kept at 37°C and
continuously oxygenated, 95 % O2 / 5 % CO2 and circulated by gas flow.
Before the experiments were started, tissues were equilibrated for 20‐40
min in the chambers to achieve steady state conditions in PD, with a
replacement of 37°C mannitol or glucose buffer at 20 min. The Isc, TER
and PD were monitored using one pair of Ag/AgCl‐electrodes (Ref 201,
Radiometer, Copenhagen, Denmark) with 3M NaCl / 2 % agar‐salt
bridges and one pair of current‐giving platinum electrodes. The chamber
experiments were performed in open circuit conditions, and a four
electrode system was used as previously described 228. The electrodes
were coupled to an external six channel electronic unit with a voltage
controlled current source. PD, Isc and TER were obtained as described by
Karlsson et al 229. Data sampling was computer controlled via an A/D D/A
board (Lab NB, National Instruments, USA) by a program developed in
Lab View (National Instruments). Every second minute, direct pulses of
1.5, ‐1.5, 3, ‐3 and 0 μA with duration of 235 ms were sent across the
mucosal specimens and the voltage response was measured. In each
53
measurement the mean voltage response of eight recordings was
calculated. By this procedure the influence of AC disturbances of 25‐100
Hz were eliminated. A linear least‐squares fit was performed of the
current (I)‐voltage (U) pair relationship: U = PD + TER x I. From the slope
of the line, TER was obtained, and PD from the intersection of the
voltage. Tissue conductance was then calculated by inverting the TER
values. All parameters were calculated during the 30‐90 min period.
Monitoring of FAE tissues revealed a higher TER compared to VE
specimens. This probably refers to a thicker tissue in FAE segments due
to the Peyer’s patches. Further, we observed a lower TER in biopsies
compared to surgical specimens which probably is due to a thinner
subepithelial tissue layer in biopsies. Conventional TER measurements,
as used in Paper I‐IV, give good assessments of changes over time, but
miscalculations of TER, and also Isc, can be made when comparing
different patients or epithelial types. Techniques that separate epithelial
from subepithelial resistance are needed to fully elucidate differences in
TER between different tissues.
4.4.3. Permeability markers
HRP and 51Cr‐ EDTA
In all Papers, we chose to use the 45 kD protein antigen HRP as a marker
of protein uptake. HRP is known to, under normal circumstances, be
taken up through the cells via macropinocytosis 158;159, and has the
antigenic potential to initiate immune responses in humans. HRP is easy
to detect and has previously been used for permeability studies in Ussing
chambers. One more advantage of HRP is its possibility to be detected by
electron microscopy.
As a paracellular marker we chose to use the inert probe 51Cr‐ EDTA. The
EDTA molecule is known to pass between the cells via the paracellular
54
route. The binding of EDTA to the radioactivity labelled Cr is very strong,
which assures that the Cr passage is equal to the passage of EDTA, and no
Ca2+ can bind to EDTA to give detergent effects. Similar to HRP, 51Cr‐ EDTA
is a widely used permeability marker in Ussing chambers.
HRP and 51Cr‐EDTA were added to the mucosal side to a final
concentration of 10‐5 M and 34 μCi/ml, respectively. Serosal samples (300
μl) were collected at 0, 30, 60 and 90 min after start. An aliquot from each
sample was saved for HRP analysis and the remainder was placed in a
gamma‐counter for 51Cr‐EDTA measurements. Permeability was
calculated during the 30‐90 min period for both markers. 51Cr‐EDTA
permeability was given as Papp (apparent permeability coefficient; cm/s x
10‐6), and HRP permeability presented as transmucosal flux (pmol/h/cm2).
E. coli K‐12 and HB101
Since we wanted to study bacteria that under normal circumstances do
not invade the mucosa, we decided to use bacteria from non‐pathogenic
E. coli strains. Furthermore, increased numbers of E. coli has been found
in Crohn’s disease mucosa. At Molecular Probes, The Netherlands,
chemically‐killed FITC‐conjugated E. coli K‐12 BioParticles are available.
These bacteria are killed with paraformaldehyde in a way that stops their
reproduction but retains antigenicity. They have previously been used
for phagocytosis studies 230 and should be a good model of bacterial
uptake. Chemically‐killed E. coli K‐12 were used in all papers.
In addition, in Paper II and III, we used live green fluorescent protein‐
incorporated E. coli HB101 (One Shot® TOP10 Competent Cells,
Invitrogen, CA, USA). For details on the incorporation, see Paper II.
Bacteria were added to the mucosal side to a final concentration
corresponding to 1.0x108 CFU/ml, i.e. the normal concentration of
bacteria in the ileal lumen. After 90 and 120 min, serosal compartments
55
were collected and analysed at 488 nm in a fluorimeter (Cary Eclipse,
Varian) where 1 unit refers to 1.5x106 CFU/ml.
Doxantrazole, CRH and VIP
In Paper IV, tissues were exposed to neuropeptides and mast cell
stabiliser in the serosal chamber. We chose to study the effects of CRH
and VIP peptides, since their involvement in stress and permeability
changes in VE have been described previously. In the chambers 10 μM of
the mast cell stabiliser doxantrazole (Sigma‐Aldrich) was added. After 20
min, HRP and 51Cr‐EDTA, or E. coli K‐12 was added to the mucosal side
while in the serosal chambers 1 μM CRH (Sigma C‐3042), or 0.1 μM VIP
(Sigma V‐3628) were added. Serosal samples were collected at 0, 30, 60,
90 and 120 min, with replacement of Krebs buffer containing the blockers
at starting concentrations.
4.5. In vitro co‐culture model of FAE In Paper II we used a modification 231 of the co‐culture model of FAE
originally described by Kerneís et al. 98. The model involves co‐culture of
Peyer´s patch lymphocytes and intestinal epithelial cells, that is thought
to trigger the conversion of the epithelial cells to M cells. We obtained the
model FAE by adding 5x105 Raji B cells (ATCC, ML, U.S.A) to the
basolateral chamber of 14‐day‐old Caco‐2 cell monolayers. The co‐culture
was then maintained for 4‐5 days before onset of experiments.
To investigate the mechanisms behind the increased HRP uptake in FAE,
we decided to inhibit the transcellular pathway. For this matter, we used
the amiloride analogue 5‐(N‐ethyl‐N‐isopropyl)‐amiloride (EIPA), which
inhibits the sodium‐hydrogen exchange at the apical membrane, thereby
reducing the uptake 232. Previous studies have shown that EIPA inhibits
transcellular HRP uptake via pinocytosis, but not via clathrin‐coated pits
56
or via receptor‐mediated uptake 233;234. In the experiments of Paper II,
HRP, with or without EIPA, was added to the apical side of the cells,
samples were withdrawn at regular time intervals, and stored at ‐20°C
until analysed by confocal microscopy.
To study the mechanisms involved in the increased bacterial uptake in
FAE we decided to inhibit the microtubule and actin filaments. For this
purpose we used cytochalasin D, which is an F‐actin polymerization
inhibitor, and colchicine, which is known to block the microtubule
transport 163. E. coli K‐12 were added to the apical side of the cells, with or
without inhibitors. After 20 min, the cell monolayers were washed, fixed
in 4 % formaldehyde, washed again and stored for visualisation via
confocal microscopy.
4.6. Immunohistochemistry In Paper IV, immunohistochemistry was used to identify the
neuropeptides VIP and CRH, and their receptors in rat FAE. Control
sections were obtained from consecutive sections present on the same
slide as the samples, which ensured a negative control for background
and unspecific binding. The controls were added secondary, but not
primary antibody. To confirm the nerve‐mast cell interaction observed in
the physiological experiments, mast cells were co‐stained with May‐
Grünvald/Giemsa, which stains the nucleus and granule of the mast cells,
respectively.
During antibody optimisation it was revealed that the rat tissue had very
high levels of endogenous peroxidase, disturbing the actual peptide
staining. However, when blocking tissues with 10 % hydrogen
peroxidase in methanol, the endogenous peroxidase activity could be
diminished. This rather tough treatment affected the histology resulting
in partly damaged tissue with epithelium separated from underlying
57
tissue. The immunohistochemical procedure was performed according to
Fig. 15.
Tissue
A
Antigen
Primary ab
Secondary ab
biotin
streptavidinBC
AB
D
Tissue
A
Antigen
Primary ab
Secondary ab
biotin
streptavidinBC
AB
D
Fig. 15. The immunohistochemical procedure used to identify expressions of
neuropeptides and their receptors in rat tissue.
To augment the antigen expressions in the tissue, we used a primary
antibody followed by a biotinylated secondary antibody. Further, we
utilised the ABC technique, where complexes that bind to the biotin
molecule are built up to enhance the antigen expressions. The
expressions were visualised by DAB staining.
4.7. Microscopy
Light microscopy
In Paper I, rat tissues were stained with May‐Grünvald / Giemsa in order
to count the number of mast cells. Cells identified as mast cells were
counted in three stressed rats and three controls, three tissues per rat.
Slides were coded, which ensured an objective evaluation.
58
In Paper IV, light microscopy was used to identify neuropeptides and
their receptors in normal rat tissue. Co‐staining for mast cells was done
with May‐Grünvald / Giemsa.
Confocal laser scanning microscopy
In Paper I‐III, confocal microscopy was used to study bacterial uptake
into rat and human tissue. Confocal microscopy is a variant of light
microscopy with many advantages compared to the regular light
microscope 235. For example, in a confocal microscope the illumination
and detection is focused on the same point of the tissue, resulting in
improved resolution and reduced degree of out‐of‐focus information.
Thereby information can be collected from very thin specimens.
Tissues in Paper I‐III were prepared for confocal microscopy as described
in the respective paper.
Transmission electron microscopy
In Paper I‐III, electron microscopy was used to study the passage routes
for HRP and E. coli in rat and human tissue. The electron microscope uses
a focused beam of electrons instead of light to create an image of the
specimen. As electrons have much shorter wavelengths than light, it is
possible to achieve magnification several thousands times higher than in
a light microscope. When electrons pass through the specimen,
interactions of heavy atoms will cause electrons to loose energy and
deviate from its original direction. These interactions are then detected
and transformed into an image.
Tissues for HRP studies were fixed in 2 % glutaraldehyde, incubated
with DAB, postfixed in 1 % osmium tetroxide, embedded in Epon plastic,
sectioned and stained with lead citrate and uranyl acetate. Tissues for
bacterial passage studies were processed in the same way as for HRP,
except for incubation with DAB.
59
5. RESULTS
Detailed descriptions of the results are given in the respective paper.
Paper I
Rats were submitted to acute or chronic stress to study the effects of
stress on the FAE, and thereby elucidate if the FAE barrier function could
be modulated by stress. Since this was the first study of FAE, we
optimised the Ussing chamber technique for studies of FAE and Peyer’s
patches before experiments started. Our results showed that FAE is
responsive to both acute and chronic stress, illustrated by changes in
electrophysiology and permeability. Acute stress increased the Isc,
conductance, and HRP passage. Chronic stress enhanced the Isc and
passage to HRP and E. coli. In addition, results revealed a significantly
augmented permeability in FAE compared to VE. After chronic stress the
HRP passage in FAE was over three times higher than in VE (Fig. 16).
P<0.05
P<0.005
VE FAE0
25
50
75
100
125
HR
P flu
x (p
mol
/h/c
m2 )
P<0.05
P<0.005
VE FAE0
25
50
75
100
125
HR
P flu
x (p
mol
/h/c
m2 )
Fig. 16. Rats were submitted to stress (filled bars) or non stress (open bars).
Segments of villus epithelium (VE) and follicle‐associated epithelium (FAE) were
mounted in Ussing chambers and horseradish peroxidase (HRP) passage was
measured over time. Results are given as the 30‐90 min passage.
60
The enhanced uptake in FAE was further emphasised by a significantly
enhanced E. coli passage compared to VE, with a passage six times higher
in FAE than in VE after stress. Moreover, after 120 min exposure in
Ussing chambers it was increased more than 30‐fold in FAE of stressed
rats compared to unstressed (Table 1). The bacterial uptake was
confirmed by confocal microscopy.
Table 1. Effects of chronic stress on bacterial passage. Rats were submitted to
stress or non stress (controls). Segments of villus epithelium (VE) and follicle‐
associated epithelium (FAE) were mounted in Ussing chambers. Passage of
chemically‐killed E. coli K‐12 was studied over time. aP < 0.005 vs. controls
FAE, bP < 0.05 vs. stress VE, cP < 0.05 vs. controls VE.
Group, tissue 120 min passage
Sress, FAE 47.9 (17.6 ± 94.7)a, b
Control, FAE 1.4 (0.9 ± 5.1)
Stress, VE 7.8 (6.4 ± 19.3)c
Control, VE 5.0 (3.0 ± 5.8)
Electron microscopy revealed ultrastructural changes in FAE induced by
chronic stress. This was indicated by areas of abnormal epithelial cell
morphology with vacuoles. Within the FAE, M cells with internalised
HRP in direct contact with lymphocytes could be identified (Fig 17 a, b).
61
X5000
L
MM
EE EE
X5000
aa bb EE
EE
X5000
L
MM
EE EE
X5000
aa bb EE
EE
Fig. 17 a, b. M cells (M) between enterocytes (E) in the follicle‐associated
epithelium of rat ileum. L = lymphocyte. Arrows indicate internalised
horseradish peroxidase molecules.
In conclusion, we found that chronic stress modulated the barrier function
of FAE allowing uptake of normally non‐invasive luminal
microorganisms. An enhanced bacterial uptake may lead to an increased
antigen load and presentation in the dome area of the Peyer’s patches.
Thereby a local inflammatory reaction could be initiated, eventually
leading to intestinal inflammation. This may have implications for stress‐
related events in IBD.
Paper II
In this paper we wanted to establish a model for studies of human FAE
and Peyer’s patches.
Initially we developed a technique for the identification of FAE in ileal
surgical specimens from patients with colonic cancer by
transilluminating the stripped mucosa from below in a dissection
microscope. We were also able to identify FAE in biopsies, using similar
techniques. Further, we could show that surgical specimens and biopsies
62
can be considered equal regarding permeability, as Ussing experiments
showed similar permeability and electrophysiology in both techniques.
Passage of HRP was higher in FAE than in VE (Fig. 18). The same pattern
was seen in transmucosal passage of E. coli K‐12 and E. coli HB101 (Fig.
19). The permeability to 51Cr‐ EDTA was equal in VE and FAE.
HR
P flu
x (p
mol
/h/c
m2 )
FAEVE0
20
40
60
80
100P < 0.001
HR
P flu
x (p
mol
/h/c
m2 )
FAEVE0
20
40
60
80
100P < 0.001
Fig. 18. Horseradish peroxidase (HRP) passage in villus epithelium (VE) and
follicle‐associated epithelium (FAE) of human ileum.
0
1
2
3
4
5
6
K-12
P < 0.01
HB101
P < 0.05VEFAE
Bac
teria
lpas
sage
(uni
ts)
0
1
2
3
4
5
6
K-12
P < 0.01
HB101
P < 0.05VEFAE
Bac
teria
lpas
sage
(uni
ts)
Fig. 19. E. coli K‐12 and HB101 passage in human villus epithelium (VE) and
follicle‐associated epithelium (FAE). Median (25‐75th interquartile range).
63
In vitro co‐culture experiments and electron microscopy revealed actin‐
dependent, mainly transcellular, uptake of E. coli K‐12 into FAE.
Furthermore, the increased HRP passage in FAE was inhibited by
blocking macropinocytosis.
In conclusion, we found an enhanced antigen and bacterial uptake, but
similar paracellular permeability, suggesting functional variations mainly
of transcellular transport in the FAE. Our results show that human ileal
FAE is functionally distinct from regular VE, making it more prone to
interaction with, and transport of, antigens and bacteria.
Paper III
This paper was designed to characterise the FAE barrier function in
patients with Crohn’s disease. We used patients with colonic cancer or
healthy volunteers as non‐inflammatory controls, and patients with
ulcerative colitis as inflammatory disease controls.
Initial morphological studies of paraffin‐embedded tissues showed that
the FAE of Crohn’s disease was prone to interact with bacteria.
Fluorimetric measurements showed increased transmucosal passage in
Crohn’s disease of both non‐pathogenic E. coli strains. The passage of K‐
12 was almost doubled in Crohn’s disease (4.9 [4.2‐5.3] units) compared
to both ulcerative colitis (2.7 [2.1‐3.8]) and controls (2.8 [2.4‐3.4]), P <
0.002. The HB101 passage was also significantly increased in Crohn’s
disease (6.7 [6.5‐11.8]) compared to both ulcerative colitis (3.5 [2.9‐5.3])
and controls (4.6 [4.0‐5.3]), P < 0.002. In VE, there was no difference in
flux between the patient groups for any of the strains.
Bacterial exposure induced changes in FAE conductance, suggesting that
the E. coli affected barrier function of Crohn’s disease mucosa. Following
bacterial uptake, there was an increased percentage of E. coli co‐localising
with dendritic cells in the mucosa of Crohn’s disease (45 [20‐67] %)
64
compared to controls (14 [0‐33] %), P < 0.0001, and augmented tissue
release of TNF‐α.
In conclusion, patients with Crohn’s disease, but not those with ulcerative
colitis, exhibited an increased transmucosal uptake of non‐pathogenic E.
coli across the FAE, compared to non‐inflammatory controls. Moreover,
we found a higher amount of dendritic cells in Crohn’s disease and
enhanced release of TNF‐α. Our findings may have implications for the
loss of tolerance to the normal gut flora in Crohn’s disease patients.
Paper IV
Rats were submitted to acute stress to elucidate the mechanisms behind
the stress‐induced increase in FAE permeability shown in Paper I. Prior
to stress, rats were intraperitoneally injected with blockers for substance
P (anti‐NK‐1R), CRH (anti‐CRH‐R) or mast cells (doxantrazole).
Results showed inhibition of stress effects in FAE when blocking the mast
cells and receptors for CRH prior to stress. In FAE, the stress‐induced
increases in permeability to HRP (56.0 ± 14.4 pmol/h/cm2 vs. controls 6.6 ±
1.9) and E. coli K‐12 (23.3 ± 2.8 units vs. controls 4.3 ± 0.7) were
diminished by anti‐CRH‐R and doxantrazole, however, the anti‐NK‐1R
had no significant effect.
In VE, the increased E. coli K‐12 passage after stress (7.1 ± 1.6 vs. controls
2.1 ± 0.4) was diminished by doxantrazole and anti‐CRH‐R, whereas
there was no effect of anti‐NK‐1R. The increased HRP passage after stress
(18.9 ± 6.9 vs. controls 6.6 ± 3.6) was only blocked by doxantrazole. Our
results further showed that stress effects on permeability were mimicked
by exposing both VE and FAE tissues to CRH and VIP in Ussing
chambers. When also pretreating with doxantrazole, these effects were
inhibited.
65
The physiological results were confirmed by the findings of CRH and
VIP, and their receptors, within the Peyer’s patches, SED, and adjacent
villi. Expressions of VIPR1 and CRH‐R1 were found on mast cells located
in the adjacent villi and surrounding the follicles, and more seldom
inside the follicles and close to the FAE (Fig. 20 a, b)
2
2
1
1
FAEFAE
FollicleFollicle
FollicleFollicle
a b
2
2
1
1
FAEFAE
FollicleFollicle
FollicleFollicleFollicleFollicle
a b
Fig. 20. Mast cells with vasoactive intestinal peptide receptor 1 (VIPR1) and
corticotropin‐releasing hormone‐receptor1 (CRH‐R1) expression, respectively, in
rat Peyer’s patches. (a) VIPR1 expression on mast cells (arrows, 1‐2) at follicle
margins. Arrowheads indicate mast cells negative for VIPR1, FAE = follicle‐
associated epithelium. (b) A mast cell with CRH‐R1 staining inside the follicle
(arrow).
66
VIPR2 and CRH‐R2 were expressed to a lower extent. Staining for VIP
and CRH peptide revealed immunoreactive mast cells mostly in the
adjacent villi and at follicle margins.
In conclusion, we found that the stress‐induced increase in FAE uptake of
antigen and bacteria was prevented by blocking the mast cells and
receptors for CRH. Furthermore, VIP mimicked stress‐induced
permeability changes, and the changes were abolished by stabilising the
mast cells. Our results may be of importance for the pathogenesis in
stress‐related intestinal disorders, and give clues to new therapeutic
approaches.
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6. DISCUSSION
Several studies have shown an enhanced uptake across the FAE and
Peyer’s patches in animals 81;158. In fact, FAE is considered as an important
entry site for antigens as well as bacteria. The earliest observable lesions
in the development of recurrent Crohn’s disease are initiated as
microscopic erosions at the FAE 7;11. Crohn’s disease is further associated
with an increased permeability and immune response to bacteria 33;65;116,
and the disease course can be altered by environmental stress 63. Thus, we
hypothesised that the barrier dysfunction seen in Crohn’s disease could
be generated by alterations in the FAE.
Initially, we wanted to elucidate if the FAE barrier was sensitive to stress.
When designing Paper I, no animal or human studies were present
investigating the effects of stress on the FAE barrier. However, we knew
from previous studies that acute and chronic stress in rats could
modulate the VE barrier, as indicated by enhanced permeability,
increased mucin production, bacterial attachment, mast cell hyperplasia,
mithochondrial swelling and initiation of inflammation 202‐206;208;210;211. With
this in mind, we decided to submit rats to acute and chronic
physiological stress to evaluate the effects on FAE. Our results revealed
that the FAE barrier was sensitive to stress, shown by changes in
electrophysiology and increased uptake of luminal antigens and bacteria.
Hereby we could, for the first time, demonstrate an enhanced
permeability in FAE after stress, and also higher permeability increase in
FAE compared to VE.
In a follow‐up study we wanted to confirm the increased FAE
permeability in humans. Results showed a considerably more
pronounced bacterial and antigen uptake also in human FAE, compared
to VE. Since we could show that stress increased FAE permeability, and
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stress is a contributing factor to IBD and Crohn’s disease 63;64, we
speculated that there could be an altered FAE function in patients with
Crohn’s disease. Our results showed that patients with Crohn’s disease
displayed an enhanced bacterial passage in FAE compared to non‐
inflammatory and inflammatory controls.
In the present studies, we found an enhanced antigen and bacterial
permeability in FAE compared to VE in both rats and humans. In
addition, we found that stress further increased the permeability in FAE.
The HRP passage was increased almost four times in FAE after stress
compared to 1.8 times in VE, and bacterial uptake increased 30 times in
FAE after stress, as compared to only 1.6 times in VE. This suggests that
FAE is more vulnerable to stress than VE, and it might be speculated that
a major part of the bacterial translocation that occurs in stress may be due
to invasion across the FAE. When comparing the permeability in Crohn’s
patients with that in non‐inflammatory controls, a higher bacterial
permeability in FAE was seen. However, the HRP passage was similar,
suggesting different mechanisms of the FAE barrier dysfunction during
stress and inflammation.
The higher passage in FAE compared to VE in the normal situation
probably refers to the different surface characteristics 81, making FAE
more accessible for luminal antigen and bacteria. In addition, the
presence of M cells within the FAE contributes to the increased
permeability 80. However, the fact that the FAE permeability increases
after stress, and is enhanced in Crohn’s disease, indicates that something
occurs in the epithelium that further facilitates for luminal antigens and
bacteria to cross the barrier. Since a higher M cell number has been found
during inflammation 90;175, the increased permeability in Crohn’s disease
could, at least partly, be due to an increased M cell number within the
FAE. One could further speculate that the numbers are also increased
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after chronic stress, which could explain the enhanced permeability.
Thus, it would have been highly interesting to verify the M cells number
in that FAE tissue investigated in our studies, but as already mentioned,
there is no reliable rat or human M cell marker, which hampers the
possibility to identify the M cells present in our FAE tissue investigated.
Contradictory to the hypothesis that the increased permeability is due to
M cells in the FAE, is one paper 236 showing an M cell independent
mechanism. In a co‐culture model of mice dendritic cells and human
epithelial cells, it was shown that dendritic cells directly can sample
luminal antigen by unzipping tight junctions and extending their
dendrites through the intact epithelium. However, this observation has
not yet been reproduced in human tissue.
The importance of FAE in permeability might be questioned due to its
smaller surface area compared to VE. The VE contains numerous folded
villi that increase the surface area considerably compared to FAE.
However, the smaller surface area is highly compensated by the larger
capacity of the FAE cells to sample luminal content, and the importance
of the underlying Peyer’s patches for initiation of immune responses.
When antigen and bacteria enter the FAE and M cells, they are
transported across the cells, and delivered to antigen‐presenting cells in
the Peyer’s patches to induce inflammatory responses. Lymphocyte‐
containing M cell pockets decrease the travelling distance from the apical
to the basolateral side, which further improves the passage in FAE. In VE,
the major part of entering content is degraded on their way through the
cells via for example lysosomal events, and only a minor amount reaches
all the way through intact.
From our results it may be suggested that in Crohn’s disease, antigen and
bacteria enter the FAE barrier at a higher rate due to a disturbed barrier
70
function. The question is whether the disturbed barrier is caused by a
primary epithelial defect, or if it is the other way around and the
increased permeability is a consequence of an early immune activation.
In the first theory, it could be speculated that patients with Crohn’s
disease have a genetically driven disrupted barrier function leading to
increased uptake of luminal antigens and bacteria, which are
phagocytosed and presented to immune cells that starts to produce
inflammatory mediators. After secretion, cytokines and chemokines
attract other inflammatory cells that start to produce even more
inflammatory mediators. Finally this could lead to further destruction of
the epithelial cells, by the inflammatory mediators, affecting epithelial
defence and control of permeability. This theory is strengthened by
studies showing increased permeability in healthy relatives of patients
with Crohn’s disease 18;20;21.
The second theory says that immune cell activation and inflammation
precede the barrier disruption. The importance of immune cell activation
in barrier dysfunction has been confirmed by several studies. For
example TNF‐α and IFN‐γ have shown to increase both paracellular and
transcellular permeability 170;237;238 and studies have shown that the barrier
dysfunction in Crohn’s disease is TNF‐α‐dependent 170;239;240. Furthermore,
it has been demonstrated that monocytes from patients with Crohn’s
disease secrete TNF‐α upon bacterial stimulation 140, and mast cells of
Crohn’s disease mucosa are major producers of TNF‐α 241;242. A study
speaking in favour of inflammation coming first is that 54 % of healthy
relatives to Crohn’s disease patients have subclinical inflammation,
measured as levels of calprotectin 243. Moreover, in a rat model of
intestinal inflammation it was shown that the rats developed
inflammation without getting increased permeability 244.
Both of the theories make sense. It could be that in Crohn’s disease the
epithelial barrier is initially to some extent disrupted, for example due to
71
an abnormal response to bacteria. This leads to increased passage,
immune activation and subsequently elevated levels of for example TNF‐
α, IFN‐γ and IL‐4 142;170;237;238 that further destroy the barrier and increase
the permeability.
The importance of the luminal microflora in Crohn’s disease is well
documented 41;42. For example studies have shown increased number of
adherent‐invasive E. coli in Crohn’s disease mucosa 43;44. The significance
of bacteria in intestinal inflammation is further strengthened by the fact
that rats susceptible for intestinal inflammation did not develop IBD
when bread under germfree conditions 49.
The novel findings in Paper III, an increased uptake of non‐pathogenic
bacteria in FAE despite an equal permeability to 51Cr‐EDTA and HRP,
point to the importance of FAE in Crohn’s disease and highlight the
importance of FAE‐bacterial interactions leading to inflammation. Since
the FAE tissues of patients with ulcerative colitis showed no increase in
bacterial passage, the enhanced bacterial transport may be specific to the
FAE of Crohn’s disease. What are the mechanisms behind this increase?
Possible explanations could be the involvement of receptors, referred to
as pattern‐recognition receptors (PRR), that recognise the so called
pathogen‐associated molecular patterns (PAMPS), such as
lipopolysaccharide (LPS) and peptidoglycans, 245 The two major groups
of PRRs are the NOD‐containing proteins and toll‐like receptors (TLRs).
The best characterised NOD proteins relevant to the intestinal physiology
are NOD1 and NOD2, where NOD2 recognises structures in a wider
range than NOD1, and is more restricted to the small intestine. Although
several potential signalling pathways involved in inflammation and
innate immunity linked to NOD2 activation have been demonstrated, the
physiologic functions are less well understood. Under normal conditions
the NOD2 expression is low, however, during inflammation the
72
expression increases 246, which could suggest a role for altered microbial
sensing in the increased bacterial uptake in Crohn’s disease. Mutations in
the NOD2 gene lead to inhibited activation of the NOD2 protein,
however, since none of the Crohn’s disease patients included in our
study carried the mutation, one can presume that the NOD2 protein was
unaffected.
TLR‐4 is the best studied TLR and is essential for the recognition of LPS,
and further limiting LPS responsiveness. A TLR‐4 polymorphism and
increased apical expression of TLR‐4 has been found in both Crohn’s
disease and ulcerative colitis 247;248. The overexpression of TLR‐4 could
result in LPS hyper‐responsiveness leading to consecutive
proinflammatory cytokine secretion which in turn stimulates TLR‐4
expression and further inappropriate signalling in the presence of
luminal LPS. It is known that TLR‐4, is upregulated in FAE and M cells of
mice 249, and may be involved in regulation of uptake of bacteria and
microparticles 250. Therefore, the role of TLR‐4 in the FAE dysfunction in
Crohn’s disease should be elucidated in further studies.
Another way in which TLRs are involved in regulation of tolerance to
commensal bacteria is through their effect on dendritic cells. After
sampling of luminal content, dendritic cells migrate across the epithelium
and by stimuli from bacteria via TLR signalling, maturation is induced 236;251. Dendritic cells are located in the SED for phagocytosis and
presenting of antigen 94;252. It has been shown that they can squeeze in
between epithelial cells through the tight junctions to sense and sample
luminal microbes 236. Our findings in Paper III of increased bacterial
uptake into dendritic cells in Crohn’s disease mucosa suggest that
dendritic cells may play an active role in the immune cell‐bacterial
interaction leading to inflammation in Crohn’s disease.
73
Our results, demonstrating a disrupted FAE barrier function after stress
in rats gave rise to Paper IV where we wanted to elucidate the role of
mast cells and neuropeptides in the increased uptake. The significance of
mast cells in stress‐induced mucosal dysfunction of VE has been clearly
demonstrated by several groups 202;204;211;212, and we could also confirm
this in Paper I, where we observed a 3‐fold increase in mast cell number
in stressed rats compared to controls. Numerous studies have
highlighted the role of CRH in stress 207;215;216, and some of them have
demonstrated inhibited effects of CRH when blocking the mast cells,
however, the importance of mast cells and CRH in stress‐induced FAE
permeability has not been reported. Our results showed inhibited stress
effects when blocking the receptors for CRH, and mimicked stress‐effects
on permeability when exposing FAE to CRH peptide in Ussing chambers.
In addition, when stabilising the mast cells with doxantrazole, the effects
of both stress and CRH were inhibited, which points to that CRH is
released during stress and increases permeability via activation of mast
cells.
In addition, we could, in Paper IV, for the first time, identify VIP as a
regulator of mucosal permeability. FAE exposure to VIP in Ussing
chambers resulted in increased paracellular permeability and
transcellular passage to antigen and bacteria. Furthermore, we found VIP
peptide and VIP receptors both in and close to mast cells at follicle
margins and in adjacent villi. The involvement of VIP in permeability has
previously only been described briefly. In a co‐culture model of human
submucosa containing the submucosal neuronal network and human
polarised colonic monolayers, the paracellular permeability was
increased by electrical stimulation of submucosal neurons 218. The effects
of the neuron activation were blocked by a VIP receptor antagonist, and
reproduced by VIP peptide. This, together with our results regarding
74
VIP‐induced increased permeability, suggest an important modulatory
role for VIP in the regulation barrier function.
In addition to the importance of mast cells and neurons during stress,
their roles in IBD have also been highlighted. Degranulation of mast cells,
and increased numbers, have been found in mucosa from patients with
ulcerative colitis 253 as well as Crohn’s disease 254. Moreover, elevated
levels of histamine have been measured in gut lumen of Crohn’s disease
patients 255.
There are only a few studies present regarding the consequences of
neuronal changes on mucosal function in IBD. However, changes in
neuropeptide innervation 256, and altered VIP 257;258 and substance P 259;260
expressions, have been found in mucosa from patients with ulcerative
colitis and Crohn’s disease. There are no reports on immunoreactivity to
CRH in Crohn’s disease, however, in mucosal inflammatory cells of
ulcerative colitis, increased CRH expressions have been observed 261.
Even if our results demonstrate a role of VIP in FAE barrier function
during stress, it can not be directly applied to the permeability changes
seen in FAE of Crohn’s disease. However, several studies have shown a
link between stress and IBD 191;194;199;200, and the fact that a disrupted
barrier function can be induced in healthy rats, by submitting them to
stress, suggests that stress may be implicated in the initiation,
perpetuation, or exacerbation of the inflammation seen in IBD.
Consequently, it could be speculated that VIP is important also in
regulating the permeability in Crohn’s disease, not at least considering
the increased VIP expression found in Crohn’s disease mucosa. However,
further studies are needed to elucidate the role of VIP in FAE barrier
function of Crohn’s disease.
75
In conclusion, this thesis presents novel insights into regulation of the
FAE barrier, as well as into the pathophysiology of Crohn’s disease by
demonstrating a previously unrecognised defect of the FAE barrier
function in ileal Crohn’s disease. Further studies in a rat IBD model
would be highly valuable to better understand the mechanisms involved,
and the role of FAE in the interplay between stress and intestinal
inflammation.
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7. CONCLUSIONS
• The FAE barrier can be modulated by stress as illustrated by
enhanced antigen and bacterial passage and ultrastructural
changes after acute and chronic stress in rats.
• The more pronounced increase of transmucosal passage in FAE
compared to VE after stress suggests that FAE is more stress‐
reactive and prone to interact with antigen and bacteria
• Human FAE in biopsies and surgical specimens can be identified
by transilluminating the mucosa from below in dissection
microscope, and the techniques can be considered equal
regarding permeability measurements in Ussing chambers.
• Human FAE exhibits a substantially higher antigen and bacterial
passage compared to VE. In vitro experiments revealed an actin‐
dependent passage of E. coli and HRP uptake via
macropinocytosis.
• Patients with Crohn’s disease demonstrate a higher transmucosal
uptake of E. coli K‐12 and HB101 compared to non‐IBD controls
and patients with ulcerative colitis, suggesting that the FAE
barrier is altered in Crohn’s disease by disease‐specific
mechanisms.
• Following bacterial uptake, patients with Crohn’s disease reveal
a higher percentage of E. coli internalised by dendritic cells
compared to non‐IBD controls.
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• The stress‐induced increase in FAE permeability seen in rats is
regulated by CRH and mast cells. Further stress effects on
permeability can be mimicked in vitro by VIP, and the effects are
abolished by blocking the mast cells.
• In rats, mast cells are present mainly in the adjacent villi, but also
in follicles and SED. The neuropeptides CRH and VIP and their
receptors are expressed in mast cells within these regions.
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8. TACK Det finns många personer som på ett eller annat sätt hjälpt och stöttat mig i mitt avhandlingsarbete. Av dem vill jag framförallt tacka; Johan D Söderholm, min huvudhandledare, som gav mig chansen att göra detta. Ditt positiva tänkande och uppmuntran har gjort att det hela tiden har känts roligt att forska! Ann‐Charlott ʺLottaʺ Ericson, min biträdande handledare, som enträget har suttit med mig vid de olika mikroskopen trots att det ibland känts hopplöst. Men Murphy’s lag till trots så löste vi alla tekniska problem och hade riktigt roligt på kuppen. Alla medförfattare, speciellt tack till Elisabet Gullberg och Per Artursson för trevligt och givande samarbete. Ylva Braaf, för ovärderlig hjälp i Ussinglabbet, ibland kan man undra vem som blev mest stressade, vi eller råttorna? Eva Sjödahl för mycket värdefull hjälp med histologi. Conny Wallon för att du hjälpte mig så mycket i början och för hjälp med insamling av patientmaterial. Ida Schoultz, min kollega och med tiden mycket goda vän, för ditt stora stöd och uppmuntran. Utan dig på jobbet hade allt varit mycket tråkigare! Tack också för att du släpat ut mig till fikarummet ibland när jag inte kunnat slita mig från datorn, de avbrotten har nog varit mer än välbehövliga... Sa’ad Y Salim, my colleague and dear friend, for always helping me out whenever I need it, especially during my maternity leave. Thanks also for your encouragement and for always being such a good friend. Femke Lutgendorff och Johan Junker, mina rumskompisar under senare delen av avhandlingsarbetet, och mina rumskompisar under den tidigare delen, Henrik Blomqvist och Hanna Olausson, för att ni bidragit till trevliga stunder på kontoret och diskussioner både vad gäller forskning och fritid. Lena Trulsson, min kollega och goda vän, för intressanta diskussioner och goda råd både vad gäller forskning och annat i livet. Tack även till Staffan Smeds för att du inspirerade mig till att själv börja forska och ge mig in i den här världen.
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Alla andra på KEF som hjälpt mig, framförallt Pia Karlsson som sett till att jag kan köra mina försök dag efter dag utan att behöva lägga tid på disk och beställningar, samt Håkan Wiktander, Iréne Cavalli‐Björkman och Kerstin Hagersten för all god service! All personal på Djuravdelningen, särskilt tack till Dan Linghammar och Jan Szymanowski för praktisk hjälp och goda råd. All personal på Kirurgen som på olika sätt har hjälpt mig i mitt forskningsarbete. Framförallt alla kirurger, ingen nämnd ingen glömd, för hjälp med insamling av operationspreparat, och sköterskor för assistans vid biopsiprovtaging, och sist men inte minst Britt‐Marie Johansson och Ulla Svensson Bater för hjälp med många praktiska saker genom åren. Tack även till alla patienter och friska frivilliga för att ni har ställt upp med material till min forskning! Personal på Patologen som har tagit sig tid att hjälpa till vid utskärning av operationsmaterial. Rakel Martinsson, Markus Jansson, Robert Berglund, och Tina Betmark, mina studenter, som under examensarbeten har hjälpt till med delar av det laborativa. Alla mina vänner för att ni har stått ut med mig under våren. Jag lovar att ta igen alla inställda luncher och träffar när detta är över :o) Alpha, min själsfrände, för allt ditt tålamod med mig senaste tiden då jag bara jobbat, jobbat och jobbat. Utan all din hjälp och ditt stöd hade jag aldrig orkat. Det här är din avhandling lika mycket som min. Tack även till dig Mathilda, min lilla solstråle, för att du finns, och ger mig perspektiv på livet. Min övriga familj, framförallt mamma, pappa och svärmor för all hjälp med Mathilda när inte tiden räckt till. Det är inte helt lätt att skriva en avhandling med en liten i knäet, även om det faktiskt tidvis gått det också….
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9. SVENSK SAMMANFATTNING Crohns sjukdom är en kronisk inflammatorisk tarmsjukdom av okänd orsak. Det tidigaste tecknet på Crohns sjukdom är mikroskopiska sår i det s.k. follikelassocierade epitelet (FAE) som täcker ansamlingar av immunceller i tarmen. FAE är specialiserat för att fånga innehåll från tarmen och transportera det till underliggande immunvävnad. Denna funktion är viktig för att inducera skyddande immunsvar, men den utgör också en ingångsväg för sjukdomsalstrande bakterier. Crohns sjukdom är associerat med ett kraftigt ökat immunsvar mot bakterier, och sjukdomsförloppet kan ändras av stress. Det övergripande syftet med avhandlingen var att studera effekterna av stress på FAE samt att undersöka rollen av FAE vid utvecklingen av tarminflammation, särskilt vid Crohns sjukdom. Inledningsvis studerades effekterna av psykologisk stress på FAE. Stressade råttor uppvisade ökad genomsläpplighet av bakterier efter stress, och passagen var högre i FAE än i vanligt epitel. Efterföljande experiment visade att stressförändringarna i slemhinnan regleras via kortikotropinfrisättande hormon och mastceller. Vidare visade det sig att vasoaktiv intestinal peptid kunde efterlikna stressens effekter på genomsläppligheten, och att detta kunde förhindras genom att blockera mastcellerna. Studier av tunntarmsslemhinna från patienter med icke‐inflammatorisk tarmsjukdom och friska kontroller visade en högre passage av bakterier i FAE än i vanligt epitel. Hos patienter med Crohns sjukdom var bakteriepassagen genom FAE betydligt ökad jämfört med kontroller. Resultaten från detta avhandlingsarbete visar att stress kan förändra upptaget av bakterier från tarmen via FAE, med mekanismer som innefattar kortikotropinfrisättande hormon och mastceller. Detta har gett nya kunskaper kring regleringen av slemhinnebarriären. Vidare presenterar denna avhandling nya insikter i sjukdomsuppkomsten vid Crohns sjukdom genom att påvisa en tidigare okänd defekt i barriärfunktionen i FAE.
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