Dissertation Hornickel Korr - elib.tiho-hannover.de · Bereich Histologie und Embryologie...

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Tierärztliche Hochschule Hannover

Investigations on the innate immunity

of the esophagus epithelium

of domesticated mammals

INAUGURAL-DISSERTATION

zur Erlangung des Grades einer

DOKTORIN DER VETERINÄRMEDIZIN

-Doctor medicinae veterinariae-

(Dr. med. vet.)

Vorgelegt von

Isabelle Nina Hornickel

aus Hagen

Hannover 2009

Wissenschaftliche Betreuung: Univ.-Prof. Dr. rer. nat. habil. Wilfried Meyer

Stiftung Tierärztliche Hochschule Hannover

Anatomisches Institut

Bereich Histologie und Embryologie

Bischofsholer Damm 15

30173 Hannover

1. Gutachter: Univ.-Prof. Dr. rer. nat. habil. Wilfried Meyer

2. Gutachter: Univ. Prof. Dr. med. vet. habil. M. Hewicker-Trautwein

Tag der mündlichen Prüfung: 8. Mai 2009

Diese Arbeit wurde durch die H. Wilhelm Schaumann Stiftung (Hamburg) finanziell

unterstützt.

Für meine Mama

Gott hat uns nicht gegeben den Geist der Furcht, sondern der Kraft und der Liebe und der Besonnenheit.

(Neues Testament, 2. Timotheus 1, 7)

Table of contents

V

Table of contents

TABLE OF CONTENTS............................................................................................. V

ABBREVIATIONS..................................................................................................... IX

FIGURES .................................................................................................................. XI

TABLES.................................................................................................................. XIII

1 INTRODUCTION ..............................................................................................1

2 LITERATURE...................................................................................................3

2.1 Macroscopic anatomy of the esophagus: Overview .............................................. 3

2.2 Histological structure: Overview.............................................................................. 3

2.3 Innate immunity: General aspects ........................................................................... 8

2.4 Antimicrobial peptides: Overview .......................................................................... 15

2.4.1 ß-defensins ............................................................................................................ 18

2.4.2 Cathelicidin ............................................................................................................ 22

2.4.3 Biological activity of antimicrobial peptides............................................................ 24

2.5 Toll-like receptors .................................................................................................... 25

2.6 C-type lectin receptors (ß-glucan receptors) ........................................................ 28

2.6.1 Collectins and ficolins: General overview .............................................................. 28

2.6.2 Mannan-binding lectin (MBL) ................................................................................. 29

2.6.3 Ficolins................................................................................................................... 30

2.6.4 Structure of collectins and ficolins.......................................................................... 31

2.6.5 Functional features of the collectins and ficolins: Activation of the complement

system.................................................................................................................... 32

2.6.6 Pathogene specificity of collectins and ficolins ...................................................... 35

2.6.7 Dectin-1.................................................................................................................. 39

2.7 Lysozyme.................................................................................................................. 41

2.8 Dendritic cells .......................................................................................................... 42

Table of contents

VI

2.9 Questions addressed in this study ........................................................................ 45

3 MATERIAL AND METHODS......................................................................... 46

3.1 Animals ..................................................................................................................... 46

3.2 Sampling ................................................................................................................... 46

3.3 Tissue Fixation ......................................................................................................... 48

3.3.1 Bouin’s solution (after BÖCK 1989) ....................................................................... 48

3.3.2 Calcium acetate-buffered formalin (after LILLIE and FULLMER 1976).................. 48

3.3.3 HOPE® fixation (after OLERT et al. 2001) .............................................................. 48

3.3.4 Fixation after KARNOVSKY (1967)........................................................................ 49

3.3.5 Liquid nitrogen sampling ........................................................................................ 49

3.4 Paraffin embedding.................................................................................................. 49

3.4.1 Paraffin embedding for samples fixed in Bouin’s solution and Ca-acetate buffered

formalin................................................................................................................... 49

3.4.2 Paraffin embedding HOPE® ................................................................................... 50

3.5 Standard EPON embedding for Karnovsky fixed tissue ...................................... 51

3.6 Sectioning................................................................................................................. 51

3.7 Histological staining ................................................................................................ 52

3.7.1 Hematoxylin-Eosin staining of Bouin and formalin-fixed tissue.............................. 52

3.7.1.1 H.E. staining of HOPE® fixed tissue ............................................................... 53

3.7.2 Trichrome staining (Masson-Goldner) (after BÖCK 1989) ..................................... 55

3.8 Microscopical evaluation ........................................................................................ 56

3.8.1 Light microscopy .................................................................................................... 56

3.8.2 Cryo scanning electron microscopy (Cryo-SEM) ................................................... 56

3.9 Immunohistochemistry............................................................................................ 57

3.9.1 Primary antibodies.................................................................................................. 57

3.9.2 Secondary antibodies............................................................................................. 59

3.9.2.1 Two step indirect method with peroxidase linked secondary antibodies ........ 59

3.9.2.2 Use of a labelled streptavidin biotin Complex (LSBC).................................... 61

3.9.2.3 Immunofluorescence (IF) ............................................................................... 62

3.9.3 Immunohistochemistry: General procedure ........................................................... 64

Table of contents

VII

3.9.4 Controls.................................................................................................................. 66

3.9.5 Preliminary tests for the establishment of the primary antibodies.......................... 66

3.9.5.1 Demasking methods tested............................................................................ 67

3.9.6 Antibody dilutions and visualisation systems applied ............................................ 71

3.9.7 Statistical analyses ................................................................................................ 72

4 RESULTS.......................................................................................................73

4.1 Light and electron microscopical observations ................................................... 73

4.1.1 Demonstration of microorganisms with cryo SEM ................................................. 73

4.1.2 General esophageal structure................................................................................ 77

4.2 Fixation experiment: Influences of different fixation media on IHC results....... 81

4.2.1 ß-defensin 2 and -3 reactions ................................................................................ 81

4.2.2 Cathelicidin reactions............................................................................................. 86

4.2.3 Toll-like receptor 2 reactions.................................................................................. 88

4.2.4 Lysozyme reactions ............................................................................................... 91

4.3 IHC results for the substances of the innate immune system: Species

comparison............................................................................................................... 93

4.3.1 Demonstration of ß-defensin 2............................................................................... 93

4.3.2 Demonstration of ß-defensin 3............................................................................... 97

4.3.3 Demonstration of cathelicidin............................................................................... 102

4.3.4 Demonstration of Toll-like receptor 2 ................................................................... 105

4.3.5 Demonstration of ß-glucan receptors................................................................... 108

4.3.6 Demonstration of lysozyme.................................................................................. 109

4.3.7 Demonstration of Langerhans cells ..................................................................... 112

5 DISCUSSION ...............................................................................................113

5.1 Fixation experiment ............................................................................................... 113

5.1.1 Influences of fixation on structure preservation ................................................... 113

5.1.2 Influences of fixation on immunohistochemical results ........................................ 117

5.2 Defence mechanisms of the mammalian esophagus: Species comparison ... 124

5.2.1 Antimicrobial peptides.......................................................................................... 124

5.2.2 Toll-like receptor 2 ............................................................................................... 129

5.2.3 ß-glucan receptors ............................................................................................... 133

5.2.4 Lysozyme............................................................................................................. 137

Table of contents

VIII

5.2.5 Langerhans cells .................................................................................................. 138

5.3 Microbial colonisation of the esophagus ............................................................ 142

5.4 Conclusions............................................................................................................ 145

6 ZUSAMMENFASSUNG............................................................................... 147

7 SUMMARY .................................................................................................. 150

8 REFERENCES............................................................................................. 152

Abbreviations

IX

Abbreviations

AB Antibody

AP Antimicrobial peptides

APC Antigen presenting cell

BC B-cell

BCR B-cell receptor

ß-GR ß glucan receptor

BSA Bovine serum albumin

CD Cluster of differentiation

CLR C-type lectin receptor

CAP Cationic antimicrobial peptide

CAT Cathelicidin

CFU Colony forming units

CRD Carbohydrate recognition domain

CTL C-type lectin receptor

DC Dendritic cell

FBD Fibrinogen binding domain

Fig. Figure

GalNac N-acetylgalactosamin

GlcNac N-acetyl-D-glucosamin

hBD-2 human ß-defensin 2

hBD-3 human ß-defensin 3

IHC Immunohistochemistry

ITAM immunoreceptor tyrosine-based activation-like motiv

LB Lamellar body

LC Langerhans cell

LN2 Liquid nitrogen

LPS Lipopolysaccharide

m monoclonal

MAC Membrane attacking complex

MCG Membrane coating granules

MDC Myeloid dendritic cell

MHC Major histocompatibility complex

mRNA messenger ribonucleic acid

Abbreviations

X

NF-κB Nuclear factor of kappa light polypeptide gene enhancer

NGS Normal goat serum

NHS Normal horse serum

NRS Normal rabbit serum

NK Natural killer cell

p polyclonal

PAMP Pathogen associated molecular pattern

PBS Phosphate buffered saline

PDC Plasmacytoid dendritic cell

PRR Pathogen recognition receptor

RT-PCR Reverse transcriptase polymerase chain reaction

SEM Scanning electron microscopy

SP-A Surfactant protein A

SP-D Surfactant protein D

Tab. Table

TEM Transmission electron microscopy

TGF Transforming growth factor

TLR Toll-like receptor

Figures

XI

Figures

Fig. 2.1 General histological structure of the mammalian esophagus according to the

great three nutrition groups 7

Fig 2.2: Danger model of innate immunity 12

Fig 2.3: Shia-Matsuzaki-Huang model 21

Fig 3.1: Principle of the two step indirect method 61

Fig 4.1: Microorganisms on the esophagus epithelium of the horse 73

Fig. 4.2: Microorganisms on the esophagus epithelium of the goat 74

Fig. 4.4: Microorganisms on the esophagus epithelium of the rat, surrounding

an excretory duct of a secretory gland 75

Fig 4.5: Microorganisms and mucus covering the esophagus epithelium

of the pig 75

Fig. 4.6: Microorganisms and mucus covering the esophagus epithelium of the

dog 76

Fig. 4.7: Bacteria and fungi without mucus covering on the esophagus epithelium

of the cat 76

Fig. 4.8: Histological structure of the esophagus of the herbivorous horse (left)

and the omnivorous dog (right) 78

Fig. 4.9: Histological overview of the esophagus of the carnivorous cat 78

Fig. 4.10: Semithin sections of the esophagus epithelium of the herbivorous

horse (left) and sheep (right) 79

Fig. 4.11: Semithin section of the esophagus epithelium of the omnivorous

mouse 79

Fig. 4.12: Semithin section of the esophagus epithelium of the carnivorous cat 80

Fig. 4.13: Demonstration of fixation differences for hBD-2 in the equine

esophagus 83

Fig. 4.14: Demonstration of fixation differences for hBD-2 in the

ovine esophagus 84

Fig 4.15: Demonstration of fixation differences for TLR2 in the

equine esophagus 90

Fig 4.16: hBD-2 reaction in the esophagus epithelium of the rat 94

Fig. 4.17: IgG rabbit isotype control for hBD-2 for the esophagus of the rat 94

Fig. 4.18: hBD-2 reaction in the esophagus epithelium of the dog 95

Fig. 4.19: Rabbit IgG isotype controle for hDB-2 in the esophagus of the dog 95

Figures

XII

Fig. 4.20: hBD-2 reaction in the esophagus epithelium of the cat 96

Fig 4.21: Differences in staining intensities in Bouin-fixed samples between the

three nutrition groups for hBD-2 97

Fig 4.22: Demonstration of hBD-3 in the esophagus epithelium of the cattle 98

Fig. 4.23: Demonstration of hBD-3 in the ovine esophagus epithelium 99

Fig. 4.24: Negative control for hBD-3 in the ovine esophagus 99

Fig. 4.25: Positive control for hBD-3 100

Fig. 4.26: Demonstration of hBD-3 in the esophagus epithelium of the pig 100

Fig. 4.27: IgG rabbit isotype control for hBD-3 100

Fig 4.28: Differences in staining intensities in Bouin-fixed samples between

the three nutrition groups for hBD-3 101

Fig. 4.29: Demonstration of CAT in the esophagus epithelium of the horse 103

Fig. 4.30: IgG mouse isotype control for CAT 103

Fig. 4.31: CAT reaction in the ovine esophagus epithelium 104

Fig. 4.32: CAT reaction in the esophagus epithelium of the dog 104

Fig. 4.33: CAT reaction in the esophagus of the cat 104

Fig. 4.34: Negative control for CAT 104

Fig. 4.35: Detection of TLR2 in the esophagus epithelium of the horse 106

Fig. 4.36: Detection of TLR2 in the esophagus epithelium of the dog 106

Fig. 4.37: Detection of TLR2 in the esophagus epithelium of the cat 107

Fig. 4.38: Differences in staining intensities in Bouin-fixed samples between the

three nutrition groups for TLR2 108

Fig. 4.39: Lysozyme reaction in the esophagus epithelium of the mouse 110

Fig. 4.40: IgG rabbit Isotype control for lysozyme 110

Fig. 4.41: Differences in staining intensities in Bouin-fixed samples between

the three nutrition groups for lysozyme 111

Fig. 4.42: Demonstration of langerin + LCs in the esophagus epithelium of

the horse 112

Tables

XIII

Tables

Table 2.1: In vitro antimicrobial activity of antimicrobial peptides in animals 23

Tab 2.2: Comparison of animal collectins and ficolins and their antimicrobial

activity 37

Tab. 3.1: Domesticated mammalian species used 47

Table 3.2: Overview fixation steps HOPE® 50

Tab. 3.3: Primary antibodies 58

Tab. 3.4: Overview of secondary detection systems 63

Tab 3.5: General immunohistochemistry protocol, comparison of

fixation techniques 65

Tab. 3.6: Tested antibody dilutions, demasking methods and

visualisation systems for Bouin and formol-fixed material 69

Tab. 3.7: Overview of tested antibody dilutions and visualisation systems

on HOPE® fixed tissue 70

Tab. 3.8: Antibody dilutions applied for established antibodies 71

Tab. 4.1: IHC results of the ß-defensins 2 reactions according to

the different fixation solutions 84

Tab 4.2: IHC results of the ß-defensins 3 reactions according to


Tab 4.3: IHC results of the CAT reactions according to


Tab 4.4: IHC results for Toll like receptor 2 according to


Tab. 4.5: IHC results for lysozyme according to


Introduction

1

1 Introduction

The innate immune mechanisms present in the esophagus epithelium of

domesticated mammals have, until now, not been in the focus of any studies. The

esophagus, as the first part of the digestive tract, has often been neglected in

research. In contrast there is a huge amount of information available on innate

defence mechanisms of the following parts of the gastrointestinal tract (STOKES and

WALY 2006, LOTZ et al. 2007, SCHENK and MUELLER 2008, SINGH et al. 2009).

The only studies available on the immune defence mechanisms of the esophagus

have been conducted in humans (AL YASSIN and TONER 1975, HOPWOOD 1995).

We aim to shed some light on innate immune parameters present in the esophagus

of nine domesticated mammalian species. We grouped the animals in order to get a

more differentiated idea on variations in the expression intensity of such defence

mechanisms, comparing influences of different nutrition types. Thus, we studied

representatives from the groups of herbivores, omnivores and carnivores.

The esophagus epithelium is confronted with mechanical strain, chemical food

additives and pathogens during the transfer of food. Such incriminatory influences

may result in a necessity for fast responding innate defence mechanisms. Therefore,

the first step of our study is to demonstrate microorganisms in the esophagus by

relevant microscopical techniques, such as cryo scanning electron microscopy (cryo

SEM). It is a matter of particular interest to detect such microorganisms in order to

answer the question whether innate defence mechanisms are required or not. The

colonisation of the human intestine with commensal bacteria and the resulting

benefits for the organism have been extensively studied (MACKROWIAK 1982,

ZILBERSTEIN et al. 2007, NEISH 2009). On the contrary, clearly less is known about

commensals in the esophagus.

We will focus on several components of the innate immune system, including

pathogen recognition receptors (PRRs), which are able to identify socalled pathogen

associated molecular patterns (PAMPs). One group of PRRs present on epithelial

cells are Toll-like receptors (AKIRA 2003, CARIO et al. 2002, 2007), which have

already been identified in humans and various mammalian species. For another, the

group of ß-glucan receptors, which are soluble PRRs (UEMURA et al. 2002,

Introduction

2

HOLMSKOV et al. 2003, VAN DE WETERING 2004), are of interest. Furthermore,

we intend to detect antimicrobial peptides (APs). APs occur in several animals and

are produced by different cells, e.g. intestinal cells (GANZ and LEHRER 1998,

BEVINS et al. 1999, SELSTED and OULETTE 2005, LINDE et al. 2008).

Additionally, we want to concentrate on the cellular components of innate immunity.

Cells of the innate immune system ingest foreign antigen, process it and present it on

their surface to trigger a further immune response. Such cells are named antigen

presenting cells (APCs), from which we intend to study a special subgroup, the

Langerhans cells (LCs). Such cells bear similarities with dendritic cells (DCs) and

fulfil a surveillance function in different epithelial tissues (CLARK et al. 2000, DE

CARVALHO et al. 2005). All possible parameters of the innate immune system will

be identified by immunohistochemistry (IHC).

Another aim of this study is to compare three different fixation media and their

influence on the preservation quality of structure and antigens in the esophagus

epithelium. Therefore, all samples collected will be fixed in Bouin´s solution, Ca-

Formol and the just recently established HOPE® solution (OLERT et al. 2001).

Influences on the IHC results obtained will be compared and discussed in detail.

Literature

3

2 Literature

2.1 Macroscopic anatomy of the esophagus: Overview

The digestive tract consists of the oral cavity, pharynx, alimentary channel and

accessory organs. According to NICKEL et al. (2004), the alimentary channel can be

divided into three different sections: Foregut, midgut and hindgut. The foregut

comprises the esophagus as well as the stomach, and the midgut includes the small

intestine (intestinum tenue), whereas the hindgut consists of the caecum and the

large intestine with colon and rectum.

The esophagus is a musculo-membranous tube, which extends from the laryngeal

part of the pharynx to the stomach. It passes through the thorax and runs within the

mediastinum. DE NARDI and RIDDELL (1991) classify the human esophagus to

have four parts, whilst NICKEL et al. (2004) divide the mammalian esophagus into

three parts: A cervical, a thoracic and an abdominal portion.

2.2 Histological structure: Overview

The esophagus is a muscular channel that connects the laryngopharynx with the

stomach and transports liquids along with previously masticated food. It shows the

same histological pattern as the other hollow organs of the digestive tract.

According to LIEBICH (2004), EURELL and FRAPPIER (2006) and SAMUELSON

(2007), the esophagus is composed of four tunics, which are named from the inside

out as follows:

1. Tunica mucosa = mucous membrane or mucosa

2. Tela submucosa (better: Lamina submucosa as part of the T. mucosa,

MEYER, personal communication)

3. Tunica muscularis

4. Tunica serosa (Tunica adventitia outside of the coelomic cavity)

The Tunica mucosa can be subdivided into three layers (LIEBICH 2004, EURELL

and FRAPPIER 2006, SAMUELSON 2007).

The inside layer is the Lamina epithelialis. It is composed of a stratified squamous

epithelium, in which the degree of keratinisation intensity varies between the different

Literature

4

species (EURELL and FRAPPIER 2006, SAMUELSON 2007). Only EURELL and

FRAPPIER (2006) describe the epithelium of cats and dogs as “nonkeratinised” and

that of horses, ruminants and pigs as “keratinised” system. SAMUELSON (2007)

takes also the view that the epithelium of the pig is “nonkeratinised”.

The further characterisation of the epithelium depends on the previously mentioned

definitions. The stratified and squamous epithelium can be subdivided into three

layers (stratum basale, stratum spinosum, stratum superficiale or corneum).

SAMUELSON (2007) emphasises that the term “nonkeratinised” is a misnomer, since

some keratinisation can occur in this tissue.

Unfortunately, LIEBICH (2004) and SAMUELSON (2007) divide the keratinised

stratified squamous epithelium into five parts (stratum basale, stratum spinosum,

stratum granulosum, stratum lucidum and stratum corneum). However, MEYER

(1986) calls attention to the fact that the stratum lucidum is a layer which only occurs

in mechanically strained specific body regions (e.g. foot pad), and cannot be

identified in the stratum corneum from the ultrastructural point of view. Due to own

findings, a modern classification of the lamina epithelialis of the esophagus should be

as following:

1. Stratum basale

2. Stratum spinosum

3. Stratum granulosum

4. Stratum corneum (superficiale)

A detailed description of the structure of a typically keratinised stratified squamous

epithelium is given by several authors (MEYER 1986, 2009; EURELL and

FRAPPIER 2006; WELSCH 2006, SAMUELSON 2007).

The basal layer (stratum basale) consists of a single layer of cuboidal to columnar

cells, which develop basal protrusions with hemidesmosomes that improve the

attachment within the basement membrane and to the lamina propia. The nucleus of

the basal cells is located centrally or apically.

The stratum spinosum consists of one to several layers of cells with a central nucleus

and many desmosomal contacts, whereby cell morphology varies between cuboidal

Literature

5

or flat roundish. This structure results in high resistance to stretching. At the same

time the intercellular spaces between the cells and their processes may provide

conduits for substances secreted by the cells.

The stratum granulosum is characterised by the presence of highly basophil, dense

and amorphous material in the cytoplasm, which can be referred to as keratohyalin

granules. Keratohyalin surrounds the cytofilaments and fills out nearly the entire cell

body. The granules mainly consist of profilaggrin, which is the precursor of filaggrin.

This protein plays an important role in keratin filament aggregation during the

process of keratinisation. Due to their lipid components and the abundance of such

granules, it is often difficult to distinguish the nucleus. The oldest cells of the stratum

granulosum undergo a necrobiotic process. The “typical” keratinisation process

begins already in this layer as a combination of necrosis, water loss and the packing

and release of glycolipids containing granules (membrane coat granules). Afterwards

the cells are flattened and organised in one to several layers (lamellae).

Within the stratum corneum, which may consist of 10 to 50 lamellae, the

keratinocytes now are strongly compressed, and, together with desmosomes and

lipid intercellular deposits, create a tight seal. The lower part of the str. corneum is

directly attached to the stratum granulosum and can be named as stratum corneum

conjunctum. Additionally, MEYER (2009) and SAMUELSON (2007) depict the more

loosely structured outermost layer system of the epithelium as stratum corneum

disconjunctum, in which the seal function has become clearly weaker. The following

process of cell abrasion is termed desquamation.

The second layer of the Tunica mucosa is the Lamina propia. It consists of tightly

interwoven collagen and elastic fibres and many free cells (fibroblasts, fibrocytes,

lymphocytes, etc.). The Lamina propia rests on the third layer of the Tunica mucosa,

the Lamina muscularis mucosae. SAMUELSON (2007) mentions that this layer of the

Lamina mucosa is missing in the dog, and EURELL and FRAPPIER (2006) include

pigs into a species group in which the lamina muscularis mucosae is not present.

The Tela submucosa, better Lamina submucosa (of the Tunica mucosa), consists of

a more or less loose connective tissue containing arteries, veins, large lymph

vessels, nerves (Plexus nervorum submucosus) and seromucous glands (EURELL

Literature

6

and FRAPPIER 2006). Along the full length of the esophagus, seromucous glands

are only present in dogs, whereas in pigs they exist primarily along the cranial half of

the esophagus. The Lamina submucosa normally also contains lymphatic cells,

which in the pig can especially be found around the glands. Herbivorous and

carnivorous species show such gland type only at the pharyngeal – esophageal

junction.

The Tunica muscularis is composed of two layers: an inner circular bundle and an

outer longitudinal one (LIEBICH 2004, EURELL and FRAPPIER 2006, SAMUELSON

2007). The distribution of striated and smooth muscle types within this tunic is

variable according to the species groups.

The authors agree that the Tunica muscularis of dogs consists entirely of smooth

muscle. LIEBICH (2004) and EURELL and FRAPPIER (2006) claim that in the horse

striated muscle tissue can be found in the last third, whereas SAMUELSON (2007)

maintains that the transition area of striated and smooth muscle is located more

caudally. Only EURELL and FRAPPIER (2006) and SAMUELSON (2007) mention

that pigs have mixed smooth and striated muscle tissues in the medium third part of

the esophagus. Herbivorous ruminants show the striated muscles system over the

whole length of the organ. In the cat, the same muscle construction seems possible.

The esophagus is surrounded by a Tunica serosa, containing a one-layered

epithelium. In most species, the serosa can only be found in the thorax. EURELL and

FRAPPIER (2006) specify that a Tunica serosa can be found in the abdominal

section of the equine esophagus, which is very long in comparison to that of other

species. Additionally, a Tunica serosa is present at a very small portion of the feline

and canine esophagus. At the cranial part outside the thorax, the esophagus is

covered by a Tunica adventitia.

Fig. 2.1: General histological structure of the mammalian esophagus according to the great three nutrition groups Above: horse - herbivorous, central: pig – omnivorous, below: cat – carnivorous; LE = lamina epithelialis, TMu = Tunica mucosa, TM = T. muscularis, TS = T. serosa; drawing by C.I. von Stemm

Literature

7

Literature

8

2.3 Innate immunity: General aspects

There are basically two categories of immunity in mammals: innate immunity and

adaptive immunity.

Innate immunity presents the first line of defence against any pathogenic agent and

is capable of reacting within hours (JANEWAY et al. 2005, TIZARD 2008). The

chemical and cellular immune mechanisms of the innate immune system are evoked,

if an invading microorganism manages to break through the primary physical barrier

of the body (for example the skin).

Innate immunity is based on the fact that microorganisms consist of other

components than the regular cells of the body (TIZARD 2008). JANEWAY (1989)

termed these components on microorganism, which enable the immune system to

recognize them, pathogen-associated molecular patterns (PAMPs). Socalled

“sentinel cells” (macrophages, mast cells and dendritic cells) express receptors on

their surface, which can bind PAMPs on bacteria, fungi, and viruses. These receptors

are called pattern-recognition receptors (PRRs). Different types of PRRs were

developed to identify various microbial molecules. As microorganisms show a wide

variety of structures, cells use PRRs which detect highly conserved molecules found

in a lot of different microorganisms.

A good example of the variety is the composition of cell membranes in bacteria.

Walls of Gram-positive bacteria are mainly made of peptidoglycans and also contain

lipoteichoic acids. Gram-negative bacteria consist of peptidoglycans covered by a

layer of lipopolysaccharide (LPS) and yeasts are covered by a mannan-rich

carbohydrate wall. All these structures present the PAMPs.

The best studied and presumably most important PRRs are the Toll-like receptors

(TLR). TAKEDA et al. (2003), AKIRA (2003) and TIZARD (2008) mention the

existence of 10 different human or mammalian TLRs (TLR1 – TLR10), although

LOTZ et al. (2007) demonstrates 11 TLRs in mice. The various TLRs are capable of

identifying many different ligands of pathogens. For example, TLR2 is able to

recognize peptidoglycan, bacterial lipoprotein, some LPS and necrotic cells, whereas

TLR3 specialised on double-stranded viral DNA. TLRs are expressed by the already

mentioned “sentinel cells”, as well as epithelial cells (KOELLISCH et al. 2005). TLRs

may either be expressed on cell surfaces (TLR2, 4 and, 5) or in cells on endosomal

membranes (TLR3, 7 and, 9) (TIZARD 2008). After TLR activation through

Literature

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pathogens a signal is passed to the cell triggering the production of numerous

cytokines and antimicrobial peptides (BIRCHLER et al. 2001). Detailed descriptions

about the occurrence and function of TLRs will follow later.

TLRs are of great importance for innate immunity and the recognition of pathogens.

Recently conducted studies indicate that also other, “non-TLR”, PRRs play a pivotal

role in this process. In this context the ß-glucan or C-type lectin-receptor family is

of outstanding interest.

ß-glucans are multibranched glucose polymers. They represent one of the most

important structural cell wall components of bacteria and fungi (KAPTEYN et al.

1995; LIPKE and OVALLE 1998). Structurally, ß-glucans are composed of ß (13)

linkages and additional ß (16) branches. Due to their ability to recognize

pathogens, ß-glucan receptors are members of the PRRs group. Another name for

this receptor group is C-type lectin receptors (CLRs). The CLRs are a large family of

proteins that possess one or more structurally related C-type lectin-like domains in

their carbohydrate recognition domain. The CLRs have been divided into 17 groups

and often mediate fungal binding, uptake and killing (ZELENSKY and GREADY

2005).

One representative of this group of PRRs are the collectins (LU et al. 2002;

HOLMSKOV et al. 2003), which can also be described as soluble patter-recognition

molecules. They form the group III of CLRs (ZELENSKY and GREADY 2005).

Collectins are proteins that recognize certain carbohydrate moieties, and thus belong

to the family of the lectins. Lectins are generally described as proteins capable of

binding carbohydrates (TIZARD 2008). Of this group, mannose-binding lectin (MBL),

surfactant protein A (SP-A), surfactant protein D (SP-D), collectin and conglutinin are

the most important. The collectins belong to the Ca2+-dependent (C-type) lectin

superfamily, and are characterised by the presence of C-type carbohydrate

recognition domain, which enables them to recognize certain sugar moieties on

pathogens (CRD) (WEIS et al. 1998).

Furthermore, another group of PRRs often discussed in the same context as

collectins is the group of the ficolins. Ficolins possess a different type of lectin

domain called the fibrinogen-like binding region (FBG) domain (ICHIJO et al. 1993;

LE et al. 1997). Unlike the collectins, they bind to carbohydrate moieties in a Ca2+

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10

independent manner (LU et al. 2002). Even though the collectins and ficolins bear no

significant similarities in their amino acid sequences, they are often portrayed

together, as they destroy microorganisms via similar effector systems (LU et al. 2002;

HOLMSKOV et al. 2003). Upon recognition of the infectious agent, collectins and

ficolins initiate the lectin pathway of complement activation through attached serine

proteases (MASPs) (HOLMSKOV et al. 2003). A detailed description of this process

will follow later.

Another ß-glucan receptor belonging to group V of the CLRs is Dectin-1. BROWN

and GORDON (2001), HERRE et al. (2004), REID et al. (2004), ZELENSKY and

GREADY (2005), BROWN (2006), KANAZAWA (2007) and TAYLOR et al. (2007)

characterise Dectin-1 as a C-type lectin-like receptor that is capable of recognising

and responding to fungal pathogens, such as Candida species (BROWN and

GORDON 2001; HERRE et al. 2004; REID et al. 2004; BROWN 2006; KANAZAWA

2007; TAYLOR et al. 2007).

One group of effector molecules of the innate immune system are peptides with

antimicrobial activity, thus called antimicrobial peptides (APs). At first scientists

speculated that these peptides were only produced by immune cells, such as

neutrophils and macrophages (GANZ and LEHRER 1994), but very soon studies

demonstrated their production by epithelial cells, for example in the gastrointestinal

or the urinary tract (SCHONWETTER et al. 1995; LEHRER and GANZ 1999). In our

study, we focus on two substances: the defensins and the cathelicidins (CATs), as

these are widely distributed throughout the animal and plant kingdom (GANZ and

LEHRER 1999; LEHRER and GANZ 2002). Antimicrobial peptides are part of the

innate immunity by directly destroying or covering microorganisms (TIZARD 2008).

Moreover, enzymes participate in the first line of defence. An enzyme targeting

microbial structures is lysozyme. It destroys the peptidoglycans of Gram-positive

bacteria by cleaving the bond between N-acetyl glucosamin and N-acetyl muraminic

acid (NIYONSABA and OGAWA 2005; TIZARD 2008).

The body has to cope with several different antigens, which can principally be

allocated to two groups. The first group includes foreign microorganisms, such as

bacteria, that invade the body. Their antigens are called exogenous antigen. In order

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to activate cells of the acquired immunity, these antigens need to be processed by

specialised antigen-presenting cells (APCs). One function of the APC is to

incorporate exogenous antigens, process them and then present them on their

surface to lymphocytes (DE CARVALHO 2005).

The second group of antigens that the body has to deal with are endogenous

antigens. These differ from the previously mentioned ones, as they are made in the

body (TIZARD 2008). For example, virus antigen can be found intracellularly, as it

forces the cells to produce new viral proteins, and thus is produced endogenously.

Molecules which present antigen on the cell surface of APCs are called major

histocompatibility complex (MHC). Exogenous and endogenous antigens are

handled via two different types of MHC molecules. The presentation of endogenous

antigen is controlled by MHC I, whereas exogenous antigen is presented by MHC II.

The two MHCs are processed within the APCs in a very different way, and obtain

certain characteristics during this process (JANEWAY et al. 2005, TIZARD 2008).

MHC molecules enable APCs to come into contact with lymphocytes. T-lymphocytes

mainly interact with APCs, whereas B-lymphocytes are usually activated by T-cells.

MHC molecules are not the only receptors expressed on cell surfaces, T- and B- cells

also carry characteristic surface molecules. One unique glycoprotein, which only

exists on T- lymphocytes, is the T-cell receptor (TCR). This immunoglobulin has

similar heterodimer functions as PRRs. Due to additional surface molecules, T cells

can be assigned to two groups: CD4 positive T-lymphocytes and CD8 positive T-

lymphocytes.

CD4+ T-cells fulfil regulatory tasks and are synonymously named T helper cells.

CD8+ T-cells are also called cytotoxic T cells and function by directly killing

pathogenes.

Antigen is presented to the T cell through a MHC molecule. CD4+ cells respond to

MHC II molecules, whereas CD8+ cells only detect the antigen together with a MHC I

molecule (JANEWAY et al. 2005; TIZARD 2008).

The cellular part of innate immunity is mainly conveyed via APCs. APCs are located

in areas with a high density of antigens, thus they are present at epithelial surfaces

and on the mucosa of the alimentary tract and other organs (CLARK et al. 2000; DE

CARVALHO 2005; TIZARD 2008).

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Three cell types capture and process exogenous antigen: Dendritic cells (DCs),

macrophages, and B cells (BCs) (Fig. 2.2).

Fig 2.2: Danger model of innate immunity. Different infectious and noninfectious molecular structures (PAMPs and endogenous alarm signals, respectively) constitute indicators known as danger associated molecular patterns (DAMPs). The DAMPs activate the innate immune system through pattern recognition receptors (PRRs) and NFkB-signaling, leading to production of host defence effector molecules (HDPs). N, neutrophils; E, eosinophils; B, basophils; Mo, monocytes; DC, dendritic cells; Ma, mast cells; MØ, macrophages; LPS, lipopolysaccharide; LTA, lipoteichoic acid; CpG, DNA with cytosine and guanine separated by a phosphate; TLR, toll-like receptor; NLR, nod-like receptor; NF-kB, nuclear factor kB; HDPs, host defence peptides; PAMP, pathogen-associated molecular patterns. (LINDE et al. 2008)

As macrophages and BC are unable to activate naïve T cells, and are only capable

of stimulating memory T cells, the functional niche of triggering naïve T cells is

occupied by dendritic cells (DCs).

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The origin of DCs is the bone marrow stem cell and the population of DCs can be

divided into two subpopulations. The myeloid DC (MDCs), which can be found in the

tissues are derived from blood monocytes. In contrast to that, the plasmacytoid DCs

(PDCs) are found in blood and lymphoid organs, and are derived from lymphoid

precursors (CELLA et al. 1997). Furthermore, one can differentiate immature and

mature DCs. Immature DCs are antigen trapping cells which have not interacted with

a pathogen so far (TIZARD 2008). After encountering an antigen, the cells are

activated and mature rapidly or die within days. This activation is triggered by PAMPs

and the TLRs act as PRRs. Hence the DC is now called mature DC, bears an

antigen and migrates to lymph nodes to trigger a T cell response. Upon antigen

presentation via MHCs, expression of co-stimulatory molecules increases at the cell

surface (for example CD86).

In summary, immature DCs are efficient in antigen processing and poor at activation

of naïve T cells, whereas mature DCs loose the capacity to take up proteins but have

the ability to present processed antigen to activated lymphocytes. DCs are the only

cells capable presenting antigen to naïve T cells and triggering their response, whilst

all other APC can only stimulate memory T cells. Therefore DCs occupy a very

unique functional niche in the immune system.

In this study we concentrate on Langerhans cells (LCs), which are immature MDCs

discovered by Paul Langerhans in the year 1868. They have been found in the

epidermis and in the stratified squamous epithelia that line mucous membranes in

humans (AL YASSIN and TONER 1975; DE FRAISSINETTE et al. 1989) and

animals (MEYER 1986) as well as in the esophagus of mice (BOCK 1974; ROWDEN

et al. 1977) and chicken (PEREZ-TORRES et al. 2002).

The hallmark feature of LCs is the presence of typical disc- or cup-shaped

cytoplasmatic organelles termed Birbeck granules (BIRBECK et al. 1961).

LCs remain in an immunological immature state in mucosal tissue and epidermis,

where they can efficiently identify, take up and process antigen, including microbial

antigens. After capturing antigens they emigrate from the epithelial tissue and mature

to professional antigen-presenting cells capable of initiating a primary T-cell mediated

response in the regional lymph node (BANCHEREAU et al. 2000).

As explained above, dendritic cells are characterised by their CD receptors. As we

wanted to detect immature dendritic cells, we tried to identify CD1a positive DCs by

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immunohistochemical staining, as various authors (ELDER et al. 1993; CLARK et al.

2000) found out that surface epithelium DCs express CD1a, whilst DCs in non-

epithelial tissue lack CD1a expression. Additionally, we conducted experiments

applying antibodies against langerin, another characteristically cell surface marker of

LCs (ROMANI et al. 2003; MIZUMOTO and TAKASHIMA 2004; NFON et al. 2008).

The presence of LCs in the human esophagus epithelium was reported over 30 years

ago (AL YASSIN 1975; GEBOES et al. 1983; TERRIS and POTET 1995), whereas

later studies could also prove the existence of CD1a positive LCs in the human

esophagus (ZAVALA et al. 2002).

Macrophages can detect microorganisms via TLRs and respond by producing

cytokines (for example interleukins and tumor necrosis factor). Another characteristic

of macrophages is the ability to directly destroy ingested material by lysosomal

proteases and oxidants. Although killing is a helpful ability, it also results in less

antigen being processed and presented on the cell surface by MHCs.

Macrophages are only able to activate sensitised T-cells, as they are unable to

engage in prolonged interactions with T-cells. Thus they cannot activate naïve T-

cells, which have not encountered an antigen yet (JANEWAY et al. 2005; TIZARD

2008).

B-lymphocytes are covered with 200,000 to 500,000 antigen receptors, the socalled

B-cell receptors (BCRs). Each BCR consists of multiple peptide chains and can be

divided into a signalling component and an antigen-binding site. These two

components work closely together, so that the stimulation of a B-lymphocyte results

in the production of antibodies by the cell. Binding of an antigen to a BCR is the

essential first step in triggering a B cell response. However, this is insufficient to

trigger antibody formation, as co-stimulation by T helper cells is necessary. The B cell

captures antigen with the BCR, processes it, and by presenting antigen in MHCs to a

T-cell receives co-stimulation from this cell. This whole process results in the

production of antibodies, which are actually soluble BCRs. Due to the high specificity

of BCRs towards antigens, the BCs play a larger role in the secondary immune

response, when T cells are already sensitised and easier to stimulate.

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Consequently B cells have two functions: Responding to antigen by producing

antibodies and acting as professional antigen presenting cell (JANEWAY et al. 2005;

TIZARD 2008).

Even though the esophageal mucosa is the first site after the oral cavity to come into

contact with environmental factors, such as pathogenic microorganisms, chemical

irritants or food additives, there is very little information available about the defence

mechanisms in the esophagus. Most knowledge about the presence of an effective

immunological system stems from studies of the resorptively strongly active intestinal

tract.

In the following chapters, a more detailed review of literature is given concerning the

immune mechanisms which might be involved in the esophagus epithelium.

2.4 Antimicrobial peptides: Overview

Antimicrobial peptides (APs) are some of the most important effector molecules of

innate immunity. Due to their ability to interact with various pathogens in several

ways, they have been the centre of attention for many scientists.

As precise molecular techniques were developed in the past 20 years, the isolation

and identification of different peptides allowed a detailed description of their structure

and functions (GANZ and LEHRER 1994).

Antimicrobial peptides have been isolated from various species. They could not only

be segregated from humans and animals but also from plants (DE FERNANDEZ

CALEYA et al. 1972), fungi and bacteria (GANZ and LEHRER 1999; LEHRER and

GANZ 1999). Numerous authors showed great interest in two types of APs:

Defensins and cathelicidins (CATs). YANG et al. (2002), BROGDEN et al. (2003),

SMET (2005), KELLY et al. (2005); HANCOCK (2006), YANG et al. (2007) and

LINDE et al. (2008) point out that particularly those two groups of peptides are of

growing medical interest, due to their therapeutic potential as endogenous antibiotics

and immune stimulants (YANG et al. 2002). Hence it is hoped that antimicrobial

peptides could be possible alternatives to antibiotics and might be another approach

to diminish the number of antibiotic-resistances. Due to their broad range of

immunomodulatory properties, other fields of application might be found.

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Generally APs are small, cationic or anionic and amphipathic peptides, which are

very heterogeneous in length, sequence and structure (SMET 2005). GANZ and

LEHRER (1999) argue that only gene encoded, ribosomally synthesised, polypeptide

antimicrobial substances of mammals with a length of up to 100 amino acid residues

can be defined as APs. Peptides synthesised by fungi and bacteria incorporate

atypical amino-acids and are nonribosomally synthesised.

The Italian Triest Database (at http://www.bbcm.univ.trieste.it/) lists approximately

900 different sequences of APs, which have been identified to this date. The peptides

are put into groups according to similarities in charge, sequence homology, functional

similarity and 3-dimensional structure (NICOLAS and MOR 1995; NISSEN-MEYER

and NES 1997; LEHRER and GANZ 1999). Two categories are distinguished: the

first group are anionic APs and the second one are cationic APs (ELLISON et al.

1985; BROGDEN et al. 2003).

Anionic APs are rich in aspartatic and glutamic acids and have been detected in

cattle, especially in the bronchoalveolar fluid (CAVERLY et al. 2001). They are

effective against Gram-negative and Gram-positive bacteria (BROGDEN et al. 2003),

as they are able to penetrate the outer cell membrane. The peptides attach to

ribosomes and inhibit the ribonuclease activity.

Compared to anionic APs, more attention is paid to cationic APs, especially in

veterinary medical research, as they are very common in domesticated animals.

HANCOCK (2006) classifies cationic AP into four structural groups: α-helical peptides

(for example cathelicidins), ß-sheet peptides with 2-4 disulfid bonds (for example α-

and ß-defensins), loop peptides with one disulfid bridge (for example bactenecin) and

extended peptide structures rich in arginine, glycine, histidine, praline and

tryptophan.

The effect of cationic APs mainly depends on their (tertiary) structure; hence their

structural features are of direct interest (SIMA et al. 2003).

BROGDEN et al. (2003) and TIZARD (2008) depict the defensins as peptides which

are 16-40 residues in size, contain cystein, have two or more disulfide bonds and

form a stabilised ß-sheet structure. α-defensins and ß-defensins are the major

members of this group. They differ in the number of residues (ß-defensins have

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slightly more), variation in cystein linkage and in distribution. Whereas α-defensins

have been detected in the azurophil granules in humans and smaller mammalian

species, such as guinea pigs, rabbits, rats and mice (GANZ and LEHRER 1994), ß-

defensins can primarily be found in epithelial cells of humans and a number of

animals. In humans, α-defensin expression in the intestinal tract is highly restricted to

Paneth cells, which are an epithelial cell lineage unique to small intestine. Expression

in other epithelial cell lineages is absent (SELSTED and OUELLETTE 2005). In

contrast to α-defensins, ß-defensins are expressed in enterocytes of the small and

large intestine (O'NEIL et al. 1999). According to AMBATUPUDI and DEANE (2008),

LINDE et al. (2008) and VAN DIJK (2008), ß-defensins are expressed by

domesticated mammals (cattle, goat, sheep, pig, horse, dog, and cat), wild mammals

(e.g. wallabies) and birds.

θ-defensins are the third group of APs and have been identified recently. In contrast

to α- and ß-defensins, which are flat triple-stranded ß sheets, they are double-

stranded circular molecules (BOMAN 2003). As the other defensins are widely

distributed across species, θ-defensins are only traced in granulocytes of Old World

monkeys, such as the rhesus macaque or orang-utans (SELSTED and OUELLETTE

2005).

In cathelicidins (CATs), a determing factor which led to their name is the similarity of

their proregions to cathelin, a 12-kDa protein from porcine leukocytes (ZANETTI et al.

1995; TOMASINSIG and ZANETTI 2005). In a large number of species, CATs could

be detected. Besides in humans, LINDE et al. (2008) sum up that CATs have also

been isolated from the guinea pig, rat, mouse, rabbit (GALLO et al. 1997; IIMURA et

al. 2005), dog, horse (SCOCCHI et al. 1999), sheep (MAHONEY et al. 1995;

HUTTNER et al. 1998), goat (SHAMOVA et al. 1999), pig (ZANETTI et al. 1994) and

birds. Mostly they can be found in the neutrophils of the previously mentioned

animals. Additionally, they have been detected in the testis, inflamed skin (GANZ and

LEHRER 1998), epithelial cells of the colon (HASE et al. 2002) and gingival

epithelium of humans (DALE and FREDERICKS 2005).

The range of microbicidal activity of cathelicidins is highly diverse. They do not only

possess activity against Gram-negative and Gram-positive bacteria (GENNARO and

ZANETTI 2000), but also against fungi (RYAN et al. 1998), and various parasites

(SCHONWETTER et al. 1995).

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2.4.1 ß-defensins

ß-defensins were discovered in the early 1990´s as antimicrobial peptides in

epithelial cells of the airways in cattle (DIAMOND et al. 1991). During the last 25

years ß-defensins have also been detected in epithelial surfaces of the skin,

respiratory tract, urogenital tract and gastrointestinal tract. For a long period of time

only two human ß-defensins, ß-defensin-1 and -2 (hBD-1 and -2) were known (GANZ

and LEHRER 1998), however, another two ß-defensins were discovered later on.

Human ß-defensin 3 (hBD-3) has been isolated from human keratinocytes, epithelial

cells of the respiratory tract (HARDER et al. 2001; WEHKAMP et al. 2003) and

recently from the placenta, endometrium, pharynx, and from intestinal epithelial cells

(DHOPLE et al. 2006; SALZMAN et al. 2007; MUKHERJEE et al. 2008). In the

intestinal epithelial cells, the Paneth cells are the major producers of defensins

(DANN and ECKMANN 2007). Moreover, the peptide was identified in stratified

squamous epithelia, such as the epidermis; it was also detected in the epithelial root

sheath of hair follicles and their glands of wild mammals (MEYER et al. 2003;

MEYER and SEEGERS 2004).

In the year 2001, hBD-4 was discovered, and it is proven that it is expressed in

several tissues (testis, gastric antrum, uterus, lung, kidney). hBD-4, -5 and -6 are the

latest three defensins being isolated. hBD-4 and -5 are specifically expressed in

human epididymidis, and hBD-6 was found in Escherichia coli (HUANG et al. 2008).

ß-defensins can either be constitutively expressed or are induced by several

pathogens. SALZMANN et al. (2007) mentioned that in general ß-defensin

expression is inducible at sites of inflammation or infection, whereas DALE and

FREDERICKS (2005) distinguished more precisely between the different groups of ß-

defensins: They pointed out that hBD-1 is constitutively expressed in epithelial cells

in lots of tissues (integument, gut, urinary tract), whereas hBD-2 and -3 is up-

regulated by bacteria and proinflammatory stimuli. A remarkable finding was that

hBD-2 is also expressed in human uninflammed gingival tissue (DALE and

FREDERICKS 2005). The authors suggested that the high level of hBD-2 expression

is a result of the exposure of the tissue to commensal, nonpathogenic bacteria.

Regarding this fact, it can be assumed that this ß-defensin has a normal surveillance

function (DALE and FREDERICKS 2005). The same findings were obtained by

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(VORA et al. 2004) for intestinal epithelial cells, as they described hBD-2 production

being induced by activation of TLR through commensal bacteria in the intestine.

Moreover hBD-2 is expressed in human uninflammed skin. It can be localised in the

uppermost layers and the stratum corneum of the epidermis. It has been suggested

that hBD-2 is synthesised and stored in the lamellar bodies of the keratinocytes of

the stratum spinosum and granulosum. Differentiation of the keratinocytes and

barrier disruption leads to release of hBD-2. As a result, hBD-2 can be found in the

intercellular spaces of the stratum corneum (HUH et al. 2002; OREN et al. 2003).

JIA et al. (2000) and GARCIA et al. (2001) independently confirmed or discussed

hBD-3 expression in keratinocytes, as well as in epithelia of the gastrointestinal and

respiratory tract, including the human esophagus epithelium. Additionally it was found

in tonsils, trachea, placenta, adult heart and skeletal muscle.

Due to economic interest, researchers mainly focus on production animals and

studies conducted on companion animals are scarce. LINDE et al. (2008) summed

up the occurrence of ß-defensins in different species.

In cattle, ß-defensin was found in the trachea, lung, tongue, mammary gland and

intestine. ß-defensin has also been detected in the ovine and caprine respiratory and

gastrointestinal tract.

Pigs evoked the interest of scientists due to the fact that the antibiotics, used as

growth promoters in sub-therapeutic levels for many decades, were prohibited in the

European Union in 2006, thus resulting in the necessity for alternative strategies.

VELTHUIZEN et al. (2008) isolated ß-defensin 2 from the porcine intestine and

studied its effect and up-regulation upon infection with different pathogens. They

found out that ß-defensin 2 is expressed in the intestine, and that ß-defensin 2 is

upregulated upon infection with Salmonella typhimurium. Additionally, LINDE et al.

(2008) demonstrated that ß-defensin 2 is also expressed in liver and kidney, though

the peptide is mostly present in the ileum.

Information on innate immune mechanisms in companion animals is rare. Up to now

ß-defensins have only been found in the canine testis (SANG et al. 2005).

As mouse and rat are the most common species used as scientific animal models,

the occurrence of defensins has been intensively studied in these species. Thus,

defensins have been detected in almost every organ (BALS et al. 1999).

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Interestingly, the mouse is the only species until now in which expression of ß-

defensin has been reported in the esophagus epithelium (JIA et al. 2000).

ß-defensins interact with pathogens. The microbicidal properties of defensins can be

explained by their ability to interact with a bacterial surface. Membrane disruption

through pore-forming activities is the major mechanism. This mode of action is

described in detail by the Shia-Matsuzaki-Huang model (Fig. 2.3). MATSUZAKI et al.

(1999), YANG et al. (2000) and ZASLOFF et al. (2000) agree on the fact that the

microbicidal activity of defensins is due to their cationic and amphiphilic nature. The

composition of the cell membrane of microorganisms is characterised by an

abundance of negatively charged phospholipids and an absence of cholesterol. This

fact allows defensins to insert themselves into the phospholipid membranes. The

hydrophobic regions are within the lipid membrane and the cationic region is located

on the outside. Thus, a carpet-like structure is formed by defensins on the microbial

surface. Subsequently the contiguity of the microbial membrane is disrupted and

pores are formed. This leads to the complete collapse of the bacterial membrane.

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Fig 2.3: Shia-Matsuzaki-Huang model. The model displays the general consensus for HDPs’ antimicrobial mode of action. 1: Host is initially exposed to microorganisms. 2: The innate immune response involves recruitment of cationic HDPs, which are immediately attracted toward the anionic microbial membrane. 3: The HDPs form a carpet-like structure on the microbial membrane, instituting channel formations. 4: The channels lead to pore-formation membrane destabilization and microbial demise. HDP, host defence peptides. (modificated after LINDE et al. 2008)

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2.4.2 Cathelicidin

CAT is the second member of the group of antimicrobial peptides, which needs to be

discussed. CATs can be put into the following groups: Peptides with α-helical or

extended helical or loop structure (RAMANATHAN et al. 2002). These groups have

been identified in humans, monkeys, mice, rats, guinea pigs, rabbits, sheep, cattle,

pigs, dogs and horses (RAMANATHAN et al. 2002; BROGDEN et al. 2003; LINDE et

al. 2008). In vitro, the CATs show a wide range of antimicrobial activity against Gram-

positive and Gram-negative bacteria as shown in Tab. 2.1. CATs either kill the

pathogens directly, by disruption of the cytoplasmatic membranes leading to lysis of

the target cell, or bind lipopolysacchrides. The mechanism of killing is similar to that

one of the ß-defensins (compare 2.4.1).

The first product of CAT producing cells is an inactive pro-form. This pro-form is

activated by proteolytic cleavage processes mediated via an elastase in cattle and

pigs (PANYUTICH et al. 1997), and proteinase-3 in humans (SORENSEN et al.

2001).

In horses, cattle, pigs, dogs, rabbits, and mice CAT is synthesised by neutrophils

(MIRGORODSKAYA et al. 1993; SCOCCHI et al. 1999; RAMANATHAN et al. 2002;

SANG et al. 2007). In addition to myeloid derived cells, CAT is expressed in other

cell types. For example, in humans it is present in the testis (MALM et al. 2000) and

in squamous epithelia, as in the oral mucosa and the skin (RAMANATHAN et al.

2002; BRAFF et al. 2005; DALE and FREDERICKS 2005; NIYONSABA et al. 2006).

Concerning the epidermis, it was shown that CAT resides in keratinocytes granules

(MCGs, LBs) of the superficial epidermal layers in humans and mice (BRAFF et al.

2005).

CAT is also present in human airway epithelial cells (BALS et al. 1998). In the human

intestinal tract, CAT was detected in surface epithelial cells of the stomach and

intestine (HASE et al. 2002, 2003). According to the results for the human intestine,

IIMURA et al. (2005) found that CAT is also expressed by murine intestinal cells. The

distribution pattern for CAT in humans was partly corroborated for murine species. In

mice, CAT was constitutively expressed largely in the colon, but was not detected in

the stomach. Only little expression was found in the small intestine. In contrast to

IIMURA et al. (2005), GALLO et al. (1997) detected CAT in the intestine and also in

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the stomach of mice. Moreover CAT was found in the testis and spleen of these

animals (GALLO et al. 1997).

With the aim to explore the functional importance of CAT in early intestinal innate

antimicrobial defence, IIMURA et al. (2005) infected CAT knockout mice with a

pathogen bacterium (Citrobacter rodentium). Their results emphasised the

antimicrobial importance of CAT, as it was demonstrated that CAT knockout mice

developed greater surface epithelial cell damage and systemic infection, in

comparison to normal mice.

As described above, the different types of ß-defensins can be either constitutively

expressed or their expression is increased due to microbial pressure. Concerning

CAT, HASE et al. (2002) mentioned its expression in the intestine independent of the

presence of microbiota, although it was modestly upregulated by enteroinvasive

microorganisms. Canine CAT is predominantly expressed in myeloid precursor cells

and to a lower extent in the spleen, liver and testis. Minimal expression was also

found in the kidney, lung and intestinal tissue (SANG et al. 2007).

Table 2.1: In vitro antimicrobial activity of antimicrobial peptides in animals (after LINDE 2008)

Species Peptide In vitro antimicrobial activity

ß-Defensins Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Staphylococcus

aureus, Aspergillus spp., Candida spp., Mannheimia

haemolytica, Mycobacterium paratuberculosis

Cattle

Cathelicidin E. coli, K. pneumonia, Salmonella typhimurium, Leptospira interrogans

Dog ß-Defensins/

Cathelicidin

E. coli, K. pneumoniae, C. albicans, Salomonella enteritidis, S. typhimurium, Staph. aureus, Listeria monocytogenes

Goat Cathelicidin E. coli, P. aeruginosa, Listeria monocytogenes

ß-defensin Corynebacterium sp., Staph. intermedius Horse

Cathelicidin E. coli, Streptococcus equinus, K. pneumonia, Serratia marcescens

ß-defensins E. coli, S. typhimurium, L. monocytogenes, C. albicans

Pig

Cathelicidin E. coli, S. typhimurium, Staph. aureus, Actinobacillus pleuropneumonia,

Pseudomonas aeruginosa, C. albicans ß-defensins Mannheimia haemolytica Sheep

Cathelicidin E. coli, S. typhimurium, P. aeruginosa, Staph. aureus, St. epidermidis, C. albicans

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2.4.3 Biological activity of antimicrobial peptides

Antimicrobial peptides are the frontiers of innate immunity, thus participating in

several modes of action. RAMANATHAN et al. (2002), BROWN (2006) and LINDE et

al. (2008) recapitulate the different functions of APs as further described:

Protective effects of the peptides have been attributed to direct killing of bacteria. The

functional feature of this effect is represented by the Shia-Matsuzaki-Huang model

(Fig. 2.3). However, many of these mechanisms were analysed in vitro, but GANZ

and LEHRER (1998, 1999) state that under physiological salt conditions ß-defensins

have reduced antimicrobial activities. It is likely that the direct antimicrobial effect

occurs mainly on the mucosal epithelia, where concentration of salt is lower.

There is evidence that CAT and ß-defensins show a synergistic effect. NAGAOKA et

al. (2000) found out that in contrast to ß-defensins, CAT is resistant to high salt

conditions. The authors presumed that the various resistances are due to the

different structure of CAT and ß-defensins. They concluded that ß-defensins cannot

function as antimicrobial molecules by themselves. In fact they are working

synergistically with CAT by increasing membrane permeabilisation of target cells.

The second function of APs is their ability to act as immune system modifiers, i.e. as

chemoattractants for immune cells enhancing their function (NIYONSABA et al.

2002). For example, they increase the cytotoxicity of natural killer cells (SCOTT and

HANCOCK 2000). Additionally, they exhibit anti-inflammatory properties by

suppressing bacterial-induced cytokine production and binding lipopolysaccharide

(LPS). As a result, LPS fail to induce tumor necrosis factor production in macrophage

cell lines thereby preventing endotoxemia.

Furthermore, cathelicidins and defensins promote cell proliferation, vasculogenesis

and wound healing (MURPHY et al. 1993; AARBIOU et al. 2004; CHAVAKIS et al.

2004).

APs can even act at the interface of innate and adaptive immunity, as it is supposed

that they support the differentiation of certain cell lineages. For example, BIRAGYN

et al. (2004) demonstrated that ß-defensin 2 simulates dendritic cell maturation and

increases the expression of co-stimulatory molecules (CD40, CD80 and CD86).

Thus, APs can be seen as a connecting link between innate and adaptive immunity.

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2.5 Toll-like receptors

Toll-like receptors (TLRs) play a pivotal role in host defence, as they recognize

microbial ligands and are members of the group of PRRs. As already mentioned, 11

mammalian TLRs have been identified. Characteristically they show three common

structural features: a divergent ligand-binding extracellular domain with leucin-rich

repeats, a short trans-membrane region, and a highly homologous cytoplasmatic toll /

interleukin-1 receptor domain, which is essential for the initiation of signalling

cascades (AKIRA 2003).

TLRs have been detected on various cell types. In our study we want to put the focus

on the expression of TLRs on the epithelium of the esophagus. In the literature,

several authors discussed the occurrence of TLRs in a number of organs. Most of the

studies conducted concentrate on TLR expression by human intestinal cells, as many

authors are of the opinion that TLRs play an important role in the pathogenesis of

inflammatory bowel disease (RAKOFF-NAHOUM et al. 2004; VORA et al. 2004;

CARIO and PODOLSKY 2006). Furthermore, the existence of TLRs on human

keratinocytes (KOELLISCH et al. 2005; NAGY et al. 2005; SUMIKAWA et al. 2006;

BUECHAU et al. 2008), the cornea (KUMAR et al. 2006), lung epithelial cells

(BIRCHLER et al. 2001; DROEMANN et al. 2003) and vaginal epithelial cells has

been described (PIVARCSI et al. 2005).

The function of the TLRs as PRRs results in an activation of defence mechanisms of

the cell. As already mentioned previously, one of the first line defence mechanisms is

the production of antimicrobial peptides. Several authors assumed a correlation for

the expression of TLRs and the production of antimicrobial peptides (APs) by the cell

(BIRCHLER et al. 2001; VORA et al. 2004; SUMIKAWA et al. 2006; BUECHAU et al.

2008). BIRCHLER et al. (2001) were the first to mention that TLRs mediate induction

of the synthesis of APs. They found out that human lung epithelial cells constitutively

express TLR2 and produce ß-defensin 2 in response to bacterial lipoprotein.

Subsequently many studies on different epithelial cell types followed.

In the intestine, triggering of TLRs is mainly caused by commensal bacteria, and the

crucial function of the TLRs in the intestine is to provide epithelial homeostasis and

integrity (HOOPER et al. 2001; RAKOFF-NAHOUM et al. 2004).

Not all of the 12 different TLRs seem to be activated under normal steady-state

conditions; moreover TLR2 and TLR4 are in the focus of attention and seem to

contribute to the production of APs (VORA et al. 2004). TLR2 is required for the

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recognition of bacterial lipopeptide, and, in combination with TLR6, for the recognition

of peptidoglycan and lipoteichoic acid, which are components of Gram-positive

bacteria (TAKEDA et al. 2003). Additionally, TLR2 is able to detect the yeast cell wall

component zymosan (AKIRA 2003). TLR4 mediates the recognition of

lipopolysaccharides (LPS), found on the outer membrane of Gram-negative bacteria

(AKIRA 2003).

In order to connect the activation of TLRs and the resulting production of APs,

signalling pathways are necessary. Signals from TLRs can be mediated into the cell

via different pathways. The nuclear factor kappa-B (NF-κB) pathway plays the key

role in this manner (VORA et al. 2004). NF-κB is a transcription factor and possesses

an important role in immunity. It is localised in the cell cytoplasm and is hold in its

inactive form through its association with the unphosphorylated IκB proteins, thus

NF-κB cannot move to the nucleus or activate genes. One way to activate the

transcription factor is via triggering TLRs with PAMPs. This results in an alteration of

the shape of the TLR. Subsequently the TLR binds several adaptor molecules, of

which the myeloid differentiation primary response gene 88 (MyD88) is the most

important one; following various kinases are activated. Through their activation in the

last step, the IκB protein is phosphorylated leading to its destruction and the release

of the active NF-κB (O'NEILL 2006).

TLR activation can be a potent stimulus of AP production, but also for the synthesis

of other factors, such as interleukin 6 or tumor necrosis factor. These substances are

involved in cytoprotection and tissue repair in the intestine (RAKOFF-NAHOUM et al.

2004) and other organs, for example in the lung (WARD et al. 2000).

As already mentioned previously, correlation between the occurrence of TLRs on an

epithelium and the resulting production of APs has not only been described for

intestinal epithelium, but also for other organs. The expression of TLR2 and a

resulting production of APs has additionally been observed in human keratinocytes of

the skin (KOELLISCH et al. 2005; NAGY et al. 2005; SUMIKAWA et al. 2006;

BUECHAU et al. 2008), in lung epithelial cells (BIRCHLER et al. 2001), in vaginal

epithelial cells (PIVARCSI et al. 2005) and also in corneal epithelial cells (KUMAR et

al. 2006). Not all APs seem to be triggered by the activation of TLRs, as from most

studies conducted can be concluded that mainly ß-defensin production is

upregulated by the activation of TLR2 or TLR4 (BIRCHLER et al. 2001; VORA et al.

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2004; NAGY et al. 2005; KUMAR et al. 2006; SUMIKAWA et al. 2006). Concerning

intestinal epithelial cells, MUKHERJEE et al. (2008) mentioned that hBD-2 is

normally low in cultured epithelial cells and can be induced in response to microbial

influence in a TLR2 dependent manner.

Only for the skin a relationship between TLR2 expression and enhanced cathelicidin

production was shown (BUECHAU et al. 2008).

Most of the observations mentioned beforehand have been obtained from human

epithelial cells, whereas publications about TLRs expression and their functional

relationship to APs for animals are sparse. For the dog, TLR expression has been

reported in different uninfected tissues. Thus, TLR4 was detected in the epithelium of

the lung, small intestine, cornea and renal tubules by performing

immunohistochemistry (WASSEF et al. 2004). mRNA of TLR4 was found in

peripheral blood leukocytes (PBL), in the spleen, stomach and small intestine, and

was moderately expressed in the liver. However, it was not detected in the kidney,

large intestine and skin (ASAHINA et al. 2003). Applying polymerase chain reaction

(PCR), mRNA of TLR2 was found in blood monocytes, lymph nodes, lung, liver,

spleen, bladder, pancreas, small intestine and skin of the dog (ISHII et al. 2006), and

also in canine heart tissue (LINDE et al. 2007). Intriguingly in one study it was

mentioned that a dysregulation of TLR2 and TLR4 may contribute to the

pathogenesis of inflammatory bowel disease (IBD) in dogs, which is characterised by

a chronic inflammation of the small intestine (SWERDLOW et al. 2006).

TLR9 occurrence in canine tissue has been maintained by real-time PCR for lymph

nodes and spleen (HASHIMOTO et al. 2005). The wide spread of TLRs in the dog

might also indicate the occurrence of APs in the mentioned tissues (LINDE et al.

2007).

Descriptions about feline TLRs are made in context with the feline immunodeficiency

virus (FIV), and the authors primarily mention TLR expression on lymphocytes

(IGNACIO et al. 2005). Additionally, mRNA of feline TLR4 was highly expressed in

lung, bladder and peripheral blood monocytes, moderately in kidney, liver, spleen

and large intestine, and with low levels in pancreas and small intestine (ASAHINA et

al. 2003).

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Selected bovine and ovine tissues have been tested for the occurrence of various

TLRs. As a result, quantitative real-time PCR confirmed expression of 10 TLRs within

the ovine jejunum, Peyers patches and lymph nodes. Whereas TLRs 3, 5 and 6 were

abundant in the jejunum, all TLRs had been detected in bovine skin; here TLRs 2

and 7 were most abundant (MENZIES and INGHAM 2006).

In cattle, a relationship between ß-defensin 5 production and TLR2 and TLR4

expression in mastitis infected mammary glands has been observed (GOLDAMMER

et al. 2004).

Occurrence of TLR2 in pigs has been verified for epithelial cells lining the

tracheobronchial and intestinal tracts, bile ducts in the liver and renal tubules.

Additionally it was found in the basal cell layer of the epidermis (ALVAREZ et al.

2008). Furthermore, TLR4 mRNA has been detected in tissue samples of the bone

marrow, thymus, lymph node, spleen, brain, liver, kidney and ovary (ALVAREZ et al.

2006).

Information about the expression of TLRs in equine tissue is scarce, although TLR9

was detected in the spleen and lymph nodes (ZHANG et al. 2008). Furthermore,

tissue expression profiles showed that TLR3 was highly expressed in the kidney,

duodenum, spleen and liver, and moderately expressed in bone marrow, lung, and

skin (SANG et al. 2008).

In summary, TLR signalling pathways might be involved in commensal-induced

antimicrobial peptide production, therefore helping to prevent pathogenic bacteria

from break through the epithelial barrier. TLR distribution is consistent with a

surveillance function at entry sites, allowing early detection of microbial invasion.

2.6 C-type lectin receptors (ß-glucan receptors)

2.6.1 Collectins and ficolins: General overview

Collectins and ficolins are pathogen recognition molecules, which bind to

oligosaccharide structures on the surface of microorganisms, subsequently leading to

the killing of bound microorganisms through complement activation and

phagocytosis.

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Collectins and ficolins are members of the group of lectins, which are in general

described as carbohydrate-binding proteins. They serve as weapons against

pathogens by being activating by the invaders (FUJITA et al. 2004).

Both groups bear no significant similarity in amino acid sequences. Nevertheless,

they have similar domain organisations and, functionally, both collectins and ficolins,

mediate microbial killing through similar effector systems. This is the reason why both

groups and their representatives are described in one chapter.

2.6.2 Mannan-binding lectin (MBL)

The existence of a molecule with the ability to recognize the yeast cell surface

component was first reported by KAWASAKI et al. (1978). They isolated a molecule

from rabbit liver and named it mannan-binding protein. The human variant of this

molecule is today known as mannan-binding lectin (MBL) or mannose-binding lectin.

MBL is, beside surfactant proteins A and D (SP-A and SP-D), the best studied family

member of the collectins. The greatest amounts of MBL are produced by the liver

and are secreted into the blood (SASTRY and EZEKOWITZ 1993; LU et al. 2002;

WAGNER et al. 2003; VAN DE WETERING et al. 2004). SP-A is mainly synthesized

by alveolar type II cells and unciliated bronchial epithelial cells (Clara cells) in the

lung (MENDELSON et al. 1991; WONG et al. 1996; MADSEN et al. 2003). SP-D was

detected in alveolar type II cells and also the production by Clara cells has been

proven (CROUCH et al. 1992; VOORHOUT et al. 1992). The extrapulmonary

production of both surfactant proteins has been demonstrated in various organs.

Besides epithelial cells of the skin, small intestine, urinary tract, and the collecting

duct of the kidney, even in the esophagus epithelium of human SP-D was detected

by IHC. It was concluded that SP-D is generally present on mucosal surface and is

not restricted to a subset of cells in the lung (MADSEN et al. 2000) . The same is

right for SP-A. PCR on trachea, prostata, pancreas, thymus, salivary glands and the

colon led to positive results, whereas immunohistochemical experiments revealed no

positive results in humans (MADSEN et al. 2003).

Results presented for animals differ from those of humans. SP-A was found in murine

lung, uterus and gastric mucosa (RUBIO et al. 1995; AKIYAMA et al. 2002), in the

porcine Eustachian tube (PAANANEN et al. 1999), in the rabbit middle ear and

maxillar sinus (DUTTON et al. 1999).

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In contrast to SP-A and SP-D, the extrahepatic biosynthesis of MBL has not been

reported so frequently for other organs.

In rodents, rabbits, pigs and rhesus monkeys two forms of MBL have been identified,

named as MBL-A, which can be found in serum, and MBL-C, that is detectable in

liver (SASTRY et al. 1991; AGAH et al. 2001). For rodents, RT-PCR showed that the

liver is the major site of expression for both MBL genes. Both were also detected in

the kidney, brain, spleen, and muscle, whereas MBL-A is exclusively expressed in

the testis. MBL-C was solely detected in the small intestine of murine species. These

findings were supported also on the protein level by positive immunohistochemical

reactions (UEMURA et al. 2002; WAGNER et al. 2003). As the non-hepatic MBL-C

expression was highest in the small intestine, the authors concluded that this MBL

type plays a role in the defence against microbial invasion and colonisation in the

intestinal tract. It was also reasoned that MBL might be the counterpart of secretory

IgA of the aquired immune system in the intestine (WAGNER et al. 2003).

In contrast to the above mentioned species, for humans, chimpanzees and chicken

only one form of MBL has been characterised (HANSEN and HOLMSKOV 1998).

Due to the fact that MBL deficiency is one of the most common immune-deficiencies

in humans (TURNER 2003; GUPTA and SUROLIA 2007), the extrahepatic sources

of MBL have been studied extensively in this species; for example mRNA could be

localized in the small intestine and the testis (SEYFARTH et al. 2006).

Also in chicken the occurrence of MBL was corroborated. Likewise it was detected in

extrahepatic organs, for example in the caecal tonsils of healthy chickens (NIELSEN

et al. 1998; LAURSEN and NIELSEN 2000).

2.6.3 Ficolins

Ficolins were originally discovered in the porcine endometrium, where they were

identified as transforming growth factor (TGF)-ß1-binding proteins (ICHIJO et al.

1991). In a broad spectrum of species ficolins were identified, such as in humans

(MATSUSHITA et al. 1996), mouse (FUJIMORI et al. 1998), pig, and hedgehog

(OMORI-SATOH et al. 2000), and the South African clawed toad (Xenopus leavis)

(KAKINUMA et al. 2003).

In humans, three types of ficolins, named H-ficolin, L-ficolin and M-ficolin have

been found. The hepatocytes are the primary source of L-ficolin and H-ficolin

(MATSUSHITA et al. 1996), but the latter substance H is also synthesised by the bile

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duct epithelial cells, and in the lung by alveolar type II cells and ciliated bronchial

epithelial cells (AKAIWA et al. 1999).

The third form of ficolins, M-ficolin, is produced by monocytes (HASHIMOTO et al.

1999) and also detectable in the lung (FUJITA et al. 2004). Whereas H-ficolin and L-

ficolin are considered to be serum proteins, this does not apply to M-ficolin. It has

been demonstrated that this lectin is expressed on the surface of peripheral blood

monocytes. These results suggest that M-ficolin plays a role in innate immunity by

acting as a PRR for pathogens on monocytes (FUJITA et al. 2004).

Mice, as well as rats, have two ficolin forms, named ficolin-A and ficolin-B. Ficolin-A

is highly expressed in liver and spleen (FUJIMORI et al. 1998). OHASHI and

ERICKSON (1998) were the first to identify ficolin-B and observed its expression in

bone marrow cells and the spleen. Just recently, ficolin-B was found to be expressed

by peritoneal macrophages (RUNZA et al. 2008).

Also in pigs two forms of ficolins have been identified. They have been named as

porcine ficolin-α and –β (ICHIJO et al. 1993). The expression of ficolin-α mRNA

could be verified for the lung, liver, uterus, spleen and bone marrow (OHASHI and

ERICKSON 1998). Ficolin-β has also be detected in the cattle, whereby tissue

expression was observed in bone marrow and spleen (LILLIE et al. 2005).

2.6.4 Structure of collectins and ficolins

The collectins own their name, due to the fact that their structure is based on

collagenous regions as well as on lectin domains (TURNER 2003). Basically

collectins are built of structural subunits, each composed of three identical

polypeptide chains (timer). The number of trimeric units per collectin molecule varies

among the collectins.

In a single peptide subunit, four structural domains can be distinguished: A N-

terminal cystein rich domain, a collagen domain, a coiled neck domain and a C-type

lectin domain, which represents the carbohydrate recognition domain (CRD)

(TURNER 2003; VAN DE WETERING et al. 2004; GUPTA and SUROLIA 2007).

The ficolins lack the coiled neck domain and possess a different type of lectin

domain, called fibrinogen-like domain (FBG) (LU and LE 1998; FUJITA et al. 2004;

RUNZA et al. 2008). This unique feature resulted in their name “ficolins” (ICHIJO et

al. 1993).

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Generally spoken, the structure of collectins and ficolins can be described as follows:

Three polypeptides fold together to form the structural subunit, and three to six of

these subunits join to form a mature protein (FUJITA et al. 2004).

Due to this arrangement, the tertiary structure of MBL and ficolins reminds of a

bouquet of flowers, constructed of octadecamers of six trimeric subunits (LU et al.

2002; HOLMSKOV et al. 2003).

Through the collagen like domain, the collectins and ficolins are associated with

serine proteases (FUJITA et al. 2004; GUPTA and SUROLIA 2007). They are of

great functional importance according the activation of the complement pathway and

the resulting destruction of microorganisms.

2.6.5 Functional features of the collectins and ficolins: Activation of the

complement system

Aiming the elimination of microorganisms, the collectins cooperate with phagocytes

and humoral factors, including the complement.

The complement system is a defence mechanism activated by innate and acquired

immune mechanisms. It consists of serum proteins together with an associated group

of cell membrane proteins. When complement proteins bind to the surface of

microorganisms, enzymatic pathways are activated, and in the next step

microorganisms are destroyed. In healthy animals, these pathways are inactive, but

they can be evoked either by the presence of antibodies on the surface of organisms

or by recognition of the complex carbohydrate moieties on the surface of infectious

agents (TIZARD 2008). The complement system consists of proteins, which are

either labelled numerically with the prefix C (for example C1 or C2) or designated by

letters of the alphabet. The complement proteins are mainly synthesised in the liver

(C3, C6; C8 and B), but some are also made by macrophages (C2, C3, C4, C5, B, D,

and P). A number of the proteins can be found free in the serum, whereas others are

cell-bound.

The complement cascade can be activated through three distinct pathways, which

are named the classical, alternative and lectin pathways.

The activation of the classical pathway requires pre-sensitisation of microorganisms

with antibodies, and is thus part of the acquired immune system. The first component

of this pathway is a multimolecular protein complex called C1, which consists of three

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proteins: C1q, C1r and C1s. C1 can be described as a complex of C1q, which is

associated with C1s and C1r. C1q is able to recognize the Fc region of IgG and IgM

immunoglobulins bound to an antigen. Subsequently recognition leads to the

formation of a membrane attack complex (MAC) and microbial lysis. This occurs via

the activation of several complement components and the initiation of a potent

protease (C3 convertase).

In contrast to the classical pathway, both, the alternative and the lectin pathway are

independent of clonal immunity and for that reason parts of innate immunity.

The alternative pathway is triggered when microbial cell walls come into contact

with complement components in the bloodstream. The complement protein C3 plays

a key role within this pathway, as it binds to acceptor groups on many pathogens and

marks them for destruction by immune cells.

C3 spontaneously breaks down into two fragments called C3a and C3b, even under

normal conditions in healthy animals. This process is called tick-over. C3b is

constructed of a highly reactive carbonyl group, which irreversibly binds to the target

cell surface. The target cell is not necessarily a pathogen, such as a bacterium, but

can also be a normal cell of the body. To avoid activation of the alternative pathway

by normal cells, the further activity of C3b is controlled by two factors, named factor H

and I. Factor H interacts with normal cell surfaces and glycoproteins rich in sialic acid

and other neutral or anionic polysaccharides, resulting in the destruction of C3b by

the activation of factor I. Consequently in a healthy individual, factors H and I destroy

C3b as fast as it is generated. Due to the fact that the components of bacterial cell

walls, such as lipopolysaccharides (LPS), lack sialic acid, factor H cannot bind to

C3b. Factor I remains inactive, and the C3b persists. Subsequently the pathogen is

marked by C3b and accessible for recognition by phagocytes, which then destroy the

cell. The process of marking a pathogen, in this case with a complement factor, is

called opsonisation. Furthermore, also the formation of a membrane attack complex

(MAC), which inserts itself into a microbial cell membrane resulting in osmotic lysis of

the pathogen, can be triggered by bound C3b. Binding of C3b leads to activation and

cleaving of several other complement factors, in which factor C5 plays an important

role (JANEWAY et al. 2005; TIZARD 2008).

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In our study the lectin pathway is of major interest, as it is activated by MBL and

also by ficolins, which bind to the surface sugar residues on microorganisms (IKEDA

et al. 1987; HOLMSKOV et al. 1994; EPSTEIN et al. 1996; LU 1997; MATSUSHITA

and FUJITA 2001; VAN DE WETERING et al. 2004; PHANEUF et al. 2007).

The discovery that MBL activates complement independently of antibodies was made

in 1987 by IKEDA et al. (1987). This finding was noticed to be a major breakthrough

for the understanding of its role in innate immunity (HOLMSKOV et al. 2003).

The ligands for the collectins are bacterial cell wall components like lipoteichoic acid

of Gram-positive and LPS of Gram-negative bacteria (POLOTSKY et al. 1996;

HOLMSKOV et al. 2003; GUPTA and SUROLIA 2007).

As collectins coat microorganisms, they act as opsonins. Subsequently specific

interaction between the collectins and receptors on phagocytic cells may result in

increased association, uptake and killing of the pathogens (MCNEELY and

COONROD 1994; PIKAAR et al. 1995; OFEK et al. 2001). In case of MBL it either

opsonises microorganisms directly or it can lead to opsonisation through complement

activation and deposit of complement factor C3 (PETERSEN et al. 2001;

HOLMSKOV et al. 2003).

MBL circulates in complex with serine proteases, known as mannan-binding lectin

associated serine proteases (MASPs). Three types of MASPs have been indentified

until now: MASP-1 (MATSUSHITA and FUJITA 1992), MASP-2 (THIEL et al. 1997)

and MASP-3 (DAHL et al. 2001). If MBL binds to the surface of microorganisms,

MASPs are activated to recruit and cleave complement factors. MASP-2 has been

shown to cleave C4 and C2 (THIEL et al. 1997). By the splitting of C4 and C2 a cell-

bound complex called C4bC2b is generated, which is able to activate the C3

convertase. As a result, great amounts of C3b and C3a are created. C3a acts as a

peptide mediator for inflammatory cells, whereas C3b binds the surface of

microorganisms and functions as an opsonin, resulting in cell marking for destruction

through phagocytes. Furthermore, C3b attaches to C5 and the C5 convertase is

generated. It subsequently cleaves C5 into C5a and C5b. C5a also, like C3a, is an

important mediator for inflammatory cells. C5b functions as an activator for the

synthesis of the MAC (JANEWAY et al. 2005; TIZARD 2008). The MASP-1 is

responsible for the direct cleavage of C3 (MATSUSHITA and FUJITA 1995). The

function of MASP-3 still remains unclear, and no relevant substrate for this serine

protease has been identified by now (GÁL et al. 2007).

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Also ficolins activate the lectin complement pathway, by association of the molecules

with MASPs (MATSUSHITA et al. 2000; RUNZA et al. 2008).

It needs to be emphasised that the lectin pathway and the classical pathway are not

completely isolated from each other; neither in structure of the activating molecules,

nor in function (FUJITA et al. 2004).

The lectin-MASP complex is structurally and functional equivalent to C1, which is

composed of the serine proteases C1r and C1s and the recognition subcomponent

C1q. This complex represents the first component of the classical complement

pathway.

In the MBL pathway, MASP-2 is the enzyme part, like C1s in the classical pathway,

which cleaves the complement components C4 and C2 to form the C3 convertase

C4bC2b (THIEL 1992; THIEL et al. 1997; FUJITA et al. 2004). Besides, MASP-1 is

capable of cleaving C3 directly (DAHL et al. 2001; ROSSI et al. 2001), with a

resulting activation of the alternative pathway (MATSUSHITA and FUJITA 1995).

By means of structure, C1q and MBL also show great similarity. As already described

previously, MBL is organised in form of a flower bouquet, consisting if six trimeric

subunits. The structure of C1q consists of six globular heads, each connected by a

strand to a central fibril-like region, composed of collagen-like triple-helical structure

(KISHORE and REID 2000).

One difference between MBL and C1q structure results from the fact that MBL is

composed of three identical polypeptide chains, respectively, whereas C1q is

composed of three different chains (FUJITA et al. 2004)

However, it still can be resumed that the overall structure of C1q is similar to that of

MBL and ficolins (HOLMSKOV et al. 2003; VAN DE WETERING et al. 2004).

Furthermore, the structure of MASP family members associated with MBL and

ficolins resembles that of C1r and C1s, as they both consist of six domains,

composed of proteins and proteases (FUJITA et al. 2004).

2.6.6 Pathogene specificity of collectins and ficolins

As already mentioned previously, collectins and ficolins bind to carbohydrates with

their lectin domain. The lectin domain of MBL is the CRB, respectively named FBG in

ficolins.

With its CRD, MBL binds carbohydrates with 3- and 4-hydroxyl groups in the

pyranose ring in the presence of calcium. Thus MBL ligands for MBL are mannose,

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glucose, L-fucose, N-acetyl-mannosamine and N-acetylglucosamine (LU et al. 2002;

HOLMSKOV et al. 2003; FUJITA et al. 2004). With this wide range of ligands, MBL is

able to recognize various types of bacteria.

Most carbohydrate structures in animals are terminated by sugars not recognized by

the collectins, e.g., galactose or sialic acid. Mammalian cells do not present the

pathogen-associated molecular patterns characteristically for microorganisms

(HOLMSKOV et al. 2003).

In contrast to collectins, ficolins do not show such a wide variety of sugar binding

capacity. Ficolins mainly bind to N-acetyl-D-glucosamine (GlcNAc) and N-

acetylgalactosamine (GalNAc) (LE et al. 1997, 1998; LU et al. 2002; FUJITA et al.

2004). For humans it has been shown that L-ficolin recognizes cell wall components

of Salmonella typhimurium.

Binding capacities of ficolins and collectins have been discussed controversial

(POLOTSKY et al. 1996; LYNCH et al. 2004). The latter authors observed that L-

ficolin, but not MBL or H-ficolin, specifically binds to lipoteichoic acid, common for

Gram-positive bacterial cell walls. In contrast to this opinion, POLOTZKY et al. (1996)

argues that also MBL shows binding capacity for lipoteichoic acid of Gram-positive

bacteria.

In the literature, various descriptions of the recognition capacity of MBL for different

microorganisms can be found. Generally spoken, common microbial structures

recognized by the collectins are lipopolysaccharides (LPS) and mannan-like high

mannose structures. Due to the fact LPS is a major glycolipid component on the

outer membrane of Gram-negative bacteria, these are ideal ligands for collectins (LU

et al. 2002; HOLMSKOV et al. 2003; VAN DE WETERING et al. 2004; GUPTA and

SUROLIA 2007). Moreover, MBL has been shown to interact with a wide variety of

Gram-positive bacteria, and various types of their cell wall component lipoteichoic

acid (LTC) (POLOTSKY et al. 1996). LU et al (2002) sums up that the wide range of

bacteria recognized by collectins highlights the importance of collectins in innate

immunity.

Even though binding of collectins and ficolins to bacteria has been reviewed

intensively for humans (HOLMSKOV et al. 2003; VAN DE WETERING et al. 2004;

GUPTA and SUROLIA 2007), information about their role in innate immunity of

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animals is sparse. While most authors only casually mention binding of ficolins and

collectins concerning animals, LILLIE et al. (2005) summarise the variety of binding

sites of collectins and ficolins to microorganisms for animals in detail. For MBL-A, the

authors observed binding to pathogenic bacteria species which infect pigs, such as

different serotypes of Heamophilus parasuis, Actinobacillus suis and Actinobacillus

pleuropneuminiae (APP).

Also, porcine ficolin-α binds to APP (BROOKS et al. 2003). Furthermore, porcine

ficolin-α binds to LPS from E.coli, Salmonella typhimurium, Salmonella enteritidis,

Pseudomonas aeruginosa and to LTA from Staphylococcus pyogenes, Bacillus

subtilis and Staphylococcus aureus (RUNZA et al. 2008).

Concerning binding sites of MBL-C, LILLIE et al. (2005) mention pathogen MBL

interaction with the following bacteria: Neisseria spp., Salmonella typhimurium,

Yersinia enterocolica, Pseudomonas aeruginosa, Escherichia coli, Mycobacterium

bovis and the yeast Candida albicans.

In order to offer a better overview, all microorganisms recognized by MBL and ficolins

are listed in Tab. 2.2.

Tab 2.2: Comparison of animal collectins and ficolins and their antimicrobial activity (modified after LILLIE et al. (2005) and RUNZA et al. (2008))

Lectin Species distribution Pathogen interaction

MBL-A (serum MBL) Pig, cattle, rat, mouse, rhesus monkey

Actinobacillus suis, A.pleuropneumoniae, Haemophilus parasuis

MBL-C (liver MBL) Pig, cattle, rat, mouse, rabbit, chimpanzee and

chicken

Neisseria spp., Salmonella typhimurium,

Yersinia enterocolica, Pseudomonas aeruginosa,

Escherichia coli, Mycobacterium bovis,

Candida albicans Ficolin-α Pig A. pleuropneumoniae,

Bacillus subtilis, E. coli, S. typhimurium, S. enteritidis, Pseudomonas aeruginosa,

Staphylococcus aureus Staph. pyogenes

Ficolin-β Pig, cattle Not determined Ficolin A Rat, mouse Not determined Ficolin B Rat, mouse Not determined Ficolin L Humans S. typhimurium, E. coli,

Staph. aureus, Streptococcus pyogenes,

Strep. agalactiae

Literature

38

For animals, there is still little known about the roles of the several collectins and

ficolins in viral infections. Concerning MBL, no information was available up to now;

only for SP-A and SP-D increased expression has been observed in lambs infected

with parainfluenza virus type 3 (GRUBOR et al. 2004).

In contrast to animals, detailed descriptions of target pathogens, beside bacteria, are

available from several studies for humans.

Thus not only bacteria were recognized by MBL in humans, but also fungi. Especially

the noncapsulated form of Cryptococcus neoformans (SCHELENZ et al. 1995) and

Candida albicans (NETH et al. 2000) were mentioned in this connection.

Regarding infections with viruses, it was an outstanding finding that MBL binds to the

human immunodeficiency virus (HIV) (HAURUM et al. 1993), and as well to influenza

virus (READING et al. 1993). Other collectins, such as SP-A and SP-D, detect

herpes simplex virus and the non-enveloped rotavirus (HOLMSKOV et al. 2003).

Concerning infections with parasites, binding of MBL to Leishmania species in

humans was observed (GREEN et al. 1994). Leishmania is an intracellular parasite,

mainly infecting macrophages, and in case of this infection it is considered that

binding of MBL and covering with complement factors provides no protective effect,

but in fact stimulates the uptake of the parasite via complement receptors on the

surface of macrophages (SANTOS et al. 2001).

In contrast to MBL, for ficolin no other binding sites, except those for bacteria, have

been described in the literature.

The general function of collectins, thus, is to protect the host by recognising PAMPs

on microorganisms and eliciting appropriate responses by activation of multiple

processes of innate immunity (HOLMSKOV et al. 2003; VAN DE WETERING et al.

2004).

Literature

39

2.6.7 Dectin-1

Dectin-1 is a C-type lectin receptor (CTL) and a member of the larger family of

pathogen recognition receptors (HERRE et al. 2004; GANTNER et al. 2005;

TAYLOR et al. 2007). The C-type lectins recognize sugar ligands through the

carbohydrate recognition domain (CRD) in a Ca2+ dependent manner. Dectin-1 is the

most recently characterised ß-glucan receptor as it was identified in humans in 2001

(BROWN and GORDON 2001). In the animal kingdom it was first identified in mice

(RYAN et al. 2002).

Human Dectin-1 differs from the murine receptor, as in humans two major isoforms

exist (WILLMENT et al. 2001; GRUNEBACH et al. 2002). The isoforms are named ß-

glucan receptor-A (ßGR-A) and ß-glucan receptor-B (ßGR-B). Concerning the

structure, they differ by the presence and absence of a stalk region, but functionally

both recognize ß-glucans of fungi (WILLMENT et al. 2001).

Dectin-1 is expressed on a variety of human leukocytes including monocytes,

macrophages, eosinophils, neutrophils and NK cells (STEELE et al. 2003;

GANTNER et al. 2005); DCs as well express Dectin-1. For our study, the fact that

Dectin-1 was detected on Langerhans cells of the human epidermis is of outstanding

interest (ARIIZUMI et al. 2000; YOKOTA et al. 2001; GAVINO et al. 2005), as the

esophagus epithelium bears similarity to the epidermis.

The receptor is also expressed on B cells and a subpopulation of T cells. WILLMENT

et al. (2005) conclude that this cell expression demonstrates that human Dectin- 1 is

not myeloid restricted. In mice, Dectin-1 is detectable on macrophages (WILLMENT

et al. 2005); REID et al. (2004) found out that in this species Dectin-1 is also

expressed on neutrophils, macrophages in the splenic red and white pulp, alveolar

macrophages, Kupffer cells of the liver, macrophages and on DCs in the lamina

propia of gut villi. The authors concluded that this expression pattern is consistent

with its role in pathogen surveillance. Supporting the results in humans, also in the

murine epidermis Dectin-1 positive LCs were detected.

In bovine breeds, mRNA transcripts of Dectin-1 were demonstrated in bone-marrow

cells, monocytes, macrophages, NK-cells and DCs (WILLCOCKS et al. 2006). Also in

sheep, Dectin-1 mRNA was found, as in the spleen, lung, kidney and skin

(NALUBAMBA et al. 2007).

Literature

40

As already mentioned previously, Dectin-1 is a C-type lectin receptor. A characteristic

for this family is the extracellular carbohydrate-recognition domain (CRD). The

cytoplasmatic part of the Dectin-1 receptor contains an immunoreceptor tyrosine-

based activation-like (ITAM-like) motif (BROWN and GORDON 2001; BROWN

2006). Between the extracellular CRD and the cytoplasmatic ITAM a stalk

transmembrane region is located. Functionally the ITAM-region is of great interest.

The ITAM can be phosphorylated and activated by ß-glucan binding in the CRD.

Through the phosphorylation of the cytoplasmatic ITAM motif, Dectin-1 is able to

mediate intracellular signalling, which results in a variety of cellular responses. For

example, the production of several cytokines and chemokines (TNF, IL2, IL6, IL10,

IL-12 and IL23) is triggered and the respiratory burst system is activated (GANTNER

et al. 2003; BROWN 2006; WILLMENT and BROWN 2008).

There is evidence that Dectin-1 collaborates with TLR-2 in recognising

microorganisms. GANTNER et al. (2003) describe how co-expression of Dectin-1

and TLR-2 results in an increasing production of TNF and IL-12. Both receptors

activate the NF-κB signalling way and mediate the production of TNF and IL-12

(DENNEHY and BROWN 2007).

Dectin-1 is capable to recognize fungal cell wall components (BROWN and

GORDON 2001; HERRE et al. 2004; DENNEHY and BROWN 2007; KANAZAWA

2007; TAYLOR et al. 2007). In vitro analysis showed that Dectin-1 possesses the

ability of identifying a wide variety of fungal species, including Aspergillus, Candida,

Coccidioides, Pneumocystis and Saccharomyces (GANTNER et al. 2005; STEELE et

al. 2005; BROWN 2006).

The important role of Dectin-1 in fungal infections in vivo is underlined by the results

of recent studies. All experiments mentioned in the following were accomplished with

mice. STEELE et al. (2005) found out that blockage of Dectin-1 during intratracheal

infection with Aspergillus fumigatus reduced lung inflammatory responses

significantly and increased fungal burdens. Via infecting a Dectin-1 knock-out mice

with Candida albicans, it was proven that lack of Dectin-1 results in an enhanced

susceptibility to systemic Candida albicans infection (TAYLOR et al. 2007).

Literature

41

2.7 Lysozyme

Lysozyme is a peptidoglycan N-acetylmuramoylhydrolase. It is a member of the class

of enzymes which mainly destroys the cell wall of Gram-positive bacteria. In this

connection, lysozyme is capable to cleave the bond between N-acetylglucosamine

and N-acetylmuramic acid of the peptidoglycan in the bacterial cell wall.

NIYONSABA and OGAWA (2005) explain why Gram-positive but not Gram-negative

bacteria are preferred targets of lysozyme: The natural substrate of lysozyme is

peptidoglycan. Gram-positive bacteria are composed of up to 40 layers of

peptidoglycan, whereas Gram-negative only have a single layer of peptidoglycan

surrounded by an outer membrane (MASSCHALCK et al. 2002). As a result, Gram-

negative bacteria are protected by their specific outer membrane, which acts as

barrier preventing contact with lysozyme.

The major cellular sources of lysozyme are neutrophilic granulocytes and

macrophages, where it is detectable in the lysosomes (DUSZYK 2001). Furthermore,

lysozyme is present in various tissues and secretion fluids of glands. So it was

detected in the skin, epithelial cells of the trachea and in Paneth cells of the small

intestine of humans (DUSZYK 2001; NIYONSABA and OGAWA 2005; WEHKAMP et

al. 2006). Concerning expression in Paneth cells, the highest amounts of lysozyme

were found in the duodenum, where it was also expressed by the Brunner`s glands.

In the jejunum and ileum, lysozyme appeared only in smaller amounts (WEHKAMP et

al. 2006).

Analyses of the distribution pattern of lysozyme in the human epidermis revealed that

it is expressed much more in the upper than the basal cells layers (NIYONSABA and

OGAWA 2005). Lysozyme was also identified in the skin of different wild mammals,

including dolphins. Here it was mostly found in cells and intercellular spaces of the

stratum spinosum and str. corneum (MEYER et al. 2003; MEYER and SEEGERS

2004).

Interestingly, lysozyme was also detected in human esophageal submucosal glands

(HOPWOOD et al. 1986). The authors described positive immunohistochemical

reactions for the substance in the duct epithelium cells.

Secretions containing lysozyme are saliva, tears, or urine, and secretions of cervical

glands (REITAMO et al. 1977; NIYONSABA and OGAWA 2005)

Literature

42

2.8 Dendritic cells

As mentioned previously, the ability of DCs to integrate a variety of environmental

signals, like the conflict with microorganisms, either commensal or pathogenic,

together with their ability to respond with an immunological reaction, makes them

cells of particular relevance and importance in the mucosal environment.

Mainly two ways of responding towards an environmental signal are possible in the

mucosal tissue: On one hand, an active defensive response to mucosal pathogens is

feasible, and on the other hand, a “tolerant” response to harmless environmental

agents, such as commensal bacteria and food-derived antigens, is the opposite type

of reaction (HAVERSON and RIFFAULT 2005).

Antigen presentation via human LCs is possible due to their expression of MHC class

II molecules, and upon the expression of CD1 molecules and langerin (SALAMERO

et al. 2001; PENA-CRUZ et al. 2003; HUNGER et al. 2004; NFON et al. 2008).

Beside MHC class molecules, which present peptide antigens towards T-cells, also

the family of the more recently discovered CD1 molecules initialises a T-cell receptor

(TCR) activation. This results in a T-cell defence reaction against other, non-peptide

lipid and glycolipid antigens, which are presented via CD1 molecules on APCs

(PORCELLI and FRANK 1995; SUGITA et al. 2000; PENA-CRUZ et al. 2003;

HUNGER et al. 2004; MOODY et al. 2004; SUGITA et al. 2004; BARRAL and

BRENNER 2007; MORI and DE LIBERO 2008). The CD1 molecules are a lineage of

cell-surface glycoproteins related in structure and evolutionary origin to the MHC-

encoded antigen-presenting molecules (KNOWLES and BODMER 1982; CALABI et

al. 1989).

Due to sequence comparisons, CD1 proteins were classified into two separate

groups over decades: human CD1a, -b and -c form group 1, and the significant more

divergent CD1d defines group 2 (PORCELLI and FRANK 1995). More recently

conducted studies implicate the existence of a third group of CD1 molecules, which is

named CD1e. This molecule has low homology with the other CD1 proteins, and thus

is not included in CD1 group 1 or 2. A supplementary difference to the other CD1

molecules, which are expressed on the cell-surface, is the fact that CD1e is

expressed in DC without reaching the plasma membrane (ANGENIEUX et al. 2000).

CD1 molecules belonging to group 1 mainly present microbe-derived lipid antigens to

CD4 and CD8 positive T cells to clear microbial infection, whereas CD1d activates a

Literature

43

specialised T cell subset known as natural killer cells (BENDELAC et al. 1995;

ROSAT et al. 1999; PENA-CRUZ et al. 2003; HUNGER et al. 2004; MIZUMOTO and

TAKASHIMA 2004).

Although CD1 molecules show homology to MHC molecules, their atomic structures

of the antigen-binding sites and their routes of intracellular trafficking differ from the

ones described for MHC molecules (SUGITA et al. 2004; VAN DEN ELZEN et al.

2005; VAN RHIJN et al. 2005; MORI and DE LIBERO 2008).

The epitopes for CD1a, CD1b and CD1c are located on various cells, as they can be

detected on myeloid dendritic cells, LC and B cells. CD1c is expressed on 20-50% of

the B cells from blood, tonsil and spleen (SMALL et al. 1987; TIZARD 2008) and,

together with CD1b, on dendritic cells in a variety of tissues including the skin, liver,

kidney, lung, and lymphoid organs in humans (PORCELLI and FRANK 1995;

JANEWAY et al. 2005).

CD1a is the specific marker molecule for LCs in the skin, which express this molecule

in great amounts (ELDER et al. 1993; CLARK et al. 2000; PENA-CRUZ et al. 2003;

MIZUMOTO and TAKASHIMA 2004; HUSSEIN 2008). Other authors also allude the

occurrence of CD1a positive DCs in the epithelium of different tissues, such as the

bronchus (TAZI et al. 1993), conjunctiva (YOSHIDA et al. 1997), gingiva

(CRAWFORD et al. 1989), vagina and cervix (MILLER et al. 1992). This tissue

distribution might be due to the fact that antigen-presentation via CD1a plays a

pivotal role at sites constantly exposed to external pathogens (PENA-CRUZ et al.

2003).

Beside Birbeck granules, detectable in LCs and CD1a, expressed as surface

molecule on LCs, langerin (CD207), a C-type lectin, is another characteristical

molecule on LCs (VALLADEAU et al. 2000; ROMANI et al. 2003; MIZUMOTO and

TAKASHIMA 2004), whereby the three molecules show a functional relationship

(VALLADEAU et al. 1999, 2000). The C-type lectin langerin, exclusively expressed

by LCs, recognizes mannose residues via its single carbohydrate recognition domain

(CRD) and captures glycolipid antigens. The complex of glycolipids and langerin is

internalised and delivered to the Birbeck granules inside the LC. In the Birbeck

granules also CD1a, which has been internalised from the cell surface, can be

detected. The interrelation between CD1a and langerin can be explained by the fact,

that CD1a is coupled with lipid moieties, collected by langerin on the cell surface

Literature

44

inside the Birbeck granules. Finally, the loaded CD1a molecule is transported back to

the cell surface to present lipid antigen to T cells (VALLADEAU et al. 2000).

In the literature various statements about the occurrence of DCs and LCs in animals

can be found. Most studies mention LC appearance in the skin (ROMANI et al.

2003). Just recently a study showed the existence of langerin positive LCs in the

porcine epidermis (NFON et al. 2008), corroborating former information (MEYER

1986). In mice it is notable that langerin is also expressed by non-cutaneous, non-

Langerhans cell derived dendritic cells in lymphoid tissue and the lung (DOUILLARD

et al. 2005; SUNG et al. 2006).

Most other reports about the occurrence of LCs in animals concentrate on the

detection of CD1a positive (CD1a +) LCs. For dogs expression of CD1a + LCs in skin

was just recently maintained (VAN BEECK et al. 2008); also in cats, CD1a + LCs

were detected (MARCHAL et al. 1997).

Description of LC cell-like dendritic cells in the intestinal tract of animals, including the

esophagus, can be found very rarely. Only one study confirmed the presence of LCs

in the esophagus of mice, verified by ultrastructural observations (ROWDEN et al.

1977). Furthermore, Langerhans cell-like dendritic cells have been identified at the

mucosal site of the chicken esophagus (PEREZ-TORRES et al. 2002).

Literature

45

2.9 Questions addressed in this study

1. Is the esophagus epithelium of domesticated mammals colonised by

microorganisms?

2. Do any mechanisms of innate immunity exist in the esophagus epithelium of

domesticated mammals?

3. Are the APs, hBD-2, -3 and CAT, produced by the epithelium?

4. Is TLR2 expressed on epithelial cells? Can a co-expression with APs be

demonstrated?

5. Is it possible to detect L-ficolin, MBL and Dectin-1 in the esophagus

epithelium?

6. Is the enzyme lysozyme produced by epithelial cells?

7. Are CD1a(+) and/or langerin(+) LCs detectable in the esophagus of

domesticated mammals?

8. Where can such innate defence mechanisms be identified in the esophagus

epithelium?

9. Do differences exist in the expression intensity of innate defence mechanisms

in herbivorous, omnivorous and carnivorous species?

10. Are structure preservation and IHC results influenced by the three different

fixation media (Bouin`s solution, Ca-Formol, HOPE®) applied?

Material and Methods

46

3 Material and Methods

3.1 Animals

Esophagus samples were collected from 10 domesticated mammalian species. The

three great nutrition groups were considered: Omnivores (pig, dog, mouse, rat),

carnivores (cat) and herbivores (goat, sheep, rabbit, cattle, horse).

One group of samples had already been taken during earlier studies on the

oesophagus epithelium of the mentioned mammals, and was available fixed and

embedded. These samples had been obtained from the Institute for Pharmacology,

Toxicology and Pharmacy of the University of Veterinary Medicine Hannover, the

abattoir in Gleidingen (Hannover) and several practitioners in the surroundings of

Hannover.

The other group of samples for this study was compiled from different other institutes

of the University of Veterinary Medicine Hannover, again the abattoir in Gleidingen

(Hannover) and small animal practitioners in Hannover.

Goat and horse samples were collected with the help of the Institute for Anatomy.

The samples of porcine esophagi were provided from the Institute for Nutrition and

the canine esophagi were supplied from the Institute for Parasitology. Sheep, rabbit,

rat and mouse samples were taken with support of the Institute for Physiolology and

the Institute for Pharmacology, Toxicology and Pharmacy. Feline oesophagi were

collected from a veterinary surgery in Hannover and the Small Animal Clinic of the

University of Veterinary Medicine Hannover; the abattoir in Gleidingen (Hannover)

provided bovine oesophagi.

3.2 Sampling

The esophagus of the different freshly dead animals was opened by careful

dissection. Following, six to seven small pieces (maximum 1 cm x 1 cm) were

excised and immediately transferred into the different fixation solutions.


47

Normally the samples were taken from the medium part of the esophagus (compare

also SCHOENNAGEL 2005).

Especially for samples fixed in HOPE® and Karnovsky solutions it was important to

control that the samples were not larger than 8 mm x 8 mm for HOPE® and 5 x 5 mm

for Karnovsky fixation, as penetration into the material would have been too slowly or

incomplete.

Tab. 3.1: Domesticated mammalian species used Nutrition type Species Number and gender

Horse 11 (6 geldings, 5 females)

Cattle 4 (females)

Goat 4 (1 male, 3 females)

Herbivores

Sheep 10 (2 males, 8 females)

Dog 9 (2 males; 2 females; 5

subadult males)

Pig 15 (females)

Mouse 9 (3 males, 6 females)

Omnivores

Rat 8 (2 males, 6 females)

Carnivores Cat 8 (4 males, 4 females)


48

3.3 Tissue Fixation

3.3.1 Bouin’s solution (after BÖCK 1989)

The following chemicals were mixed:

1500 ml of saturated picric acid

+ 500 ml of 37 % formaldehyde (Merck)

+ 100 ml glacial acetic acid 99-100 %

The samples remained in the solution for 48 hours. Afterwards they were washed

several times in 70% ethanol with the addition of some drops of ammonia (conc.) to

remove the picric acid, and stored in 80% ethanol before running through an

ascending concentration of ethanol for embedding.

3.3.2 Calcium acetate-buffered formalin (after LILLIE and FULLMER

1976)

Calcium acetate-buffered formalin is a mixture of the following chemicals:

20 g Ca-acetate monohydrate (Merck)

+ 100 ml 37 % formaldehyde solution (Merck)

+ 900 ml de-ionised water

The samples remained in the solution for at least 24 hours, and later were washed

and stored in 70% ethanol.

3.3.3 HOPE® fixation (after OLERT et al. 2001)

The HOPE® fixation solutions are ready for use, and therefore no mixing of basic

chemicals is necessary. The complete HOPE® protocol is described in Tab. 3.2. Two

different fixation solutions were employed.

1. Incubation in HOPE I solution for 12-72 h at 0-4°C in the refrigerator

2. Removal of HOPE I solution

3. Incubation in 5 ml of an ice cold HOPE II / acetone solution (100 ml

acetone + 100µl HOPE II) for 2 h at 0-2°C on ice in the refrigerator.


49

3.3.4 Fixation after KARNOVSKY (1967)

In order to prepare semithin sections and for future electron microscopical

observations, esophagus samples of each species were also fixed in Karnovsky`s

solution.

The following substances were mixed:

20 g para-formaldehyde (Merck) were dissolved in 250 ml distilled water

+ 100 ml glutaraldehyde (Merck)

+ 150 ml of a 0.2 molar cacodylate buffer (Serva)

The solution has to be stored at 4°C. The tissue pieces have to be very small (5mm x

5mm) to obtain good fixation and must be left in the solution for at least 24 hours.

3.3.5 Liquid nitrogen sampling

Liquid nitrogen samples were taken in order to analyse the surface of the esophagus

epithelium applying cryo scanning electron microscopy (Cryo-SEM). Therefore, small

pieces (2 x 4 x 8 mm) of the esophagus were transferred into liquid nitrogen (LN2) (-

180 °C) immediately after dissection, and then stored separately in small cavities of a

covered aluminium block, that was kept in LN2 until use.

3.4 Paraffin embedding

3.4.1 Paraffin embedding for samples fixed in Bouin’s solution and Ca-

acetate buffered formalin

Both types of samples were initially cut into small pieces and put into marked plastic

capsules for paraffin embedding. Afterwards, the Bouin-fixed samples were stored in

80 % ethanol and then dehydrated in ascending concentrations of ethanol in order to

facilitate paraffin penetration into the tissue.

In contrast to the Bouin-fixed samples, the formalin-fixed material was stored in the

fixation medium, before it was rinsed in tap water over night and then transferred into

70% ethanol.


50

The following steps were identical for both sample types.

1. 24 hours 80% ethanol

2. 2 hours 96% ethanol

3. 2 hours isopropanol (abs.)

In order to remove the ethanol from the tissue, the samples in the capsules were

immersed in xylene (Riedel-de Haen) for 2 hours for two times, before they were

transferred to 60°C paraffin I (Paraplast, Sherwood) over night. The next day, the

capsules were stored in paraffin II for 4 hours and for another 4 hours in paraffin III.

In the last step, they were embedded with fresh 60°C paraffin in moulds, before

hardening at 4°C in the refrigerator.

3.4.2 Paraffin embedding HOPE®

After the HOPE II/acetone solution had been discarded, the samples were incubated

with freshly prepared cold acetone over 2 hours. This step was repeated twice, each

incubation lasting 2 hours.

Acetone is a dehydrating solution and removes the HOPE solution from the tissue.

After dehydration, the specimens were directly transferred into low melting paraffin

(52-54°C), which was prepared and stored in a warming cupboard. The first

incubation in paraffin I was carried out at 54°C for 12-16 hours; the second one in

paraffin II required 4 hours. It is important to avoid a trapping of air bubbles in the

paraffin. In the last step, the tissues were embedded in paraffin and the blocks were

stored in the refrigerator to cure until sectioning.

Table 3.2: Overview fixation steps HOPE® Reagent Incubation time

HOPE I 12-72 hours

HOPE II / acetone 2 hours

Acetone 2 hours

Acetone 2 hours

Acetone 2 hours

Low melting paraffin (54°C) I 12-16 hours

Low melting paraffin (54°C) II 4 hours


51

3.5 Standard EPON embedding for Karnovsky fixed tissue

In comparison to paraffin embedded tissue, less shrinkage and less separation of the

tissue layers in EPON embedded material occurs. The synthetic material is much

harder than paraffin and it is possible to prepare semithin (0.2-1 µm) and ultrathin

slides (about 80 nm), which can be used for transmission electron microscopy (TEM).

For embedding, the tissue samples were dehydrated in ascending concentrations of

ethanol. In the next step, the samples were incubated in propylenoxide for 30 to 60

minutes. Following they were put in a propylenoxide-Epon mixture, and finally in an

absolute Epon mixture for another 30 to 60 minutes. After preparing a fresh Epon

mixture and adding the polymerisation enhancer dimethyl-amino-phenol (DMP-30),

the small samples were blocked in rubber moulds. Polymerisation was carried out in

an incubator at 36°C during the first day; the temperature was increased to 45°C on

the second day and to 60°C on the third day.

3.6 Sectioning

The procedure of sectioning is rather similar for most of the different samples

embedded in different media. After hardening in the refrigerator, the paraffin blocks

were removed from the metal moulds and sectioned with a rotation microtome

(Reichert Jung). The sections were not thicker than 5 µm, for the

immunohistochemical experiments conducted.

For the sections of paraffin embedded material only one water bath was necessary

with a temperature of approximately 50°C to allow stretching; for sections of the

HOPE®-fixed material, however, two water baths were needed. In the latter case, the

sections were collected in the first water bath, which had to be cold water, and the

second water bath needed to be tempered at 35-39°C. One by one section had to be

dipped in the heated water baths and just a short period of stretching is required.

After stretching, the sections were transferred on adhesive slides and dried in an

incubator. HOPE® sections are sensitive against heat, and therefore were dried

overnight at 37°C, whereas the other slides were dried for 3-4 hours in a warming

cupboard (50°C).


52

The Epon series embedded samples were treated slightly different. In order to

prepare semithin sections (0.5 µm) an ultra-microtome (Ultracut E, Leica) with a

diamond knife was used.

3.7 Histological staining

3.7.1 Hematoxylin-Eosin staining of Bouin and formalin-fixed tissue

In order to evaluate the quality of tissue preservation, all collected samples were

stained in the H.E. and Masson Goldner trichrome stains.

For a good differentiation of nuclei and cytoplasm, the preferred method was H.E..

After this procedure, nuclei are blue and the cytoplasm is pink, the nuclear and cell

boundaries are well defined (LILLIE and FULLMER 1976). The H.E. staining

represents a dichromatic staining, which is composed of the nucleus dye hematoxylin

and the acidic cytoplasm dye eosin (BÖCK 1989). The originally herbal dye

hematoxylin is activated after a process of acid cleaning.

Before starting staining, hemalaun after DELAFIELD had to be prepared. Therefore,

4 g of hematoxylin (Merck) were dissolved in 25 ml absolute ethanol and in the next

step 400 ml of 10% ammonium aluminium sulfate (Merck) were added. After four

days of ripening, 100 ml of glycerin (Merck) and 100 ml of methanol were added.

Over several months, the hemalaun had to oxidize and to be aerated, before it was

ready for use.

To make the ready-to-use cytoplasm dye, an acetic 1 % eosin (Merck) solution with

96 % ethanol was prepared.

For the paraffin embedded material it is necessary to remove the surrounding wax

from the tissue before any other treatment is possible. This process is called

deparaffinisation. Therefore the slides were immersed in xylene two times for 15

minutes, whereby the paraffin was extracted. Subsequently, water had to be brought

back into the tissue to allow staining in an aqueous milieu.

The process of rehydration is achieved with the help of descending concentrations of

ethanol:


53

1. 2 minutes isopropanol (abs.)

2. 2 minutes 96 % ethanol



Following the slides were brought in de-ionised water, before staining was started.

The following staining protocol was used (BÖCK 1989):

1. 8 minutes hematoxylin (after DELAFIELD)

2. 10-15 seconds in 0.1 % HCL

3. 15 minutes under running tap water

4. 5 min in Eosin (Annex) in distilled water with 5 drops acetic acid glacial

(Riedel-de Haen)

5. flushing in 70 % ethanol





10. 2 minutes ethanol (abs.)


12. 5 minutes xylene


Finally the slides were covered with the synthetic resin EUKITT® (Kindler).

3.7.1.1 H.E. staining of HOPE® fixed tissue

The HOPE® fixed tissue needs also the procedure of deparaffinisation. This process

differs slightly from that one described for the Bouin and formalin-fixed material.

Deparaffinisation is achieved via an incubation in 60°C isopropanol (abs.), and

rehydration is accomplished in 70% acetone at 2-8°C.

To obtain the ideal conditions for deparaffinisation, cuvettes with isopropanol were

placed in a warming cupboard until they had reached the correct temperature. The

same procedure was conducted for the cuvettes with cold 70% acetone, as they


54

were stored in the refrigerator until they had achieved the suitable temperature. To

guarantee good deparaffinisation and rehydration, the following protocol was applied:

1. 10 minutes 60°C temperate isopropanol (abs.)

2. 3 minutes twice fresh 60°C isopropanol (abs.)

3. 10 minutes twice fresh 70% cool (2-8°C) acetone

Incubation times in the dyes for H.E. staining are normally shorter for HOPE®-fixed

material than for the other two fixation methods. Another important aspect is to keep

attention to the fact that HOPE®-fixed tissue is sensitive for any influence of acid. For

this reason, no acetic acid was added to the eosin dye. Furthermore, the immersion

times in the ascending concentrations of ethanol had to be different. The following

protocol was used:

1. Flushing in de-ionised water twice

2. 5 minutes de-ionised water

3. 2-4 minutes hemalaun after DELAFIELD

4. short washing in de-ionised water

5. 1-2 minutes under running tap water to perform bluing reaction

6. 2-4 minutes eosin

7. Washing in de-ionised water twice

8. Dipping in 70% isopropanol twice

9. Dipping in ethanol (abs.) twice

10. Incubation for 10 minutes in ethanol (abs.)

11. Brief washing in xylene (Riedel-de Häen)


Finally slides were removed, dip-dried and mounted with Eukitt® (Kindler).


55

3.7.2 Trichrome staining (Masson-Goldner) (after BÖCK 1989)

This staining is normally used for the demonstration of connective tissue (green),

which can be distinguished from muscle tissue (red).

Only the Bouin and formol fixed samples were stained after Masson-Goldner, as the

acidic substances used are too aggressive for the HOPE® fixed material and no

satisfactory results could be expected.

After deparaffinisation, the tissue was stained as follows:

1. 10 minutes hemalaun (after Delafield)

2. 10 seconds 0.1% HCL

3. 15 minutes running tap water

4. 5 minutes acid-fuchsin-ponceau [0.2 ponceau de xyline (Merck) + 0.1 g acid

fuchsin (Merck) + 300 ml de-ionised water + 0.6 ml acetic acid glacial]

5. 5 minutes 1 % acetic acid

6. 10 minutes phosphotungstic acid-orange-G [10 g phosphotungstic acid (Fluka)

+ 5g orange-G (Fluka) + 250 ml de-ionised water] to bleach the connective

tissue


8. 5 minutes lightgreen [0.5 g lightgreen (Merck) + 250 ml de-ionised water + 0.5

ml acetic acid glacial]




12. 2 minutes ethanol (abs.)


14. two times 5 minutes xylene

15. mounting with EUKITT® (Kindler)


56

3.8 Microscopical evaluation

3.8.1 Light microscopy

After staining with H.E. and/or the Masson-Goldner trichrome procedure, the Bouin,

Ca-Formol and HOPE® fixed esophagus samples were analysed with a light

microscope [Zeiss Photomicroscope II with a digital camera (Olympus DP70)]. After

this inspection for structure quality, it was decided which samples would allow

immunohistochemical experiments.

3.8.2 Cryo scanning electron microscopy (Cryo-SEM)

For analysis, one piece of the esophagus samples stored in LN2 was transferred in

the LN2 reservoir of a cryo sample loading device (Bal-Tec) and carefully mounted in

a specially designed clamping device on top of an object table. By means of a LN2-

cooled vacuum cryo transfer system (cryo shuttle VCT 100, Bal-Tec), in which the

sample was kept in an atmosphere of evaporising nitrogen for about 15 sec

(temperature lower than -180 °C); the loaded object table was rapidly transferred in a

freeze-etch / freeze-fracture unit (MED 020, Bal-Tec). Therein it was placed on a cryo

stage, where it was kept under high vacuum (5 x 10–6 mbar) at a minimum

temperature of -150 °C. After appropriate positioning of the stage, the esophagus

sample was hit once with a blade, thus creating a freeze-fracture from the outermost

to the deeper skin layers. Minor amounts of ice were removed from the sample

surface by gentle sublimation (-120 °C for 5 min, 1.7x10-4 mbar). Subsequently, the

surface of the sample was sputter-coated with gold palladium (40 sec), whereby all

samples were sputtered twice and from opposite directions (40 mA, 2.5 x 10-2 mbar,

argon). Using the shuttle, the coated sample was transferred onto the cryo stage

(Bal-Tec) of an adapted LEO 1430 vp scanning electron microscope (Zeiss) and

inspected at minimum vacuum of 9 x 10-6 mbar with an accelerating voltage of 10 kV.

The findings were documented the help of a digital camera.

For the demonstration of microorganisms on the surface of the esophagus it was

very important to rely on careful and artifact-free tissue processing (see also MEYER

et al, 2007). In this context, cryo SEM was the most suitable approach due to the

following reasons: Biological material, both plant and animal tissue, consist to a large

amount of water, which might occupy up to 98% of total tissue volume. Most types of

tissues, particularly those with hydrated structural elements, like several forms of


57

connective tissue, are heavily altered under conditions of conventional SEM

preparations that apply alcoholic dehydration and critical point drying (CPD). The

abdication of chemical fixation, dehydration and solvents during cryo SEM

processing means that delicate structures are maintained without shrinkage and

without loss of soluble sample components. The latter feature is especially crucial

studying the localisation and distribution of extra- and intracellular aqueous or

colloidal material, such as, additionally, the mucus cover of the esophagus surface.

In contrast to other methods, which to variable extents are associated with the

generation of structural artifacts, the use of cryo freezing techniques instead of

chemical fixation allows to attain an ultrastructure which is as close as possible to its

natural state.

3.9 Immunohistochemistry

Immunohistochemistry is the localisation of antigens or proteins in tissue sections by

the use of labelled antibodies as specific reagents through antigen-antibody

interactions that are visualised by a colour marker, such as fluorescent dyes,

enzymes, or colloidal gold.

Five important representatives produced by the innate immune system were chosen:

Antimicrobial peptides, ß-glucan receptors, Toll-like receptors, lysozyme and

Langerhans cells. In order to detect the latter groups, different primary antibodies

were used.

As another aim of this thesis, different fixation methods (Bouin`s solution, Ca-

buffered formalin, HOPE® fixation) and their influences on immunohistochemical

reations were compared.

3.9.1 Primary antibodies

For the demonstration of the various parts of the innate immune system of the

esophagus epithelium several primary antibodies were applied (see Tab. 3.3). All

antibodies mentioned were diluted with PBS and 1 % bovine serum albumin (BSA)

as an additive until the suitable functional concentration had been reached. The

antibodies were stored at 4 °C and the PBS / 1% BSA mixture prepared at -20 °C

until use.


58

Tab. 3.3: Primary antibodies AP: Antimicrobial peptide, ß-GR: ß-glucan receptor, LC: Langerhans cell, m: monoclonal, p: polyklonal

Primary antibody

Origin Specificity Clonality, isotype

Vendor

ß-defensin-2 (AP)

rabbit human, mouse p Biologo,

Kronshagen, Germany

ß-Defensin-3 (AP)

rabbit human, mouse p Biologo,

Kronshagen, Germany

Cathelicidin (LL37/CAP18)

(AP) mouse human

m, IgG1

HyCult Biotechnology,

Sanbio, Beutelsbach,

Germany

CD1a (clon O 10)

(LC) mouse human

m, IgG1

Biologo, Kronshagen,

Germany

Langerin (CD 207)

(LC) rabbit mouse, rat p


Germany

Dectin-1 (ß-GR)

goat mouse p Biologo,

Kronshagen, Germany

L-Ficolin (clon GN4)

(ß-GR) mouse human

m, IgG1


Germany

MBL (clon 3E7)

(ß-GR) mouse human

m, IgG1


Germany

Toll-like-receptor 2

mouse human m,

IgG2a


Germany

Lysozyme rabbit human p DAKO,

Golstrup, Denmark


59

3.9.2 Secondary antibodies

As secondary detection systems two different types of visualisation were tested. The

first one is known as direct method. It is a one step staining technique and involves

a labelled antibody (for example FITC labelled). The antibody reacts directly with the

antigen in the tissue. Due to the fact that this method is rather insensitive, as little

amplification signal occurs, the indirect method is the technique of choice. For the

indirect method an unlabelled primary antibody is applied, which reacts with the

tissue antigen. To amplify the signal of the reaction, a labelled secondary antibody is

used (PEARSE 1985). These antibodies can be labelled with various substances. In

our study we applied antibodies labelled with a fluorescent dye (FITC), and

streptavidin-biotin and/or with horseradish peroxidase (HRP).

3.9.2.1 Two step indirect method with peroxidase linked secondary antibodies

To detect primary antibodies produced in the mouse or rabbit, we used the EnVision®

systems (DAKO). This visualisation technique is a two-step indirect staining method

(Fig. 3.1). The secondary antibody is conjugated with a horseradish peroxidase

(HRP) labelled dextran polymer. It is important to use a secondary antibody, which

has a strong affinity to the first antibody. Subsequently, the second antibody must be

directed against the immunoglobulin isotype of the species in which the first antibody

has been raised. For example, a primary antibody raised in the mouse must be

incubated with a secondary antibody raised in the rabbit, reacting as an anti mouse

immunoglobin.

To visualise the reaction, a 3,3`-diaminobenzidine (DAB) chromogen solution is used.

If the peroxidase linked secondary antibody has bound to the primary antibody,

peroxidase reduces DAB and a brownish colour reaction can be observed. As a

catalyst for this reaction, hydrogen peroxide (H2O2) is needed. It is important to notice

that mainly in granulocytes, mast cells and erythrocytes an endogenous peroxidase

activitivity can be observed. The easiest way to block the endogenous peroxidase is

to incubate the samples in an 80 % ethanol solution with H2O2 (197 ml 80 % ethanol

+ 3 ml 30% H2O2) for 30 minutes during the process of rehydration. As the

rehydration of HOPE® samples is accomplished in cold 70 % acetone, a blocking of

the endogenous peroxidase has to be conducted after the step of rehydration

through a 10 minutes incubation in de-ionised water and H2O2 (197 ml de-ionised

water + 3ml H2O2).


60

Two types of DAKO visualisation systems were applied, depending on the origin of

the primary antibody. EnVision® mouse was used to detect antibodies with mouse

specificity, while EnVision® rabbit was employed to visualise the binding of a primary

anti-rabbit antibody. The kits contained the peroxidase labelled secondary antibody,

DAB and a HRP substrate buffer.

A general immunohistochemical protocol can be found in Tab. 3.5. The visualisation

step for EnVision® is described in detail as following; the procedure is equal for all

three fixing methods:

1. Deparaffinisation, rehydration and blocking of the endogenous peroxidase

2. Washing in phosphate-buffered saline (PBS) or de-ionised water

3. Demasking (3.9.5.1)

4. Washing in PBS

5. Blocking of electrostatic binding sites (3.9.3)

6. Incubation of primary antibody over night at 4°C

7. Washing in PBS

8. Visualisation:

Incubation with secondary peroxidase linked antibody EnVision® rabbit

or mouse over 45 minutes

Washing in PBS to remove excess antibody

Incubation with DAB (40µl DAB + 1000 µl HRP substrate buffer)

over 5 minutes.

9. Rinsing in running tap water and/or PBS

10. Dehydration in ascending concentrations of ethanol

11. Mounting with Eukitt® (Kindler)

Furthermore, another two step indirect visualisation technique was utilised, as not all

primary antibodies used derived from the mouse or rabbit. The antibody to detect

Dectin-1 was extracted from goats, thus a secondary anti-goat antibody was needed.

The antibody applied was peroxidase labelled, similar to the one described above,

but as no visualisation kit was available, the binding of the secondary antibody had to

be visualised with a self-prepared DAB solution. Therefore DAB (Sigma) as a powder

was dissolved in 200 ml PBS and filtered. 14 µl of 30% H2O2 were added and the

slides were incubated in the solution over 10 minutes under constantly stirring.


61

Fig 3.1.: Principle of the two step indirect method DAB: 3,3`-diaminobenzidine

3.9.2.2 Use of a labelled streptavidin biotin Complex (LSBC)

This method uses the high affinity of avidin for biotin and is described as a three layer

technique. Avidin is a large glycoprotein and can be labelled with peroxidase. Avidin

shows a high electrostatical binding-affinity to the tissue and contains carbohydrate

groups, which show affinity to tissue lectins. These problems were eliminated through

the development of streptavidin, an avidin derived from Streptococcus avidini. Biotin

is a low molecular weight vitamin, which can be conjugated to antibodies.

As already mentioned previously, this method is of a three layer type. The first layer

is the primary antibody. The second layer is a biotinylated secondary antibody,

whereas the third layer is an enzyme-streptavidin conjugate (HRP-streptavidin) to

replace the complex of avidin-biotin peroxidase. The reaction is visualised by

applying the substrate DAB. All substrates mentioned and secondary antibodies were

included in the visualisation kit from BioGenex/DCS.

The blocking of the endogenous peroxidase is conducted as described under 3.9.2.1.

Primary

antibody

Secondary Peroxidase labelled antibody

DAB colour reaction

Epitope


62

According to the fact that the steps before and after visualisation equal those ones

described before and in Tab. 3.5 the following protocol is restricted to the step of

visualisation:

Incubation with a biotinylated secondary antibody over 40 minutes

Washing in PBS 3x5 minutes to remove excess antibody

Incubation with HRP-streptavidin for 40 minutes

Washing in PBS 3x5 minutes to remove excess enzyme

Visualisation with DAB (250 µl DAB substrate buffer + 2250 µl de-ionised

water + 2 drips liquid DAB + 1 drip H2O2), incubation for 5 minutes

3.9.2.3 Immunofluorescence (IF)

The third visualisation technique used was immunofluorescence. To identify the

primary antibodies, secondary antibodies conjugated with fluorescein isothiocyanate

(FITC) were applied. FITC was first introduced by John L. Riggs in 1960 as a stable

yellow powder and today is available as commercial product (PEARSE 1985). The

principle of IF is that of a relatively fast decreasing light emission of molecules, which

have been activated via the absorption of energetic rays. The light emission is visible

in the fluorescence microscope, where FITC shows a green-yellowish reaction, using

a specific filter [Zeiss Axioskop equipped with an epifluorescence device (FITC filter

combination: BP450-490, FT510, LP520)]. As the reaction intensity decreases very

fast, reaction analysis has to be made immediately and pictures have to be taken.

Different FITC labelled antibodies depending on the origin of the first antibody were

used and are shown in Tab. 3.4. In contrast to the other visualisation systems, FITC

needs no blocking of endogenous peroxidase, as no chromogen is used. The slides

were mounted with Mowiol (Sigma Aldrich), in order to obtain a longer lasting

reaction. The Mowiol solution was self prepared after the following protocol:

1. 2.4 g Mowiol (Sigma Aldrich)

+ 6.0 g glycerol (99.5%) (Sigma)

+ 6 ml aqua dest.

The components were mixed with a magnetic stir bar for 4 hours

2. In the next step, 12 ml of 0.2 M tris-buffer pH 8.5 [for 100 ml: 0.88 g TRIZ

hydrochlorid powder + 1.75 TRIZ base powder (Sigma)] were added.


63

3. This mixture was put in a 50°C water bath and stirred until the components

had been dissolved

4. The solution was filled into an Eppendorf tube and centrifuged for 15 minutes

at 5000 G, in order to remove air bubbles.

5. Aliquots of Mowiol were frozen at -18°C until use.

The procedure until the incubation with the secondary antibody was the same as

described under 3.9.2.1 and 3.9.2.2, after removing excess antibody through

washing in PBS, the next step followed:

Dilution of the secondary FITC labelled antibody 1:200 with PBS

Incubation over 40 minutes at room temperature

Rinsing in PBS once, followed by 3x5 minutes PBS

Mounting with Mowiol

During incubation and rinsing, the wet chamber and cuvettes were covered with

aluminium foil to avoid exposure to light.

Tab. 3.4: Overview of secondary detection systems AB: Antibody, FITC: Fluorescein isothiocyanate, HRP: Horseradish peroxidase

Labelled second. antibody or

enzyme

Origin and species specificity

Method Vendor

Second. AB labelled with a HRP conj. to a

dextran polymer

Goat-anti mouse or rabbit

Two step indirect method

DAKO Hamburg, Germany

Secondary AB labelled with peroxidase

Rabbit or horse- anti goat


Vector

Biotinylated secondary AB

HRP marked streptavidin

Multi Link® against mouse, rabbit, guinea

pig and rat

Three step method: Labelled

StreptAvidin-Biotin method (LSAB)

BioGenex San Remo

USA

FITC labelled secondary AB

Horse- anti mouse or rabbit or

Rabbit- anti goat


Vector


64

3.9.3 Immunohistochemistry: General procedure

Before starting any reaction on the paraffin embedded material, deparaffinisation and

rehydration needs to be accomplished as described under 3.7.1 / Tab. 3.5. The

blocking of the endogenous peroxidase activity was already described under 3.9.2.

During fixation, especially in formalin and Bouin-fixed tissue, it occurs that protein-

cross links are formed and methyl-bridges are accumulated during the formaldehyde

reaction with amino acids. This fact might result in a reduced immuno reactivity of the

protein (epitope masking). To decrease the amount of protein cross links, an epitope

demasking might be necessary to avoid wrong negative reactions. Epitope

demasking can also be called antigen retrieval and several techniques can be

applied. In our study, four different types of pre-treatments were tested until the

suitable method for the respective antibody was established.

To reduce background staining due to hydrophobic interactions between the antibody

and tissue, normal serum was put on the slides over 20 minutes before the

incubation with the primary antibody. Proteins of the normal serum bind the various

charges of the tissue, and thus the following primary antibody is able to react

specifically with the tissue. We used normal goat (NGS), normal rabbit (NRS) and

normal horse serum (NHS), depending on the origin of the secondary antibody.

Before use, the normal serum was inactivated in a 56°C water bath for 30 minutes. In

order to avoid background staining, due to the proteins in the normal serum, the

normal serum was diluted 1:500 with PBS (500 µl normal serum + 2000 µl PBS).

According the same reason, the primary antibody was diluted with 1% bovine serum

albumin (BSA) (Sigma).

The tissue was incubated with the diluted primary antibody over night at 4°C to

ensure a good linkage of the primary antibody. Afterwards the reaction was

visualised applying the different detection systems (3.9.2).

To give an overview of the general approach of immunohistochemistry, the

procedure is described for Ca-formol and Bouin-fixed material in comparison to

HOPE® in Tab. 3.5.


65

Tab 3.5: General immunohistochemistry protocol, comparison of fixation techniques Abs.: Absolute, AR: Antigen retrieval, min: Minutes, IF: immunofluorescence, NHS: Normal horse serum, NGS: Normal goat serum, NRS: Normal rabbit serum, PBS: Phosphate buffered saline, RT: room temperature, TEC: Tris-EDTA-citrate buffer

Step Formol-Ca and Bouin-

fixed tissue HOPE® fixed tissue

Deparaffinisation 2 x10 min. xylene 2 min. isopropanol

2x10 min. 60°C isopropanol

2x3 min 60°C isopropanol

Rehydration + blocking endogenous peroxidase

2 min. ethanol 30 min. 80%

alcohol+H2O2 2 min. 70 % alcohol

2x10 min. 4°C acetone

Washing 3x5 min. PBS 2x3 min. de-ionised water 5 min. de-ionised water

Demasking

TEC-buffer Citrat buffer AR buffer (DAKO) Trypsin

Blocking endogenous peroxidase

10 min. de-ionised water+ H2O2

Washing Short rinse in PBS 3x5 min PBS Blocking electrostatic binding sites

20 min NGS, NRS or NHS

20 min NGS, NRS or NHS

Incubation of primary antibody

Over night at 4°C in suitable dilution

Over night at 4 °C in suitable dilution

Washing 3x5 min. PBS 3x5 min. PBS

Visualisation EnVision® BioGenex® IF

EnVision® BioGenex® IF

Washing

For IF: 1x rinse in PBS, 3x5 min PBS

Others: 5 min PBS, 10 min. running water

For IF: 1x rinse in PBS, 3x5 min PBS

Others: 5 min PBS, 2x10 min PBS

Rehydration

Only necessary for EnVision® and BioGenex®

2 min. 70% alcohol 2 min. 80% alcohol 2 min. abs. alcohol 2 min. isopropanol 2x5 min. xylene

Only necessary for EnVision® and BioGenex® 2x rinse in 70%

isopropanol 2x rinse in ethanol

(abs.) 10 min ethanol (abs.) rinse in xylene 5 min. xylene

Mounting

For EnVision® and BioGenex®:

Eukitt® (Kindler) For IF: Mowiol

For EnVision® and BioGenex®: Eukitt® (Kindler)

For IF: Mowiol


66

3.9.4 Controls

For all immunohistochemical experiments positive, negative and isotype controls

were attendantly conducted on all animal species studied.

As references for the tissues needed as a positive control the antibody datasheets of

the vendor were used. Positive controls were conducted if the reference tissue

needed was available. Thus, the following positive control tissues were applied:

Murine skin (langerin), equine skin (CD1a, hBD-2 and -3) and equine liver (MBL and

L-ficolin).

To conduct the negative controls, the first antibody was omitted and instead the

tissue was incubated with PBS / 1% BSA.

In order to exclude the possibility of non-specific binding of the Fc-part of the

respective primary antibody, isotype controls were conducted. Therefore, the primary

antibody was omitted and replaced by a IgG mouse (Sigma) or rabbit (Sigma)

antibody depending on the origin of the first antibody. The respective protein

concentrations of the antibodies were considered. This means, that the IgG mouse or

rabbit antibody was applied in the same protein concentration, as the primary

antibody tested.

3.9.5 Preliminary tests for the establishment of the primary antibodies

For the primary antibodies listed in Tab. 3.3, initially immunohistochemical

preliminary tests were conducted. Therefore various demasking methods and

different antibody dilutions were tested and controls were accomplished. The first

preliminary tests on the animal esophagi were conducted on pig, mouse / rat, horse,

cat and goat tissue, in order to get an impression of the reactivity within the different

nutrition types. If the suitable antibody had been established, the

immunohistochemical experiments were performed for all species and esophagi

collected. If it was not possible to establish an antibody during the first preliminary

tests for the species mentioned, the same tests were applied on the sheep, dog and

rabbit esophagus. In the next step, different pre-treatments and visualisation systems

were considered, as described in Tabs. 3.6 and 3.7.


67

3.9.5.1 Demasking methods tested

Basically two types of antigen retrieval techniques can be distinguished: heat induced

epitope retrieval and proteolyse induced epitop retrieval. In our study we tested

trypsin as proteolyse induced epitope retrieval, whereas the others ones were heat

induced.

Trypsin:

200 ml distilled water were heated in a warming cupboard until it had a temperature

of approximately 37°C. Following 0.2 g of trypsin (Sigma) and 0.2 g calcium-chloride

powder (Merck) were added, and the pH was adjusted to 7.6 with diluted NaOH

(Merck). The solution was transferred to a warm cuvette and the slides were

incubated for 30 minutes in a warming cupboard at 37°C.

Heating pre-treatment with citrate buffer:

To use citrate buffer as a pre-treatment, two stock solutions had to be prepared. The

first one (stock solution A) comprised 0.1 M citric acid. To prepare 0.1 M citric acid,

21g citric acid powder (Riedel-de-Haen) were added to 1000 ml de-ionised water. For

the second stock solution (stock solution B), 0.1 M sodium citrate was prepared. In

this case 29.4 g sodium citrate powder was mixed in 1000 ml de-ionised water.

In the following step, 18 ml of stock solution A were added to 82 ml of stock solution

B and mixed with 1000 ml de-ionised water (pH 6).

Finally the solution was heated to 96-98°C and the slides were incubated in the

solution for 30 minutes.

Heating pre-treatment with Tris-EDTA-citrate buffer (TEC):

900 ml of distilled water and 100 ml TEC stock solution (composition below) were

filled in a beaker. A magnetic stir bar and an empty slide holder were put into the

solution. In the next step, the beaker was put on a heating plate and under constant

stirring the solution was heated up to 96°C. Following, the slide holder with the slides

was placed into the hot solution. The slides were left in the solution for 30 minutes at

96-98°C.

TEC stock solution:

1. 2.5 g Tris-base (MW 121.1) (Sigma)

2. 5.0 g EDTA (Riedel-de-Haen)

3. 3.2 g Tri-Natrium-Citrat (Merck)


68

4. dissolve in 800 ml de-ionised water

5. adjust to pH 7.8 with NaOH

fill up to 1000 ml with de-ionised water

Heating treatment with DAKO target retrieval solution (TRS):

The TRS from DAKO was tested with pH 6 and pH 9 (TRIS EDTA base). 7 ml of the

TRS stock solution pH 6 or 9 were mixed with 63 ml distilled water (1:10). This

solution was filled into a beaker and heated under constant stirring to 96°C. Than the

slides were put into the hot solution and heated at 96-98°C. It was important to keep

attention to heating time, as the pre-treatment of the slides in the pH 9 solution

should not exceed 20 minutes. Slides in the pH 6 solution were pre-treated for 30

minutes. Finally, a cooling down period of maximum 15 minutes followed.


69

Tab. 3.6: Tested antibody dilutions, demasking methods and visualising systems for Bouin and formol-fixed material. VS: Visualising system, 1: DAKO EnVision rabbit (two-layer method), 2: DAKO EnVision mouse, 3: DAB as pure substance, 4: BioGenex (LSAB), 5: Immunofluorescence, AB: antibody, TEC: Tris-EDTA buffer

Antibody Antibody dilution, tested demasking methods and visualising systems ß-defensin 2 1:50, 1:100, 1:250, 1:350,

1:450 no pre-treatment, VS: 1

1:250, 1:350 TEC-buffer VS: 1

ß-defensin 3 1:50, 1:100, 1.250, 1:500, 1:1000, 1:1200, 1:1500 no pre-treatment, VS: 1

1:1000, 1:1200, 1:1500 TEC-buffer, VS: 1

Cathelicidin 1:10, 1:20, 1:30, 1:40, 1:50no pre-treatment, VS: 2

1:20, 1:30 TEC-buffer, VS: 2

CD1a 1:10, 1:20, 1:50 no pre-treatment, VS: 2,4 + 5

1:10, 1:20 TEC-buffer, VS: 2, 4 +5

1:10, 1:20 Trypsin VS: 2+4

1:10, 1:20 DAKO TRS pH 6+9 VS: 2+4

Dectin-1 1st AB: 1:10, 1:20 2nd AB: 1:200 No pre-treatment, VS: 3+4

1st AB:1:10, 1:20 2nd AB: 1:200 TEC- buffer , VS: 3+5

1st AB: 1:10, 1:20 2nd AB: 1.200 Trypsin, VS: 3

1:10, 1:20, DAKO TRS pH 6+9 VS: 3+5

1:10, 1:20 citrate buffer VS: 3

Langerin 1:10, 1:20 No pretreatmentVS: 1

1:10, 1:20 TEC-buffer VS: 1

L-Ficolin 1:10, 1:20 No pre-treatment, VS: 2,4+5

1:10, 1:20 TEC-buffer, VS: 2,4+5

1:10, 1:20 Trypsin, VS: 2

1:10, 1:20 DAKO TRS pH 6+9 VS: 2,4+5

1:10, 1:20 citrate buffer, VS: 2

Mannose-binding-lectine

1:10, 1:20 No pe-treatment VS: 2, 4+5

1:10, 1:20 TEC-buffer VS: 2,4+5

1:10, 1:20 Trypsin VS: 2

1:10, 1:20 DAKO TRS pH 6+9 VS: 2+5

1:10, 1:20 Citratebuffer VS: 2

Toll-like-receptor 2 1:10, 1:20 No pre-treatment VS: 2

1:10, 1:20 TEC-buffer VS: 2

1:10, 1:20 Trypsin VS: 2

Lysozyme 1:50, 1:75, 1:100 No pretreatment, VS: 1

1:50, 1:75, 1:100 TEC-buffer ,VS: 1

1:50, 1:75, 1:100 Trypsin, VS: 1


70

Tab.3.7.: Overview of tested antibody dilutions and visualisation systems on HOPE® fixed tissue VS: Visualisation system, VS 1: DAKO EnVision® rabbit (two-layer method), VS 2: DAKO EnVision® mouse, VS 3: DAB as pure substance, VS 4: BioGenex (LSAB), VS 5: Immunofluorescence

Antibody Antibody dilutions, visualisation systems ß-defensin 2 1:350, 1:450, 1:700, 1:1000, 1:1500

VS: 1+4

ß-defensin 3 1:1200, 1:1800, 1:2000, 1:3000, 1:4000 VS: 1+4

Cathelicidin 1:50, 1:150, 1:200, 1:300 VS: 2

CD1a 1:10, 1:50 VS: 2

1:10 VS: 5

Dectin-1 1:10, 1:50 VS: 3

1:10 VS: 5

L-Ficolin 1:10, 1:20, 1:100 VS: 2

1:10 VS: 5

Mannose-binding-lectine 1:10, 1:20 VS: 2

1:10 VS: 5

Toll-like-receptor 2 1:10, 1:20, 1:50 VS: 2

Lysozyme 1:200, 1:300, 1:400, 1:500, 1:1000, 1:2000, 1:3000, 1:5000 VS: 1


71

3.9.6 Antibody dilutions and visualisation systems applied

After conducting the preliminary tests it was decided which dilution of the

primary antibody seemed to ensure the best results (compare Tab. 3.8), and

also the advantages or disadvantages of a pre-treatment were considered.

Furthermore, the different results of the visualisation systems were compared

and it was decided which system had to be the suitable one.

As some primary antibodies could not be established in an acceptable way,

experiments were accomplished on the whole animal material only for the

established antibodies. Due to the fact that the reactivity of the established

primary antibodies for lysozyme, ß defensin-2 and -3, cathelicidin and TLR2

could not be improved through application of a pre-treatment, it was decided to

conduct the experiments without pre-treatments.

The two layer visualisation system EnVision® rabbit and EnVision® mouse from

DAKO seemed to be the most suitable visualisation system, because with the

three layer technique of BioGenex, colour intensity was not satisfactory and

false positive reactions on HOPE® fixed material could be observed.

Tab. 3.8: Antibody dilutions applied for established antibodies Dilution with PBS+1% BSA

Antibody Dilution for Bouin and formol-fixed tissue

Dilution for HOPE® fixed tissue

ß defensin-2 1:350 1:1500

ß defensin-3 1:1200 1:1200

Cathelicidin 1:30 1:150

Toll-like receptor 2 1:10 1:20

Langerin (CD207) 1:20 Not conducted

Lysozyme 1:50 1:2000


72

3.9.7 Statistical analyses

To describe the immunohistochemical results, a semiquantitative scale was

used. The staining intensity was classified using six levels (0: no reaction, 0.5:

very weak reaction, 1: weak reaction, 1.5: mild reaction, 2: medium reaction,

2.5: strong reaction, 3.0: very strong reaction).

For the evaluation of the statistical significance we used the computer program

SAS. Basically two different statistical tests were applied. For analysing the

results found for the various substances and receptors in the three fixation

solutions (Bouin`s solution, Ca-formol, HOPE®), the signed rank test was

applied, as it was considered that the results found did not show normal

distribution. The three different fixation solutions were compared with each

other, considering each of the five layers of the esophagus epithelium. All data

obtained from the experiments were pooled, considering the species and

fixation solution applied. The results were expressed as standard error of mean,

and values of p < 0.05 were regarded as significant difference.

The comparison of the nutrition groups was accomplished applying the

Wilcoxon test. All species were allocated to one nutrition group (herbivorous,

carnivorous, or omnivorous). Regarding all five layers of the epithelium, the

results of the different groups were compared with each other. P-values < 0.05

demonstrated a statistical significant difference between the nutrition groups.

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73

4 Results

4.1 Light and electron microscopical observations

4.1.1 Demonstration of microorganisms with cryo SEM

The demonstration of a microbial occupation of the surface of the esophagus

epithelium was realised using cryo SEM. The pictures revealed a one layer

colonisation of the epithelium with bacteria and fungi in each species studied

(Figs. 4.1 to 4.7). The species considered were: Horse, cattle, goat, pig, rat,

dog and cat. Species related differences in the density of microorganisms found

could not be observed.

Fig 4.1 Microorganisms on the esophagus epithelium of the horse Predominantly bacteria (fungus marked as: ►, bacteria marked as: →)

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Fig. 4.2: Microorganisms on the esophagus epithelium of the goat High magnification. Larger balls are fungi and smaller ones are bacteria. Star like structures are ice crystals due to sublimation processes during cryo SEM.

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Fig. 4.4: Microorganisms on the esophagus epithelium of the rat, surrounding an excretory duct of a secretory gland Field of bacteria is marked by: →, a fungus is marked by: ►

Fig 4.5: Microorganisms and mucus covering the esophagus epithelium of the pig

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76

Fig. 4.6: Microorganisms and mucus covering the esophagus epithelium of the dog Bacterium is marked by: →

Fig. 4.7: Bacteria and fungi without mucus covering on the esophagus epithelium of the cat Bacterium is marked by a: → and field of fungi is marked by a: ►

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4.1.2 General esophageal structure

In order to compare the structural composition of the esophagus epithelium, all

tissue samples collected were stained with H.E. and Masson-Goldner trichrome

stain. The procedure was the same for all fixation media applied. The HOPE®

fixed samples were only stained in H.E.. The protocol applied slightly differed

from the one used for samples fixed in Ca-formol and Bouin. Before IHC

experiments were started, the integrity of the tissue was evaluated. The best

results regarding structure preservation were obtained from the Bouin-fixed

samples. Almost all samples revealed very good tissue integrity. The HOPE®

fixed samples exhibited a slightly diminished quality of structure preservation. A

separation of the lamina epithelialis and the lamina propia could be observed in

some of these samples. Additionally, in some cases the epithelium rolled up. In

a number of the Ca-formol fixed esophagus samples, tissue preservation was

relatively bad, whereby the different layers (lamina epithelialis, lamina propia

and Tunica muscularis) appeared as completely separated from each other.

The structure of the esophagus epithelium of the species studied was mainly

evaluated using semithin sections stained with toluidine blue. In this connection,

differences between the three nutrition groups could be demonstrated. The

epithelium of the herbivorous species was more prominent, in comparison to

that one of the omnivorous and carnivorous species studied. This fact

concerned all layers, but was most obvious in the stratum corneum. While the

carnivorous cat exhibited a very thin stratum corneum, better named stratum

superficiale, as almost no keratinisation occured in this species, the stratum

corneum of the herbivorous animals was thicker. Furthermore, the corneocytes

of the latter seemed to be organised in dense layers. This structural feature was

most obvious in the equine esophagus epithelium. The stratum corneum of the

omnivorous pig was even more prominent, compared to conditions in the cat. In

contrast to the stratum corneum of the herbivorous species, the pig did not

show such a dense organisation.

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Fig. 4.8: Histological structure of the esophagus of the herbivorous horse (left) and the omnivorous dog (right) The LS of the canine esophagus contains seromucous glands, Staining: Masson Goldner, TS: Tunica serosa, TM: Tunica muscularis, LS: Lamina submucosa, LP: Lamina propia, LE: Lamina epithelialis

Fig. 4.9: Histological overview of the esophagus of the carnivorous cat Staining: Masson Goldner, TS: Tunica serosa, TM: Tunica muscularis, LS: Lamina submucosa, LP: Lamina propia, LE: Lamina epithelialis

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Fig. 4.10: Semithin sections of the esophagus epithelium of the herbivorous horse (left) and sheep (right) The layers of the stratum corneum exhibit a clearly lamellar structure Staining: Toluidine blue, SB: Stratum basale, SS: Stratum spinosum, SG: Stratum granulosum, SC: Stratum corneum

Fig. 4.11: Semithin section of the esophagus epithelium of the omnivorous mouse Macrophages can be seen in the Lamina propia, Staining: Toluidine blue, SB: Stratum basale, SG: Stratum granulosum, SC: Stratum corneum

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Fig. 4.12: Semithin section of the esophagus epithelium of the carnivorous cat Keep attention to the rather loose structure of the SC Staining: Toluidine blue, SB: Stratum basale, SS: Stratum spinosum, SG: Stratum granulosum, SC: Stratum corneum

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4.2 Fixation experiment: Influences of different fixation

media on IHC results

4.2.1 ß-defensin 2 and -3 reactions

Human ß defensins-2 (hBD-2) and human ß defensins-3 (hBD-3) are both

members of the group of antimicrobial peptides, and thus will be discussed

together.

Concerning hBD-2, for the samples fixed in Bouin`s solution and Ca-formol

equal dilutions of the primary antibody were used (hBD-2: 1:350). On the

contrary, higher dilutions were applied for the HOPE® fixed samples, as such

samples exhibited too strong reaction intensities with lower dilutions (hBD-2:

1:1500). Regarding hBD-3, equal antibody dilutions for all three fixation media

were used.

The comparison of the expression patterns of the two hBD types within the

lamina epithelialis of the esophagus revealed several differences as related to

the different fixation solutions. Those differences are shown in detail in Tabs.

4.1 and 4.2, and Figs. 4.13 and 4.14. A statistically relevant variation of the

staining intensity for hBD-2 could be observed primarily in the horse and the

sheep. Intriguingly, the comparison of the results found for Bouin and Ca-formol

fixed samples in the equine esophagus showed significant differences regarding

all layers (p-value < 0.05), except the stratum corneum (Fig. 4.13). Whereas in

the Ca-formol fixed tissue normally no positive reaction was observable in the

layers, an obviously positive reaction could be shown for the Bouin-fixed

samples in all layers. These findings were confirmed for hBD-3 (p-value < 0.05),

apart from the fact that the stratum basale showed a very weak positive reaction

in the Ca-formol fixed samples. Comparing the results for hBD-2 of the Bouin

and HOPE® fixed samples in the equine esophagus revealed a statistically

significant difference for the stratum spinosum (lower and upper layer) (p-value

< 0.05); again the Bouin samples exhibited a stronger reaction. The same fact

was noticed for hBD-3, besides a difference demonstrated in the stratum basale

and granulosum (p-value < 0.05). Concerning the sheep, a statistically relevant

difference for hBD-2 could be demonstrated comparing the results of the Bouin

and Ca-formol fixation as well as comparing the Bouin and HOPE® fixation (p-

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82

value < 0.05) (Fig. 4.14). Interestingly, for both comparisons a difference was

shown for the same layers (stratum corneum, stratum granulosum, stratum

basale). Again, the HOPE® fixed samples exhibited a weaker reaction than the

Bouin samples. The results of hBD-3 expression in the sheep comparing Bouin

and Ca-Formol fixed samples equalled the ones described for hBD-2. It is a

remarkable finding, that statistically significant differences observed in the horse

and the sheep could be corroborated for hBD-2 and -3 in the same

localisations. The same finding was made analysing the IHC results of the

porcine esophagus. In this regard, a reaction difference for hBD-2 and -3 was

found for the stratum basale and the str. granulosum (p-value < 0.05)

The reaction comparison between the Ca-formol and HOPE® fixed samples

revealed no statistically relevant differences (p-values > 0.05).

SB SS (l) SS (u) SG0

1

2

3

Ca-FormolBouin`s solution

HOPE

* **

** *

Sta

inin

g in

ten

sit

ies

Fig. 4.13: Demonstration of fixation differences for hBD-2 in the equine esophagus. Shown data: Mean and standard deviation. Applied antibody dilutions for the primary antibody: Bouin`s solution and Ca-Formol: 1:350, HOPE®: 1:1200 (SB: Stratum basale, SS (l): lower Stratum spinosum, SS (u): upper Stratum spinosum, SG: Stratum granulosum, *: p-value < 0.05)

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SB SG SC0

1

2

3

4

Ca-Formol

Bouin`s solution

HOPE*

*

**

**

Sta

inin

g i

nte

ns

itie

s

Fig. 4.14:Demonstration of fixation differences for hBD-2 in the ovine esophagus. Shown data: Mean and standard deviation. Applied antibody dilutions for the primary antibody: Bouin`s solution and Ca-Formol: 1:350, HOPE®: 1:1200) (SB: Stratum basale, SG: Stratum granulosum, SC: Stratum corneum, *: p-value < 0.05)

Tab. 4.1: IHC results of the ß-defensins 2 reactions according to the different fixation solutions (B= Bouin’s solution, F= Ca-formol; H= HOPE®, l= lower, u= upper, n.d.= not determined; Reaction intensities: 0.5 = very weak, 1 = weak, 1.5 = mild, 2 =medium, 2.5 = strong, 3 = very strong)

Tab 4.2: IHC results of the ß-defensins 3 reactions according to the different fixation solutions (B= Bouin’s solution, F= Ca-formol; H= HOPE®, l= lower, u= upper, n.d.= not determined; Reaction intensities: 0.5 = very weak, 1 = weak, 1.5 = mild, 2 =medium, 2.5 = strong, 3 = very strong)

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Layers Species

Str. corneum Str. granulosum Str. spinosum (u) Str. spinosum (l) Str. basale

B 0,5 B 1,5 B 1,5 B 2 B 2 F 0 F 0 F 0 F 0 F 0 Horse

H 0 H 1 H 0,5 H 0,5 H 1 B 1,5 B 0 B 0,5 B 0,5 B 2 F 1 F 0 F 0 F 0 F 0,5 Cattle

H 0,5 H 0,5 H 1 H 1 H 1 B 2 B 2,5 B 2 B 2 B 2,5

F 0,5 F 0 F 0 F 0,5 F 0,5 Goat

H 1 H 2,5 H 2 H 1 H 1 B 2 B 2,5 B 0,5 B 0,5 B 2 F 0 F 0 F 0 F 0 F 0

Herbivorous

Sheep

H 0 H 0 H 0 H 0 H 0 B 0,5 B 1,5 B 0 B 0 B 2 F 1,5 F 0 F 0 F 0 F 0,5 Pig H 0 H 1 H 1 H 1,5 H 1

B 2,5 B 0,5 B 1 B 2 B 2,5 F 2 F 0 F 0 F 0 F 0,5 Dog H 0 H 1 H 1 H 1 H 0 B 1 B 1 B 0 B 0,5 B 1,5 F 0 F 0 F 0 F 0 F 0 Mouse H 1 H 1,5 H 1,5 H 1,5 H 1 B 1 B 3 B 2 B 2 B 2,5 F 1 F 1 F 0 F 0 F 0,5

Omnivorous

Rat n.d. n.d n.d n.d n.d. B 3 B 0 B 0 B 1 B 2 F 2 F 0 F 0 F 0 F 1 Carnivorous Cat H 1 H 1 H 1 H 2 H 0,5

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Layers Species

Str. corneum Str. granulosum Str. spinosum (u) Str. spinosum (l) Str. basale

B 0 B 0,5 B 2 B 2 B 2 F 0 F 0 F 0 F 0 F 0,5 Horse

H 0 H 1 H 0,5 H 0 H 0,5 B 1,5 B 1,5 B 0 B 1 B 2 F 1 F 0 F 0 F 0 F 0 Cattle

H 0 H 1 H 1 H 1 H 1 B 1 B 0 B 1 B 1,5 B 2 F 0 F 0 F 0 F 0 F 0 Goat

H 2 H 2 H 1,5 H 1 H 0,5 B 1 B 2 B 0,5 B 0 B 1,5 F 0 F 0 F 0 F 0 F 0

Herbivorous

Sheep

H 0 H 0,5 H 1 H 0,5 H 0,5 B 0 B 1 B 0 B 0 B 1,5 F 1 F 0 F 0 F 0 F 0 Pig H 1 H 1,5 H 0,5 H 1 H 0,5 B 1 B 0,5 B 1 B 2 B 2,5 F 0 F 0,5 F 0 F 0 F 0 Dog H 0 H 3 H 2,5 H 1,5 H 0 B 1 B 0,5 B 0 B 1 B 1,5

F 0,5 F 0 F 0 F 0 F 0,5 Mouse H 2 H 1 H 0,5 H 0,5 H 0,5

B 2,5 B 0,5 B 1 B 1 B 2 F 2 F 0 F 0 F 0 F 1

Omnivorous

Rat n.d. n.d n.d n.d n.d.

B 1,5 B 1 B 1 B 2 B 3 F 0 F 0 F 0 F 0 F 0 Carnivorous Cat H 1 H 1 H 0,5 H 0 H 0

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4.2.2 Cathelicidin reactions

The third member of the group of antimicrobial peptides analysed in this

dissertation was CAT. In this case the following antibody dilutions were used:

1:30 for Bouin and Ca-formol fixed samples and 1:150 for the HOPE® fixed

samples (compare Tab. 3.8), as the staining intensity in the latter was too

intensive in a lower dilution.

Comparable to the results described for the ß defensins, statistically relevant

differences again were observed for the equine, ovine and porcine esophagus

epithelium (compare Tab. 4.3). The differences found for the ovine tissue were

limited to the stratum basale and stratum granulosum (p-value < 0.05). It should

be emphasised, that these findings were also made comparing the Bouin

fixation with the Ca-formol fixation, and comparing Bouin with the HOPE®

fixation. In both combinations, the results varied at the same location.

Furthermore, these results confirmed those ones described for hBD-2 and -3.

The differences mentioned resulted from the fact that in the Ca-formol and

HOPE® fixed sections almost no positive IHC reaction could be observed.

The only statistically significant difference between the fixation methods in the

horse was detected comparing Bouin fixation with Ca-formol fixation, and

concerning the lower stratum basale (p-value < 0.05). The statistical analysis of

the results for the Bouin and HOPE® fixed sections of the porcine esophagus

only revealed a difference for the stratum basale (p-value < 0.05).

The comparison of the Ca-formol and HOPE® results revealed no statistical

disparity (p-values > 0.05).

Tab 4.3: IHC results of the CAT reactions according to the different fixation solutions (B= Bouin’s solution, F= Ca-formol; H= HOPE®, l= lower, u= upper, n.d.= not determined; Reaction intensities: 0.5 = very weak, 1 = weak, 1.5 = mild, 2 = medium, 2.5 = strong, 3 = very strong)

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Layers Species

Str. corneum Str. granulosum Str. spinosum (u) Str. spinosum (l) Stratum basale

B 0 B 1 B 1 B 1 B 2,5 F 0 F 1 F 1 F 0 F 2 Horse

H 0 H 1 H 1 H 1 H 2 B 0 B 0 B 0 B 0,5 B 2 F 0 F 0 F 0 F 0 F 0,5 Cattle

H 0 H 0,5 H 1 H 1 H 1,5 B 0 B 0,5 B 1 B 1 B 2 F 0 F 0 F 0 F 1 F 1,5 Goat

H 0 H 3 H 1,5 H 2,5 H 2,5 B 0 B 1,5 B 0,5 B 0,5 B 2 F 0 F 0 F 0 F 0 F 0

Herbivorous

Sheep

H 0 H 0 H 0 H 0,5 H 0 B 0 B 0,5 B 0 B 0 B 3 F 0 F 0 F 0 F 0 F 0,5 Pig H 0 H 1,5 H 1 H 2 F 2

B 0,5 B 0,5 B 1,5 B 1,5 B 2,5 F 0,5 F 0 F 0,5 F 0 F 0 Dog H 0 H 2 H 2,5 H 2 H 3 B 1 B 1,5 B 1,5 B 2 B 2 F 0 F 0 F 0 F 0,5 F 0,5

Omnivorous

Rat n.d. n.d n.d n.d n.d. B 0 B 0,5 B 1 B 1 B 2 F 0 F 0 F 0 F 1 F 1,5 Carnivorous Cat H 0 H 2 H 2,5 H 2 H 2

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4.2.3 Toll-like receptor 2 reactions

Interestingly, differences according to the fixation solutions used were found for

the equine and ovine esophagus epithelium. These findings are comparable to

those described for the APs. In the case of TLR2, statistically significant

differences for the equine esophagus were identified comparing Bouin fixation

with Ca-formol and HOPE® fixations (compare Tab. 4.4 and Fig. 4.15). It is

important to mention, that the HOPE® sections were incubated with an antibody

diluted 1:20, and for the ones of Bouin and Ca-formol fixed tissue a dilution of

1:10 was applied. Such differences were obvious in the stratum basale and the

stratum spinosum, comparing Bouin and Ca-formol fixation (p-value < 0.05).

Bouin-fixed samples exhibited a stronger staining intensity (Fig. 4.15). The

same differences were observed, comparing Bouin with the HOPE® fixation. In

this case, additionally, a significant difference was observed for the stratum

granulosum of the equine esophagus (p-value < 0.05). Reaction staining

conducted on Bouin-fixed tissue produced the strongest staining intensity in all

layers, except the stratum granulosum (compare Tab. 4.4). In the latter layer,

the HOPE® fixation method resulted in a stronger staining. In contrast to the

comparison of Bouin fixation with Ca-formol and HOPE® fixation, the

comparison of Ca-formol with HOPE® fixation only rarely showed relevant

differences for the substance studied. Regarding TLR2, a difference could be

observed for the equine esophagus. This difference was evident in the stratum

granulosum (p-value < 0.05). Intringuingly, in this case HOPE® fixed samples

showed the stronger reaction intensity.

In the ovine esophagus epithelium, a difference was apparent in the stratum

basale comparing Bouin fixation with Ca-formol and HOPE® fixation. These

observations are comparable to those for the APs studied (compare 4.2.1 and

4.2.2).

Additionally, statistically significant differences were identified in the stratum

basale of the caprine and canine esophagus. Regarding the fixation media, the

latter observations were made in the canine esophagus comparing the Bouin-

fixed samples with the Ca-formol fixed ones. The samples fixed in Bouin`s

solution exhibited the stronger reaction. In the caprine esophagus, the

difference was noticeable comparing the Bouin and HOPE ® fixation. Again the

Bouin-fixed samples revealed the stronger staining intensity.

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89

SB SS (l) SS (u) SG0

1

2

3

Ca-Formol

Bouin`s solution

HOPE

**

**

**

**

Sta

inin

g i

nte

nsi

ties

Fig 4.15: Demonstration of fixation differences for TLR2 in the equine esophagus. Shown data: Mean and standard deviation. Applied antibody dilutions for the primary antibody: Bouin`s solution and Ca-Formol: 1:10, HOPE®: 1:20) (SB: Stratum basale, SS (l): lower Stratum spinosum, SS (u): upper Stratum spinosum, SG: Stratum granulosum, *: p-value < 0.05)

Tab 4.4: IHC results for Toll like receptor 2 according to the different fixation solutions (B= Bouin’s solution, F= Ca-formol; H= HOPE®, l= lower, u= upper, n.d.= not determined; reaction intensities: 0.5 = very weak, 1 = weak, 1.5 = mild, 2 = medium, 2.5 = strong, 3 = very strong)

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90

Layers Species

Str. corneum Str. granulosum Str. spinosum (u) Str. spinosum (l) Stratum basale

B 0 B 0 B 2 B 2,5 B 2,5 F 0 F 0 F 0,5 F 0,5 F 0,5 Horse

H 0 H 2 H 0,5 H 0 H 0,5 B 0 B 0 B 0,5 B 1 B 2 F 0 F 0 F 0 F 0 F 0 Cattle

H 0 H 1 H 2 H 1 H 0,5 B 0 B 0 B 1 B 1 B 2 F 1 F 0 F 0 F 0,5 F 0,5 Goat

H 0 H 2 H 0 H 0 H 0 B 0 B 0 B 0 B 1,5 B 2 F 0 F 0 F 0 F 0,5 F 0,5

Herbivorous

Sheep

H 0 H 0,5 H 1 H 0,5 H 0,5 B 0 B 1 B 0 B 0,5 B 1 F 1 F 0 F 0 F 0 F 1 Pig H 0 H 1 H 0,5 H 0 F 0 B 0 B 0 B 0 B 1 B 2 F 0 F 0 F 0 F 0 F 0 Dog H 0 H 1 H 0,5 H 0,5 H 0,5 B 0 B 0 B 0,5 B 1 B 2 F 0 F 0 F 0 F 0 F 1

Omnivorous

Rat n.d. n.d n.d n.d n.d. B 0 B 0 B 1 B 1 B 2 F 0 F 0 F 0 F 0 F 0,5 Carnivorous Cat H 0 H 1 H 0,5 H 0,5 H 0,5

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4.2.4 Lysozyme reactions

The only difference between the fixation solutions used was found for the

equine esophagus (Tab. 4.5). The lysozyme antibody for the Ca-formol and

Bouin fixed samples was applied in a 1:50 dilution and for the HOPE® fixed

ones in a 1:2000 one. In contrast to most of the other substances studied, a

difference was also identified comparing the Ca-formol fixation with the HOPE®

fixation. In this case, a disparity appeared in the upper stratum spinosum (p-

value < 0.05). Surprisingly, the samples fixed in Ca-formol exhibited the

stronger reactions. The comparison of Bouin and HOPE® revealed a difference

between the stratum basale and lower stratum spinosum (p-values < 0.05).

Again, the staining intensity in the HOPE® fixed samples was weaker. For the

Bouin - Ca-formol comparison, a difference was only shown for the stratum

basale (p-value < 0.05), where the Bouin-fixed tissue revealed stronger

reactions. The other species studied revealed no disparities (p-values > 0.05).

Tab. 4.5: IHC results for lysozyme according to the different fixation solutions (B= Bouin’s solution, F= Ca-formol; H= HOPE®, l= lower, u= upper, n.d.= not determined, reaction intensities: 0.5 = very weak, 1 = weak, 1.5 = mild, 2 = medium, 2.5 = strong, 3 = very strong)

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Layers Species

Str. corneum Str. granulosum Str. spinosum (u) Str. spinosum (l) Str. basale B 0,5 B 1,5 B 1,5 B 1,5 B 2,5 F 0 F 2 F 2 F 1 F 1 Horse

H 0 H 1,5 H 0,5 H 0 H 1 B 0 B 0 B 0 B 1 B 2,5 F 2 F 0,5 F 1,5 F 2 F 1 Cattle

H 0 H 0,5 H 1 H 0,5 H 0,5 B 0 B 1 B 1 B 1 B 1

F 0,5 F 0,5 F 0 F 0 F 0,5 Goat

H 2 H 2 H 0 H 0 H 2 B 0 B 2 B 0 B 0,5 B 1,5 F 1 F 1 F 0,5 F 0,5 F 1

Herbivorous

Sheep

H 0 H 2 H 0,5 H 1 H 1 B 1 B 1 B 0 B 0 B 2

F 0,5 F 0 F 0 F 0,5 F 0,5 Pig H 1 H 1,5 H 1 H 0 H 2 B 0 B 0,5 B 0,5 B 1,5 B 2 F 1 F 1 F 1 F 0 F 2 Dog H 0 H 1,5 H 1 H 1 H 1 B 0 B 0 B 0 B 0 B 2

F 0,5 F 0,5 F 0,5 F 0,5 F 1 Mouse H 1,5 H 2 H 1,5 H 1,5 H 1 B 0 B 0 B 0 B 0 B 2

F 0,5 F 0,5 F 0,5 F 0,5 F 1

Omnivorous

Rat n.d. n.d n.d n.d n.d. B 3 B 0 B 0 B 1 B 2,5 F 1 F 0 F 1,5 F 2 F 2 Carnivorous Cat H 0 H 1 H 1 H 1 H 1,5

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93

4.3 IHC results for the substances of the innate immune

system: Species comparison

4.3.1 Demonstration of ß-defensin 2

Due to the fact that the Bouin-fixed esophagus tissues revealed the best structural

and most reliable IHC results, the following descriptions will primarily focus on

observations made after using this fixation medium. However, significant findings

from HOPE® or Ca-Formol fixed tissue concerning the distribution pattern of the

analysed substances will also be mentioned.

As we aimed to compare the occurrence and distribution pattern of the substances

studied between the three important nutrition groups (herbivorous, omnivorous,

carnivorous), the description of the findings will be accomplished focussing on

each group. In order to identify significant differences between the three nutrition

types, statistical tests were applied (compare 3.9.7), and each layer of the

epithelium was considered.

Almost all herbivorous species studied displayed the same distribution and

reaction pattern for hBD-2 within the esophagus epithelium: A strong reaction was

observed in the stratum basale, upwards the staining intensity decreased in the

stratum spinosum but was up-regulated again in the stratum granulosum, except

in the cattle, where no reaction in the stratum granulosum could be found. The

goat exhibited the strongest reaction intensities in all layers. Within the cells, the

positive staining was limited to the cytoplasm. In all herbivorous species, a positive

reaction could be detected in the stratum corneum, even though the horse only

showed a weak staining. The positive staining in the stratum corneum was limited

to the most luminal part, and the reacting granules seemed to cover the epithelium

in the form of a thin layer. The cells of the stratum corneum were completely

negative.

In the omnivorous species, the distribution pattern of hBD-2 was almost the same

as demonstrated for the herbivorous animals. The strongest reactions were found

in the esophagus epithelium of the laboratory rat (Fig. 4.16). In contrast to the

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94

other omnivorous species, the dog showed no increase, but a decrease of staining

intensity in the stratum granulosum (Fig. 4.18).

The cat, as a typical carnivorous animal, not surprisingly displayed a distribution

pattern of hBD-2 comparable to that one of the dog, despite the fact that no

staining was observed in the upper stratum spinosum and the stratum granulosum

(Fig. 4.20).

Fig 4.16: hBD-2 reaction in the esophagus epithelium of the rat Very strong reaction intensity in the SG, Bouin fixation SB: Stratum basale, SS: stratum spinosum, SG: Stratum granulosum, SC: Stratum corneum

Fig. 4.17: IgG rabbit isotype control for hBD-2 for the esophagus of the rat The IgG rabbit isotype control was applied in the same protein concentration as the primary antibody, Bouin fixation SB: Stratum basale, SS: stratum spinosum, SG: Stratum granulosum, SC: Stratum corneum

Results

95

Fig. 4.18: hBD-2 reaction in the esophagus epithelium of the dog Keep attention to the decrease of staining intensity in the SG Bouin fixation, SB: Stratum basale, SS: Stratum spinosum, SG: Stratum granulosum, SC: Stratum corneum

Fig. 4.19: Rabbit IgG isotype control for hDB-2 in the esophagus of the dog The isotype control was applied in the same protein concentration as the primary antibody. Bouin fixation, SB: Stratum basale, SS: Stratum spinosum, SG: Stratum granulosum, SC: Stratum corneum

Results

96

The comparison of the herbivorous and the omnivorous species revealed no

statistically significant difference in the staining intensity of the esophagus samples

fixed in Bouin`s solution (p-value > 0.05). In contrast, significant differences could

be observed comparing herbivorous and omnivorous species with the carnivorous

cat (Fig. 4.21). Differences in staining intensity were found for the stratum

corneum and the stratum granulosum, comparing herbivorous species and the cat

(p-value < 0.05). In the stratum corneum, the cat displayed a stronger reaction,

whereas almost no reaction was observed in the stratum granulosum of the

esophagus of this species. The comparison of the omnivorous animals with the cat

exhibited a significantly stronger reaction only in the stratum corneum of the cat (p-

value < 0.05).

In contrast to the Bouin-fixed tissue, a difference between the herbivorous and

omnivorous species was observed for the HOPE® fixed samples. Statistically

significant differences were found for the stratum corneum and the lower and

upper stratum spinosum (p-value < 0.05). The omnivorous species studied reacted

slightly stronger. However, no differences were found comparing the herbivorous

Fig. 4.20: hBD-2 reaction in the esophagus epithelium of the cat Bouin fixation, SB: Stratum basale, SS: Stratum spinosum, SG: Stratum granulosum, SC: Stratum corneum

Results

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or omnivorous animals with the carnivorous cat (p-value > 0.05), as related to the

Bouin-fixed material.

SG SC0

1

2

3

4

*

* Herbivorous species

Omnivorous species

Carnivorous species

*

Sta

inin

g i

nte

nsi

ties

Fig 4.21: Differences in staining intensities in Bouin-fixed samples between the three nutrition groups for hBD-2. Data shown: Mean and standard deviation of staining intensities. (SB: Stratum basale, SS: Stratum spinosum, *: p- value < 0.05)

4.3.2 Demonstration of ß-defensin 3

Concerning the herbivorous species, the cattle and the sheep expressed almost

the same distribution pattern of hBD-3 (compare Tab 4.2). Therefore, the most

prominent expression of this antimicrobial peptide (AP) was observed in the

stratum basale (Fig. 4.22 and 4.23). Within the stratum spinosum, only for the

lower part of this stratum of the cattle a slightly positive reaction was observed,

whereas the upper part of the stratum spinosum reacted completely negative in

both species. An up-regulation of reaction intensity was demonstrated in the

stratum granulosum of the esophagus epithelium in the cattle and the sheep. The

sheep showed a slightly weaker staining in the stratum corneum. In the cattle, the

reaction intensity was consistently strong within both layers. Similar to the

distribution of hBD-2, a reaction of the cells in the stratum corneum was absent in

all species, but the epithelium surface was covered by a thin layer of granules

reacting positively for hBD-3. In the esophagus epithelium of the horse and the

goat, the hBD-3 reaction was also mostly strong within the stratum basale. This

degree of reaction intensity remained constant in the stratum spinosum of the

horse, and appeared significantly diminished in the stratum granulosum and

stratum corneum. In the last two layers only a weak reaction could be

Results

98

demonstrated. The goat displayed almost the same distribution pattern, albeit of

the fact that in this species no positive reaction staining was found in the stratum

granulosum.

In the omnivorous species, the pig expressed a distribution of hBD-3 similar to the

sheep, as both species exhibited a strongly positive reaction in the stratum basale

and no staining in the stratum spinosum, before the staining was up-regulated

again in the stratum granulosum and remained constant in the stratum corneum

(Fig 4.26). The mouse and the rat displayed a strong reaction in the stratum

basale, a slightly weaker one in the stratum spinosum and stratum granulosum,

before the staining intensity increased again in the stratum corneum. Interestingly,

the reaction in stratum corneum of the rat was the most prominent one of all

species.

The cat and the dog showed comparable results as the reaction intensity

decreased continuously from a very strong one in the stratum basale towards a

weak one in the stratum granulosum; the stratum corneum again displayed a

stronger reaction. Comparing the reaction intensities of the different species, it is

of great interest that the cat showed the strongest reaction intensities of all the

species studied in the stratum basale.

Fig 4.22: Demontration of hBD-3 in the esophagus epithelium of the cattle Bouin fixation, SB: Stratum basale, SS: Stratum spinosum, SG: Stratum granulosum, SC: Stratum corneum

Results

99

Fig. 4.23: Demonstration of hBD-3 in the ovine esophagus epithelium hBD-3 is up-regulated in the SG Bouin fixation, SB: Stratum basale, SS: Stratum spinosum, SG: Stratum granulosum, SC: Stratum corneum

Fig. 4.24: Negative control for hBD-3 in the ovine esophagus First antibody was omitted and replaced by PBS+1% BSA. Bouin fixation, SB: Stratum basale, SS: Stratum spinosum, SG: Stratum granulosum, SC: Stratum corneum

Results

100

Fig. 4.25: Positive control for hBD-3 As positive control tissue equine skin was used and the primary antibody was applied in the same concentration as for the reactions on the esophagus tissue (1:2000), a hair follicle (with hair) in the skin is marked by: →, Bouin fixation

Fig. 4.26 Demontration of hBD-3 in the esophagus epithelium of the pig hBD-3 is up-regulated in the SG, SB: Stratum basale, SS: Stratum spinosum, SG: Str granulosum, SC: Str. corneum

Fig. 4.27: IgG rabbit isotype control for hBD-3 Demonstrated for the porcine esophagus, IgG and hBD-3 antibody were applied in the same protein concentrations, for abbr. see Fig. 4.26

Results

101

The comparison of the three nutrition types displayed similar results, as described

for the hBD-2 reactions. Statistical analysis comparing the results for the Bouin-

fixed tissue of the herbivorous and omnivorous animals studied showed no

differences between these two nutrition groups (p-value > 0.05). Only in the

stratum basale of the herbivorous animals and the carnivorous cat a significant

variation in staining intensity could be observed (p-value < 0.05), the cat exhibiting

the strongest reaction (Fig. 4.28). The same situation was found comparing the

omnivorous species with the cat, but the cat, additionally, showed a significantly

stronger reaction in the lower stratum spinosum (p-value < 0.05).

Comparable to the results found for hBD-2 with the HOPE® fixed samples, a

statistical difference for hBD-3 was also observed for the herbivorous and

omnivorous species (p-value < 0.05), even though this variation was only present

in the stratum corneum. However, in contrast to the herbivorous animals, a slightly

positive reaction could be noticed for the omnivorous species in this layer.

SB SS (l)0

1

2

3

4

**

*

Herbivorous species

Omnivorous species

Carnivorous species

Sta

inin

g i

nte

nsi

ties

Fig 4.28: Differences in staining intensities in Bouin-fixed samples between the three nutrition groups for hBD-3. Data shown: Mean and standard deviation of staining intensities. (SB: Stratum basale, SS (l): lower Stratum spinosum, *: p- value < 0.05)

Results

102

4.3.3 Demonstration of cathelicidin

All herbivorous species reacted strongly positively for the AP cathelicidin (CAT) in

the stratum basale. In the horse, the reaction intensity decreased to a weak one in

the stratum spinosum and remained stable up to the stratum granulosum, before it

decreased in the stratum corneum (Fig. 4.29). The goat almost showed the same

reaction pattern, despite the fact that the reaction within the upper stratum

spinosum and the stratum granulosum was very weak (Fig. 4.31). The cattle and

the sheep both displayed a very weak reaction in the lower stratum spinosum,

before the staining disappeared in the upper stratum spinosum. In contrast to the

cattle, the sheep reacted strongly positive for CAT within the stratum granulosum.

The positive reactions were limited to the cytoplasm of the cells. Concerning the

stratum corneum, no positive reaction was demonstrated within the cells covering

the epithelium.

The pig was the species that showed the strongest reaction intensity of all

mammals studied within the stratum basale. Interestingly, in many pigs the

positive reactions were exclusively evident in this layer of the esophagus

epithelium, whereas in the other species the AP again was identified within the

stratum granulosum. Both, the dog and the rat displayed a strong reaction within

the stratum spinosum. In the stratum granulosum of the rat the intensity remained

constantly high, whereas it decreased in this layer of the canine esophagus (Fig.

4.32). Intriguingly, the last two mentioned species were the only ones reacting

positively for CAT within the stratum corneum cells, showing a thin layer of

granules reacting positively for CAT on the epithelium.

The distribution pattern of CAT in the canine epithelium was the same as detected

for the esophagus epithelium of the cat, despite the fact that the latter species

exhibited no positive reaction within the stratum corneum (Figs 4.32 and 4.33).

Results

103

Fig. 4.29: Demonstration of CAT in the esophagus epithelium of the horse The reaction intensity in SB is strong. Bouin fixation, SB: Stratum basale, SS: stratum spinosum, SG: Stratum granulosum, SC: Stratum corneum

Fig. 4.30: IgG mouse isotype control for CAT Demonstrated for the equine esophagus epithelium. Bouin fixation, SB: Stratum basale, SS: stratum spinosum, SG: Stratum granulosum, SC: Stratum corneum

Results

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Fig. 4.31 CAT reaction in the ovine esophagus epithelium Bouin fixation, SB: Stratum basale, SS: stratum spinosum, SG: Stratum granulosum, SC: Stratum corneum

Fig. 4.32: CAT reaction in the esophagus epithelium of the dog Bouin fixation, SB: Stratum basale, SS: stratum spinosum, SG: Stratum granulosum, SC: Stratum corneum

Fig. 4.33: CAT reaction in the esophagus of the cat, Bouin fixation, abbreviations compare Fig. 4.32

Fig. 4.34 Negative control for CAT Shown for the esophagus of the cat, Primary was omitted and replaced by PBS+1% BSA

Results

105

Similar to the other APs, for CAT no variation between herbivorous and

omnivorous species could be detected in Bouin-fixed samples (p-value > 0.05). In

contrast to the ß-defensins, no difference between the three nutrition types could

be observed for any of the layers.

The reactions found in the HOPE® fixed samples slightly differed from those ones

observed for Bouin-fixed material. Comparing the herbivorous with the omnivorous

animals a significant difference between the nutrition groups was found (p-value <

0.05). This variation was particularly present in the stratum granulosum and the

stratum spinosum. The comparison of the other nutrition types corroborated the

results found for Bouin-fixed tissue. The goat, particularly, showed a very strong

reaction in these layers compared to the omnivorous species.

4.3.4 Demonstration of Toll-like receptor 2

In all herbivorous species studied a strongly positive reaction for TLR2 was

observed in the stratum basale (Fig. 4.35). This reaction slightly diminished

towards the lower stratum spinosum in all the herbivorous animals, nevertheless in

the horse this reduction occurred in the upper stratum spinosum. The more luminal

esophagus layers, the stratum granulosum and the stratum corneum, generally

displayed negative reactions in the herbivorous group.

Resembling the situation in the herbivorous animals, in the omnivorous species a

strong reaction for TLR2 was generally discernable in the stratum basale (Fig.

4.36). Likewise, a reduction of reaction intensity towards the stratum spinosum

was found in all the omnivorous species studied. A significant finding was detected

for the pig, exhibiting a weak reaction within the stratum granulosum, whereas all

the other animals showed no reaction within this layer. It should be emphasised,

that it was not possible to detect TLR2 in the stratum corneum of any of the

species studied.

The positive reaction for TLR2 in the cat was limited to the stratum basale and the

stratum spinosum (Fig. 4.37). Comparable to the results found for the APs, the cat

and the dog displayed the same distribution of reaction patterns for TLR2 within

the esophagus epithelium.

Results

106

Fig. 4.35: Detection of TLR2 in the esophagus epithelium of the horse Strong reaction intensity in the SB, Bouin fixation, SB: Str. basale, SS: Str. spinosum, SG: Str. granulosum, SC: Str. corneum,

Fig. 4.36: Detection of TLR2 in the esophagus epithelium of the dog Bouin fixation, SB: Stratum basale, SS: stratum spinosum, SG: Stratum granulosum, SC: Stratum corneum

Results

107

For the Bouin-fixed samples, in four of the five layers of the esophagus epithelium

a statistically significant difference was demonstrated for the herbivorous animals

compared to the omnivorous species (p-value < 0.05) (Fig. 4.38). All layers,

except the stratum corneum, exhibited differences. Primarily the herbivorous

animals showed distinct reactions in the different layers. A stronger reaction in the

omnivorous species was exclusively evident for the stratum granulosum. As

already mentioned, the pig was the only species showing a distinct reaction in this

layer. For the herbivorous and carnivorous species studied a difference was only

found for the lower stratum spinosum (p-value < 0.05), which reacted slightly

stronger in the herbivorous species. The statistical analysis of the omnivorous and

the carnivorous cat revealed a difference between these two nutrition types in the

upper stratum spinosum and stratum granulosum (p-value < 0.05). As the pig was

the only species exhibiting a positive reaction in the stratum granulosum, the cat,

of course, showed the weaker staining intensities. In contrast, the reaction

intensities in the upper stratum spinosum were higher in the cat compared to the

omnivorous species. In contrast, in HOPE® fixed samples no differences between

the three nutrition groups could be observed.

Fig. 4.37: Detection of TLR2 in the esophagus epithelium of the cat Bouin fixation, SB: Stratum basale, SS: stratum spinosum, SG: Stratum granulosum, SC: Stratum corneum

Results

108

SB SS (l) SS (u) SG0.0

0.5

1.0

1.5

2.0

2.5* Herbivorous species

Omnivorous species

Carnivorous species** *

**

*

Sta

inin

g i

nte

ns

itie

s

Fig. 4.38: Differences in staining intensities in Bouin-fixed samples between the three nutrition groups for TLR2. Data shown: Mean and standard deviation of staining intensities. SB: Stratum basale, SS (l): Lower stratum spinosum, SS (u): Upper stratum spinosum SG: Stratum granulosum, *: p- value < 0.05

4.3.5 Demonstration of ß-glucan receptors

L-ficolin

For L-ficolin no positive reaction could be observed in none of the species studied,

whereby various pre-treatments and three different visualisation systems had been

tested, as described under 3.9.2 and 3.9.5. The fact of negative reactions involved

all three fixation methods. However, not only the epithelium showed any positive

reaction, but also the other layers of the esophagus exhibited no staining.

MBL

Comparable to the results described for L-ficolin also the experiments for the

detection of MBL did not produce positive reactions. Bouin-fixed samples as well

as Ca-formol and HOPE® fixed material reacted completely negative. Similar to L-

ficolin, it was not possible to demonstrate MBL in one of the esophagus layers.

Results

109

Dectin-1

Experiments conducted on the esophagus tissue of all studied species did not

show positive results, pertaining to all fixation solutions. None of the esophagus

layers showed a positive reaction.

4.3.6 Demonstration of lysozyme

Appling IHC for lysozyme revealed very strong positive reactions within the

stratum basale of the horse and the cattle. The staining intensity diminished

towards the lower stratum spinosum in both species. The weak reaction in the

cattle disappeared towards all following upper layers. In the horse, the reaction in

the lower stratum spinosum was slightly stronger and continuous up to the stratum

granulosum. A positive reaction within the stratum corneum of the herbivorous

animals was only demonstrable for the horse, which showed a weak reaction. In

the stratum basale of the goat and the sheep, the reaction intensity for lysozyme

was slightly reduced, in comparison to the other herbivorous animals. The goat

expressed the same weak reaction intensity throughout all layers, except for the

stratum corneum where the reaction decreased. Comparable to the distribution

pattern of CAT, the reaction intensity of lysozyme in the sheep again increased in

the stratum granulosum, after it had decreased in the stratum spinosum.

In the mouse and the rat, the positive reaction for lysozyme was limited to the

stratum basale, whereas the other layers showed no positive staining (Fig. 4.39).

A significant positive reaction in the pig was demonstrated in the stratum basale.

The stratum spinosum reacted negatively, the stratum granulosum and stratum

corneum only showed a weakly positive staining. A continuous decrease of

reaction intensity towards the lumen was demonstrated for the esophagus

epithelium of the dog and this species displayed no positive reaction within the

stratum corneum.

In the cat the most prominent reaction was found in the stratum basale and also in

the stratum corneum. It should be emphasised that the cat expressed the

strongest reaction of all species in the stratum corneum. The layers between the

stratum basale and the stratum corneum of the cat generally displayed no positive

reaction.

Results

110

The comparison of the three nutrition groups revealed several differences

regarding Bouin-fixed samples (Fig. 4.41). Significant variations could be identified

between the herbivorous and the omnivorous animals in the stratum granulosum

and the lower and upper part of the stratum spinosum (p-value < 0.05). The

herbivorous species exhibited stronger reactions within these layers. Another

statistically significant difference was observed in the stratum granulosum,

comparing the herbivorous animals and the carnivorous cat (p value < 0.05). Again

the herbivorous species exhibited the stronger reaction. Furthermore, a significant

difference for the stratum corneum was demonstrated comparing herbivorous

species with the carnivorous cat (p-value < 0.05). Interestingly, the cat showed a

much stronger reaction in this layer than the herbivorous animals. Such result was

also observed comparing omnivorous species and the cat (p-value < 0.05). In the

Fig. 4.39: Lysozyme reaction in the esophagus epithelium of the mouse Bouin fixation, SB: Stratum basale, SS: Stratum spinosum, SG: Stratum granulosum, SC: Stratum corneum

Fig. 4.40: IgG rabbit Isotype control for lysozyme Demonstrated for the murine esophagus. The IgG antibody was applied in the same protein concentration as the lysozyme antibody, Bouin fixation, SB: Str. basale, SS: Str. spinosum, SG: Str. granulosum, SC: Str. corneum

Results

111

latter, another difference was found for the lower stratum spinosum, again the cat

here displayed a stronger reaction intensity.

The immunohistochemical experiments conducted using the HOPE® fixed samples

brought about results consistent for the three nutrition types. Only in the upper

stratum spinosum a significant difference between the herbivorous and

omnivorous species was detected (p-value < 0.05). The omnivorous species

displayed slightly stronger reactions.

SS (l) SS (u) SG SC0

1

2

3

4

*

* Herbivorous species

Omnivorous species

Carnivorous species

*

**

**

Sta

inin

g i

nte

nsi

ties

Fig. 4.41: Differences in staining intensities in Bouin-fixed samples between the three nutrition groups for lysozyme. Data shown: Mean and standard deviation of staining intensities. SS (l): Lower Stratum spinosum, SS (u): Upper Stratum spinosum SG: Stratum granulosum, SC: Stratum corneum, *: p- value < 0.05

Results

112

4.3.7 Demonstration of Langerhans cells

Langerin

Langerin (+) LCs were sporadically detected in the esophagus of the mouse and

the horse (Fig. 4.42). In both animal species such cells were organised within the

stratum basale. In the other species studied no positive reactions for langerin (+)

LCs could be demonstrated.

CD1a

Applying an antibody against the surface molecule CD1a of Langerhans cells did

not produce positive results. In contrast to langerin, none of the species studied

reacted positively. The results did not differ between the three various fixation

solutions used.

Fig. 4.42: Demontration of langerin + LCs in the esophagus epithelium of the horse A langerin (+) LCs is marked by a: →, Bouin fixation

Discussion

113

5 Discussion

5.1 Fixation experiment

5.1.1 Influences of fixation on structure preservation

One aim of this thesis was to compare three different fixation media and to

evaluate their particular influence on the preservation of the structure of the

esophagus tissue and on the reactions of the IHC experiments performed. The

three fixation media used were: Bouin’s solution (after BÖCK 1989), Calcium

buffered formalin (after LILLIE and FULLMER 1976) (Ca-formol) and the HOPE®

fixation technique (after OLERT 2001). The first two fixation solutions are for the

most part formalin based (additionally Bouin`s medium is supplemented with picric

acid). Due to the fact that formalin is a hazardous substance and bears several

other disadvantages, e.g. masking of antigen epitopes by cross-linking proteins,

new alternatives are more and more in the focus of attention (OLERT et al. 2001;

BUESA 2008). We demonstrated that all samples fixed in Bouin`s solution

revealed an excellent tissue structure. In contrast, approximately 35 % of the

HOPE® fixed samples showed a decrease in the quality of tissue preservation.

This means in detail that tissue integrity was not always guaranteed. For example,

the epithelium of the esophagus was separated from the lamina propia and

revealed distinct clefts. In approximately 60 % of the Ca-formol fixed samples, the

quality of structure preservation was substantially reduced. The integrity of the

epithelium and the lamina propia was destroyed. An ideal fixation should preserve

the original structure of the tissue as good as possible and should be able to

provide an equivalent close to the natural state (BÖCK 1989). This state can be

reached by fast penetration of the fixation fluid into the tissue, thus avoiding

autolysis and make feasible rapid conservation (PEARSE 1985; BÖCK 1989).

Under these circumstances the best preservation of the structure as well as

continuity of the esophagus tissue were obtained using Bouin`s fixation medium.

The HOPE® fixation showed the next best results, followed by the relatively

unsatisfactory results obtained after Ca-formol fixation. OLERT et al. (2001)

claimed good formalin-like structure preservation for HOPE® fixed samples. The

Discussion

114

comparison of our results with the findings of the latter authors emphasises a

clearly controversial state on this matter. Unlike the fixation media we used,

common formalin (not buffered formaldehyde in aqueous solution) was applied in

the latter study. On one hand we can state, that tissue preservation of HOPE® was

even superior to Ca-formol fixation, but, on the other hand, it was not as good as

Bouin fixation. Studies conducted on HOPE® fixed human placenta demonstrated

that this fixation can result in a structural quality that is superior to cryo sections

(BLASCHNITZ et al. 2008). The comparison of HOPE® and formalin fixation was

also subject to other studies. ANDREI et al. (2008) observed that structural

alterations in the human uterine cervix, such as nuclear distortion and epithelial

clefts, were minimal in HOPE® fixed samples. The findings of a study comparing

structure preservation of ovine lung tissue fixed in 10% neutral buffered formalin

(NBF) or Bouin`s solution displayed no differences in the structure preservation

between the two fixatives used (BENAVIDES et al. 2006).

Specimen collection

The process of fixation is influenced by several different factors. The first critical

aspect is specimen collection, whereby several factors have to be taken into

account to obtain representative results. It was important to keep the time period

between tissue removal and its immersion in the fixation solution as short as

possible. In our study, all samples were obtained from freshly dead animals. Due

to the fact that the samples fixed in the solution HOPE® I had to be very small, the

preparation of this material took some time. On the one hand, this fact could

explain the slightly reduced structure quality of the HOPE® fixed samples, on the

other hand, however, the time period between sampling and immersing the tissue

was short (< 15 minutes), and the samples were constantly cooled on an icepack.

As the Bouin and Ca-formol samples were treated identically, the tissue collection

and preparation could not cause the reduced quality of structure preservation of

the Ca-formol fixed samples observed.

Discussion

115

Chemical characteristics of the fixation media

An additional fact, which has to be taken into account, is the chemical composition

of the fixation solutions used. To some extent, Bouin and Ca-formol fixation are

both based on formalin, which belongs to the chemical group of aldehyds. Fixation

with these chemicals is a complex process including a rapid penetration that stops

autolysis, followed by covalent binding and cross-linking. These three parts of

fixation proceed simultaneously, but at very different rates (penetration is 12 times

faster than binding) (BÖCK 1989; BUESA 2008). In contrast to Ca-formol, Bouin`s

solution additionally contains picric acid and glacial acetic acid. Bouin`s solution

has the advantage of penetrating into the tissue more rapidly, and therefore it

produces good tissue structure excluding shrinkage (LILLIE and FULLMER 1976).

This fact is in accordance with our findings, as the use of Bouin`s solution resulted

in the best morphological preservation of all tissue samples collected. The fast

penetration rate of Bouin`s solution is perhaps the most relevant explanation for

our findings. In this context, it is particularly difficult to explain the fixation process

of the HOPE® fixed samples, as the selling company (DCS) offers no detailed

description of the ingredients of the HOPE® I solution (also named protection

solution) and the HOPE® II solution. OLERT et al. (2001), who introduced the

HOPE® fixation into histological research, stated that HOPE® I solution is a

hyperosmolar mixture of different amino acids (10-100 mMol), with a pH of 5.8-6.4,

and works like an immersion fixative. In the second step, the tissue is immersed

into a mixture of HOPE® II and acetone (absolute), followed by an incubation in

acetone (absolute). Acetone acts as a dehydrating agent, according to the

specifications of the manufacturer, and replaces the increasing ethanol

concentrations used for the embedding of Bouin or Ca-formol fixed tissue. The

HOPE® solutions are considered to protect the tissue from protein cross-linking

occurring during dehydration and incubation in the low melting paraffin. From our

point of view, the HOPE® fixation seems to be based mainly on acetone fixation.

Acetone fixates tissue by coagulation of the proteins present. Pure acetone lacks

popularity as routine fixative as it does not preserve tissue structure as good as,

for example, formaldehyde (WERNER et al. 2000). A negative side effect of using

acetone is that it can cause strong shrinkage artefacts, as also confirmed by our

observations. After a histological staining, HOPE® fixed tissue often equally

Discussion

116

displayed shrinkage artefacts, such as the epithelium rolling up. As acetone is a

potent dehydration medium, a solid crust at the periphery of the tissue might be

formed and cause diminished penetration rates of the paraffin. This aspect might

be an explanation for the lower quality of tissue structure of the HOPE® fixed

samples compared to the Bouin fixed samples.

Influence of the temperature

Another critical aspect is the temperature during fixation. Whereas fixation in Ca-

formol and Bouin`s solution are fairly independent of temperature influences and

can be mainly conducted at room temperature, the first steps of the HOPE®

fixation have to be done under constant cooling. In general, low temperatures

retard autolysis, but also decrease the diffusion rate and thus prolong penetration.

In our experience, it can be concluded that it is particularly important to have a

temperature of not more than 4°C during the incubation in HOPE® I and HOPE® II

solutions. Even only slightly higher temperatures resulted in a reduced quality of

tissue preservation.

Influence of the duration of fixation

Another important aspect influencing structure quality is fixation duration. For

formalin based solutions, a time of less than 24 hours generally results in a

mixture of formalin and ethanol fixation. An interruption of formalin fixation before it

is completed will lead to cross-linking only at the tissue periphery. Coagulation

occurs near the centre, caused by the ascending ethanol solutions during tissue

dehydration, or the centre of the tissue sample remains unfixed (WERNER et al.

2000). In our case, the possibility of a fixation period which was too short and

caused reduced tissue quality of the Ca-Formol fixed samples can be neglected,

as we incubated the samples for 30-40 hours. Bouin`s fixative is suitable for a

longer fixation without causing over fixation (BÖCK 1989; MEYER personal

communication); however, tissue fixed in Ca-formol is susceptible for such

problem. Over fixation may lead to excessive cross-linking and cause irreversible

damage of epitopes. Moreover, this damage of epitopes affects the reactions of

IHC experiments (compare 5.1.2) (WERNER et al. 2000; VAN ALSTINE et al.

Discussion

117

2002; BLASCHNITZ et al. 2008). In the HOPE® fixed esophagus tissue, over

fixation did not seem to be much of a problem. However, we incubated the tissue

12-72 hours according to the instruction of the manufacturers. To evaluate the

possible influence of over fixation on the HOPE® fixed samples, further

experiments with varying incubation times are necessary.

According to the structural quality of the esophagus tissues, it can be concluded

that the samples fixed in Bouin`s solution produced the best results followed by

the HOPE® fixed samples. Regarding the latter findings, some tissue damage

might be due to wrong handling of the tissue (tissue pieces may have been too

large). Another reason for the slightly reduced quality of structure preservation

might be the influence of acetone, in particular on the esophagus tissues, as

experiments on human placenta and uterus revealed good tissue preservation

(ANDREI et al. 2008; BLASCHNITZ et al. 2008). The esophagus is composed of

several different tissues types including accurately defined cell structures and

functions. For example, the epithelium contains a great amount of keratins

(SCHOENNAGEL 2005), in contrast to other organs. Thus, the slightly reduced

tissue preservation in our study might be a result of these particular tissue

characteristics. According to the diminished structural quality of Ca-formol fixed

tissue, it might be presumed that the penetration rate of the fluid is rather slow in

comparison to picric acid and acetic acid (glacial) that are added to Bouin`s

solution. As already emphasised earlier (LILLIE and FULLMER 1976; JAMES and

TAS 1984; PEARSE 1985), a mixture of different components is the still the best

way to achieve high quality tissue preservation.

5.1.2 Influences of fixation on immunohistochemical results

One aim of this thesis was to discuss the influence of fixation on the quality of the

IHC results obtained from the esophagus epithelium. In this context we provide the

first comprehensive study on the fixation of the mammalian esophagus, comparing

the use and effects of three different fixation solutions. One central question of our

study was, whether or not the staining intensities of an IHC reaction in the five

layers of the esophagus epithelium differed depending on the fixation media used.

Therefore, the staining intensity was evaluated with a semi quantitative scale

system (0= no reaction up to 3= very strong reaction). This comparison was

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controlled by statistical analyses of the semiquantitative data obtained. The

findings can already be summarised to a first general statement: Our study

demonstrated no general significant differences in the staining intensities between

the fixation media used, even though a difference could be shown in certain layers

of the esophagus epithelium in certain species for some antibodies. Discussing

and evaluating the reasons for these results is nevertheless of great importance.

Although the IHC findings did not differ significantly between the three fixation

media applied, it is, firstly, important to consider the influences of fixation on the

structural preservation of the esophagus tissue (compare 5.1.1). Only samples

with the best preservation quality were taken into the IHC procedure. Such feature

was evaluated after routine H.E. staining, and only tissue sections of high

structural quality were used for the IHC experiments. In this connection, almost all

samples fixed in Bouin`s solution could be taken. In contrast, only approximately

40% of the Ca-formol and 65% of the HOPE® fixed samples were usable. Due to

the great quantity of the samples collected, tissue sections from almost every

animal species used were available for IHC analyses.

Influences on the quantity of antibodies

One astonishing finding of several former studies was that the quantities of the

primary antibodies could be reduced using HOPE® fixed tissue, in comparison to

formalin-fixed tissue. These observations are corroborated by findings of

GOLDMANN et al. (2003) and ANDREI et al. (2008). According to our results, in

most cases it was possible to reduce the required primary antibody amount usally

by 50-80% and for lysozyme even by 97.5% compared to samples fixed in Bouin`s

solution or Ca-formol (compare Tab 3.8). Such feature emphasises economical

advantage, however, the question why HOPE® fixation offers this opportunity has

still to be clarified.

Principles of fixation

In contrast to the two well established fixatives Bouin`s solution and Ca-formol, the

HOPE® fixation solution is not formalin based. Formalin fixation was introduced

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more than 100 years ago by the German medical practitioner F. Blum (1893). He

already demonstrated that formaldehyde forms methylene compounds with amino,

amide and hydroxyl groups, thus affecting the solubility and reactivity of proteins

(BLUM 1893, 1896). Therefore, it can be concluded that the formation of such

methylene cross-links (PUCHTLER and MELOAN 1985) is absent in HOPE® fixed

tissue (OLERT et al. 2001). Nevertheless, the primary characteristic of a fixative is

to stabilise tissue structure in order to protect against shrinking effects during the

use of concentrated organic solvents. OLERT et al. (2001) argue that the direct

application of organic solvents would be an ideal option, as the accumulation of

denaturing events that takes place during fixation could be circumvented. This

technique is rarely applied due to the incalculable negative affects of organic

solvents on the tissue. Although the exact mechanism of the HOPE® fixation still

needs to be elucidated, its aim is to protect the tissue from the influences of the

dehydration solution (acetone) used and to reduce the number of protein cross-

links. For this purpose, the tissue is directly transferred into a special protection

solution (ingredients not listed in detail) to weaken the negative influences of

acetone. Earlier studies already recommended acetone for an optimal retention of

antigenic activity in embedded tissue (KAKU et al. 1983). The authors stated that

the major disadvantage of acetone fixation is a considerable hardening and

shrinkage of the material. Such negative side effects should be prevented in the

HOPE® fixation system via previous incubation in a so-called protection solution.

As a consequence the reduction of protein-cross links in HOPE® fixed specimens

might directly result in improved preservation of antigenicity. In contrast, formalin

fixation results in a loss or decrease of antigenicity, due to methylene cross-links

of reactive sites on proteins making certain epitopes inaccessible for antibodies.

This drawback can be partly overcome by techniques based on heat denaturation

or enzymatic digestion, which facilitate the retrieval of antigens (WERNER et al.

2000; CARRASCO et al. 2004). Some authors argue that this time consuming

procedure could be avoided using the HOPE® fixation, as less antigen masking

occurs (GOLDMANN et al. 2003; ANDREI et al. 2008). Due to the fact that the

established antibodies used in our studies did not require any pre-treatment, we

cannot corroborate or refute these statements concerning the HOPE® fixation. It is

important to emphasise that in our study the attempt to establish some of the

antibodies in the esophagus (MBL, L-ficolin, Dectin-1 and CD1a) remained

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inconclusive after the use of all three fixatives. Due to the fact that the primary

antibodies for MBL and L-ficolin showed positive results on the equine liver

(positive control), such antibodies revealed a cross-reactivity at least for this

species. Hence, it can be concluded that MBL and L-ficolin are not present in the

esophagus epithelium of the horse. Regarding the other species, we cannot totally

refute the existence of the latter ß-glucan receptors, as it is possible that the

applied antibodies exhibit no cross-reactivities. These findings correspond with

observations made by BLASCHITZ et al. (2008), who argued that inadequate

antibodies lacking specificity cannot lead to satisfying IHC results and that the

HOPE® technique will not solve such problems. In conclusion, HOPE® did not

result in a direct advantage concerning the establishment of antibodies in our

study, which might be related to the applied antibodies. Nevertheless, supportive

evidence is provided that the improved preservation of antigenicity of HOPE®

fixation was directly related to the effect that the HOPE® specimens required only

50-2.5% of the amount of primary antibodies in comparison to the other two

fixation solutions. The influence of mild pre-treatments (for example heating in

citrate buffer) on HOPE® fixed esophagus tissue needs to be evaluated in future

experiments.

The main advantage of HOPE® fixation is, as discussed by BLASCHITZ et al.

(2008), that it provides the possibility of applying cryo-compatible antibodies to

paraffin sections. It has been demonstrated that antibodies in the human placenta

immunolocalised their antigens on cryo sections and on HOPE® fixed but not on

formalin-fixed paraffin sections. These results were related to the improved

preservation of soluble proteins in HOPE® fixed samples, in comparison to

samples fixed by the formalin method. The authors emphasised the benefit of the

HOPE® technique for cases when only cryo-compatible antibodies are available. In

our study most of the antibodies applied were established for use on paraffin

sections. Only the antibody designed for the detection of the collectin MBL had

been tested on cryo sections and not on paraffin ones. Our findings could not

corroborate the findings of BLASCHITZ et al. (2008), as no positive reactions

could be demonstrated on HOPE® fixed samples for MBL. The missing cross-

reactivity of the antibody in other species, than the horse, has to be taken into

account. Nevertheless, the HOPE® fixation can be looked upon as an

advantageous tool for fixation when discussing the aspect of saving time. The

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HOPE® protocol is shorter than the protocol used for the other two fixatives, owing

to the fact that the dehydration step (increasing ethanol concentrations) and the

incubation time in paraffin is more time-consuming for the Bouin and Ca-formol

fixed samples. The dehydration step is limited to the incubation in acetone for the

HOPE® fixed samples.

In spite of the possible advantages of the HOPE® fixation procedure, it is of great

importance to discuss the HOPE® fixation more critically. One aspect to be kept in

mind is that the ingredients of the HOPE® solutions are not listed or defined in

detail by the selling company. It is only mentioned that the solutions contain amino

acids (OLERT et al. 2001). An interaction of amino acids with antigens is

conceivable and could result in a change of epitope structure, which could

influence the immunoreactivity of the antigens to be detected. A second critical

feature is that the tissue sections prepared should not be stored for more than 7

days (personal communication DCS) before starting an IHC experiment. During

our IHC experiments, we tried to use freshly prepared tissue sections, as false

negative reactions were observed when using tissue sections prepared eight

weeks earlier. On the contrary, tissue sections of Bouin and Ca-formol tissue could

be used for IHC over years, without any recognisable negative influence on the

immunoreactivity (MEYER personal communication). A possible explanation for

the shorter usability of the HOPE® sections could be a decreased number of

protein cross-links than in formalin-fixed material, yielding a diminished stability of

proteins and tissue structure. Due to this fact it is also important to keep the

sections and paraffin blocks refrigerated until use after HOPE® fixation.

Another fact disqualifying HOPE® from routine diagnostics is the necessity of

refrigerating the samples immersed in HOPE® solution. In contrast, there is no

need to refrigerate formalin-fixed samples.

Differences in reaction intensities

In some cases significant differences between the fixation media could be

determined. Such differences were most obvious in the equine and ovine

esophagus epithelium, revealing a diminished reaction intensity in the HOPE®

fixed samples. A decrease of staining intensity was observed, particularly after

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analysing the results for ß-defensin 2 and 3 and TLR2. As already mentioned

before, in some cases the amount of primary antibody necessary could be

reduced by more than one third for the HOPE® sections, in comparison to the

Bouin and Ca-formol sections. Concerning the ß-defensins, it can be presumed

that this reduction was too high, resulting in a weaker reaction in the equine and

ovine esophagus epithelium compared to the other fixation media. Due to the

great quantity of species studied, it was particularly difficult to determine a suitable

antibody dilution for all species.

Regarding the results found for Ca-formol fixed esophagus tissue in our study, one

possible explanation for the bad quality of structure preservation and

immunoreactivity of some of the Ca-buffered formol fixed tissues could be the high

pH level of the solution. Ca-bufferd formol has a pH level of 7.1 – 7.3 and Bouin`s

solution fixes at a pH level of about 6.0. PUCHTLER and MELOAN (1984)

emphasised that maximum tissue fixation occurs in the pH range 4 to 5.5, no

increase in tissue stabilisation was observed above pH 5.5. They concluded that

the increased amount of formaldehyde bound at higher pH levels only blocks

numerous reactive groups. We can support the latter findings and assume that the

reduced antigenicity of the Ca-formol fixed samples is due to the higher pH-level of

the latter solution. Furthermore, an over fixation of the tissue in Ca-formol could

affect the stability of epitopes and result in diminished immunoreactivity (WERNER

et al. 2000). However, the aspect of over fixation is negligible, as we incubated the

samples for not more than 48 hours.

The positive and negative effects of various fixatives on the IHC based detection

of antigens have been discussed extensively in the literature. Supportive evidence

for our findings that fixation in Bouin`s solution leads to the best structural and IHC

results has been supplied by several authors (BEDOSSA et al. 1987; SMITT et al.

1993; CARRASCO et al. 2004). SMITT et al. (1993) agreed that Bouin`s fixative

obtained the best structural and IHC reactions for fixed human cerebellum in

contrast to formalin. The effect of fixation on ovine lung tissue was studied by

CARRASCO et al. (2004). They demonstrated that Bouin`s solution proved to be

the most suitable fixative for structural and immunohistochemical studies.

BEDOSSA et al. (1987) investigated the influences of fixation on human liver

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tissue and also emphasised that Bouin`s solution was the best fixative for their

studies. Several other authors discussed the negative effect of formalin fixation on

various tissues (PUCHTLER and MELOAN 1985; ARNOLD et al. 1996; VAN

ALSTINE et al. 2002). ARNOLD et al. (1996) found that neutral buffered formalin

was generally the poorest fixative (in comparison to ethanol and Bouin`s solution)

for maintaining antigen recognition by IHC. VAN ALSTINE et al. (2002) discussed

the possible effects of over fixation on tissues fixed in formalin in detail. They

concluded that an incubation exceeding three days results in a substantially

decreased sensitivity of an immunohistochemical test. Our samples were only

fixed for a maximum of 48 hours. In a more recently conducted study, BUESA

(2008) summarised the advantages and disadvantages of formalin fixation. On the

one hand the author argued that formalin fixation has been well established for

more than 100 years and is rapid as well as economic. Furthermore, he stated that

most antibodies are optimised for use on formalin-fixed paraffin embedded tissue

(FFPET), and that cross-links of proteins are reversible. On the other hand, the

author stresses the carcinogenic potential of formalin. From our point of view this

fact is one of the only really important advantages of the HOPE® fixation method,

because its media probably do not contain hazardous ingredients.

We can conclude that the HOPE® fixation technique offers some advantages,

especially concerning the costs of antibodies and time. If the HOPE® protocol is

well established in a laboratory, the preservation of structure is to some extend

comparable to samples fixed in Bouin`s solution, although differentiated structural

information cannot be obtained. In order to choose HOPE® as a serious alternative

to other fixatives, the ingredients of the solutions should be clearly defined to

achieve a critical evaluation of results obtained.

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5.2 Defence mechanisms of the mammalian esophagus:

Species comparison

5.2.1 Antimicrobial peptides

We focused our immunohistochemical studies on several parameters of the innate

immune system, in order to understand whether or not parts of the innate system

participate in the mucosal defence of the mammalian esophagus epithelium.

Although constitutive, this system is activated by the presence of microorganisms

and their products, providing a rational for a potential bio defence strategy (NEISH

2009). Besides our aim to detect receptors of the innate immune system

expressed by epithelial cells (TLRs) or on sentinental cells (LCs), our intent was to

detect substances produced and secreted by the epithelial cells (APs, ß-glucan

receptors, and lysozyme).

Mucosal defence strategies in the human esophagus

Information about esophageal mucosal defence is sparse, even in humans.

Nevertheless, HOPWOOD (1995) demonstrated several interesting aspects of

esophageal defence, e.g. various defence mechanisms can be elicited by reflux

damage. The author argued that chemical damage wrought by proteinases is

countered by cell turnover, intercellular barriers and pH management.

Furthermore, the presence of epithelial growth factor (EGF) is an added defence

feature that acts when ulcers are formed. For our study, the evidence of an

“intercellular barrier” is of particular interest. HOPWOOD (1995) demonstrated that

the intercellular space between the cells in the stratum spinosum was not empty,

as it appeared to be in electron microscopy, but showed the presence of

“mucosubstances”. However, during his studies the characteristics and functions

of this material remained unclear. In contrast to these findings, we confirmed the

presence of APs as intercellular material between the layers of the esophagus

epithelium of the nine mammalian species studied. This might be due to the fact

that the substances accumulate in large amounts between cells of the stratum

spinosum only in the human esophagus (HOPWOOD 1995), but are generally

present in smaller amounts in other layers. In our study we used highly sensitive

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IHC to detect APs, so that it was possible to give a detailed description of their

distribution in the esophagus epithelium.

Additionally, we provide the first artefact-free electron microscopical pictures (cryo-

SEM) of the surface of the esophagus epithelium, thus verifying the existence of a

one-layered microbial colonisation in all species studied. To be able to tolerate and

benefit from these microorganisms, the eukaryotic host must monitor the

microbiota and control their number and composition. One possible way to control

colonisation with commensal and potentially pathogenic bacteria is the production

of APs, which are part of the first line of defence (INAMOTO et al. 2008; NEISH

2009).

As APs are found in various epithelial tissues (DALE and FREDERICKS 2005;

BRAFF and GALLO 2006) and due to their antimicrobial and immunoregulatory

activity, scientists perceive APs as promising candidates for new therapeutic

drugs, also in veterinary medicine (LINDE et al. 2008; SANG and BLECHA 2009).

In accordance with the sites expressing APs, our discussion focuses on two other

organ systems. To start out, an analysis of the AP expression pattern in other

parts of the digestive tract is of interest. As a matter of fact APs are involved in the

control of the physiological microbial flora and mucosal homeostasis (BEVINS et

al. 1999; IIMURA et al. 2005; LOTZ et al. 2007; SALZMAN et al. 2007;

MUKHERJEE et al. 2008; VELDHUIZEN et al. 2008). For example, the production

of APs (mainly α-defensin) in the small intestine of humans is restricted to Paneth

cells located in the intestinal crypts, whereas in the large intestine CAT and ß-

defensins contribute to the antimicrobial barrier (LOTZ et al. 2007). Interestingly

enough, reduced numbers of Paneth cells and a decrease in the α-defensin

production were noted in Crohn`s disease patients (WEHKAMP et al. 2005).

Thereafter the specific construction of the esophagus epithelium has to be taken

into account. In most herbivorous and omnivorous species the esophagus shows a

more or less keratinised stratified squamous epithelium, which is not really

comparable to the one of the epidermis of the skin. The latter organ presents a

reliable mechanical, as well as an immunological barrier to infection, displaying a

considerable capacity of innate immunity. Due to constant attacks of

microorganisms and remarkable mechanical strain, research focussed on the

expression of APs in the epidermis (OREN et al. 2003; BRAFF et al. 2005; ELIAS

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2005; ELIAS and CHOI 2005; BRAFF and GALLO 2006). Whenever the

occurrence of this substance group is discussed, it is of great interest whether the

APs are produced constitutively or inducible at the site of inflammation. BRAFF et

al. (2005) found that CAT is constitutively expressed by neutrophils in the skin and

inducible in keratinocytes in response to infection. In contrast to this, ELIAS and

CHOI (2005) argued that hBD-2 and -3, and CAT are expressed at low levels in

unperturbed skin, but occur in higher levels in healing wounds and in inflammatory

dermatoses. Our results demonstrated that hBD-2 and -3 and CAT are expressed

constitutively in the esophagus epithelium of the species studied. It is

inconceivable that all animals were infected, and thus the constitutive production

of the considered APs is necessary to maintain the homeostasis of the esophagus

epithelium. None of the animal species revealed negative results, only the reaction

intensity differed between the species. Due to our findings we agree with ELIAS

and CHOI (2005) and can transfer their findings demonstrated for the skin to a

certain extent to the esophagus epithelium of the domesticated mammals studied.

Nevertheless, additional experiments analysing the influence of infection by certain

pathogens need to be conducted, whereby the up-regulation of APs could be a

likely effect.

Comparison of expression sites in the esophagus epithelium

In the skin, APs are normally stored in so-called lamellar bodies (LBs) or

membrane coating granules (MCGs), respectively, but were also found in the

intercellular space (OREN et al. 2003; ELIAS and CHOI 2005). While the glycolipid

contents of the LBs are well known, the additional AP contents were a surprise.

OREN et al. (2003) gave a detailed description on the expression sites of hBD-2 in

the epidermis. They performed immunogold labelling and showed that hBD-2 is

predominantly located in the spinous and granular layers. Dense labelling was

demonstrated for the LBs of the keratinocytes and some additional staining was

seen in the intercellular space. In contrast, ELIAS and CHOI (2005) described a

different expression pattern of hBD-2 and additional hBD-3 and CAT in the

epidermis. Conducting IHC, they showed that all three APs were predominantly

localised in the outer epidermis. At this site they appeared in LBs and in the

intercellular spaces of the stratum corneum. The authors argued that the APs are

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perfectly positioned to intercept pathogenic microorganisms when they attempt to

penetrate the epidermis in between the corneocytes. With regard to our own

findings from the esophagus epithelium, we could not corroborate the results of

OREN et al. (2003) and ELIAS and CHOI (2005). One contrasting finding was the

observation in all species and APs studied, that constantly strong to very strong

reaction intensities occurred in the cells of the stratum basale. Due to the fact that

this layer has the highest rate of proliferation, we presume a direct connection to

AP production intensities. With regard to the three nutrition types studied, a

statistically significant difference could be shown for hBD-3 in the stratum basale.

Interestingly enough, the carnivorous cat exhibited a slightly stronger reaction in

comparison to the herbivorous and omnivorous species analysed. The increased

production of hBD-3 in the feline stratum basale could be considered a reaction

due to diminished preventative mechanical protection properties of the esophagus

epithelium. The feline esophageal epithelium is only weakly keratinised, and

therefore cannot function as a reliable mechanical barrier against invading

microorganisms. Thus, the epithelium has to be protected by larger amounts of

APs. Supporting evidence for this theory is provided by the results observed for

the stratum corneum. What is more, statistically relevant differences were found in

this layer when comparing herbivorous and omnivorous species with the

carnivorous cat. This time hBD-2 was expressed in greater amounts in the

carnivorous cat. Moreover, hBD-3 was expressed at a higher level in the cat, even

though the difference between the nutrition groups was not obvious from the

statistical point of view. Consequently, the feline esophagus epithelium is

intensively protected by ß-defensins. Due to its reduced mechanical protection by

the weakly keratinised epithelium and the constant influence of microorganisms, ß-

defensin production is remarkable and has to fulfil a distinct protective task.

Presumably hBD-2 is more effective on the epithelial surface, and hBD-3 functions

in the more basal part of the feline esophagus epithelium. This explanation and the

exact amount of APs produced remains to be examined in future experiments (for

example by the application of densitometry). On the one hand, these observations

are to some extent astonishing, as one would expect the herbivorous species to

exhibit the strongest reactions, due to their rough-textured food and the resulting

high mechanical strain. On the other hand, our findings showed the influence of

other epithelial properties, such as a regulated AP production. A more keratinised

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epithelium protects the mucosa from invading microorganisms mechanically, and a

less keratinised epithelium is protected by an increased production of ß-defensins.

It remains to be elucidated why the reaction intensity of all three APs decreased

from the stratum basale towards the lower and upper stratum spinosum. One

explanation could be that APs are stored in vesicles, comparable to the LBs

(MCGs) of the epidermis (OREN et al. 2003; ELIAS and CHOI 2005), and are not

detectable by the primary antibody, due to the compact packing. The idea of

substance transport in vesicles corresponds with findings of INAMOTO et al.

(2008), who investigated the epithelial response towards the attachment of

indigenous bacteria in the small intestine of the rat. One of their observations was

that defensins and lysozyme are transferred via cellular vesicles into the lumen,

where they fused with the membrane of an invaginated bacterium. However, in

some cases an increase in reaction intensity could be observed towards the

stratum granulosum of the esophagus. Concerning hBD-2, this was true for the

goat, sheep, pig, mouse and rat. Regarding hBD-3, the phenomenon was

demonstrated for cattle, sheep, pigs and mice, and for CAT an increase was

observed in the ovine and porcine esophagus epithelium. Analysing the species

that showed the increase mentioned makes the existence of a correlation between

these results and the nutrition type a realistic feature. All of the species are

herbivorous or omnivorous and because of the enormous mechanical strain on

their esophagus epithelium, in comparison to the esophagus of the carnivorous

cat, APs have to be stored in the stratum granulosum. This “layer” of APs in the

latter stratum probably indicates some kind of barrier function. An intriguing finding

is that such a “barrier” was constantly detectable in the porcine esophagus

epithelium. As shown by the analysis of the structure of the esophagus epithelium,

the pig revealed a rather loose and varying organisation of the stratum corneum.

Such feature may result in an increased penetration of microorganisms. This

specific structural aspect explains the necessity of a protective layer of APs. Due

to the fact that the esophagus epithelium of the cat is protected by a layer of hBD-

2 covering the surface, the storage of APs in the stratum granulosum is not

necessary in this species. For CAT, the strongest reaction intensity was seen in

the pig, mainly in the stratum basale. One explanation could be that the

commensal flora of the porcine esophagus especially triggers this AP production.

LINDE et al. (2008) showed in in-vitro experiments that CATs are produced by the

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porcine organism and demonstrated its high effectiveness against: E. coli,

Salmonella typhimurium, Staphylococcus aureus, Actinobacillus pleuropneumonia,

Pseudomonas aeruginosa, and Candida albicans. It remains to be elucidated via

microbial differentiation, whether these microorganisms also belong to the

commensal flora of the porcine esophagus surface.

LINDE et al. (2008) showed in-vitro activity of equine ß-defensins against the

following microorganisms: Corynebacterium spp. and Staphylococcus intermedius.

These two Gram-positive cocci were not found in the microbial flora of the

esophagus of the horse (MEYER et al. 2009). Furthermore, LINDE et al. (2008)

described in-vitro activity of CAT against the following bacteria: Escherichia coli,

Streptococcus equinus, Klebsiella pneumonia and Serratia marcescens. In

contrast to the afore-mentioned results for ß-defensins, this spectrum of

antimicrobial activity is more consistent with the findings made by MEYER et al.

(2009) concerning the actual microbial status of the equine esophagus. These

authors demonstrated a high occupation with the Gram-negative facultative

anaerobic rods E.coli. However, E.coli is an ubiquitous bacterium and cannot be

made out as specific for the equine species or generally explain the expression of

CAT in the equine esophagus. The results indicate that the characteristic microbial

flora of the esophagus of the different domesticated mammals still needs to be

analysed more intensively.

With regard to our own findings and the literature studied, it can be concluded that

the diverse repertoire of APs is most likely a key factor in allowing the esophagus

mucosal surface to maintain homeostasis concerning the diverse colonising

bacterial populations.

5.2.2 Toll-like receptor 2

Toll-like receptors (TLRs) comprise a class of transmembrane pattern recognition

receptors that play a key role in microbial recognition, induction of antimicrobial

genes and control of adaptive immune responses (JANEWAY and MEDZHITOV

2002). Eleven different TLRs have been identified in mice (LOTZ et al. 2007) and

ten in humans (AKIRA 2003; JANEWAY et al. 2005). One aim of this study was to

provide the first proof of TLR expression in epithelial cells of the esophagus of the

species studied. After an analysis of the literature available, we decided to put our

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focus on TLR2, as several authors emphasised the occurrence of this TLR on

intestinal epithelial cells of the human and murine gut (CARIO and PODOLSKY

2006; CARIO et al. 2007; TIZARD 2008), and on epidermal keratinocytes

(KOELLISCH et al. 2005; LEBRE et al. 2007). For the intestine it was

demonstrated that TLRs are involved in tissue regeneration and inflammation, as

well as in controlling the integrity of the intestinal epithelial barrier (RAKOFF-

NAHOUM et al. 2004; CARIO and PODOLSKY 2006; CARIO et al. 2007).

Regarding the skin, LEBRE et al. (2007) showed that TLRs actively contribute to

the induction of an immune response.

In this dissertation thesis the existence of TLR2 on epithelial cells of the

esophagus was verified by IHC. Thus, we provide the first comprehensive

evidence for the expression of the latter TLR in the nine different species studied.

So far no information concerning the expression of TLRs in the esophagus

epithelium was available from other mammals including humans. In our study we

were able to demonstrate the existence of TLR2 in all species, which indicates a

constitutive expression of TLR2. These observations are in line with the findings of

CARIO et al. (2007) who emphasised the functional relevance of TLR2 to control

tight junction associated intestinal barrier integrity and to balance mucosal

homeostasis. Furthermore, a constitutive expression of TLR2 was observed on

human keratinocytes in the skin (LEBRE et al. 2007). A constitutive expression of

TLRs in cells creating an epithelial barrier against invading microorganisms is

comprehensible as these PRRs enable the organism to continuously screen the

microbial composition, readily recognize potential imbalances and rapidly initiate

innate immune responses.

In regard to the specific locations of TLR expression in the esophagus epithelium,

it could be observed in all species that the staining intensity of TLR2 was very high

in the stratum basale and decreased towards the stratum spinosum. The cells of

the stratum granulosum and the stratum corneum showed no positive reaction.

Only in the porcine esophagus a positive staining was observed in the stratum

granulosum. It appears logical that the stratum corneum cells do not express

TLR2, as it can be assumed that such cells do not possess the full ability to

respond, i.e. by the production of antimicrobial products. The fairly isolated

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expression of TLR2 in the basal part of the epithelium may have a functional

background. It is conjecturable that TLR expression at this site of the esophagus

epithelium is correlated with high production of APs in the stratum basale

(compare 5.3.1). Several authors described a functional relation between the

expression and activation of TLRs and the inducible production of APs via a TLR

dependent pathway (BIRCHLER et al. 2001; VORA et al. 2004; SUMIKAWA et al.

2006; BUECHAU et al. 2008; LINDE et al. 2008). The findings of the latter authors

correspond with our results. It was only possible within the scope of this study to

demonstrate a co-expression of TLR2 and APs, leaving the evaluation of the direct

functional relation of both to further studies. However, we could not accurately

differentiate which AP is induced due to a TLR2 response. BIRCHLER et al.

(2001) argued that only hBD-2 was induced via a TLR2 dependent pathway, whilst

hBD-3 was not inducible via TLR2. Later, the findings of BIRCHLER et al. (2001)

were confirmed for intestinal and epidermal cells (VORA et al. 2004; KUMAR et al.

2006; MUKHERJEE et al. 2008). To our knowledge, only SUMIKAWA et al. (2006)

demonstrated a TLR2 dependent hBD-3 production in human keratinocytes.

Nevertheless, a TLR2 dependent expression of CAT was only demonstrated for

the skin (BUECHAU et al. 2008). It is an interesting finding that TLR2, hBD-2 and -

3 and CAT expression was shown for the stratum granulosum of the porcine

esophagus. This feature might indicate a TLR2 triggered production of the latter

APs in the stratum granulosum. Further studies should concentrate on the

expression of TLR4 in the esophagus epithelium, as this TLR is considered to

participate in the surveillance of the intestinal tract, and co-expression with APs

has already been demonstrated (CARIO et al. 2000; CARIO et al. 2002; VORA et

al. 2004).

Functions of TLR2 in the esophagus epithelium

Besides being responsible for triggering an innate immune response, i.e. induction

of AP production, TLRs are thought to fulfil further tasks to help maintain epithelial

homeostasis.

RAKOFF-NAHOUM et al. (2004) demonstrated the functions of TLR2 by

simulating intestinal injury and inflammation in TLR2 and MyD88 (TLR signalling

protein) knock-out mice. First, they showed a dysregulation of proliferation and

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132

differentiation in the intestinal epithelium in the absence of TLR signals. TLR

absence resulted in an increased number of proliferating cells. Secondly, the

authors demonstrated a decreased production of cytoprotective and reparative

factors (IL-6 and TNF) in such knock-out mice. IL-6 and TNF play a pivotal role in

protecting the epithelium from injury and in the initiation of repair responses.

Moreover, it was shown that the production of such cytokines is dependent on the

presence of commensals, which stimulate TLRs. It can be assumed that the loss

of commensal-mediated cytoprotection of the intestinal barrier, due to TLR2 /

MyD88 dysfunction, results in the destruction of epithelial homeostasis. After the

breakdown of epithelial integrity normally innocuous bacteria, such as Escherichia

coli, can cross the Tunica mucosa, invade the blood stream, and cause fatal

systemic infection (JANEWAY et al. 2005). Besides functioning as PRRs, TLR2

might also act as regulatory feature of the epithelial integrity in the esophagus

epithelium, i.e. in case of injury. This could be one explanation for our finding that

herbivorous mammals revealed stronger reaction intensities to TLR2, in

comparison to the omnivorous and carnivorous species studied. The epithelium of

the herbivorous species is exposed to higher mechanical stress than that of the

other two nutrition groups. It remains to be elucidated in future experiments, if IL-6

and TNF are also present in the esophagus epithelium of the animal species

studied, in order to support or refute the latter theory.

Since TLRs play a pivotal role in innate immunity and are the front liners in

pathogen recognition, we hypothesised that TLRs are also expressed in the

esophagus epithelium. Furthermore, we aimed to reveal whether a co-existence

with APs occurs, as previously shown for other epithelial surfaces. After

demonstrating a colonisation of the esophagus surface with microorganisms by

cryo SEM, we had strong evidence that PRRs are in fact necessary in the

esophagus epithelium. In this context we were clearly able to demonstrate TLR2

expression in the nine species studied. Moreover, we can support our hypothesis

of a simultaneous expression of TLR2 and the APs hBD-2, -3, and CAT. It has to

be taken into account that TLR2 is stimulated by the commensal bacteria and

induces the production of APs by the epithelial cells. Furthermore, the regulation of

antimicrobial responses by TLR2 activation could prevent over production of

antimicrobial proteins that could interfere with intestinal homeostasis. More

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specific information about the functional relationship of TLR2 and the APs has to

be gained from future experiments, also meaning that the signal pathway of TLR2

(e.g. NF-κB) in the epithelial cells of the esophagus has to be studied in more

detail.

5.2.3 ß-glucan receptors

The ß-glucan receptors studied in this thesis comprise MBL, L-ficolin and Dectin-1

and, like all ß-glucan receptors, belong to the group of lectins. Lectins are able to

recognize carbohydrate moieties, the unique “sugar codes”, on pathogens with

their characteristic lectin binding domain, hence acting as PRRs (LU et al. 2002;

GUPTA and SUROLIA 2007). Such lectin molecules are divided into further

groups. MBL belongs to the group of collectins, L-ficolins is member of the ficolin

group and Dectin-1 is another C-type lectin receptor, specific for recognising

fungal invaders. The structural and functional features of each group have been

extensively described in the literature chapter of this thesis. Therefore we will

waive a discussion of these aspects in detail at this point.

There was substantial evidence that collectins and ficolins are predominantly

found at the interface of body and environment, i.e. at epidermal (MEYER et al.

2008) and mucosal surfaces (DELAY 1999; UEMURA et al. 2002; WAGNER et al.

2003; VAN DE WETERING et al. 2004; LILLIE et al. 2005). Regarding these

findings, we aimed to support or refute the presence of such molecules of innate

immunity in the esophagus epithelium of the nine domesticated mammals studied.

We conducted several IHC experiments to demonstrate ß-glucan receptors in the

esophagus epithelium of the various species. However, all our experiments ended

without a positive result despite the fact that various antigen retrieval techniques

and different visualisation methods were tested. The positive controls on equine

liver revealed a distinct staining for MBL and L-ficolin. Hence, it can be resulted

that at least in this species the latter ß-glucan receptors are not present. In order

to totally refute their occurrence in the other species studied, the cross-reactivity of

the antibodies applied needs also to be elucidated on positive control tissue.

Furthermore, a new fixation technique, the HOPE® fixation, was employed, which

we hypothesised would result in better antigen preservation than the formalin

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134

based fixations (compare 5.1.2). However, even the putative improvement in

antigen preservation did not bring about any positive results concerning ß-glucan

receptors. On one hand, the results described are not surprising, as to this day no

substantial evidence for the occurrence of the ß-glucan receptors examined in the

esophagus was provided for humans. Only the collectin surfactant protein D (SP-

D) could be shown here by IHC (MADSEN et al. 2000). On the other hand, the

collectin MBL has already been identified in the intestinal tract of mice and pigs.

UEMURA et al. (2002) and WAGNER et al. (2003) demonstrated MBL in the

murine small intestine by IHC and PCR, and DELAY (1999) also proved a positive

reaction for the latter collectin in the intestinal crypts of the pig. The expression on

mucosal surfaces suggests that MBL has a general function in innate immunity in

the gastrointestinal tract (VAN DE WETERING et al. 2004). The results of

UEMURA et al. (2002) actually revealed that MBL, which is secreted from villous

epithelial cells, acts as a humoral immune factor in the intestine, comparable to the

secretory IgA. The latter authors found the maximum expression of MBL in the

jejunum, which they characterised as relatively sterile in comparison to the colon

and argued that MBL, particularly, may be able to contribute to this sterility. These

findings do not correspond with our own observations. Following the

argumentation of UEMURA et al. (2002), one would consider MBL to be

expressed extremely high in the esophagus to maintain sterility. We consider that

a certain “antigenic pressure” is required for the production of this substance.

However, such level is not reached in the esophagus, and thus, MBL is not

produced by the epithelial cells of the esophagus. The esophagus reveals only a

thin layer of microorganisms on its luminal surface (compare 5.4). Consequently,

ß-glucan receptors may not be required to complement the innate defence

mechanisms as a first line of defence in the esophagus. These findings contradict

the theory of other authors, who characterised ß-glucan receptors as molecules of

an early immune response (HOLMSKOV et al. 2003; GUPTA and SUROLIA 2007;

SCHOREY and LAWRENCE 2008). The hypothesis that a ß-glucan receptor

response is only required at sites exposed to a high number of pathogens, is

consistent with recent observations from the integument of marine and freshwater

aquatic mammals, whereby MBL, L-ficolin and Dectin-1 were demonstrated

(MEYER et al. 2008). Staining for L-ficolin was generally the strongest and most

obvious in all layers of the epidermis, except in the stratum basale. Concerning

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135

MBL, the stratum basale showed a weak reaction. Additionally, positive reactions

for this lectin were observed in macrophages. Positive staining for Dectin-1 was

limited to secretory cells and the epidermis of the capybara. The authors

emphasised that in marine or freshwater ecosystems the skin has to cope with a

multitude of microorganisms. It is a remarkable finding that the sparsely-haired

capybara was the only species exhibiting a positive staining for Dectin-1. This

result indicates that Dectin-1 might be the ß-glucan receptor that is activated due

to increased pathogen pressure. Another reason could be that the skin of the

capybara is strained with a high number of fungi, for which Dectin-1 is the

specialised PRR. In this context, the pathogen specificity of the different ß-glucan

receptors should be taken into consideration. The primer ligand for MBL is LPS,

the major cell wall component of Gram-negative bacteria (HOLMSKOV et al. 2003;

VAN DE WETERING et al. 2004; LILLIE et al. 2005). L-ficolins have a wider range

of pathogen specificity and interact with Gram-negative and positive bacteria

(RUNZA et al. 2008). In contrast, Dectin-1 primarily recognizes the cell wall

components of fungi (WILLMENT and BROWN 2008). MEYER et al. (2009)

isolated bacteria from the equine esophagus. The genera found most frequently

comprised Gram-positive Streptococci, Gram-negative E. coli, Actinobacillus spp,

and Prevotella spp. and yeasts. LILLIE et al. (2005) and RUNZA et al. (2008)

studied the distribution of MBL and ficolins between the species and their

interaction with pathogens in various animals (compare also Tab. 2.2).

Unfortunately, they did not provide information concerning the horse. One finding

corresponding with the microbial flora of the equine esophagus is that the authors

demonstrated antimicrobial activity of MBL and ficolins against Actinobacillus spp.

and Escheriachia coli. The rather low occupation of the equine esophagus with

Candida spp. may indicate that Dectin-1, the ß-glucan receptor specific for fungi, is

rarely required. There are two principal explanations as to why the horse revealed

no positive reaction for any ß-glucan receptors. The first one being that bacterial

antigens are very species specific. Hence, as an example, the comparison of

porcine and equine Actinobacillus spp. is not tenable scientifically. Eventually, the

microbiota of the esophagus of the mammals studied differed considerably from

that recognized by ß-glucan receptors. Further experiments on the composition of

the natural microflora are needed. The second explanation is that the very species

specific genotypes of lectins make it particularly difficult to predict from one

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136

mammalian species to another (FUJITA et al 2004; LILLIE et al. 2005). As already

described in the literature chapter, various subtypes of lectins are present in the

animal kingdom. The comparison of lectin orthologues from different species

showed that they differ in many aspects (RUNZA et al. 2008). As a result, cross-

reactivity for antibodies specific for humans and animals cannot be assumed.

Further investigations are necessary in order to abandon the theory of a ß-glucan

receptor occurrence in the esophagus of domesticated mammals. Other methods,

e.g. in situ hybridization or PCR, could be applied and/or species specific

antibodies should be designed.

It is important to emphasise the different functional features of the epithelium of

the small and large intestine compared to the esophagus epithelium. In contrast to

the esophagus, the other parts of the intestine fulfil a wider range of secretory and

resorptive tasks (INAMOTO et al. 2008). Regarding this fact, it can be conjectured

that the epithelial cells of the mammalian esophagus are rarely needed for the

production of ß-glucan receptors.

YOKOTA et al. (2001), GANTNER et al. (2003) and DENNEHY and BROWN

(2007) demonstrated a co-expression and collaboration of Dectin-1 and TLR2. The

observation of cooperative pathogen recognition could not be corroborated in our

study. The co-expression of both receptors results in an increased production of

TNF and IL-12. One can regard these two chemokines as not necessary for

ensuring tissue homeostasis in the esophagus epithelium.

Furthermore, Dectin-1 was detected on LCs of the skin (ARIIZUMI et al. 2000;

YOKOTA et al. 2001; GAVINO et al. 2005). Regarding the esophagus, however,

our results do not provide supportive evidence for this theory. In our study

langerin(+) LCs were identified in the equine and murine esophagus (compare

5.3.4). Although yeasts were present, the equine esophagus has not to deal with a

very high fungal burden in comparison to the skin. This may explain the missing

expression of Dectin-1 on LCs of the esophagus epithelium.

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137

5.2.4 Lysozyme

Lysozyme is an enzymatically active antimicrobial protein, which targets essential

cell wall components of microorganisms. Functionally, lysozyme is a glycosidase

that hydrolyses linkages of peptidoglycan, an important constituent especially of

the cell wall of Gram-positive bacteria. Due to its preference for peptidoglycan,

lysozyme is more effective against this bacterial group (GANZ 2004).

Besides being present in a number of secretes, including tears and saliva,

lysozyme is also actively involved in the mucosal defence of the intestinal tract

(WEHKAMP et al. 2005; INAMOTO et al. 2008; MUKHERJEE et al. 2008).

Considering these recent findings, we tried to detect lysozyme in the esophagus

epithelium of nine different domesticated mammals. Our results revealed positive

reactions in all the species studied. This observation indicates that lysozyme is

constitutively expressed in the mammalian esophagus epithelium. Our findings

correspond with the observation made in microbiologically sterile “germ-free” mice.

These laboratory animals revealed lysozyme expression in the intestine,

independent of microbial signals (HOOPER et al. 2001). However, the role of the

commensal bacteria of the esophagus in the induction of lysozyme production

remains to be elucidated. MEYER et al. (2009) described the microbial flora of the

equine esophagus. Although the bacteria detected were predominantly Gram-

negative, lysozyme still seems to play a pivotal role in the innate immune defence

of the esophagus epithelium. Due to the fact that α-haemoloytic Streptococcus

spp. were also isolated in high numbers, a lysozyme induction is likely.

The distribution pattern of lysozyme varied between the different layers of the

esophagus epithelium. The only observation made in all species was the generally

high expression of the enzyme in the stratum basale. We think that lysozyme is

produced by the cells of the basal layer and then transported to the epithelial

surface. Our findings differ from those made by NIYONSABA and OGAWA (2005),

who described the distribution pattern of lysozyme in the human epidermis. They

found lysozyme expression to be much stronger in the upper cells layers than in

the basal part. In some species, the staining intensity of lysozyme decreased

continuously from the stratum basale towards the stratum corneum, e.g. the horse

or the dog (Tab. 4.5). In others, for example in the cat, the staining persisted in the

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138

layers between the stratum basale and stratum corneum. Similar observations

have been made in the epidermis of marine mammals (MEYER et al. 2003;

MEYER and SEEGERS 2004). This phenomenon may be explained by a vesicular

transport or storage of the enzyme during epithelial development towards the

lumen. Supportive evidence for this theory is also provided by INAMOTO et al.

(2008), who described a vesicular transport of lysozyme through the intestinal

epithelium of rats. It is possible that the vesicular form of lysozyme was not

detectable by the antibody we applied. In order to corroborate this statement,

further experiments are needed, such as immunogold labelling of the secondary

antibody for TEM analysis.

As expected, in three of the five esophagus layers studied the herbivorous species

exhibited the strongest expression of lysozyme in comparison to the omnivorous

mammals. We reason that this expression pattern is a typical feature of the

esophagus epithelium of the herbivorous species being exposed to high

mechanical strain. Although the epithelium comprises thick layers of corneocytes,

this mechanical protection obviously cannot completely guarantee epithelial

integrity. As no strongly keratinised cells protect the epithelium of the carnivorous

cat, a thin layer of lysozyme covers the epithelium instead. This explains the

statistically relevant increase of lysozyme expression in the feline stratum corneum

in comparison to the herbivorous and omnivorous species.

In conclusion, lysozyme forms an innate chemical shield in the esophagus

epithelium of domesticated mammals, and is another vital player of immunity

against microbial threats.

5.2.5 Langerhans cells

An additional objective of this thesis was to support or refute the fact that cellular

mechanisms participate in innate immune mechanisms of the esophagus

epithelium of the nine different species studied. Supporting evidence concluding

that cells of the innate immune system are also actively involved in the defence of

epithelial surfaces was provided in earlier studies on the esophagus and other

epithelial tissues, such as the epidermis (AL YASSIN and TONER 1975; ZAVALA

et al. 2002; PENA-CRUZ et al. 2003; ROMANI et al. 2003; DE CARVALHO et al.

2005; HUSSEIN 2008; NFON et al. 2008). Most authors focussed their attention

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on two cell subsets of the innate immunity: Dendritic cells (DCs) and T-cells. There

is a strong interaction between these two cell types, i.e., DCs possess antigen-

presenting molecules, which stimulate a T-cell response (PENA-CRUZ et al. 2003;

SUGITA et al. 2004; VAN RHIJN et al. 2005; MORI and DE LIBERO 2008). To

identify the first instances of innate immunity, we concentrated on the detection of

a DC subgroup, the Langerhans cells (LCs). For decades LCs were thought to be

the typical DCs present in stratified squamous epithelia, such as the epidermis.

Due to modern findings, LCs can no longer be regarded as representatives of DCs

in general. Several studies encouraged the view that LCs may possess various

features that make them special and distinguish them from other types of DCs

(ROMANI et al. 2003). Principally there are three characteristical “cell markers” for

LCs: The antigen presenting molecule CD1a, the glycolipid receptor langerin

(CD207), and the tennis racket-shaped intracellular organelles, the Birbeck

granules (VALLEDEAU et al. 2000; ROMANI et al. 2003; MIZUMOTO and

TAKASHIMA 2004). We chose to detect LCs by IHC, using primary antibodies

against the CD1a and the langerin molecule. The advantage of the latter approach

is that this receptor is also expressed on immature LCs (VALLEDEAU et al. 2000).

In contrast CD1a and Birbeck granules are mainly found in activated LCs.

The presence of LCs in the human esophageal epithelium has been reported

previously (AL YASSIN 1975; GEBOES et al. 1983; TERRIS and POTET 1995;

ZAVALA et al. 2002). The authors applied electron microscopy or IHC for

identification. Our results showed a positive staining for langerin(+) LC in the

equine and mouse esophagus epithelium, whereby no positive staining was

observed in any of the nine species studied when using the CD1a antibody.

Several pre-treatments were applied to identify CD1a positive LCs, however, all

the methods tested were inconclusive. Thus, our findings only partly correspond

with the most recent findings of TERRIS and POTET (1995) and ZAVALA et al.

(2002). These authors detected LCs in the esophagus epithelium of humans by

using a CD1a antibody. Similarities can be observed in regard to the location of

LCs: Both of the latter author groups detected LCs in the stratum basale and

spinosum, which is in line with our findings. We could not corroborate the results of

AL YASSIN and TONER (1976), because these authors detected LCs in the

medial and superficial layers (stratum granulosum and str. corneum). Additionally,

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140

a positive reaction for LCs was demonstrated in the stratum basale of the feline

and equine esophagus by applying the zinciodide-osmium method

(SCHOENNAGEL 2005). Furthermore, SCHOENNAGEL (2005) showed that

CD1a(+) LCs are present in the porcine and ovine esophagus. However, our

observations are only partially in accordance with the findings of this author. A

corresponding finding is that we also observed a positive reaction in the equine

esophagus and detected langerin(+) LCs. The positive reaction for the ovine and

porcine species could not be corroborated.

The theory of a more basal orientation of the LCs seems more conclusive due to

two aspects. First, LCs are predominantly present near T-cells, which are the cells

they stimulate. T-cells are mainly found in the suprabasal cell layers of the

esophagus epithelium (TERRIS and POTET 1995; RESNICK et al. 1999). Second,

similar results for LC distribution were described in the skin, where LCs could be

detected in the basal part of the epidermis in humans (ROMANI et al. 2003), but

also in different other mammals, such as in the normal epidermis of the horse

(HAMADA et al. 1992) and the pig (MEYER 1986) by electron microscopy. In

porcine epidermis, LCs were also found via IHC, with antibodies against langerin

(NFON et al. 2008). In a recently conducted study, LCs were identified in canine

skin by IHC against CD1a surface molecules (VAN BEECK et al. 2008). Infection

correlated occurrence of LCs in the skin was described for the dog. LCs play an

important role in lupus erythematosus, atopic dermatitis (DAY 1996), and in

parasitic diseases such as demodicosis and leishmaniosis (DAY 1997; DE

CARVALHO 2005).

It remains to be elucidated, why some species reacted positive for langerin, while

none revealed a positive staining for CD1a. One theory is that the human specific

antibodies applied simply displayed no cross-reactivity with the studied species.

DE CAVALHO et al. (2005) already stated that monoclonal antibodies directed

against one species do not necessarily exhibit cross-reactivity with other species,

especially as the CD molecules are very species specific. Cells which exhibited

langerin did not simultaneously express CD1a antigen-presenting molecules on

their surface. We have to bear in mind which functional features both receptors

possess: Langerin captures glycolipids, transports these to the Birbeck granules,

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141

where CD1a is loaded and finally transported to the surface in order to present the

internalised antigen fragments to T-cells (VALLADEAU et al. 2000; HUNGER et al.

2004). This means that langerin(+) / CD1a(-) LCs are capable of internalising

circulating glycolipids with their langerin receptors into the cell. Nevertheless, they

do not behave as APCs, due to the fact they do not express CD1a on their

surface. It is possible that such cells may be able to provide a mechanism to

sequester circulating antigen and reduce its immunogenic potential (MORI and DE

LIBERO 2008). As a result, the antigenic pressure on the mucosal surface is

reduced. In case of increased antigenic pressure such cells eventually mature to

APCs and express CD1a on their surface.

One recent finding which has to be taken into account is the fact that dermal DCs

can express langerin too (NAGAO et al. 2009). Thus, the hypothesis that langerin

is a specific marker for LCs has to be abandoned. With regard to our findings this

means that the cells detected could also belong to the DC subset. NAGAO et al.

(2009) determined that LCs and langerin(+) DCs exhibit variable migratory

capabilities in vitro and repopulate distinct anatomic compartments in the skin at

diverse rates after conditional depletion in vivo. Langerin(+) DCs, in contrast to

LCs, did not require TGFbeta1 for development. Further studies on cell cultures

stimulated with TGFbeta1 have to be conducted, in order to differentiate between

langerin(+) DCs and LCs in the esophagus.

The observations of our study encourage the view that in the esophagus of murine

and equine species LCs also contribute to the first immunological barrier towards

luminal antigens. It remains to be elucidated, if such cellular mechanisms are also

present in other domesticated mammals. Species specific antibodies against the

CD epitopes of various species need to be designed for this approach.

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5.3 Microbial colonisation of the esophagus

Our study povides the first evidence of microbial colonisation on the esophagus

surface of the important domesticated mammals. By applying cryo SEM an

authentic display of the microorganisms covering the surface was achieved

(compare figures at 4.1.1). In the same way it was shown that innate defence

mechanisms are required in the esophagus where the epithelium has to control

and ward off bacterial threats. The epithelial surface revealed an occupation by a

thin layer of bacteria and fungi. Species specific differences in the quantity of the

microbiota could not be determined. In order to quantify the number of

microorganisms further studies are necessary, whereby they should begin by

establishing a standardised method for collecting samples.

The interest in the microbial composition of the regular digestive tract developed in

the 19th century; during the lifetime of Louis Pasteur, who believed that a symbiotic

relationship between man and bacteria was essential for life. The development of

experimental (animal) models allowed the research on this relationship in

absolutely germ-free mice, demonstrating the role of commensal microflora, i.e. a

function in vitamin synthesis (vitamin K and B12), the metabolism of nitrogen

compounds and lipids, and especially the participation as barrier against the

invasion of pathogenic microorganisms (MACKROWIAK 1982; ZILBERSTEIN et

al. 2007). The latter authors studied the microbial flora of the human digestive tract

in healthy volunteers. They collected samples from the mouth, esophagus,

stomach, duodenum, jejunum, ileum, colon, and rectum, and determined the flora

composition. The bacteria most frequently encountered in the human esophagus

were Streptococcus sp. (40%) and Staphylococcus sp. (20%). Furthermore, the

esophagus revealed a colonisation by Corynebacterium sp., Lactobacillus sp. and

Peptococcus sp. (all 10%). The authors claimed that the esophageal microbiota

are mainly transitory, similar to that detected in the mouth. However, they stated

that when alimentary stasis occurs, as for example in neoplasia, there is an

excessive increase in bacterial growth (LAU et al. 1981). In order to determine

whether the microbial colonisation of the esophagus of the species studied in this

thesis is only transitory or also indigenous, it is necessary to begin by

characterising the microbial flora of the oral cavity of every animal. Comparing the

microbial flora of the human esophagus (ZILBERSTEIN et al. 2007) with that of

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the only other species studied, the horse (MEYER et al. 2009), revealed no

similarities. Such finding is not surprising, since humans and horses have

completely different nutrition habits. For a comparison of the human microbiota

with the microflora of an animal species, the porcine digestive tract should be

investigated in detail, as swine are often used as models for humans also due to

similarities regarding the digestive organs.

In contrast to the esophagus, a lot of information is available on the microbial

colonisation of other parts of the human digestive tract. Information about bacterial

communities is expected to provide crucial information how to develop therapies

for various gastrointestinal diseases. Furthermore, scientists hope to generate

knowledge about the next generation of probiotic bacteria as part of functional

food (SINGH et al. 2009). The gut bacterial microbiota has established multiple

mechanisms to influence the eukaryotic host, generally in a beneficial way. The

prokaryotic genomes of the human microbiota encode a spectrum of metabolic

capabilities beyond that of the host genome, making the microbiota an integral

component of human physiology (HOOPER et al. 2001; SCHENK and MUELLER

2008; NEISH 2009). Modulating bacterial numbers and diversity is entrusted to the

gastrointestinal immune system. Although bacteria are distributed throughout the

intestine, the major concentration of microorganisms can be found in the large

intestine (SINGH et al. 2009). The diversity of species encompasses around 400-

500 different bacteria (LOTZ et al. 2007). The small intestine has microbial

communities with 1x108 CFU g-1 ileal content. Through the remainder of the

digestive tract, bacterial concentrations gradually increase, reaching 1x1011 CFU

g-1 (SINGH et al. 2009). Within the 400-500 species mentioned, 30-40 species

make up for 99% of the total population of the intestine (GUARNER 2006). The

results of several studies indicate that five genera are predominant in the large

intestine of humans: Bacteroides, Eubacterium, Bifidobacterium, Peptococcus,

and Fusobacterium (SANDBORN et al. 2003; SINGH et al. 2009). It is important to

note that although immunostimulatory structures recognized by the innate immune

system are generally produced by both, commensal and pathogenic bacteria, the

synthesis of low stimulatory hypoacytylated LPS from Bacteroides might contribute

to homeostasis (WEINTRAUB et al. 1989).

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With regard to our own observations, one difference was most obvious between

the colonisation of the intestinal tract and the esophagus, i.e. the quantity of

microorganisms detected. In contrast to the resorpative intestine, only a thin layer

of microbiota covered the epithelial surface of the esophagus. These findings are

in line with observations of LOTZ et al. (2007) from the human esophagus, which

is covered with only low to very low numbers of bacteria. As the esophagus

mucosa does not fulfil such digestive tasks, as the absorption of nutrients or

synthesis of vitamins, the colonisation with commensal bacteria here seems not

essential for nutrient absorption. As a result, much less bacteria, in comparison to

the intestine, can be observed. Nevertheless, due to the fact that we demonstrated

the existence of pattern recognition receptors in the esophagus epithelium, we

also showed that already a relatively low number of microorganisms is able to

evoke the response of the innate immune system. It is possible that such a low

number of microorganisms maintains homeostasis in the esophagus, whereby an

excessive colonisation with pathogenic bacteria may be avoided.

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5.4 Conclusions

From the results obtained in the current study, it can be concluded that a first-line

of defence mechanisms of the innate immune system contributes to maintaining a

microbial homeostasis at the surface of the esophagus epithelium of domesticated

mammals. The esophageal antimicrobial arsenal encompasses a variety of PRRs

and antimicrobial proteins and peptides.

One of the first authorities for pathogen recognition appears to be TLR2. Most

likely this receptor recognizes sugar moieties on the surface of pathogens, thus

triggering an innate immune response. Apparently one of these responses is the

production of antimicrobial peptides (Aps). We observed expression of ß-defensin

2 and -3 as well as CAT in all species studies. The co-expression of TLR2 and

APs by the epithelial cells was clearly demonstrated for all animals. Comparing the

three different nutrition groups (herbivorous, omnivorous, carnivorous) revealed

differences in the expression pattern of the TLR2 and APs studied. Regarding the

ß-defensins, it could be shown that the weakly keratinised esophagus epithelium

of the carnivorous cat compensates the diminished mechanical protection by an

increased production of ß-defensin 2 and -3. In contrast, the herbivorous species

exhibited a higher expression of TLR2 when compared to the carnivorous and

omnivorous species. We explained this observation with the functional feature of

TLR2 to trigger the production of substances that maintain epithelial integrity

(RAKOFF-NAHOUM et al. 2004). The esophagus epithelium of the herbivorous

species is confronted with enormous mechanical strain, clearly making such a

function necessary.

Another antimicrobial protein contributing to the formation of an innate epithelial

shield in the esophagus epithelium is lysozyme, detectable in the esophagus of all

species studied. In contrast to ß-defensin 2 and -3, lysozyme was expressed in

higher amounts in the herbivorous species studied. Although their epithelium is

protected by an often distinctly keratinised epithelium, the complementary

production of lysozyme seems to be required to protect the epithelium from

invading bacteria, as perforations can occur from the rough plant material (MEYER

et al. 2008, unpublished data).

Since Langerhans cells could only be proven in the murine and equine esophagus

epithelium, their importance for the surveillance of the esophagus epithelium in

healthy animals has to remain open.

Discussion

146

In regard to the fact that ß-glucan receptors could not be identified in the present

study, we believe that these PRRs do not play a key role in the epithelial defence

of the esophagus. It is conjecturable that the microbial colonisation of the

esophagus epithelium, in contrast to that of the epidermis, is too insignificant as to

trigger a ß-glucan receptor response (MEYER et al. 2008). In general, IHC offered

a good possibility to study the presence of innate defence mechanisms in the

esophagus epithelium.

In addition, the application of cryo SEM permitted the first artefact free

demonstration of microbial colonisation of the esophagus. The pictures obtained

revealed a thin layer of bacteria and fungi covering the epithelial surface. This

result can be declared as vital for justifying the necessity of innate defence

mechanisms.

The third main topic of this dissertation was the comparison of three different

fixation media. Our results indicated that the recently established HOPE® fixation

offers a good opportunity to study certain immune parameters in the esophagus

epithelium. Nevertheless, it is no serious alternative to the well established

formalin based Bouin`s solution but could be an alternative to cryosections.

Fixation in the latter medium resulted in the best preservation of tissue structure

and antigens. The frequently applied Ca-formol fixation proved to be the poorest

fixative in regard to preservation quality and maintenance of antigenicity in the

esophagus epithelium.

Zusammenfassung

147

6 Zusammenfassung

Untersuchungen zur angeborenen Immunität des Oesophagusepithels der

Haussäugetiere

Isabelle N. Hornickel

Ziel dieser Dissertation war zu klären, ob das Oesophagusepithel von

Haussäugetieren charakteristische Merkmale der angeborenen Immunität

aufweist. Im Rahmen aktueller Studien am humanen Darm wurde bereits das

Vorhandensein und die Notwendigkeit von Mechanismen der angeborenen

Immunität offenkundig. Die Autoren unterstrichen im Besonderen das

Zusammenspiel zwischen den im Darmtrakt zahlreich vorhandenen kommensalen

Mikroorganismen und einer Aktivierung der angeborenen Immunität, um die

epitheliale Homöostase zu garantieren. Im Rahmen der vorliegenden Dissertation

wurde zunächst der Frage nachgegangen, ob überhaupt eine bemerkenswerte

Besiedlung des Oesophagusepithels mit Mikroorganismen besteht. Im nächsten

Schritt sollte geklärt werden, ob und inwieweit vorhandene Bakterien und Pilze zur

Aktivierung des angeborenen Immunsystems beitragen.

Methodische Grundlage für die vorliegende Studie waren immunhistochemische

Färbungen, die qualitativ und semiquantitativ ausgewertet wurden. Als Material

dienten Oesophagusproben von verschiedenen frischtoten Haussäugetierarten mit

unterschiedlicher Ernährungsweise (Omnivore: Schwein, Hund, Maus, Laborratte,

Karnivore: Katze und herbivore: Ziege, Schaf, Kaninchen, Rind, Pferd). Als

zusätzlicher methodischer Aspekt waren die Einflüsse von drei verschiedenen

Fixationsmedien auf die immunhistochemischen Ergebnisse zu beurteilen.

Verglichen werden sollte die Verwendung von zwei formalinhaltigen Fixantien

(Bouin´sche Lösung und Ca-Acetat Formalin) mit einer neuen, möglicherweise

Antigen schonenden Fixationsmethode namens HOPE®. Nach der Fixation im

jeweiligen Medium wurden die Proben in Paraffin eingebettet. Um eine

artefaktfreie Darstellung der die Oesophagusoberfläche besiedelnden

Mikroorganismen sicher zu stellen, wählten wir die Untersuchung mittels eines

Zusammenfassung

148

Cryo Rasterelektronenmikroskop (Cryo REM). Hierzu wurden die einzelnen

Proben bis zur Untersuchung in flüssigem Stickstoff gelagert.

Die Ergebnisse lieferten den Beweis für die Existenz von Parametern der

angeborenen Immunität im Oesophagusepithel der Haussäugetiere. Es konnte

gezeigt werden, dass dem tierischen Organismus verschiedene Pathogen-

erkennende Rezeptoren zur Verfügung stehen, um eine frühe Antwort des

Immunsystems auf fakultativ pathogene Keime zu aktivieren. Als erste Instanz

einer Pathogenerkennung scheint der Toll-like Rezeptor 2 von besonderer

Bedeutung zu sein, der hauptsächlich von den Zellen des Stratum basale des

Oesophagusepithels exprimiert wurde. Zum weiteren Schutz der epithelialen

Barriere produzierten die Epithelzellen kationische antimikrobielle Peptide.

Innerhalb dieser Studie, wurden dabei Cathelizidin, sowie ß-Defensin 2 und -3

nachgewiesen. Somit konnten Ergebnisse aus Studien an humanen intestinalen

und kutanen Epithelgeweben bestätigt werden, die eine Koexpression von Toll-like

Rezeptoren und antimikrobiellen Peptiden zeigten. Das nur schwach verhornte

Oesophagusepithel der karnivoren Katze wurde vor einer Keiminvasion durch

besonders hohe Mengen an antimikrobiellen Peptiden geschützt, während im

Epithel der herbivoren und omnivoren Spezies eine mehr oder weniger starke

Keratinisierung und ein charakterischer histologischer Aufbau für einen guten

mechanischen Schutz sorgten.

Ein Nachweis der löslichen, Pathogen-erkennenden ß-glucan Rezeptoren auf den

Zellen des Oesophagusepithels der Haussäugetiere war innerhalb dieser Studie

nicht möglich. Auf Grund des Vergleichs mit anderen Arbeiten, z.B. von der Haut

mariner Säuger, lässt sich vermuten, dass die Expression dieser Rezeptoren eine

höhere Belastung mit Mikroorganismen erfordert, als es im Oesophagus der Fall

ist. Weitere Untersuchungen auf diesem Bereich sind also notwendig.

Neben den antimikrobiellen Peptiden konnte außerdem das enzymatisch aktive

Protein Lysozym in allen Spezies nachgewiesen werden, welches somit ebenfalls

einen Beitrag zur Epithelhomöostase liefert.

Zusammenfassung

149

Des Weiteren wurde ein Beweis für das Vorkommen zellulärer Abwehrinstanzen

innerhalb des Oesophagusepithels der Haussäugetiere erbracht. In diesem

Zusammenhang gelang es, Langerhans Zellen beim Pferd und bei der Maus durch

immunhistochemische Methodik zu erfassen.

Die immunhistochemischen Analysen dieser Studie wurden durch die Cryo REM

Darstellung einer dünnen Schicht von Bakterien und Pilzen auf der

Oesophagusoberfläche ergänzt.

Mit der vorliegenden Arbeit konnte außerdem ein Beitrag zur aktuellen Diskussion

über Alternativen zu formalinbasierenden Fixationsmethoden geliefert werden.

Bezüglich dieser Thematik ist festzuhalten, dass die HOPE® Fixierung in der

Grundlagenforschung eine kostensenkende Alternative darstellt, jedoch für eine

Routinediagnostik auf Grund vieler variabler Parameter nicht unbedingt geeignet

erscheint. Eine weitere Schlussfolgerung auf der Basis unserer Resultate ist

daher, dass immunhistochemische Befunde auch immer im Zusammenhang mit

der Fixierungsmethode kritisch diskutiert werden sollten. So zeigte sich in unserer

Studie, dass die immunhistochemischen Färbungsergebnisse von Ca-Formol

fixierten Proben kaum verwertbar waren, hingegen die Fixierung in Bouin`scher

Lösung gute Ergebnisse in allen Bereichen (Strukturerhaltung und

Immunhistochemie) erbrachte.

Zusammenfassend gesehen, liefert die vorliegende Dissertation eine detaillierte

Charakterisierung der Parameter der angeborenen Immunität des

Oesophagusepithels der Haussäugetiere. Die gewonnenen Erkenntnisse könnten

bei der Erforschung humaner Erkrankungen der Speiseröhre, wie zum Beispiel der

Refluxoesophagitis oder des Barrett Oesophagus, besonders im Zusammenhang

mit den vorhandenen Tiermodellen (Maus, Ratte, Schwein) von Nutzen sein.

Summary

150

7 Summary

The aim of this dissertation thesis was to support or refute the hypothesis, whether the

esophagus epithelium of domesticated mammals exhibits characteristical features of

innate immunity or not. Within the scope of recent studies on human intestinal tissue, the

occurrence and necessity of innate immune mechanisms has already been demonstrated.

The authors emphasised that the interaction between the numerously occurring intestinal

microorganisms and the activation of the innate immune system is required to maintain

intestinal homeostasis. In this context, first, we tried to answer the question if it is possible

to show a microbial colonisation of the esophageal surface. Second, it should be clarified

to which extend this colonisation contributes to an activation of innate immune

mechanisms.

The experimental basis of this study was immunohistochemical staining which was

evaluated in a qualitative and semiquantitative matter. The esophagus samples used were

obtained immediately after euthanization from nine species of domesticated mammals of

three nutriton types (omnivores: pig, dog, mouse, rat; carnivores: cat; herbivores: goat,

sheep, rabbit, cattle, horse). An additional feature of this thesis was to analyse the

influence of different fixation media on the immunohistochemical results. Two formalin

based fixation solutions (Bouin`s solution and Ca- acetate formalin) were compared with a

recently introduced, probably more gentle fixation medium, the HOPE® fixation. In order to

guarantee an artefact free demonstration of microorganisms colonising the esophagus

epithelium, we applied cryo scanning electron microscopy (cryo SEM).

We clearly confirmed the existence of several innate immune parameters in the

esophagus epithelium of domesticated mammals. In this context, various PRRs which

trigger a first response of the innate immune system were demonstrated. Toll-like receptor

2 seems to play a pivotal role in this manner. This PRR was primarily expressed on the

epithelial cells of the stratum basale. Furthermore, cationic antimicrobial peptides

contribute to the maintainance of epithelial integrity. Within this study, the antimicrobial

peptides cathelicidin and ß-defensin 2 and 3 could be demonstrated. Hence, we

corroborated results obtained from studies on human intestinal and cutanous tissue,

which showed a co-expresseion of Toll-like receptors and antimicrobial peptides. The less

keratinised esophagus epithelium of the carnivorous cat was protected from exogenous

strain by an extraordinary high amount of antimicrobial peptides. In contrast, in the

epithelium of the herbivorous and omnivorous species a stronger keratinisation and a

characteristical epithelial layer structure guaranteed an effective mechanical barrier.

Summary

151

A detection of the soluble pathogen recognising ß-glucan receptors remained

inconclusive. Based on the comparison with publications, for example from marine

mammals, it can be conjectured that an epithelial exposition to higher amounts of

microorganisms is required to trigger a production of these PRRs. This hypothesis is in

line with the rather low number of microorganisms present in the esophagus of the

animals studied.

Additionally, the enzymatically active protein lysozyme could be demonstrated in the

mammalian esophagus epithelium. Thus, it can be assumed that this protein participates

as another parameter of the innate innate immune mechanisms present in the esophagus

epithelium.

Furthermore, cellular components of the innate immune system were present in the

esophagus epithelium. In this context, we demonstrated langerin (+) Langerhans cells in

basal layers of the murine and equine esophagus.

The immunohistochemical results were complemented by observations made by cryo

SEM. It could be shown for all species studied that the esophagus epithelium is covered

by a thin layer of bacteria and fungi. However, detailed analyses of the microbial

composition for each species have still to be conducted.

Finally, a substantial contribution to the actual discussion concerning the advantage of

formalin alternatives could be provided by the present study. On the one hand, the HOPE®

fixation method offers a rather cheap alternative, as the antibody amounts could be

reduced. On the other hand, an application in routine diagnostic is not advisable, because

of several variable parameters. It can be concluded that immunohistochemical results

always have to be discussed in connection to the fixation method applied. We clearly

demonstrated a diminished immunoreactivity for Ca-formol fixed samples; satisfactory

results were obtained, particularly, from samples fixed in Bouin`s solution.

It can be summarised that the present dissertation offers a detailed characterisation of the

innate immune parameters present in the esophagus epithelium of domesticated

mammals. Our findings might also contribute to the progress of research on human

esophageal illnesses, such as reflux esophagitis or Barrett`s esophagus. In this

connection, the use of already established animal models (mouse, rat, pig) could benefit

from our results to some extent.

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a

Danksagung

Ich danke allen, die mich auf meinem Weg bis an diese Station meines Lebens

begleitet haben.

Meinen besonderen Dank möchte ich meinem lieben Betreuer Prof. Dr. Wilfried

Meyer aussprechen. Durch seine Hilfsbereitschaft, fachlich kompetente

Unterstützung und Förderung und auch persönlichen Beistand, hat er mir viele

Türen geöffnet. Er hat es geschafft, dass ich mich an meine Zeit als seine letzte

Doktorandin sehr gerne zurück erinnern werde. Der „ewige Fibroblast“ und die

gute Zusammenarbeit in ganz Deutschland werden mir in schöner Erinnerung

bleiben.

Ich danke meinen Freundinnen und Kolleginnen Marion Gähle und Doris Walter

(„Mutti“) sehr herzlich für die unglaubliche Unterstützung bei der Bearbeitung

meiner zahlreichen Proben. Darüber hinaus gilt ihnen mein Dank aber ebenso

dafür, dass ich jeden Tag gerne ins Institut gekommen bin und mit ihnen lachen,

weinen und essen (!) konnte.

Weiterhin möchte ich der besten VMTA Praktikantin der Welt, Ellen Baumeister,

für ihre ungeheuer motivierte Mitarbeit an meinem Projekt meine besondere

Anerkennung aussprechen!

Kerstin Rohn danke für ihre hervorragende und schnelle Hilfe bei der EPON

Einbettung und der Herstellung der Semidünnschnitte.

Die wundervollen zeichnerischen Darstellungen der Oesophagusstruktur in

meiner Dissertation verdanke ich der Künstlerin Caren Imme von Stemm.

Bei PD Dr. Johannes Kazca und Prof. Dr. Johannes Seeger möchte ich mich

für die Realisierung und Unterstützung bei unserem Projekt der Cryo REM

Darstellung von Mikroorganismen im Oesophagus bedanken.

PD Dr. Anke Schnapper danke ich für die fachliche Unterstützung, die

technische Hilfe bei Word und viele tolle Gespräche.

Ich bedanke mich bei der H. Wilhelm Schaumann Stiftung für die finanzielle Unter-

stützung dieser Arbeit.

b

Den folgenden Angehörigen der Stiftung Tierärztliche Hochschule Hannover und

Kollegen aus der Kleintierpraxis danke ich für ihre Hilfe bei der Erlangung von

Oesophagusproben: Prof. Dr. Wolfgang Löscher und Dr. Maren Fedowitz für die

Bereitstellung der Rattenproben, Prof. Dr. Andrea Meyer-Lindenberg und Dr.

Miriam Hemmes für die Bereitstellung der Oesophagusproben von Katzen sowie

Prof. Dr. Gerhard Breves, Prof. Dr. Josef Kamphues und Prof. Dr. Thomas

Schnieder und ihren Arbeitsgruppen für die unkomplizierte Kooperation bei der

Gewinnung einer Vielzahl von Oesophagusproben von Kleinsäugern, Schweinen

und Hunden.

Die freundliche, kompetente Hilfe bei der statistischen Auswertung von Dr. Martin

Beyerbach sei ebenfalls dankend anerkannt.

Mein besonderer Dank für die unermüdliche Hilfe bei der sprachlichen Korrektur der

Arbeit gilt meiner Freundin Emily Northrup.

Ich danke meinen „acht Mädels“ für ihren treuen Beistand und viele tolle Momente

neben der Arbeit. Ohne euch wäre das Leben nur halb so lustig und schön!

Meiner besten Freundin, Bernadette Hellmann, danke ich für ihre tiefe Freundschaft.

Wann fahren wir wieder nach Berlin?!?

Meiner Tante und meinem Onkel, Monika und Klaus Hornickel, danke ich für die

Unterstützung bei meinem Studium und dafür, dass sie mir vertrauensvoll ihren süßen

Mops überlassen.

Bei meinem Freund, David Finkenstädt, bedanke ich mich dafür, dass er immer an

mich glaubt und mich bei der Realisierung meiner Ziele unterstützt. Das tiefe Vertrauen

zwischen uns hat mir viel Kraft gegeben und diese Leistung ermöglicht. „Lieben

belebt“…, das sagte schon Goethe, und er hatte Recht.

Der Dank an meine Mama, Karin Hornickel, ist auf Grund ihrer Leistung schwer mit

wenigen Worten zusammenfassen. Ich danke ihr für ihre Liebe, ihren Stolz, ihre

Förderung und für ein Vertrauenvolles Mutter-Tochter Verhältnis in guten und in

schlechten Zeiten. Du hast mir die Werkzeuge an die Hand gegeben, die das

Wahrwerden meiner Träume ermöglicht haben.

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