Table Of ContentSTUDIES
IN
THE
EXPRESSION
AND
MODULATION
OF
MUCOSAL
ADDRESSIN
CELL
ADHESION
MOLECULE-1
(MAdCAM-1)
Thesis
submitted
for
the
degree
of
Doctor
of
Philosophy
Aftab
Ala
MB
BS
FRCP
2010
Centre
for
Hepatology
Division
of
Medicine
UCL
Medical
School
Royal
Free
Campus
University
College
London
1
SUPERVISORS
Professor
HJF
Hodgson
DM,
FRCP,
FMedSci
Sheila
Sherlock
Chair
of
Medicine
Centre
for
Hepatology
Division
of
Medicine
UCL
Medical
School
Royal
Free
Campus
University
College
London
Professor
AP
Dhillon
MA
MD
FRCP
FRCPath
Professor
of
Histopathology
Department
of
Cellular
Pathology
Royal
Free
Hospital,
London
2
Abstract
Introduction: The endothelial mucosal cell adhesion molecule (MAdCAM-1) is
considered to be critically important in recruiting lymphocytes expressing the α4β7 cell
surface integrin. In addition to its well-characterised role in the normal gastrointestinal
tract, there is emerging evidence of its role in liver and gastrointestinal inflammation. The
ability to detect MAdCAM-1 has thus far been challenging, hindering progress into studies
to explore its modulation.
Aims: (i) To characterise MAdCAM-1 in the liver and gut, (ii) establish an in vitro
model system of MAdCAM-1 and (iii) investigate the factors leading to its expression
and subsequent modulation.
Methods: I have described novel methods of detecting MAdCAM-1 by:
1. Characterising its presence in the human liver, gut and associated tissues e.g. pancreas.
2. Developing a reverse transcriptase–polymerase chain reaction (RT-PCR) technique so as
to detect MAdCAM-1 in the gastrointestinal system and thus quantify its expression using
Real-Time RT-PCR analysis.
3. Developing an in vitro cell culture system using the endothelial SVEC4-10 cells to express
and subsequently modulate the expression of MAdCAM-1.
Results: Using immunohistochemical methodology I found that in end stage chronic liver
disease, MAdCAM-1 is expressed primarily on the peribiliary plexus and lymphoid
aggregates, where it may facilitate lymphocyte egress. MAdCAM-1’s constitutive
expression was confirmed in histologically normal gut tissue and its upregulation was
demonstrated in ulcerative colitis and Crohn’s disease, particularly localised to the venular
endothelium of the lamina propria and follicular dendritic cells. MAdCAM-1 mRNA from
human gut was measured by a RT-PCR technique in which a 94 base pair product
consistent with human mucosal vascular MAdCAM-1 was detected in normal large bowel.
Real Time analysis confirmed that MAdCAM-1 was upregulated in end stage liver
disease. In a cell culture system MAdCAM-1 was shown to be upregulated by TNFα on
SVEC4-10 using immunofluoresence studies and its expression was further modulated by
steroids and anti-sense oligonucleotides.
Conclusion: The importance of MAdCAM-1 in the gastrointestinal system is emphasised
throughout. Our in vitro culture system utilising the SVEC endothelial cell line provides
the basis for studying the modulation of MAdCAM-1 expression.
3
Ethical
Approval
Approval for parts of the study involving human patients was obtained from The Royal
Free Hospital, Ethics Committee
(Ethics ID 5441, Project ID 5441)
4
Acknowledgments
I am deeply indebted to my mentor Professor Humphrey Hodgson for supporting me.
His guidance, knowledge, patience and wisdom have been invaluable before, during and
after the project.
I wish to thank Professor Amar Dhillon for his enormous enthusiasm, supervision and
expertise. I am thankful to David Brown, Korsa Khan and Ruth Jacobs for their
invaluable laboratory assistance, advice, and experience, all members of the Centre for
Hepatology, Royal Free Campus and Department of Histopathology for their assistance
whenever it was needed. I also wish to thank Dr Joseph Odin, Dr Swann Thung and Dr
Scott Friedman, for their help, advice and the use of laboratory space and materials
during my Advanced Hepatology Fellowship 2003-4, at Mount Sinai Hospital Medical
Center and Mount Sinai School of Medicine, New York, USA.
I am grateful to the Wellcome Trust, Digestive Diseases Foundation, The Tana Trust,
The British Transplant Society and The St John’s Ambulance Trust who supported me
and the study.
Finally, I thank my family without whom none of this would have been possible.
5
Table
of
Contents
Abstract
....................................................................................................................1
Ethical
Approval.............................................................................................................4
Acknowledgments.........................................................................................................5
List
of
figures................................................................................................................11
List
of
tables.................................................................................................................14
Publications
and
presentations
related
to
research....................................................15
List
of
Abbreviations....................................................................................................16
Synopsis
..................................................................................................................19
Chapter
1
The
role
of
cell
adhesion
molecules
in
leukocyte
recruitment
in
the
liver
and
gut
21
..................................................................................................................21
1.1
Background......................................................................................................22
1.1.1
Leukocyte
trafficking...........................................................................................22
1.1.2
Adhesion
molecules,
cell
trafficking
&
recruitment............................................23
1.1.2.1
Tethering
and
rolling.....................................................................................25
1.1.2.2
Activation
of
integrins...................................................................................25
1.1.2.3
Firm
adhesion
and
migration........................................................................27
1.1.3
Leukocyte
migration
into
the
liver
and
gut...................................................27
1.2
Classes
of
cell
adhesion
molecules
in
the
liver
and
gut...................................29
1.2.1
Selectins...............................................................................................................29
1.2.1.1
E-‐
selectin
(expressed
on
endothelial
cells)..................................................31
1.2.1.2
P-‐selectin
(expressed
on
endothelial
cells
and
platelets).............................31
1.2.1.3
L-‐selectin
(expressed
on
leukocytes)............................................................32
1.2.2
The
Immunoglobulin
(Ig)
Superfamily..................................................................32
1.2.2.1
Intercellular
adhesion
molecule-‐1
and
2
(ICAM-‐1,
ICAM-‐2)..........................34
1.2.2.2
Vascular
cell
adhesion
molecule-‐1
(VCAM-‐1)................................................34
1.2.2.3
Platelet
endothelial
cell
adhesion
molecule-‐1(PECAM-‐1)............................34
1.2.2.4
Mucosal
addressin
cell
adhesion
molecule-‐1
(MAdCAM-‐1).........................35
1.2.2.4.1
Expression
of
MAdCAM-‐1
during
development...........................
36
1.2.2.4.2
Regulation
of
MAdCAM-‐1
expression............................................40
1.2.2.5
Carcinoembryonic
antigen-‐related
cell
adhesion
molecule
(CEACAM-‐1).....41
1.2.3
Integrins........................................................................................................42
1.2.4
E-‐Cadherin.....................................................................................................43
1.2.5
CD44..............................................................................................................43
1.2.6
Vascular
adhesion
protein
(VAP-‐1)...............................................................44
1.3
Chemokines.....................................................................................................45
1.4
Regulation
and
synthesis
of
endothelial
adhesion
molecules.........................49
1.5
Adhesion
molecules
in
gastrointestinal
disease..............................................50
1.5.1
Gastrointestinal
infection.............................................................................50
6
1.5.2
Coeliac
Disease..............................................................................................51
1.5.3
Radiation
enteritis.........................................................................................51
1.5.4
Inflammatory
bowel
disease.........................................................................52
1.6
Entero-‐hepatic
recirculation
&
extra-‐intestinal
manifestations
of
IBD...........54
1.6.1
Extraintestinal
intestinal
manifestations
of
IBD......................................................54
1.7
Modulating
adhesion
molecule
expression
and
therapeutic
applications
of
anti-‐adhesion
therapy
in
IBD.......................................................................................56
1.8
Adhesion
molecules
in
liver
disease................................................................57
1.9
Liver
allograft
rejection...................................................................................60
1.10
Ischaemia/Reperfusion....................................................................................61
1.11
Aims
of
the
Study............................................................................................62
Chapter
2
MAdCAM-‐1
expression
in
the
liver,
gut
and
associated
tissues..............63
2.1
Overview
of
MAdCAM-‐1
expression
in
the
gastrointestinal
tract...................64
2.2
Experiment
1:
Morphological
survey
of
MAdCAM-‐1
expression
in
the
human
liver...............................................................................................................................65
2.2.1
Background...................................................................................................65
2.2.2
Hypothesis
and
Aims.....................................................................................65
2.2.3 Material
and
Methods..................................................................................66
2.2.3.1
Alkaline
Phosphatase
Anti-‐Alkaline
Phosphatase
(APAAP)
methodology.....67
2.2.3.2
Trilogy
methodology
as
a
form
of
antigen
retrieval......................................67
2.2.3.3
Immunoperoxidase
staining..........................................................................68
2.2.3.4
Dendritic
cell
and
lymphatic
vessel
staining.................................................69
2.2.3.5
Immunofluoresence
Microscopy..................................................................69
2.2.3.6
Immunohistochemical
Assessment...............................................................70
2.2.4 Results...........................................................................................................70
2.2.5
Discussion......................................................................................................80
2.3
Experiment
2:
Expression
of
MAdCAM-‐1
in
IBD.............................................85
2.3.1
Background...................................................................................................85
2.3.2
Hypothesis
and
Aims.....................................................................................85
2.3.3
Methods........................................................................................................85
2.3.3.1
Immunohistochemistry.................................................................................86
2.3.3.2
Morphometric
analysis..................................................................................86
2.3.4
Results...........................................................................................................87
2.3.5
Discussion......................................................................................................92
2.4
Experiment
3:
Expression
of
MAdCAM-‐1
in
the
human
pancreas..................96
2.4.1
Background...................................................................................................96
2.4.2
Methods........................................................................................................97
2.4.2.1
Morphological
analysis..................................................................................98
2.4.3
Results...........................................................................................................98
2.4.4
Discussion....................................................................................................103
7
Chapter
3
Developing
an
in
vitro
model
of
MAdCAM-‐1
expression........................105
3.1
Overview.......................................................................................................106
3.2
Developing
an
in
vitro
model
of
MAdCAM-‐1
expression
(8
chamber
well)..107
3.2.1
Background.......................................................................................................107
3.2.2
Aims ................................................................................................................. 107
3.2.3
Methods.............................................................................................................108
3.2.3.1
SVEC,
bEND
cells
and
Culture
Medium.......................................................108
3.2.3.2
Subculture
procedure
and
trypsinising
cells...............................................108
3.2.3.3
Immunofluoresence
staining
of
MAdCAM-‐1...............................................109
3.2.3.4
MAdCAM-‐1
and
time
course
of
its
expression
on
SVEC
and
Bend.3...........110
3.2.3.5
Image
Analysis............................................................................................110
3.2.3.6
Statistical
analysis.......................................................................................110
3.2.4
Results................................................................................................................111
3.2.4.1
Cell
viability................................................................................................111
3.2.4.2
Morphology
and
immunofluoresence
.......................................................111
3.2.4.3
Time
course
of
MAdCAM-‐1
immunofluoresence
induction
by
TNFα.........115
3.3
Developing
an
in
vitro
model
of
MAdCAM-‐1
expression
(96
well
plate).....117
3.3.1
Background.......................................................................................................117
3.3.2
Aims...................................................................................................................116
3.3.3
Method..............................................................................................................118
3.3.3.1
Image
Analysis............................................................................................118
3.3.4
Results
............................................................................................................119
3.3.5
Discussion..........................................................................................................120
Chapter
4
Qualitative
and
Quantitative
measurement
of
MAdCAM-‐1
expression
in
cells
........................................................................................................................122
4.1
Experiment
1:
Development
of
a
Reverse
Transcriptase-‐Polymerase
Chain
Reaction
(RT-‐PCR)
assay
to
assess
MAdCAM-‐1
expression
in
gastrointestinal
tissues.........................................................................................................................123
4.1.1
Background.......................................................................................................123
4.1.1.1
Human
and
Mouse
MAdCAM-‐1................................................................123
4.1.1.2
Polymerase
chain
reaction........................................................................128
4.1.2
Aim
...................................................................................................................128
4.1.3
Methods...........................................................................................................128
4.1.3.1
Reverse
Transcription................................................................................129
4.1.3.2
Polymerase
Chain
Reaction.......................................................................130
4.1.3.4
Gel
extraction
of
PCR
DNA........................................................................131
4.1.3.4
DNA
sequencing........................................................................................132
4.1.4
Results..............................................................................................................132
4.1.5
Discussion.........................................................................................................135
4.2
Experiment
2:
Developing
a
real
time
RT-‐PCR
method
to
quantify
gut
and
liver
tissue
MAdCAM-‐
1.............................................................................................137
4.2.1
Introduction
-‐
Real
time
PCR............................................................................137
4.2.1.1
Quantitation..............................................................................................137
8
4.2.2
Quantitation
of
Mouse
tissue
MAdCAM-‐1
mRNA
by
RT-‐PCR.........................138
4.2.2.1
Isolation
of
Total
RNA
from
tissue............................................................138
4.2.2.2
Reverse
Transcription...............................................................................139
4.2.2.3
Quantitative
Polymerase
Chain
Reaction.................................................139
4.2.3
Results............................................................................................................140
4.2.4
Discussion.......................................................................................................141
4.3
Development
and
use
of
a
Real
Time
PCR
assay
to
quantify
MAdCAM-‐1
mRNA
in
human
disease............................................................................................144
4.3.1
Isolation
of
Total
RNA
from
tissue..................................................................144
4.3.2
Reverse
Transcription.....................................................................................144
4.3.3
Quantitative
Polymerase
Chain
Reaction.......................................................144
4.3.4
Results............................................................................................................149
4.3.5 Discussion........................................................................................................151
Chapter
5
Modulation
of
MAdCAM-‐1
expression...................................................154
5.1
Overview.......................................................................................................155
5.2
Experiment
1:
Effect
of
modulating
MAdCAM-‐1
using
an
in
vitro
model.....156
5.2.1
Introduction..................................................................................................156
5.2.2
Methods........................................................................................................157
5.2.3
Results...........................................................................................................157
5.2.4
Discussion.....................................................................................................161
5.3
Experiment
2
Modulating
mouse
MAdCAM-‐1
expression
using
specific
antisense
oligonucleotides........................................................................................163
5.3.1
Introduction..........................................................................................................163
5.3.1.1
Binding
to
complementary
mRNA..........................................................164
5.3.1.2
Utilization
of
Rnase
H..............................................................................164
5.3.1.3
Intracellular
Anti-‐S
degradation..............................................................164
5.3.1.4
Non-‐antisense
effects.............................................................................165
5.4
Methods......................................................................................................166
5.4.1
Generation
and
design
of
antisense
oligonucleotides.................................166
5.4.2
Oligonucleotide
treatment...........................................................................167
5.4.3
Oligonucleotide
Uptake
in
endothelial
cells.................................................167
5.4.4
Quantifying
the
effect
of
different
oligonucleotides
and
varying
concentrations
on
MAdCAM-‐1
expression...............................................................168
5.5
Results........................................................................................................169
5.6
Discussion...................................................................................................172
Chapter
6
General
Discussion.................................................................................176
6.1
Role
and
expression
of
MAdCAM-‐1
in
tissues.............................................177
6.2
Other
observations
on
MadCAM-‐1..............................................................179
6.3
Detection
of
MAdCAM-‐1
expression
in
fluids..............................................182
6.4
Potential
of
imaging
MAdCAM-‐1
expression
in
inflammation.....................182
9
6.5
MAdCAM-‐1
as
a
therapeutic
target..............................................................183
6.5.1
Modulating
MAdCAM-‐1
and
α4β7
axis...........................................................183
6.5.2
Modulating
MAdCAM-‐1
using
Anti-‐MAdCAM-‐1
antibodies............................185
6.5.3
Modulating
MAdCAM-‐1
protein
synthesis:
applications
of
oligonucleotides-‐
silencing
MAdCAM-‐1
RNA.........................................................................................186
6.5.4
Chemokines
and
emerging
relationships
with
MAdCAM-‐1
and
its
modulation..................................................................................................................188
6.5.5
VAP
and
its
emerging
relationship
in
the
expression
MAdCAM-‐1..................190
6.5.6
Other
MAdCAM-‐1
related
targets...................................................................191
6.6
Future
work..................................................................................................193
References
................................................................................................................194
10
Description:SVEC4-10 using immunofluoresence studies and its expression was further
modulated by steroids and anti-sense oligonucleotides. Conclusion: The
importance
