Studies in the expression and modulation of mucosal addressin cell adhesion molecule-1

219 Pages·2011·61.43 MB·English

by  Ala, A.

STUDIES
 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