Immunoregulation of acquired ocular immunobullous disease

296 Pages·2012·7.01 MB·English

by  Geraint Williams

Immunoregulation of Acquired Ocular
Immunobullous Disease

By
Geraint Powell Williams
MBBCh, BSc, MRCOphth

A thesis presented to The University of Birmingham
for the degree of Doctor of Philosophy

Academic Unit of Ophthalmology
School of Immunity and Infection
University of Birmingham

October 2011
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Abstract

Ocular Mucous Membrane Pemphigoid (OcMMP) is a blinding immunobullous disease,
characterised by auto-antibody driven conjunctival inflammation and scarring. My hypothesis
was that progressive fibrosis in OcMMP, occurring in the apparent absence of clinical
inflammation, was driven by underlying inflammatory processes.

I observed that in OcMMP, progressive scarring did occur in the apparent absence of
clinically identifiable inflammation and I was able to improve clinical documentation by
developing and validating an objective Fornix Depth Measurer (FDM) for assessment of
scarring.

I optimised non-invasive Ocular Surface Impression Cytology (OSIC) combined with flow
cytometry to characterise conjunctival leukocytes. I found that CD8αβ+ effector memory,
cytotoxic, mucosal-homing T cells were the dominant population in health. This population
was unaltered with age but CD4+ T cells, capable of producing IFN-γ, increased.

In OcMMP, the conjunctiva was characterised by decreased CD8+ lymphocytes and an
elevation in CD45INTCD11b+CD16+CD14- neutrophils. Although neutrophils correlated with
clinical inflammation, they were even present in the absence of identifiable conjunctivitis.
This elevation was associated with progression of scarring assessed by FDM, even in the
clinically Non-inflamed eye.

These findings confirmed my hypothesis and provide a platform for quantifying neutrophils as
a biomarker of sub-clinical inflammation and their role in the scarring process.
Conjunctival Inflammation: a historical perspective

“Hyperaemia of the conjunctiva may result from many causes, as the irritation produced
by cold wind, dust, or irritating vapours, such as tobacco smoke; overwork of the eyes,
actual or relative to their capabilities…

The amount of injection varies; in the worst cases, the whole of the ocular conjunctiva
presents a network, with very irregular meshes, formed by dilated and tortuous vessel;
these can be moved over the surface of the eye by rubbing the lid over them, and they can
be emptied by slight pressure; in both respects, as well as not being especially marked in
the circumcorneal zone, differing from the dilated vessels seen in inflammation of the
cornea or iris…

In slighter cases there are very few dilated vessels, and in in some they only become
visible after prolonged use of the eyes, or after exposure to wind, etc.”

Robert Brudenell Carter and William Adams Frost.
Affections of the eyelids, lacrymal apparatus, and conjunctiva.
In Ophthalmic Surgery.
London, Cassell & Co.1887 pp117-118
Acknowledgments

First, I would like to thank my supervisors, Saaeha Rauz (SR) and John Curnow for
making this project possible. I am very grateful to them for giving me the opportunity
to undertake my PhD, assisting with the ethics process and helping me learn about
these diseases, about immunology and about research methodology in general.

Many clinical and non-clinical colleagues have been enormously kind in teaching me
and include Graham Wallace and Alastair Denniston, who I would like to thank for
their help, their willingness to share ideas about immunology and invaluable insight
and Pete Nightingale for his help with the understanding of statistics. I am also very
grateful to the Wellcome Trust for generously sponsoring my fellowship.

I am extremely grateful to my many collaborators and co-investigators including
Robert Barry, Simon Bowman, Jonas Brane, Paul Cottrell (PC), John Dart, Lindsay
Durant, Matt Edmunds, Bertus Eksteen, Simon Evans (SE), Abdul-Jabbar Ghauri
(AG), John Hamburger (JH), Pete Hampson, John Hazeldine, Kath Howlett, Imran
Khan (IK), Steve Kissane, Sherine Kottoor, Heather Long (HL), Philip Murray,
Kadambari Oswal, Annette Pachnio (AP), Cherry Radford (CR), Siobhan Restorick,
Tariq Saeed (TS), Alex Sinclair, Adam Slater, Annelise Soulier, Helen Sue
Southworth (HSS), Sreekan Sreekanthan (SS), Stephen Taylor, Paul Tomlins (PT)
and Emma Yates who have contributed to the design and application of so many
aspects of the project and the many, many patients who have been so generous in
giving their time.

Finally, I would like to thank my wife Gemma. Her patience when yet another
weekend has been spent in front of my laptop has been saint-like. Getting me to
laugh when I’ve become unbearable has kept me sane. Her support, has kept me
going. Diolch.
Table of Contents
Chapter 1: General Introduction 1
1.1 The Ocular Surface 2
1.2 The Lacrimal Functional Unit 6
1.3 Immunoregulation of the Ocular Surface 8
1.3.1 Principles of immunity and inflammation 9
1.3.2 Mucosal Immunology 17
1.3.3 Ocular Surface Immunity 24
1.4 Ageing changes and the mucosal immune system 27
1.5 Cicatricial Conjunctivitis 29
1.6 Acquired Ocular Immunobullous Disease 33
1.7 Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis 34
1.8 Mucous Membrane Pemphigoid (MMP) 37
1.8.1 Aetiology and Pathogenesis of MMP 37
1.8.2 Antibodies in MMP 38
1.8.3 Inflammation in MMP 41
1.8.4 Cicatrisation in MMP 43
1.8.5 Clinical Features and Challenges in MMP 46
1.8.6 Clinical Assessment of Disease Activity in OcMMP 48
1.8.7 Clinical Assessment of Disease Damage in OcMMP 49
1.8.8 Therapeutic Intervention in OcMMP 52
1.9 Detecting Cellular Changes in the Ocular Surface 55
1.10 My hypothesis and its context 58
1.11 Aims of the thesis 58
1.12 Objectives for the thesis 59
Chapter 2: Materials and Methods 60
2.1 Clinical Studies 61
2.1.2 Ethical Approval 61
2.1.3 Study Recruitment 61
2.1.4 Clinical Documentation 63
2.1.5 Databases 63
2.1.6 Clinical Sampling 64
2.2 Ex vivo experimentation 65
2.2.1 Haematoxylin and Eosin staining of OSIC membranes 65
2.2.2 Cell Recovery from OSIC 65
2.2.3 Preparation of Lysed Blood 66
2.2.4 Flow Cytometry 66
2.2.5 Antibody Cell Surface Marker Staining 67
2.2.6 Intracellular Cytokine and Cytolytic Protein Staining 68
2.2.7 Transcription Factor Staining 69
2.2.8 Viral peptide MHC-I Tetramer Staining 69
2.3 Statistical analysis 71
Chapter 3: Measuring Clinical Parameters in Acquired Ocular 72
Immunobullous Disease
3.1 Introduction 73
3.1.1 Defining the clinical phenotype of patients with OcMMP 73
3.1.2 Determining an objective method for recording the conjunctival 74
cicatricial process
3.1.3 Determining an optimal system for accurately phenotyping disease 75
3.2 Methods 76
3.2.1 Defining the clinical phenotype of patients with OcMMP 76
3.2.2 Determining an objective method for recording the conjunctival 79
cicatricial process
3.2.2.1 Design of a bespoke Fornix Depth Measurer (FDM; BMEC 79
Version 1)
3.2.2.2 Enhancement of the FDM for enumerating symblephara (FDM; 83
BMEC Version 2)
3.2.3 Determining an optimal system for accurately phenotyping disease 86
3.3 Results 88
3.3.1 Defining the clinical phenotype of patients with OcMMP 88
3.3.2 Optimising a system for objectively measuring the cicatricial 100
process
3.3.2.1 FDM (BMEC Version 1) 100
3.3.2.2 FDM (BMEC Version 2) 106
3.3.3 Determining an optimal system for accurately phenotyping disease 108
3.4 Discussion 110
3.4.1 Defining the clinical phenotype of patients with OcMMP 110
3.4.2 Determining an objective method for recording the conjunctival 114
cicatricial process
3.4.3 Determining an optimal system for accurately phenotyping disease 117
Chapter 4: Characterising the healthy conjunctival epithelial leukocyte 119
population
4.1 Introduction 120
4.1.1 Optimisation of mucous membrane cellular profiles detection by 120
OSIC
4.1.2 Defining resident conjunctival leukocyte populations 121
4.1.3 Age-related changes in the healthy human conjunctival epithelium 121
4.1.4 Effector Function of the dominant conjunctival epithelial CD8+ T 122
cells
4.2 Methods 123
4.2.1 Optimisation of mucous membrane cellular profiles detection by 123
OSIC
4.2.2 Defining resident conjunctival leukocyte populations and age-related 124
changes in the healthy human conjunctival epithelium
4.2.3 Effector Function of the dominant conjunctival epithelial CD8+ T 126
cells
4.3 Results 129
4.3.1 Optimisation of mucous membrane cellular profiles detection by 129
OSIC
4.3.2 Defining resident conjunctival leukocyte populations 133
4.3.3 Age-related changes in the healthy human conjunctival epithelium 137
4.3.4 Effector Function of the dominant conjunctival epithelial CD8+ T 143
cells
4.4 Discussion 151
4.4.1 Optimisation of mucous membrane cellular profiles detection by 151
OSIC
4.4.2 Defining resident conjunctival leukocyte populations and age-related 152
changes in the healthy human conjunctival epithelium
4.4.3 Effector Function of the dominant conjunctival epithelial CD8+ T 156
cells
Chapter 5: Characterising the conjunctival epithelial leukocyte populations 160
in acquired ocular immunobullous disease
5.1 Introduction 161
5.1.1 Stevens-Johnson Syndrome (SJS) and Toxic Epidermal Necrolysis 162
(TEN)
5.1.2 Ocular Mucous Membrane Pemphigoid (OcMMP) 164
5.2 Methods 165
5.2.1 SJS-TEN Study 165
5.2.2 OcMMP Study 167
5.3 Results 170
5.3.1 SJS-TEN Study 170
5.3.2 OcMMP Study 179
5.4 Discussion 202
5.4.1 SJS-TEN Study 202
5.4.2 OcMMP Study 207
Chapter 6: General Discussion 213
6.1 Introduction 214

6.2 Measuring clinical parameters in acquired ocular immunobullous disease 216

6.3 The conjunctival epithelial leukocyte population in health and in acquired 220
ocular immunobullous disease

6.4 Conclusions 228
Chapter 7: Appendices 229
7.1 Clinical Record Form 230

7.2 Publications arising from the thesis 231

Chapter 8. List of References 232