Immunopathogenesis of Primary Cicatricial Alopecia

135 Pages·2013·3.57 MB·English

Immunopathogenesis of Primary Cicatricial
Alopecia

A thesis submitted to the University of Manchester for the degree of
Master of Philosophy in the Faculty of Medical and Human Sciences

2013

Omima Abojela Eruk

School of Medicine

Institute of Inflammation & Repair

1
Table of contents Page
Table of contents 2
List of tables 7
List of figures 8
Abbreviations 10
Abstract 13
Dedication 14
Declaration 15
Copyright 15
Chapter 1: INTRODUCTION 16

1.1 Project Overview 17
1.2 Hair follicle anatomy and cycling 18
1.2.1 Functional anatomy 18
1.2.2Hair cycle 22
1.3 The bulge region 24
1.4 Epithelial hair follicle stem cells biology 24
1.5 The bulge region as a niche for the hair follicle stem cells 26
1.6 Epithelial hair follicle stem cells biomarkers 27

2
1.7 Hair follicle immunology 27
1.7.1 Mast cells 27
1.7.2 Macrophages 28
1.7.3 T-lymphocytes 30
1.7.4 Gamma-delta T cells 31
1.7.5 Natural killer and natural killer T cells 31
1.7.6 Langerhans cells 32
1.8 Hair follicle immune privilege 35
1.8.1 General features of immunoprivileged tissue sites 35
1.8.2 Immune privilege in the human hair follicle 35
1.8.2.1 Bulb immune privilege 35
1.8.2.2 Bulge immune privilege 36
1.9 Immunopathology of the hair follicle: Primary cicatricial
alopecia (PCA) 38
1.9.1 Clinical and histological classification of PCAs 40
1.9.2 Lichen planopilaris (LPP) 47
1.10 Pathogenesis of PCA, with emphasis on LPP 48
1.10.1 General principles of inflammatory PCA pathobiology 48

3
1.10.1.1 Inflammatory damage to hair follicle epithelial stem cells in LPP 49
1.10.1.2 Programmed organ deletion 49
1.10.2 Precipitating and predisposing factors in LPP 50
1.10.3 The potential role of the sebaceous gland in LPP 51
1.10.4 PPAR-gamma defect in LPP 51
1.11 Morphological and molecular characteristics of epithelial-
mesenchymal transition 53
1.12 Aims 56

Chapter 2: METHODS AND MATERIALS 57
2.1 Literature search and analysis strategy 58
2.1.1 Selection strategy of optimal hair follicle immune cell markers 58
2.1.2 Selection strategy for EMT markers 58
2.2 Buffers and Reagents 60
2.3 Human tissue sources 63
2.4 Sample Processing 63
2.4.1 Tissue sectioning 63
2.4.2 Deparaffinisation 64
2.4.3 Antigen retrieval 64
2.4.3.1 Heat induced epitope retrieval (HIER) 64

4
2.4.3.2 Proteolytic induced epitope retrieval (PIER) 65
2.5 Histological staining 65
2.5.1 Haematoxylin and eosin staining (H&E) 65
2.5.2 Toluidine blue staining protocol to identify mast cells 66
2.6 Immunohistochemical staining 67
2.6.1 Formalin-fixed samples 67
2.6.1.1 Envision method– Horseradish peroxidase on formalin fixed paraffin
embedded skin tissue FFPE (Dako- Hamburg, Germany) 67

2.6.1.2 Vectastain-ABC peroxidase method 70
2.6.2 Frozen samples 72
2.6.2.1 ABC immunostaining method on frozen normal skin tissue (Vector
Laboratories- Burlingame, Germany) 72
2.6.2.2 Envision method – Horseradish peroxidase on frozen normal skin
tissue (Dako- Hamburg, Germany) 72
2.7 Real time- quantitative polymerase chain reaction 74

2.8 Hair follicle compartment and bulge identification 75
2.8.1 Epithelial hair follicle compartment 75
2.8.2 Perifollicular mesenchyme compartment (connective tissue sheath) 78
2.8.3 Identifying of the bulge region in the hair follicle 79
2.8.4 Identifying of the bulge region in a lesional HF 80
5
2.8.5 Quantitative Immunohistomorphometry 80

Chapter 3: RESULT 82
3 Selection of optimally suited markers for studying hair
follicle immunopathology, bulge immune privilege,
and EMT 83
3.1 Regulatory T + cells 83
3.2 Natural killer and Natural killer T cells 84
3.3 Mast cell abnormalities in LPP 87
3.4 Macrophage abnormalities in LPP 92
3.5 Epithelial-mesenchymal transition (EMT) is a component of
LPP pathobiology: Preliminary evidence 96

3.6 Q-PCR results 101
Chapter 4: DISCUSSION 104
REFERENCE 112
Acknowledgements 135
Word count 29839

6
List of Tables:

Table 1.1 Definition of the corresponding anatomical terms in the hair follicle 21
Table 1.2 Characteristics of epithelial hair follicle stem cells (eHFSCs) 25
Table 1.3 The North American Hair Research Society (NAHRS) classification
of primary cicatricial alopecia (PCA) 40
Table 1.4 Clinical and histological features of PCA 42
Table 2.1 Antibodies used on formalin fixed paraffin embedded tissue,
employing the Envision kit HRP protocol (Dako-Hamburg, Germany) 68
Table 2.2 Antibodies used on formalin fixed paraffin embedded tissue
employing the Envision kit HRP protocol and the Vectastain
ABC-Peroxidase kit (Vector Laboratories- Burlingame, Germany) 71
Table 2.3 Antibodies used on frozen samples with two different kits; ABC-
peroxidase and Envision HRP kit 73
Table 2.4 Microscopic field placements for analysis: using the Keyence microscope
for analysing the defined reference region in the hair follicle 76
Table 2.5 Morphological and Immunohistochemical landmark criteria for
identifying the bulge in human HFs 79

7
List of Figures

Figure 1.1 Histomorphology of the hair follicle. 20
Figure 1.2 Normal hair cycles 23
Figure 1.3 Schematic representation of terminal hair follicle 33
Figure 1.4 Immune privilege of hair follicle bulge 37
Figure 1.5 Diagnostic signs of alopecia 39
Figure 1.6 Primary Cicatricial alopecia presented by lichen planopilaris (LPP) 47
Figure 1.7 several pathogenic factors that may be involved in hypotheses
for the development of primary cicatricial alopecia 52
Figure 1.8 Epithelial mesenchymal transition mechanisms 54
Figure 1.9 Model to describe the biological subtypes of EMT 55
Figure 2.1 Hair follicle epithelial compartments 77
Figure 2.2 Perifollicular mesenchyme analysis 78
Figure 2.3 Hair follicle bulge region identification 80
Figure 2.4 “Image J” semi-quantitative immunohistomorphometry 81
Figure 3.1 FOX P3+ cells in the normal human (healthy) skin tissue (FFPE) 84
Figure 3.2 CD161+cells in the FFPE normal (healthy) skin tissue 85
Figure 3.3 Vα24 +cells expression in the human hair follicle epithelium
in (LPP) using immunohistochemistry on frozen samples 86
8
Figure 3.4 Mast cell tryptase + cells in normal human skin tissue 88
Figure 3.5 Histological staining of LPP hair follicle using Toluidine blue 89
Figure3.6 Mast cell tryptase +cells in the 30μm perifollicular mesenchyme
compartments of LPP 90
Figure3.7 Mast cell tryptase +cells (MCT) in the 100μm perifollicular
mesenchyme compartments of LPP 91
Figure 3.8 CD163 + cells expression in the bulge epithelium of LPP hair follicle 93
Figure3.9 CD163 immune reactivity in defined 30 microns perifollicular Mesenchymal
compartments in LPP HF compared with HF from healthy scalp skin 94
Figure 3.10 CD163 +cells in defined 100 microns perifollicular mesenchymal
compartments in LPP HF compared with HF from healthy scalp skin 95
Figure 3.11 Vimentin+ cells in the bulge epithelium and perifollicular mesenchyme
adjacent to the bulge of LPP HF (30um) 97
Figure 3.12 Vimentin +cells in the bulge epithelium and perifollicular mesenchyme
Tissue adjacent to the bulge 98
Figure 3.13 E-Cadherin +cells in the bulge epithelium of LPP HF 99
Figure 3.14 E-Cadherin immune reactivity (IR) in hair follicle bulge epithelium
in LPP 100
Figure 3.15 Q-PCR of Vimentin gene expression in the bulge epithelia of the
lesional hair follicle 102
Figure 3.16 Q-PCR of E-Cadherin gene expression in the bulge epithelial of the
lesional hair follicle 103
9
Abbreviations:
α- MSH Alpha-melanocyte stimulating hormone

AD Autosomal dominant
AKN Acne keloidalis nuchae
AM Alopecia mucinosa
APC Antigen-presenting cells
APM Arrector pili muscle
AR Autosomal recessive
BMZ Basement membrane zone
CCCA Central centrifugal cicatricial alopecia
CCLE Chronic cutaneous lupus erythematosus
cDNA Complementary DNA
CLA Cutaneous leukocyte antigen
CTS Connective tissue sheath
DAB 3,3’-Diamiobenzidine
DCS Dissecting cellulitis of the scalp
dH20 Distilled water
dsDNA Double strand DNA
ECM Extracellular matrix
eHFSC Epithelial hair follicle stem cell
EM Electron microscope
EMT Epithelial mesenchymal transformation
EPDS Erosive pustular dermatosis of the scalp
Fas Factor of apoptosis stimulus
FasL Factor of apoptosis stimulus ligand
FB Foreign body
10