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1. Auflage 2008

© 2008 by Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH, Gießen Printed in Germany

ISBN 978-3-939902-84-3

Verlag: DVG Service GmbH Friedrichstraße 17

35392 Gießen 0641/24466

geschaeftsstelle@dvg.net www.dvg.net

Tierärztliche Hochschule Hannover

Anatomisches Institut

Detailed analysis of immunoprivileged tissues in

skin appendages

INAUGURAL-DISSERTATION

Zur Erlangung des Grades einer

Doktorin der Veterinärmedizin

- Doctor medicinae veterinariae -

(Dr. med. vet.)

Vorgelegt von

Katja Christina Meyer

(Hamburg)

Hannover 2008

2

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

Stiftung Tierärztliche Hochschule Hannover

Anatomisches Institut

Histologie und Embryologie

Bischofsholer Damm 15

30173 Hannover

Univ. Prof. Dr. med. habil. Ralf Paus

Universität zu Lübeck

Klinik für Dermatologie, Allergologie und Venerologie

Ratzeburger Allee 160

23538 Lübeck

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

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

Tag der mündlichen Prüfung: 18. November 2008

In liebevollem Gedenken an meinen Vater

TABLE OF CONTENTS

Table of contents

Table of contents _________________________________________________________ 5

Abbreviations ____________________________________________________________ 9

Figures _________________________________________________________________ 12

Tables __________________________________________________________________ 16

1 INTRODUCTION _____________________________________________________ 17

2 LITERATURE ________________________________________________________ 20

2.1 A short synthesis of hair follicle biology ________________________________ 20

2.1.1 Hair follicle morphogenesis___________________________________________________ 22

2.1.2 Functional anatomy of the hair follicle __________________________________________ 24

2.1.3 Hair follicle cycle____________________________________________________________ 31

2.2 Sinus hair follicle biology: overview ____________________________________ 35

2.2.1 Sinus hair follicle morphogenesis______________________________________________ 36

2.2.2 Functional anatomy of the sinus hair follicle_____________________________________ 38

1.1.1 Sinus hair follicle cycle_______________________________________________________ 42

2.3 Murine nail apparatus: overview________________________________________ 44

2.3.1 Nail morphogenesis _________________________________________________________ 45

2.3.2 Functional anatomy of the nail ________________________________________________ 46

2.3.3 Growth of the nail ___________________________________________________________ 50

2.4 Immunological background for the current study ________________________ 51

2.4.1 Innate immune system_______________________________________________________ 51

2.4.2 Acquired immune system ____________________________________________________ 52

2.4.3 Immune privilege: Definition and basic characteristics ____________________________ 53

2.5 Immune privilege in skin appendages___________________________________ 56

2.5.1 The anagen hair bulb as an immunoprivileged site_______________________________ 59

2.5.2 Downregulation of MHC class Ia and NK cells___________________________________ 61

2.5.3 Infantile human nail matrix is a site of relative immune privilege ___________________ 64

2.6 The immune privilege collapse model of AA pathogenesis _______________ 67

Collapse and restoration of IP in the anagen hair bulb in vitro __________________________ 69

2.7 The human bulge region as a site of relative immune privilege? __________ 71

TABLE OF CONTENTS

2.8 Bulge IP collapse and the pathogenesis of PCA _________________________ 73

2.9 Function of immunoprivileged sites ____________________________________ 75

2.10 Immune privilege markers _____________________________________________ 77

2.10.1 MHC class I molecules ____________________________________________________ 78

2.10.2 β2-microglobulin__________________________________________________________ 81

2.10.3 MHC class II molecules ___________________________________________________ 82

2.10.4 MHC class Ib molecules ___________________________________________________ 84

2.10.5 CD4+ and CD8+ T cells ___________________________________________________ 85

2.10.6 α-MSH and ACTH ________________________________________________________ 86

2.10.7 TGF-β __________________________________________________________________ 89

2.10.8 MIF_____________________________________________________________________ 91

2.10.9 IDO_____________________________________________________________________ 92

2.10.10 CD200 __________________________________________________________________ 95

2.10.11 Mast cells _______________________________________________________________ 96

2.10.12 ICAM-1 ________________________________________________________________ 100

2.10.13 β-defensin 2 ____________________________________________________________ 101

2.11 K(D)PT – a candidate as hair growth modulator and IP restorer in anagen hair

bulbs? _____________________________________________________________________ 103

2.12 Questions addressed in this study ____________________________________ 105

2.13 Experimental design _________________________________________________ 105

3 Materials and methods ______________________________________________ 107

3.1 Tissue collection_____________________________________________________ 107

3.1.1 Human tissue collection_____________________________________________________ 107

3.1.2 Murine tissue collection _____________________________________________________ 108

3.2 Human hair follicle isolation __________________________________________ 110

3.3 Human HF organ culture______________________________________________ 111

3.4 Full thickness human scalp skin organ culture _________________________ 114

3.5 Histological stainings ________________________________________________ 115

3.5.1 Hematoxylin-Eosin staining__________________________________________________ 115

3.5.2 Trichromatic staining _______________________________________________________ 116

3.5.3 Toluidine blue staining ______________________________________________________ 116

3.5.4 Leder`s esterase staining ___________________________________________________ 117

TABLE OF CONTENTS

3.5.5 Ki-67 / TUNEL_____________________________________________________________ 118

3.6 Immunohistochemistry _______________________________________________ 120

3.6.1 Primary antibodies _________________________________________________________ 120

3.6.2 Avidin Biotin Complex-Peroxidase____________________________________________ 121

3.6.3 EnVision®-alkaline phosphatase _____________________________________________ 123

3.6.4 Immunofluorescence _______________________________________________________ 123

3.6.5 Tyramide signal amplification (TSA) __________________________________________ 124

3.7 Histomorphometry ___________________________________________________ 125

3.7.1 Assessment of hair cycle stages _____________________________________________ 125

3.7.2 Assessment of proliferating matrix keratinocytes _______________________________ 125

3.7.3 Assessment of immunostaining intensity ______________________________________ 126

3.7.4 Assessment of MHC class I IR in isolated HFs _________________________________ 127

3.7.5 Assessment of mast cells ___________________________________________________ 128

3.7.6 Assessment of MHC class II, CD4, CD54, CD11b, mast cells and c-kit positive cells 128

3.7.7 Microscopical equipment____________________________________________________ 129

3.7.8 Statistical analysis _________________________________________________________ 129

4 Results ____________________________________________________________ 131

4.1 Immune privilege and the human hair follicle bulge _____________________ 131

4.1.1 Demonstration of MHC class Ia and β2-microglobulin expression on CD200+ cells __ 131

4.1.2 Demonstration of MHC class II+ cells in the bulge ______________________________ 137

4.1.3 Demonstration of HLA-E expression in CD200+ bulge cells ______________________ 140

4.1.4 Locally generated immunosuppressants complement the bulge IP ________________ 141

4.1.5 Demonstration of macrophage migration inhibitory factor and indoleamine-2,3-

dioxygenase______________________________________________________________________ 144

4.1.6 Influence of IFN-γ on ectopic MHC class I protein expression in the bulge ORS_____ 149

4.2 Influence of the α-MSH related tripeptide K(D)PT on human hair follicle

biology in situ under pro-inflammatory conditions ____________________________ 151

4.2.1 Influence of K(D)PT on IFN-γ induced MHC class I and II protein expression _______ 151

4.2.2 Reaction of K(D)PT on the IFN-γ induced upregulation of total mast cell numbers and

mast cell degranulation ____________________________________________________________ 155

4.2.3 Influence of K(D)PT on the hair cycle _________________________________________ 157

4.2.4 Influence of K(D)PT on hair matrix keratinocyte proliferation and apoptosis ________ 158

4.3 Immune privilege and murine sinus hair follicles _______________________ 159

4.3.1 Demonstration of MHC class I molecules______________________________________ 159

4.3.2 Demonstration of MHC class II molecules _____________________________________ 161

TABLE OF CONTENTS

4.3.3 Demonstration of CD4+ T cells ______________________________________________ 163

4.3.4 Demonstration of CD11b molecules __________________________________________ 165

4.3.5 Demonstration of TGF-β1 molecules__________________________________________ 167

4.3.6 Demonstration of mast cells _________________________________________________ 169

4.4 Immune privilege and the murine mouse nail apparatus_________________ 171

4.4.1 Demonstration of MHC class I molecules______________________________________ 171

4.4.2 Demonstration of MHC class II molecules _____________________________________ 173

4.4.3 Demonstration of CD4+ T cells ______________________________________________ 175

4.4.4 Demonstration of TGF-β1 molecules__________________________________________ 176

4.4.5 Demonstration of CD54 molecules ___________________________________________ 177

4.4.6 Demonstration of mast cells _________________________________________________ 178

4.4.7 Demonstration of β-defensin 2 _______________________________________________ 180

5 Discussion _________________________________________________________ 181

5.1 Introductory remarks _________________________________________________ 181

5.2 Methods employed ___________________________________________________ 182

5.3 Immune privilege in the human hair follicle bulge, murine nail and sinus hair

follicle _____________________________________________________________________ 186

5.3.1 The human HF bulge _______________________________________________________ 186

5.3.2 The murine sinus hair follicle and nail apparatus________________________________ 193

5.3.3 Comparison of the human HF bulge, murine sinus hair follicle and nail apparatus ___ 200

5.4 Effects of K(D)PT on the hair follicle immune system ___________________ 202

5.5 Conclusions _________________________________________________________ 204

5.6 Perspectives_________________________________________________________ 205

6 Summary___________________________________________________________ 207

7 Zusammenfassung _________________________________________________ 209

8 References _________________________________________________________ 212

9 Annex______________________________________________________________ 257

ABBREVIATIONS

Abbreviations

AA Alopecia areata

α-MSH Alpha-melanocyte stimulating hormone

ABC Avidin-biotin complex

AMP Antimicrobial peptides

AP Alkaline phosphatases

APC Antigen presenting cell

APM Arrector pili muscle

BM Basement membrane

ECM Extracellular matrix

eSC Epithelial stem cell

CAP Cationic antimicrobial peptide

CD Cluster of differentiation

CDLE Chronic discoid lupus erythematosus

CGRP Calcitonine-gene related peptide

CK Cytokeratin

Col Collagen

CTS Connective tissue sheath

DAB 3,3’-diaminobenzidine

DAPI 4’,6-diamidin-2’-phenylindol-dihydrochlorid

DC Dendritic cell

DP Dermal papilla

DTH Delayed type hyperpsensitivity

EAE Experimental autoimmune encephalitis

FGF Fibroblast growth factor

Fig Figure

FITC Fluorescein isothiocyanate

FT Follicular trochanter

GFP Green fluorescent protein

ABBREVIATIONS

h hour

HF

HLA

HM

Hair follicle

Human leucocyte antigen

Hair matrix

HS Hair shaft

ICAM Intraepithelial cellular adhesion molecule

IDO 2,3 indoleamine-dioxygenase

IFN-γ Interferon-gamma

IP Immune privilege

IR Immunoreactivity

IRS Inner root sheath

ITIM Immunoreceptor tyrosine inhibitory motif

KIR Killer cell immunoglobulin-like receptor

Kit CD117

LC Langerhans cell

MBP Myelin basic protein

MC Melanocortin

MC-R Melanocortin receptor

MHC Major histocompatibility complex

MICA MHC class I chain-related A

min Minutes

MIF Macrophage migration inhibitory factor

Mitf Microphthalmia-associated transcription factor

MK Matrix keratinocyte

mSC Mesenchymal stem cell

NaCl Sodium chloride

NaOH Sodium hydroxide

NFκκκκB Nuclear factor of kappa light polypeptide gene enhancer in B-cells

NK Natural killer cell

ORS Outer root sheath

ABBREVIATIONS

PAMP Pathogen-associated molecular patterns

PPR Pattern recognition receptors

PBS Phosphate buffered saline

PCA Primary cicatricial alopecia

PICS Perifollicular inflammatory cell clusters

PNF Proximal nail fold

PNM Proximal nail matrix

POD Programmed organ deletion

POMC Pro-opiomelanocortin

RER Rough endoplasmatic reticulum

SEM Standard error of the mean

SG Sebaceous gland

SW Sweat gland

Tab Table

TAP Transporter in antigen presentation

TBS Tris buffered saline

TCR T cell receptor

TGF-β Transforming growth factor β

TLR Toll like receptor

TSA Tyramide signal amplification

VIP Vasointestinal peptide

FIGURES

Figures

Fig. 2.1 Three dimensional diagram of the mammalian skin (FUCHS 2007)........ 20

Fig. 2.2 Terminal human HF in anagen VI ............................................................ 25

Fig. 2.3 The human HF bulge ............................................................................... 26

Fig. 2.4 HF: Keratinocyte lineages and structure.................................................. 27

Fig. 2.5 Schematic drawing of a hair bulb............................................................. 28

Fig. 2.6 Morphology of human HFs in different hair cycle stages. ........................ 32

Fig. 2.7 The hair follicle cycle. .............................................................................. 33

Fig. 2.8 Diagram of active vibrissal follicle in adult mouse.................................... 36

Fig. 2.9 Diagram of stages 1-8 in development of vibrissal follicles in mouse. ..... 37

Fig. 2.10 Murine vibrissal follicles of the snout. ...................................................... 40

Fig. 2.11 Musculature of vibrissal follicles............................................................... 42

Fig. 2.12 Mouse vibrissal follicle cycle.................................................................... 43

Fig. 2.13 Main characteristics of the nail in different animals and humans............. 46

Fig. 2.14 Schematic longitudinal section of mouse hair follicle and nail unit........... 47

Fig. 2.15 Murine nail apparatus. ............................................................................. 48

Fig. 2.16 Diagram of innate and adaptive immune system..................................... 53

Fig. 2.17 Billingham’s experiment: Survival of epidermal melanocyte allotransplants

in the host anagen HF. ............................................................................ 58

Fig. 2.18 The anagen hair bulb............................................................................... 59

Fig. 2.19 Distribution of peri- and intrafollicular CD4+, CD8+ T cells and CD1a+

cells. ........................................................................................................ 60

Fig. 2.20 Activation of NK cell activity..................................................................... 62

Fig. 2.21 The similarity of anatomical structure between HF and nail..................... 64

Fig. 2.22 Distribution of HLA-ABC, CD4+ and CD8+ T cells in the murine nail

apparatus (ITO et al. 2008b)................................................................... 65

Fig. 2.23 ´Immune privilege collapse model` of alopecia areata pathogenesis....... 67

Fig. 2.24 The human HF bulge region .................................................................... 72

Fig. 2.25 Hypothesized model of the pathogenesis of cicatricial alopecia .............. 74

Fig. 2.26 The cycle-dependency of murine HF MHC class I antigen ...................... 80

FIGURES

Fig. 2.27 The expression of MHC class I pathway molecules on the murine anagen

HF............................................................................................................ 82

Fig. 2.28 The different roles of α-MSH as immunomodulator (LUGER et al. 2000) 87

Fig. 2.29 Molecular mechanisms of IDO-induced immunosuppression .................. 94

Fig. 2.30 Stimulatory and immunosuppressive functions of mast cells. .................. 98

Fig. 2.31 Biosynthesis of POMC peptides an natural melanocortins. ................... 104

Fig. 3.1 Human scalp skin specimen .................................................................. 107

Fig. 3.2 solation of human HFs........................................................................... 111

Fig. 3.3 Hair follicles in a 24-well plate ............................................................... 112

Fig. 3.4 Isolated hair follicles .............................................................................. 112

Fig. 3.5 Experimental design of the HF organ culture......................................... 113

Fig. 3.6 Full thickness human scalp skin punch biopsies in 6-well multi well plate

........................................................................................................................ 114

Fig. 3.7 Auber’s line marked in the human HF.................................................... 126

Fig. 3.8 Reference areas for the quantitative analysis of MHC class I IR ........... 127

Fig. 4.1 IR is downregulated in the human bulge region for MHC class I ........... 132

Fig. 4.2 Schematic drawing of MHC class I IR pattern ....................................... 133

Fig. 4.3 Quantitative immunohistochemistry for MHC class I.............................. 133

Fig. 4.4 MHC class I IR is downregulated in CD200+ bulge ORS cells .............. 134

Fig. 4.5 Schematic drawing of CD200 IR pattern................................................ 135

Fig. 4.6 IR in the human hair follicle for β2-microglobulin.................................... 136

Fig. 4.7 Schematic drawing of β2-microglobulin IR pattern ................................. 136

Fig. 4.8 Quantitative immunohistochemistry for β2-microglobulin ....................... 137

Fig. 4.9 IR in the human bulge region for MHC class II ...................................... 138

Fig. 4.10 MHC class II+ cells in the human hair follicle and human bulge region . 139

Fig. 4.11 Schematic drawing of MHC class II IR pattern ...................................... 139

Fig. 4.12 HLA-E expression by CD200+ cells in the human bulge, double

Immunofluorescence ............................................................................. 140

Fig. 4.13 Schematic drawing of MHC class II IR pattern ...................................... 140

Fig. 4.14 IR in the human bulge region for α-MSH ............................................... 141

Fig. 4.15 TGF-β2 is expressed in the bulge ORS................................................. 142

FIGURES

Fig. 4.16 Schematic drawing of α-MSH IR pattern................................................ 143

Fig. 4.17 Schematic drawing of TGF-β2 IR pattern .............................................. 143

Fig. 4.18 IR is upregulated in the human bulge region for MIF............................. 144

Fig. 4.19 Quantitative immunohistochemistry for MIF........................................... 145

Fig. 4.20 IR is upregulated in the human bulge region for IDO............................. 146

Fig. 4.21 IR in different parts of the proximal ORS for IDO................................... 147

Fig. 4.22 Quantitative immunohistochemistry for IDO .......................................... 147

Fig. 4.23 Schematic drawings of MIF and IDO IR pattern .................................... 148

Fig. 4.24 Influence of IFN-γ on MHC class I expression of human HFs in full

thickness organ culture ......................................................................... 149

Fig. 4.25 Quantitative analysis of MHC class I staining intensity in the bulge....... 150

Fig. 4.26 Isolated HFs stained for MHC class I..................................................... 151

Fig. 4.27 Quantitative immunohistochemistry for MHC class I in isolated treated HFs

........................................................................................................................ 152

Fig. 4.28 Quantitative analysis of the mean number of MHC class II+ cells ......... 153

Fig. 4.29 Staining intensity of MHC class II+ cells ................................................ 154

Fig. 4.30 Staining for mast cells............................................................................ 155

Fig. 4.31 Quantitative analysis of the number of mast cells and their degranulation

ratio ....................................................................................................... 156

Fig. 4.32 Influence of K(D)PT on the hair cycle stage .......................................... 157

Fig. 4.33 Ki-67 / TUNEL staining on human HFs.................................................. 158

Fig. 4.34 IR in the murine vibrissal follicle for MHC class I ................................... 159

Fig. 4.35 Quantitative immunohistochemistry for MHC class I.............................. 160

Fig. 4.36 Schematic drawing of MHC class I IR pattern ....................................... 160

Fig. 4.37 Schematic drawing of MHC class II IR pattern ...................................... 161

Fig. 4.38 MHC class II positive cells in the murine vibrissal follicle....................... 162

Fig. 4.39 IR in the murine vibrissal follicle for CD4+ T cells.................................. 163

Fig. 4.40 Quantitative immunohistochemistry for CD4.......................................... 164

Fig. 4.41 Schematic drawing of CD4 pattern ........................................................ 164

Fig. 4.42 IR in the murine vibrissal follicle for CD11b ........................................... 165

Fig. 4.43 Quantitative immunohistochemistry for CD11b...................................... 166

FIGURES

Fig. 4.44 Schematic drawing of CD11b IR pattern................................................ 166

Fig. 4.45 IR in the murine vibrissal follicle for TGF-β1 on cryosections ................ 167

Fig. 4.46 IR in the murine vibrissal follicle for TGF-β1 on Bouin fixated specimens

........................................................................................................................ 168

Fig. 4.47 Schematic drawing of TGF-β1 pattern................................................... 168

Fig. 4.48 Demonstration of mast cells in the murine vibrissal follicle .................... 169

Fig. 4.49 Quantitative immunohistochemistry for mast cells ................................. 170

Fig. 4.50 Schematic drawing of mast cell expression pattern............................... 170

Fig. 4.51 IR in the murine nail apparatus for MHC class I .................................... 171

Fig. 4.52 Quantitative immunohistochemistry for MHC class I.............................. 172

Fig. 4.53 Schematic drawing of MHC class I expression pattern.......................... 172

Fig. 4.54 IR in the murine nail apparatus for MHC class II ................................... 173

Fig. 4.55 Quantitative immunohistochemistry for MHC class II............................. 174

Fig. 4.56 Schematic drawing of MHC class II expression pattern......................... 174

Fig. 4.57 IR in the murine nail apparatus for CD4+ T cells ................................... 175

Fig. 4.58 Schematic drawing of CD4 expression pattern...................................... 175

Fig. 4.59 IR in the murine nail apparatus for TGF-β1 ........................................... 176

Fig. 4.60 Schematic drawing of TGF-β1 expression pattern................................. 176

Fig. 4.61 IR in the murine nail apparatus for CD54............................................... 177

Fig. 4.62 Schematic drawing of CD54 expression pattern.................................... 177

Fig. 4.63 IR in the murine nail apparatus for mast cells........................................ 178

Fig. 4.64 Quantitative immunohistochemistry for MHC class I.............................. 179

Fig. 4.65 Schematic drawing of mast cell expression pattern............................... 179

Fig. 4.66 IR in the murine nail apparatus for β-defensin 2 .................................... 180

Fig. 4.67 Schematic drawing of β-defensin 2 expression pattern ......................... 180

TABLES

Tables

Tab. 2.1 Basic data on human HFs ....................................................................... 21

Tab. 2.2 HF morphogenesis in mice...................................................................... 23

Tab. 2.3 Glossary of anatomical and trichology terms........................................... 30

Tab. 2.4 Human nail apparatus – basic data ......................................................... 49

Tab. 3.1 Primary antibodies and secondary detection systems........................... 120

Tab. 3.2 Primary anti-mouse antibodies and secondary detection systems ........ 121

INTRODUCTION

17

1 INTRODUCTION

Immunologically ´privileged` sites or well-defined tissue compartments such as

present in the eye, brain, fetus, brain, and testes, have been fascinating objects of

research since the 80s and 90s. Immune privilege (IP) describes immunosuppressive

mechanisms that inhibit antigen presentation, and subsequent immune responses, in

a particular anatomical site. Immune privilege is thought to protect vulnerable tissues

with poor regenerative potential from excessive tissue damage caused by an

unrestricted immune response (MEDAWAR 1948; BILLINGHAM and SILVERS 1971;

STREILEIN 1993).

In skin appendages it has been shown previously that the proximal anagen bulb of

hair follicles (HF) (PAUS et al. 1999b, 2005; CHRISTOPH et al. 2000; ITO et al.

2004, 2007; GILHAR and KALISH 2006; GILHAR et al. 2007) and the human

proximal nail matrix (ITO et al. 2005c) are prominent sites of relative IP. In addition,

recent gene and protein expression data (MORRIS et al. 2004; TUMBAR et al. 2004;

COTSARELIS 2006b) have raised the possibility that follicular IP may not be limited

to the anagen hair bulb, but also to the epithelial stem cell region in the outer root

sheath (ORS) of HFs, termed the bulge region. So far, nothing is known about the

murine nail apparatus or vibrissal follicle.

The role of IP in the HF is currently unknown. Interestingly, collapse of IP in the

anagen hair bulb is thought to be central to the pathogenesis of the organ-specific

autoimmune condition, alopecia areata (PAUS et al. 2003). Therefore, it is possible

that IP evolved to reduce the risk of autoimmune hair-loss developing in an individual

creature, where loss of hair could threaten the ongoing survival of that individual

(PAUS et al. 2003). Since protection of bulge epithelial stem cells from immune

destruction is essential for preserving the regenerative and cycling capacity of HFs

(PAUS and COTSARELIS 1999; COTSARELIS 2006a; TIEDE et al. 2007a), it would

make sense if the bulge region also had established a relative IP. Convincing

treatment and management of alopecia areata and other hair loss disorders are still

INTRODUCTION

18

missing. Therefore, it is of paramount importance, firstly, to better characterize

immunoprivileged sites and secondly, to create substances and find mechanisms to

maintain and to restore IP.

Although limited gene expression data from isolated, human and mouse bulge-

derived cells suggested the existence of a second area of intrafollicular IP

(COTSARELIS 2006a), convincing protein evidence for this is still missing. Since IP

is a phenomenon that is based on functional protein expression patterns of entire

tissue compartments, not on gene expression patterns of individual cells in culture

(PAUS et al. 2005; NIEDERKORN 2006) we aimed in the current study to generate

protein evidence in situ [i.e. immunoreactivity (IR) evidence] that would support or

refute the hypothesis of bulge IP in human HFs. For this purpose, both routine and

increased-sensitivity immunohistochemical staining techniques were employed, and

the corresponding IR patterns were evaluated by quantitative immunohistochemistry.

This was complemented with histochemical and histomorphometric assessments. In

order to obtain functional evidence, we performed full thickness human scalp skin

organ cultures to investigate whether interferon-γ (IFN-γ), a key inducer of IP

collapse in hair bulbs, has a similar effect on the putative bulge IP.

In addition, we have evaluated a novel, synthetic α-MSH -related tripeptide [K(D)PT],

which is currently examined in different pre-clinical assay systems as a potential new

immunosuppressant with multiple clinical indication, including the possibility that it

may restore IP collapse. Therefore, the effects of K(D)PT on HF IP and biology were

examined in microdissected, organ-cultured human scalp HFs (PHILPOTT et al.

1990; BODO et al. 2007, 2008; VAN BEEK et al. 2008) under pro-inflammatory

conditions [(i.e. addition of interferon-gamma to the medium (ITO et al. 2004)].

Moreover, as an additional contribution to the charting of ´white spots` on the map of

cutaneous immunobiology, we have utilized this opportunity to clarify the IP status of

two other important skin appendages in the mammals, namely mouse sinus hair

follicles and the murine nail/claw apparatus, since these appendages still remain to

INTRODUCTION

19

be carefully characterized with respect to their IP status. The immunological

characterization of murine sinus hair follicles and nail apparatus and their IP status is

of great importance: Although vibrissal follicles differ substantially e.g. from pelage

and human terminal scalp HFs in their cycling characteristics, architecture and

innervation (DAVIDSON and HARDY 1952; MEYER 1999), they are frequently

employed in basic hair research and in drug screening assays – despite the fact that

their immunology remains largely obscure. Even less is known about the murine nail

apparatus from an immunological point of view. This may reflect the lamentable

general lack of interest in the life sciences community in something as supposedly

´profane` as nails, although many new mouse mutants display nail abnormalities and

although nail disorders in humans quite often coincide with other dermatological

disorders (CYGAN et al. 1997; AHMAD et al. 1998; GODWIN and CAPECCHI 1998;

VOLLRATH et al. 1998; KAWAKAMI et al. 2000; MECKLENBURG et al. 2004, 2005;

MOOKHERJEE et al. 2006; NAKAMURA and ISHIKAWA 2008).

In the following, after a short introduction into relevant essentials of HF, sinus hair

follicle and nail apparatus biology, the current state of research on immune privilege

in general will briefly be summarized, and key open questions will be delineated.

Subsequently, we critically discuss which relevant immune privilege markers have

been proposed in the past, with emphasis on markers that may be relevant for the

immune privilege in human HFs, and succinctly explain relevant background

information on those markers that were selected for study in the current context.

The experimental work for this thesis was performed in close collaboration with the

Department of Dermatology (Prof. Dr. R. Paus), University of Lübeck, and was

integrated into an ongoing, industry research project that exploited human HFs as an

innovative and instructive screening tool for identifying promising new drug

candidates (here: K(D)PT).

LITERATURE

20

2 LITERATURE

2.1 A short synthesis of hair follicle biology

The skin and its appendages have many different tasks to fulfill. The hair, which is

the main product of the HF, has several functions, i.e. protection against

environmental traumata, thermoregulation, social communication, mimicry and to act

as a container for sequestering and excreting unwanted compounds (PAUS and

COTSARELIS 1999; STENN and PAUS 2001). The importance of every function

depends on the mammalian species, possibly its domestication level, and the

environment. The HF is one of the most complex micro organs of the mammalian

body and the only organ, which permanently and lifelong regenerates through the so-

called HF cycle. The cycle length varies species-specific and location-specific, e.g., it

lasts years in humans and weeks in mice. But the developmental and cycle stages as

well as the basic transformations of the HF underlie the same pattern in human and

murine skin (KLIGMAN 1959) (Fig. 2.1).

Fig. 2.1 Three dimensional diagram of the mammalian skin (FUCHS 2007)

LITERATURE

21

Tab. 2.1 Basic data on human HFs Modified after PAUS et al. 2007

Total number ~ 5 000 000 (mostly vellus!) Number of scalp hair follicles

~ 100 000 Blondes: + 20% Redheads: - 20%

Average density (scalp) terminal + vellus

1135/cm2 (newborn), 615/cm2 (20-30 years), 485/cm2 (30-50 years), 435/cm2 60-80 (years) Asian: lower density; terminal only: ca 250/cm2; bald scalp (45-70 years): 330/cm2, highest density: cheek + forehead!

Hair embryology

Development progresses at fixed intervals (274-350mm) in cephalocaudal direction, first visible in eyebrow, upper lip and chin region (9th week)

Hair cycle distribution (term. scalp hair)

Anagen: 85-90%, Telogen: 10-15 %, Catagen: < 1%

Duration of hair cycle phases (terminal scalp hair)

Anagen: 2-5 years, Catagen: 2-3 weeks, Telogen: 3 months + location-specific differences (terminal moustache: 4-14 weeks, terminal arms: 6-12 weeks, terminal legs: 19-26 weeks, vellus: 6-12 weeks; premature anagen induction induced by plucking or telogen hair shafts (depilation); estrogens prolong anagen; thyroxine promotes growth, corticosteroids retard anagen onset

Number of lifetime cycles

~10-20

Physiological hair shedding rate (scalp)

~100-200/day (substantial interindividual and seasonal variations)

Hair shaft production rate (scalp)

~35mm/day, 1cm/month; hair production is not influenced by cutting/shaving; estrogens reduce hair growth rate; androgens increase hair growth rate and hair diameter in androgen-dependent sites (beard)

Hair shaft ∅ and length

Vellus: <0.03 mm; 1-2 mm Terminal: >0.06 mm; 1mm

Hair shaft structures Cuticle (outside), cortex, medulla (centre), cuticle maintains hair fibre integrity; cortex contains bulk of hair keratins and keratin-associated proteins; hair fibre strength is largely due to disulfide bonding; medulla consists of loosely connected trichocytes with large intercellular air spaces; provides insulation

Hair graying (canities)

Generally commences in the third decade of life on the temples, spreading later to crown and occiput; by the age of 50 years, 50% or the population has at least 50% grey hair

Hair patterns Pubic hair: horizontal (90% of women, 20% of men), acuminate (10% of women, 50% of men); diffuse chest hair: normally grows only in men, after puberty (until 6th decade); axillary: appears about 2 years after first pubic hairs; trichoglyphics: single, clockwise parietal present in 95% of individuals

Outer root sheath (ORS)

Outermost sheath of HF keratinocytes, merges distally into the basal layer of the epidermis and proximally into the hair bulb

Sebaceous gland (SG)

Glandular structure close to the insertion of the APM with holocrine function, lipid-filled sebocytes

Terminal hair Large, usually pigmented and medullated hair Vibrissae = Sinus hair follicle

Special sensory HFs with unique anatomy and biology, found in different regions of animal skin, but not in humans; largest and most densely innervated HFs with special sinusoid blood supply; first HFs to develop

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22

2.1.1 Hair follicle morphogenesis

HF morphogenesis is influenced and governed by a plethora of growth factors,

growth factor antagonists, adhesion molecules and intracellular signal transduction

components (BOTCHKAREV and PAUS 2003). At defined time points during fetal

(humans) and perinatal (rodents) skin development, HF morphogenesis begins from

small epithelial placodes (hair germs) in the epidermis above a mesenchymal

condensation (Fig. 2.2). In the following a rapid progress to the generation of

multicylindric, mature pilosebaceous units (vellus = primary HF sebaceous gland) in

the hominids, including the humans, or the HF complex (primary HF, apocrine tubular

gland, sebaceous gland) in the other mammalian groups occurs. These epidermal

keratinocytes are stimulated to commit HF specific differentiation, and the

mesenchymal cells, forming the dermal papilla (DP), send each other signals to

achieve progression to the next developmental stage. Thus, the epidermal pegs grow

downward into the dermis as a solid column of proliferating cells to enclose dermal

papillary cells and to construct the hair bulb. The hair bulb is the location, where rapid

proliferation and differentiation of the keratinocytes occurs. In the following, six

distinct cell compartments are formed: medulla, cortex and cuticle of the HS, the

cuticle and the Huxley and Henle layers of the inner root sheath (IRS). The latter

separates the HS from the ORS, which forms the external concentric layer of

epithelial cells in the HF (SENGEL 1976).

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23

Tab. 2.2 HF morphogenesis in mice Stage 0: Accumulation of nuclei (pre-germ), stage 1: Epidermal thickening (hair peg), stage 2: Forming of a broad column with concentrically arranged keratinocytes, stage 3: DP at the proximal end of the hair peg, stage 4: Henle’s layer of the IRS develops as a cone-shaped structure above the DP, stage 5: elongation of the IRS halfway up (hair cone), stage 6: HF reached the deep hypodermis (subcutis) and hair canal is visible, stage 7: the tip of the HS leaves the IRS and enters the hair canal, stage 8: HF acquires its maximal length and reaches the hypodermal muscle layer (panniculus carnosus) (PAUS et al. 1999b)

Eight defined gradual steps of HF morphogenesis can be distinguished, which

underlie different mechanisms and factors. In stage 0, epidermal nuclei accumulate

and form the so-called pregerm (PINKUS 1958), which develops in stage 1 into a

circumscribed epidermal thickening of enlarged keratinocytes in the basal layer of the

epidermis, termed the hair peg (DRY 1926). Forming of a broad column with

concentrically arranged epithelial keratinocytes around the follicular axis ends stage

2. At the proximal end of the column fibroblasts condensate and form the DP (stage

3). The hair peg elongates and the IRS starts to develop as a cone-shaped structure

(stage 4, 5), which in pigmented skin is visible by its melanin formation (PAUS et al.

1999b). In stage 6 the HF reaches the deep hypodermis and a hair canal is formed.

This is followed by the entering of the HS into the hair canal (stage 7). By reaching

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24

the hypodermal muscle layer (panniculus carnosus), if present (stage 6) the HF has

its maximal length and its prominent HS emerges through the epidermis (stage 7).

This event determines the end of morphogenesis and the onset of the first hair cycle.

First recognizable cyclic changes of HF activity start when the HF enters a stage of

physiological apoptosis-driven involution (catagen) (STRAILE et al. 1961;

DEPLEWSKI and ROSENFIELD 2000; STENN and PAUS 2001).

This course of morphogenesis occurs in humans but also in sparsely and densely

haired mammals, like pigs or mice (Fig. 2.2); the latter species are often used as a

model for the human skin (MEYER 1986, 2009; MEYER and GOERGEN 1986;

PAUS et al. 1999b)

2.1.2 Functional anatomy of the hair follicle

The human skin contains about 5 million HFs, of which mostly are vellus HFs.

Thereof are 100.000 HFs prominently displayed on the scalp (plus those of

eyelashes and eyebrows) (DAWBER 1997; PAUS and PEKER 2003; PAUS and

FOITZIK 2004). HFs can be divided into three different types: lanugo, vellus and

terminal (primary) HFs. Although the different types of HFs follow the same

construction principles of functional bioarchitecture, they display some structural and

pigmentary differences (DAWBER 1997; PAUS and COTSARELIS 1999). Most of

the HFs in the skin are of the vellus type. In contrary, the human scalp skin is

basically covered with terminal HFs only.

Every mature anagen scalp HF displays the shape of an inverted wine glass into

whose calyx an onion-like structure, the follicular dermal papilla is located. The

architecture of the HF is constructed on the need or key function as a fibre production

facility, whose outwards-growing hair shaft has to be carefully protected on its way up

to the skin surface. Interaction with the surrounding dermis would provoke infection

and therefore has to be prevented. The directional growth is based on guiding

structures and slippage planes: Terminally differentiated keratinocytes form a

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25

hardened inner cylinder (i.e. IRS) and guide the central hair shaft. In addition, the

companion layer of the ORS functions as a slippage plane, and facilitates the

outgrowing of the hair shaft together with the IRS (STENN and PAUS 2001;

LANGBEIN et al. 2002; PAUS and PEKER 2003).

The HF consists of eight concentric cylinders, forming the epithelial HF

compartments: ORS, companion layer (ORS), Henle’s layer (IRS), Huxley’s layer

(IRS), cuticle (IRS), as well as cuticle, cortex and medulla of the hair shaft (Fig. 2.2).

Each of these cylinders were formed from a distinct lineage of epithelial

differentiation and differ in structural proteins (e.g. hair keratins trichohyalin), enzyme

activities or adhesion and matrix molecules (POWELL and ROGERS 1997;

LANGBEIN et al. 1999, 2001).

Fig. 2.2 Terminal human HF in anagen VI Proximal HF (PAUS et al. 2007)

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26

Progeny of eSCs generate the compartments of ORS, IRS, hair matrix, and HS.

These slow-cycling, ´label retaining` eHFSC are present throughout the entire lifetime

of the HF, and are vital as a major site of eHFSCs to the regeneration and cycling

capacity of the HF (COTSARELIS et al. 1990, 1999; LYLE et al. 1999; TUMBAR et

al. 2004; OHYAMA et al. 2006; TIEDE et al. 2007a, 2007b). These eSCs reside in an

area of the outer root sheath (ORS), called the bulge (Fig. 2.3). The bulge is located

at the insertion point of the arrector pili muscle (APM) below the sebaceous gland

(SG) duct and indicates the lowermost point of the ´permanent` HF during hair

cycling (COTSARELIS 2006a) (Figs. 2.3, 2.4 A, 2.24)

Fig. 2.3 The human HF bulge The bulge region is located at the insertion point of the arrector pili muscle (APM) below the sebaceous gland (SG) duct and indicates the lowermost point of the “permanent” HF during hair cycling. In the bulge reside slow-cycling eHFSC, which are vital for the regeneration and cycling capacity of the HF. (FUCHS 2007)

In mouse and fetal human HFs a prominent swelling or protrusion of the ORS defines

the localization of the bulge, whereas in human skin the bulge is more difficult to

detect, because such a major ORS protrusion is usually very difficult to see

(COTSARELIS 2006a). In human skin, the insertion point of the APM and the

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27

recently found characteristic structure, the so-called ´follicular trochanter` can provide

a useful histological demarcation of the human bulge (TIEDE et al. 2007a).

These eSCs, from which the ORS is generated by transient amplifying cells

(COTSARELIS et al. 1990), reside permanently in the bulge region, while those

transient amplifying cells have been postulated to arise from a second population of

stem cells that have become deposited in the secondary hair germ (PANTELEYEV et

al. 2001; BLANPAIN et al. 2004; CHRISTIANO 2004) (Fig. 2.4 A) These cells

construct IRS, hair matrix and HS. However, until now convincing proof for the latter

concept is missing. In genetically engineered mice, bulge stem cells have been

demonstrated to generate all epithelial cells lineages, including IRS, hair matrix, and

HS (TAYLOR et al. 2000; OSHIMA et al. 2001; BLANPAIN et al. 2004).

Fig. 2.4 HF: Keratinocyte lineages and structure Keratinocyte lineages in the HF (A) (PAUS et al. 2007), structure of the human HF (B) (WHITING 2004)

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28

The anagen hair bulb (Fig. 2.5) is one of the key compartments of the HF and

constitutes the actual HS factory, in which one of the most rapidly proliferating cell

populations are found in any mammalian tissue: the keratinocytes of the one-layered

hair matrix. In the precortical hair matrix, which is situated above the DP, these cells

initiate their terminal differentiation to trichocytes. Later on, they receive

melanosomes from the melanocytes of the HF pigmentary unit for HS pigmentation.

During further process of differentiation into HS cuticle, cortex and medulla, the

keratinocytes express a defined set of keratins (SLOMINSKI and PAUS 1993a;

TOBIN and PAUS 2001). Matrix cells, medulla, cortex, IRS and ORS represent

ectodermal derivatives, whereas the DP, the CTS and the hyaline membrane,

separating the CTS from the ORS, are derivatives of the neural crest.

Fig. 2.5 Schematic drawing of a hair bulb DP=dermal papilla, CTS, connenctive tissue sheath, bORS=basal layer of the ORS, cl=companion layer, He=Henle layer, Hu=Huxley layer, icu=cuticle of the IRS, Cu=hair shaft cuticle, ma/co=hair shaft medulla and cortex, gp=germinative pool

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29

The diameter and volume of the DP determines the number of specialized fibroblasts

and is an indicator for its secretory power for the release of ´papilla morphogens`

(JAHODA and REYNOLDS 1996; PAUS et al. 1999b). Thus the larger the DP is the

bigger the HF and the diameter of the hair shaft. If a DP is destroyed, it can be fully

reconstituted from the proximal connective tissue sheath (CTS) of the HF (JAHODA

1992; JAHODA and REYNOLDS 1996; REYNOLDS et al. 1999), which harbors

mSCs (LAKO et al. 2002; JAHODA 2003). The exchange (so-called trafficking) of

fibroblasts between DP and the proximal CTS occurs during each telogen-anagen-

catagen transformation and results in substantial changes in DP volume and cell

content (TOBIN et al. 2003).

The angle of the hair shaft is dependent on the action of the arrector pili muscle. The

muscle is under adrenergic control, and thus involuntarily contracts in situations of

sudden stress, anxiety or anger, ´making one’s hair stand-up` (PAUS and PEKER

2003). However, in humans this capacity became less important compared to

animals. In human scalp skin, a single APM structure is shared by all the follicles

within the so-called follicular unit, a defined group of 2–4 terminal and 1–2 vellus

(HEADINGTON 1984), joining bulky cords of muscle fibres at one pole of the

follicular unit at the upper isthmus level (POBLET et al. 2002).

The follicular innervation system is responsible for the recognition and signaling of

sensitive tactile stimuli (e.g. hair shaft movements caused by wind, insects, stroking).

In addition, the follicular neural plexus may also have important trophic and

regulatory functions by the release of neurotransmitters, neuropeptides and

neurotrophins (BOTCHKAREV et al. 1997, 1998a, 1998b, 1999; PAUS et al. 1997;

PETERS et al. 1999, 2002a). The bulge and isthmus region of human HFs contain a

particularly dense network of sensory and autonomic nerves, as well as numerous

Merkel cell complexes in human HFs (but not in murine pelage HFs) (UHR 1984;

(BOTCHKAREV et al. 1997a; PAUS et al. 1997; PETERS et al. 2002).

The vasculature is similar to the innervation very densely and basket-like located

around the HF. It arises from the dermal and hypodermal vascular plexus and is

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30

formed by arterioles, capillaries and venules with numerous shunts. This perfusion

system sheathes the entire follicle, weaving through its CTS, and even inserts into

the DP of terminal HFs, in humans but also in all other mammals (MECKLENBURG

et al. 2000; YANO et al. 2001). That ensures, that all key regions of the HF have

abundant access to all essential factors and that metabolic products can be removed.

Tab. 2.3 Glossary of anatomical and trichology terms (modified after PAUS and PEKER 2003; MUELLER-ROEVER et al. 2001)

Bulb Prominent onion-shaped thickening on the proximal end of the HF, relatively undifferentiated matrix cells, melanocytes and cells from the proximal ORS

Bulge Convex extension of the distal part of the ORS, near the epidermis, location of epithelial HF stem cells and insertion of the APM

Club hair Resting HS with a hollow brush of keratinized keratinocytes on the proximal end, tightly attached to the cortical cells of the hair cortex

Connective tissue sheath (CTS)

Part of the dermal connective tissue, tightly attached to the outer side of HF, composed of fibroblasts, macrophages and connective tissue

Dermal papilla (DP) = Follicular papilla

Mesodermal part of the HF, closely packed mesenchymal cells, framed by the bulb matrix during anagen

Epithelial strand Column of epithelial cells between the germ capsule and the compact DP, laterally demarcated by the thickened glassy membrane

Follicular pigmentary unit

Melanin-producing HF melanocytes located above and around the upper on-third of the DP, transfer of eu-or pheomelanosomes to differentiating HF keratinocytes in the precortical matrix; goes largely into apoptosis during each catagen phase, regenerated from melanocyte stem cells

Secondary germ capsule = Secondary hair germ

Bag-like structure of glycogen-free cells of distal ORS, surrounding the club hair

Hair shaft (HS) Terminally differentiated HF keratinocytes (trichocytes), the HS is divided into cuticle, cortex and medulla

Hyaline membrane = Vitreous membrane = Glassy membrane

Outermost noncellular part of the HF, basal lamina and two layers of orthogonally arranged collagen fibres, separates ORS from CTS

Isthmus Middle portion of the HF extending from the sebaceous duct to the insertion of APM (bulge region)

Inner root sheath (IRS) Multilayered structure composed of terminally differentiated HF keratinocytes surrounded by the ORS, surrounds the hair up to the hair canal

Lanugo hair Fine hair on the fetal body, shed in utero or during the first weeks of life Outer root sheath (ORS)

Outermost sheath of HF keratinocytes, merges distally into the basal layer of the epidermis and proximally into the hair bulb

Sebaceous gland (SG) Glandular structure close to the insertion of the APM with holocrine function, lipid-filled sebocytes

Terminal hair Large, usually pigmented and medullated hair Vibrissae = Sinus hair follicle

Special sensory HFs with unique anatomy and biology, found in different regions of animal skin, but not in humans; largest and most densely innervated HFs with special sinusoid blood supply; first HFs to develop

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31

2.1.3 Hair follicle cycle

The HF is one of the few micro organs of the body that undergoes lifelong cycling.

HF cycling describes the morphological evidence of rhythmically re-occurring growth,

regression and tissue re-modeling events in this complex neuroectodermal-

mesodermal interaction system (PAUS and FOITZIK 2004). Originally, HF cycling is

synchronized in mammals in accordance to seasonal changes in habitant or

procreational activities (STENN and PAUS 2001). In mice, pelage HF cycling occurs

in a wave-like synchronous pattern starting from neck to tail (MUELLER-ROEVER et

al. 2001). In humans, the synchronized follicular cycling is lost after one year of life

and is replaced by a random or mosaic pattern of asynchonized hair cycling

(WHITING 2004). Such pattern type is also observed in domesticated mammals kept

under indoor conditions (MEYER et al. 1980, 2009a). The purpose for asynchronous

HF cycling in humans is not fully investigated but may include cleaning of the skin

surface of debris and parasites and excretion of chemicals by encapsulation within

trichocytes (STENN and PAUS 2001). In addition, HF cycling might serve as

regulator of paracrine or even endocrine secretion of hormones and growth

modulators produced within the follicle and secreted into the skin and / or circulation

(PAUS and COTSARELIS 1999).

The cyclic transformations from phases of rapid growth (anagen), via apoptosis-

driven regression (catagen) to relative quiescence (telogen) (DRY 1926), are

characterized by regression and proliferation activity and are influenced by numerous

of factors (e.g. growth factors, cytokines, hormones, neuropeptides) (for review

(STENN et al. 1996; PAUS and COTSARELIS 1999). 85 to 90% of all scalp HFs are

within anagen stage, which lasts for 2-6 years. The duration of hair growth

determines the length of the HF. Catagen lasts for a few weeks and is replaced by 2

to 4 months of telogen phase. Scalp HFs grow approximately between 0.3 and 0.5

mm per day, which is determined by the proliferation and differentiation of the matrix

keratinocytes (MKs) (DAWBER 1997; STENN and PAUS 2001; PAUS et al. 2007).

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32

Fig. 2.6 Morphology of human HFs in different hair cycle stages. (A) telogen HF, (B) anagen HF, (C) catagen II HF, (D) HF in stage catagen V. (FITZPATRICK 2008)

Anagen is the growth phase of the hair cycle and has been divided into 6 sub stages

(anagen I-VI) (Figs 2.6 B, 2.7) defined by specific morphologic criteria (MUELLER-

ROEVER et al. 2001). This formation of the HF displays structural and molecular

analogies to fetal HF morphogenesis (PAUS et al. 1999a). Anagen starts with the

proliferation of secondary germ cells in the bulge region and is characterized by a

massive proliferation and differentiation of keratinocytes of the hair matrix, as well as

the remodeling of perifollicular innervation, the HF immune system and the

pigmentation of the HS by follicular melanogenesis (PETERS et al. 2001). Except for

the last substages, anagen VI (the duration of which dictates the shaft length), the

length of the other anagen phases does not change substantially dependent on the

location.

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33

Fig. 2.7 The hair follicle cycle. (Modified after ALONSO and FUCHS 2006; FUCHS 2007)

Catagen is the regression phase (catagen I-VIII) (Figs 2.6 C,D, 2.7). During catagen,

the lower ´cycling` portion of each HF regresses entirely in an apoptosis- and

terminal differentiation-driven process of organ involution of the lower part of the HF

(LINDNER et al. 1997; PAUS and PEKER 2003). The earliest signs of catagen are

the termination of melanin production in the hair bulb and retraction of melanocyte

dendrites in the HF pigmentary unit. Further, it is characterized by condensation and

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34

upward movement of the DP (TOBIN et al. 1998), leaving an angiofibrotic strand or

stela indicating the former position of the anagen root (WHITING 2004).

At the end of this involution process, the HF enters into the resting phase, the so-

called telogen (Figs 2.6 A, 2.7). The resting club hair is situated at the bulge level

where the APM inserts into the HF. The telogen bulb is non-pigmented and has no

IRS. In telogen, the HF is characterized by relative quiescence. However, telogen is

considered to be much more important than the term ´resting` implies, since the

epithelial remnants of the telogen HF (distal ORS, secondary hair germ, bulge) are

engaged, e.g. in substantial biochemical activity and some degree of proliferation

(PAUS and COTSARELIS 1999; MUELLER-ROEVER et al. 2001).

In addition, during the course of cycling, substantially remodeling of both the HFs

innervation and vasculature occurs (BOTCHKAREV et al. 1997a; MECKLENBURG

et al. 2000; YANO et al. 2001). The HF transition between distinct stages of

development and postnatal cyclic regeneration is governed by a bidirectional signal

exchange between follicular keratinocytes and fibroblasts of the follicular DP, which

is supposed to be the control centre of follicle growth, initiating and terminating

anagen. Many molecular key regulators that had been involved in the regulation of

HF development are also recruited for the control of cycling. Just to point out one is

TGF-β, which induces catagen development (LITTLE et al. 1994). The HF

development is due to DP fibroblasts and its contact to hair MKs (JAHODA and

REYNOLDS 1996), which signals act on the eSCs of the follicle to initiate anagen

(bulge activation hypothesis). The stem cells are supposed to generate rapidly

dividing transient amplifying cells that migrate towards the DP for constructing a new

hair bulb (LAVKER et al. 1993).

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35

2.2 Sinus hair follicle biology: overview

Sinus hair follicles belong to the evolutionary oldest tactile sense organs of the

mammalian skin that have such a central role that they are the first developing hair

type in embryonal stage and even do exist in congenital hairlessness in mice

(HALATA 1993; MEYER 1999; MEYER and ROEHRS 1986). Sinus hair follicles are

also known as whiskers, vibrissae, vibrissal follicles, feelers or tactile, sensory or

sinus hairs and were firstly described as ´large stiff hairs (BLAND-SUTTON 1887;

BEDDARD 1902) that are pre-eminently sensory` and which differ from all other

types of hair through the presence of erectile tissue in their follicles (BOTEZAT 1897;

DANFORTH 1925b) (Fig. 2.8). Vibrissal follicles are highly sensitive

mechanoreceptive complexes that receive pressure and contact stimuli and initiate

behavior-relevant reactions through central nervous regulation. Vibrissal follicles are

important for the cognition of the environment, for social contacts between animals,

food intake. Loss of vibrissal follicles can immediately to loss of orientation and

subsequent erratic behavior (MEYER 1999).

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36

Fig. 2.8 Diagram of active vibrissal follicle in adult mouse. (DAVIDSON and HARDY 1952)

2.2.1 Sinus hair follicle morphogenesis

As in the human HF, morphogenesis of vibrissal HFs is governed by a series of

different events and is marked by a high degree of order and pattern in time and

space (DANFORTH 1925a; GRUNEBERG 1943; YAMAKADO and YOHRO 1979;

VAN EXAN and HARDY 1980): The development is more rapid than in pelage

follicles. In the 12-day embryo, the epidermal plugs of the first vibrissal follicles

appear and hairs emerge 5-6 days later. In contrast, pelage HFs start develop in the

14 day embryo and require longer to emerge (DAVIDSON and HARDY 1952).

In general, the differentiation of vibrissal follicles is almost the same as that of pelage

HFs (HARDY 1949, 1951) (Fig. 2.9). It starts with an epidermal downgrowth (stage

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37

1), formation of a pre-papillae (stage 2) and DP (stage 3) (ZIETZSCHMANN 1920)

are formed. In stage 4 a hollow cone develops by the hardening of certain cells from

the hair matrix, which then gives rise to the IRS. In stage 5 a hair canal is formed with

the condensation of cells, with strongly basophilic nuclei, on an elevation or cap of

epidermis above the follicle, followed by the occurrence of a keratinized hair shaft in

inside the cone (stage 6). This formation of a canal by keratinization from the

epidermal surface downwards distinguishes the vibrissal follicles from the pelage

HFs, in which keratinization begins as a blind cavity within the deeper layers of the

epidermis at the level of the stratum spinosum (HARDY 1949), and in which no cap

of epidermal cells and no condensation of basophilic nuclei occurs. At stage 6a this

condensation reaches the tip of the hair cone, whereas at stage 6b a canal is formed

within this area by the keratinization of cells in the centre. The hair shaft has pierced

the IRS at stage 7. Morphogenesis ends with the emerging of the hair shaft from the

skin (stage 8) and the forming of blood capillaries of the DP (DAVIDSON and

HARDY 1952).

Fig. 2.9 Diagram of stages 1-8 in development of vibrissal follicles in mouse. Stage 0: no follicles, stage 1: epidermal downgrowth and follicle plugs, stage 2: pre-papillae, stage 3: papillae, stage 4: hair cones develops by the hardening of certain hair matrix cells, stage 5: hair canal is formed, stage 6a: hair formation, stage 6b: opening of hair canals, stage 7: hair shafts in hair canals, stage 8: hairs emerged (DAVIDSON and HARDY 1952)

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38

Vibrissal follicles are much larger and stouter than those of pelage. The epidermal

plug is from the beginning on surrounded by a dermal sheath, in which few isolated

blood cells are visible at stage 8. While the follicle is growing and the DP is formed,

the follicle gets the characteristic hourglass shaped (Fig. 2.9). At stage 3 the

characteristic thickening of the ORS becomes evident, and by stage 6 the superior

and inferior swellings can be distinguished. In the newborn mouse the lower blood

sinus is differentiated with a well-developed fibrous wall and connective tissue

trabeculae, filled with blood cells. Three days later the upper sinus and ringwulst are

completely differentiated. After birth no new vibrissal follicle is added to those already

regularly arranged in rows, whereas pelage HFs continue to appear until day 5 till 8

after birth (DAVIDSON and HARDY 1952).

Later on, new four substages of vibrissa follicle development which occurred prior to

stage 1 of DAVIDSON and HARDY (1952) were described (VAN EXAN and HARDY

1980). In addition, it was found that vibrissal pattern formation is likely to be a

complex process relying on the interaction of cells and tissues (comparable to normal

HFs), rather than on unidirectional instructions from neurons to other cell types

(WRENN and WESSELLS 1984).

2.2.2 Functional anatomy of the sinus hair follicle

The major mystacial vibrissae of the mouse and rat are arranged on the snout (from

the nose to the cheek) in five ´horizontal` (rostrocaudal) rows and one ´vertical`

(dorsoventral) row which lies just caudal to the horizontal rows. Within each row a

characteristic anterior-posterior size gradient is observed: The largest vibrissae are

being located near the cheek and the smallest near the nose (OLIVER 1966b). The

location of the vibrissae follicles and the numbers in the major groups are

predetermined and constant (DANFORTH 1925a; GRUNEBERG 1943; DUN and

FRASER 1958; YAMAKADO and YOHRO 1979), except in a few mutants

(YAMAKADO and YOHRO 1979). It was also found that vibrissal follicles grew

synchronously within the same margin (IBRAHIM and WRIGHT 1975). Within this

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39

group of vibrissal follicles it is considered that the bigger follicles (so-called

´macrovibrissae`) have especially the function as a ´distance decoder`, whereas

smaller ´microvibrissae` are critically involved in object recognition (BRECHT et al.

1997).

Vibrissal follicles are similar in their histological structure compared to pelage HFs:

They consist of the same concentric cylinders, forming the epithelial HF

compartments: ORS, companion layer (CL), Henle’s layer (IRS), Huxley’s layer (IRS),

cuticle (IRS), as well as cuticle, cortex and medulla of the hair shaft (see Tab. 2.1).

On the other hand, there are many structural features which distinguish vibrissal

follicles from pelage HFs (DAVIDSON and HARDY 1952) (Fig. 2.10):

Sinus hair follicles are highly innervated sensory organs (VINCENT 1913;

WINKELMANN 1959), which are represented in the somatosensory cortex of the

brain by their cortical ´barrels` (WOOLSEY and VAN DER LOOS 1970). Each

vibrissal follicle is supplied by a large deep vibrissal nerve arising from the infraorbital

nerve (DORFL 1982), which enters the capsule in the lower third of the follicle

(MAROTTE et al. 1992). In addition, the vibrissal follicle is innervated by several

smaller superficial nerves (DAVIDSON and HARDY 1952) which arise from the

dermal plexus. The innervation is particularly dense in the larger caudal vibrissal with

about 200 myelinated and 100 unmyelinated fibres in each deep nerve (WAITE and

CRAGG 1982; WELKER and VAN DER LOOS 1986; CRISSMAN et al. 1991). These

afferents supply a variety of sensory receptors: Merkel cells in the epidermal rete

ridge collar at the mouth of the follicle and in the external root sheath at the level of

the ring sinus; longitudinal lanceolate endings in the mesenchymal sheath at the level

of the ring sinus; Ruffini and terminals and free nerve endings in the inner conical

body and in the cavernous sinus; and a few free nerve endings in the DP (ANDRES

1966; RENEHAN and MUNGER 1986; RICE et al. 1986; MUNGER 1991). Further it

was found, that for example Merkel cells contain receptors and transporter for

serotonin, substance P, calcitonin gene relating protein, vasointestinal peptide and

met-encephalin in rats (TACHIBANA et al. 2005; TACHIBANA and NAWA 2005).

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40

Vibrissal follicles have a great size: Pelage HFs are long and thin, and are of rather

uniform diameter, whereas vibrissal follicles produce longer and thicker hairs, but are

themselves larger and stouter (see e.g. MEYER 2009b).

The sinus hair follicles have an hour-glass-shaped due to a thickening of the ORS,

forming the characteristic superior and inferior swellings. The ORS consists of a

single layer of basal cells and a multi-layer (three or four) of epithelial cells. Further,

the inferior swelling of the ORS or the sometimes called ´bulge`, is considered to

reside stem cells, similar to the human bulge (SIEBER-BLUM et al. 2004).

A distinctive feature of vibrissal follicles is an IRS collar at the level of the sebaceous

gland opening, which forms a tight-fitting collar with a serrated inner margin, which is

absent from the pelage HFs of the mouse (DRY 1926), of the Australian opossum

(GIBBS 1938) and of the merino sheep (DUERDEN 1924). It may exist for the

mechanical support of any large fibres in their follicles.

Fig. 2.10 Murine vibrissal follicles of the snout. HE staining

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The medulla of vibrissal follicles has cells and air spaces resembling a honeycomb,

and is probably of the intermediate type (HAUSMANN 1924), whereas in pelage the

medulla cells are arranged in regular rows. The glassy membrane (basal membrane)

separates the epithelial ORS of the follicle from the mesenchymal sheath, and is also

passed by nerves and contains Merkel cells.

Vibrissal follicles are surrounded by a thick dermal sheath from the very early

beginning of morphogenesis, within the blood sinuses lie. All vibrissal follicles

possess blood sinuses and abundant nerve endings lying within the dermal sheath,

which are characteristic of all tactile HFs, but absent in pelage hair follicles. The

lower or venous blood sinus surrounds the lower portion of the follicle with connective

tissue bands within the cavity. The more superficial upper or Ring sinus has no

trabeculae, but contains the so-called ´ringwulst`. This ringwulst is attached to the

dermal sheath adjacent to the follicle wall, forming a collar surrounding the follicle. It

consists of connective tissue and is penetrated by nerve branches and blood

capillaries. Vibrissal DPs contain a blood supply in contrast to murine pelage DP and

are more richly innervated than those of the pelage.

In mice, each vibrissal and pelage HF consists of only one small sebaceous gland,

and lacks sweat glands. Vibrissal follicles have no smooth muscle corresponding to

the arrector pili muscle. Instead, the vibrissal HF has a group of larger striated

muscle fibres (so-called intrinsic muscles), which are solely attached to the dermal

sheath without any bony attachment (DOERFL 1982). Each of these follicular

muscles connects two adjacent follicles of the same row (Fig. 2.11).

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Fig. 2.11 Musculature of vibrissal follicles (A) Schematic drawing of two neighbouring follicles in the same row. R: rostral, C: caudal, B: fibrous band connecting the lower parts of the follicles, P: plate, the band between the rostral and caudal faces of two adjacent follicles, L: longitudinal muscular band formed by fibres of m. levator labii sup. and m. maxillolabialis, N: follicular nerve accompanied by an artery. (B) vertical section of two neighbouring vibrissal follicles. Masson-Goldner staining. Bar (B): 100µm.

1.1.1 Sinus hair follicle cycle

Similar to other human HFs and murine pelage HFs, vibrissal follicles show a

rhythmical and characteristic cycling. However, important differences exist, since

they do not dramatically undergo the extensive shortening seen during catagen in

pelage HFs and more importantly, their catagen and particularly telogen phases are

abbreviated (YOUNG and OLIVER 1976). For that reason, a different terminology

was outlined by YOUNG and OLIVER (1976), compared to the more detailed

classification used for pelage cycle (CHASE 1954).

Thus, hair cycle stages of the vibrissal follicles are divided into two distinct phases

(growth, regression), classified further into eight categories: pro-anagen (PA), very

early anagen (VEA), early anagen (EA), mid anagen (MA), late anagen (LA), early

catagen (EC), mid catagen (MC) and late catagen (LC) by their morphology of hair

bulbs and relative length of hair shafts (YOUNG and OLIVER 1976; ROBINSON et

al. 1997) (Fig. 2.12). Important to note is, that in contrast to pelage or human HFs,

termination of hair growth in the previous hair cycle and initiation of hair regeneration

in the following cycle partially overlaps. Thus, telogen is not obvious; otherwise the

sensory function of the tactile organ would be interrupted.

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Anagen follicles are categorized by the ratio of hair length of the growing hair shaft to

the fully grown club hair formed in the previous hair cycle; for early anagen the ratio

is less than one-quarter, for mid anagen between one-quarter and two-thirds, and for

late anagen over two-thirds.

Fig. 2.12 Mouse vibrissal follicle cycle (A) Diagram of vibrissal follicle growth cycle. (modified after (YOUNG and OLIVER 1976). (B)The bluish colour detects alkaline phosphatase activity during the hair cycle in the dermal papilla. PA: pro-anagen, VEA: very early anagen, EA: early anagen, MA: mid anagen, LA: late anagen, EC: early catagen, MC: mid catagen, LC: late catagen; Alcalic Phosphatase IR is labeled in blue (IIDA et al. 2007b)

In catagen, follicles have only one hair shaft (Fig. 2.12 A), because the old one

already sheds off from the follicle. The bulbs start to decrease their volume due to the

ceasing of proliferation of hair matrix cells and the upward movement of the hair shaft

base (YOUNG and OLIVER 1976) and become more longitudinally stretched and

thin in accordance with the upward movement of the club hair (Fig. 2.12 B).

Similar to human HFs, the underlying mechanisms guiding the vibrissal follicle

through the cycle have not yet been fully characterized. However, epithelial-

mesenchymal interactions are also considered to be important in hair growth

regulation (COHEN 1965; KOLLAR 1970; LINK et al. 1990; IIDA et al. 2007a). The

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cycle time was shown to be dependent on the size of the follicle and dermal papilla:

smaller vibrissal follicles have a shorter cycle time than that of the larger vibrissal

follicles. Relevant age and sex-dependent differences have not been demonstrated

(IBRAHIM and WRIGHT 1975), which confirmed the generally agreed hypothesis,

that vibrissal follicles are outside hormonal control in contrast to other HFs. It had

been demonstrated, that the complete removal of the DP ceases follicle growth

(OLIVER 1966b), and resumes after implantation of cultured dermal papilla cells

(OLIVER 1966a; JAHODA et al. 1984; HORNE et al. 1986; PISANSARAKIT and

MOORE 1986; JAHODA 1992; MCELWEE et al. 2003). Other studies also

suggested that the DP activity contributes to hair cycle changes, e.g. through

expressing different levels of alkaline phosphatases (IIDA et al. 2007b), fibronectin,

laminin or type IV collagen (JAHODA et al. 1992). Independently from normal cycling,

plucking of vibrissal follicles at any time during the cycle resulted in the induction of a

new hair (JOHNSON and EBLING 1964; IBRAHIM and WRIGHT 1978b, 1978a).

2.3 Murine nail apparatus: overview

The nail or claw unit comprises the distal-most, dorsal structure of vertebrate limbs.

The main purpose of the nail apparatus is to provide a protective covering, known as

the nail plate, over the dorsal aspect of each distal digit of the hands and feet

(ACHTEN 1968; RUNNE and ORFANOS 1981; JIARAVUTHISAN et al. 2007). In

addition, to protect the fingertips from traumatic injury, the nail plate also applies a

pressure that opposes the volar side of the terminal phalanx, which contributes to the

enhances sensory discriminatory ability of the fingertips (DE BERKER et al. 2001).

Besides this, claws or nails have applications like scratching and grooming and can

be utilized as a means of defense or attack. In higher primates including humans,

nails have developed in conjunction with the acquisition of manual dexterity

(DAWBER 1980), and are modified to become a cosmetic accessory and

occasionally are capable of conveying information about social standing of an

individual (MURDAN 2002); other mammals do not possess such flattened claws

(DAWBER 1980). Nails are considered to have developed from claws which are

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similar keratinized structures at the ends of the distal phalanx of many animals

(CLARK 1936), although important ultrastructural and biochemical differences are

present (THORNDYKE 1966).

2.3.1 Nail morphogenesis

Nail morphogenesis is governed by multiple different signals and develops from an

ingrowth of the epidermis into the dermis (PAUS and PEKER 2003) which gives rise

to a plate formed by fully keratinized (onychocytes), the nail plate. This visible part of

the nail apparatus functions as a protection of the tips of digital phalanges. The nail

apparatus is formed from an invagination of the primitive epidermis on the dorsum of

the terminal phalanges (DAWBER 1980), namely, the same primitive epidermis that

gives rise to hair, sweat glands and the stratum corneum (PAUS and PEKER 2003).

Its developmental time course is very constant and predictable so that fingernails in

the neonate have been exploited as useful parameters for estimating the gestational

age at term in human (BALAKRISHNAN and PURI 1973; PARKIN et al. 1976) and

farm animals. This first epidermal thickening appears during the ninth week of

gestation in human and by the 13th week the nail bed and nail fold possess a

granular layer with keratohyalin granules which disappear when the hard nail plate is

formed (LEWIS 1954). In mice, the nail primordium is first seen at embryonic day 15

in all but the anterior-most digit (thumb) of the mouse forelimb (KAUFMAN 1992).

The nail primordium of this vestigial digit is formed a few days later. At the level of its

proximal transverse groove, an epidermal duplication grows and forms the primordial

matrix (RUNNE and ORFANOS 1981). Before this matrix begins definitive nail

production, a thin rootless fore nail arises from the subsequent ´sole horn` area.

While this fore nail covers the nail bed, the primordial matrix differentiates and

produces the nail plate (ZAIAS 1963). The protruding nail finally pushes through the

eponychium and covers the entire nail bed. Initially, matrix and nail bed keratinize

similarly to the normal epidermis with the formation of keratohyalin; in both areas

typical parakeratotic keratinization follows at a later stage (RUNNE and ORFANOS

1981). Although the same principles of neuroectodermal-mesodermal interactions

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described for HF development may also be relevant for nail morphogenesis, only little

is known exactly. Currently, a limited number clues to the molecular controls of nail

growth does exist (PAUS et al. 2007).

2.3.2 Functional anatomy of the nail

In animals the nail is more important as a gripping device or scratching tool,

compared to humans, and shows differences in the anatomical structure (Fig. 2.13).

Sections of the nail in vertebrate groups, including primates and humans, reveal, that

the thickness of the sole horn varies, but that the nail plate is consistent (ZIEGLER,

1954).

Fig. 2.13 Main characteristics of the nail in different animals and humans. (ACHTEN 1968)

Like the HF, the nail unit is composed of keratinized epithelium and supporting

mesenchyme. The epithelial compartment can be divided anatomically into several

compartments contiguous with the epidermis (Fig. 2.14) (BADEN and KVEDAR

1993): The nail plate as a fully cornified structure and the four highly differentiated

epithelial structures: the proximal nail fold (PNF), the nail matrix, the nail bed, and the

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hyponychium (ZAIAS 1963). The intimate anatomic relationship between nails and

phalanges determines the shape and curvature of the nails, and is also responsible

for the common occurrence of bone alterations in nail diseases, and vice versa

(PAUS et al. 2007). In contrast to the skin, the nail apparatus lacks of hypodermal

tissue and pilosebaceous units or HF complexes, respectively (PAUS and PEKER

2003). The dermis consists of a fibrocollagenous network, with bundles of collagen

radiating into the periostium of distal phalangeal bones and is situated between the

nail plate and the bone (TOSTI and PIRACCHINI 1999). Because of this special

region with the underlying bone, bacterial and other infections in this region with a

rapid soft tissue spread can cause osteomyelitis of the distal phalanx and increase

the risk of peripheral neuropathy (PAUS et al. 2007).

Fig. 2.14 Schematic longitudinal section of mouse hair follicle and nail unit (A) Proliferating cells reside in the lower bulb region of the hair follicle (the matrix). Their descendents differentiate into distinct epithelial cell types arranged in concentric rings, such as outer root sheath (ORS), inner root sheath (IRS), and hair shaft (cuticle, cortex, and medulla). The matrix surrounds a group of specialized mesenchymal cells called the dermal papilla (DP), which are the source of signals required for hair follicle development. Activated, endogenous Notch1 is detected in the nuclei (red) of the matrix not adjacent to the DP and of the precursors of cortical cells. (B) The nail unit is composed of four types of epithelial cells. The matrix contains proliferating cells that undergo terminal differentiation in the keratogenous zone, producing the nail plate. The second epithelial cell types constitute the nail bed that underlies the nail plate and spans from the matrix to hyponychium. The hyponychium is the third cell type, underlying the free end of the nail plate. The last epithelial cell type is the nail fold, which is the skin enveloping the nail plate. The dorsal and ventral epidermis of the digit is indicated in orange. The bone is marked in gray. Dermal cells reside in areas marked in white. Activated Notch1 is detected in the nuclei (red) of cells in the dorsal matrix. Note that similarities in color between the hair follicle and nail do not imply that these cell types are homologous. (LIN and KOPAN 2003)

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The nail plate can be seen as equivalent to the epidermal stratum corneum, although

it is very firm attached to the nail bed and shows no desquamation. The major part of

the nail plate arises from the dorsal matrix in rodents, whereas in human the

intermediate matrix contributes the greatest mass to the nail plate (THORNDYKE

1966). Besides that, the nail bed is also supposed to contribute slightly to nail growth

(JOHNSON et al. 1991; DE BERKER and ANGUS 1996). Nail plate formation

involves keratinization and flattening of postmitotic matrix cells, nuclear loss, and

cytoplasmic condensation (BADEN and KVEDAR 1993). These events occur in a

region called the keratogenous zone located dorsally to the matrix (Fig. 2.15 B). Nail

plate cells express a subset of the hard keratins expressed in hair (LYNCH et al.

1986; HEID et al. 1988; MOLL et al. 1988). They form the major structure of the nail

unit, which in some species is quite extensive, for example in ungulates or forepaws

of mongooses (e.g. Suricata suricatta), whereas it is rudimentary, for example, in

primates.

Fig. 2.15 Murine nail apparatus. HE staining; Hyp: Hyponychium; NP: Nail plate; NB: Nail bed; P: Pad; PNF: Proximal nail fold; PNM: Proximal nail matrix; PH: 1st and 2nd Phalanx

The nail matrix keratinocytes are larger and higher proliferating than epidermal

keratinocytes (PICARDO et al. 1994; NAGAE et al. 1995) and produce soft and/or

hard keratins dependent on the location in the nail matrix (HEID et al. 1988;

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KITAHARA and OGAWA 1993, 1994, 1997). The proliferation in the proximal matrix

is, like in the HF, not restricted to cells in contact with the basement membrane (LIN

and KOPAN 2003). The nail matrix harbors melanocytes in a high density (HIGASHI

1968; HIGASHI and SAITO 1969; TOSTI et al. 1994), which are located

predominantly in the distal matrix and in suprabasal layers in contrast to epidermal

melanocytes located in the entire basal layer. In addition, Merkel cells are also found

in human nail matrix, although more in fetal than in adult nails (MOLL and MOLL

1993).

The nail fold is a specialized epidermal transition zone followed by the matrix. Distal

to the matrix the nail bed is located, a zone containing mitotically inactive cells

(ZAIAS and ALVAREZ 1968; BADEN and KVEDAR 1993). The hyponychium

connects the nail bed with the ventral epidermis of the digit. In addition, the nail

apparatus displays an abundant vascular supply with anastomoses. A network of

three systems (superficial, proximal, distal arcade) supply different parts of the nail

apparatus und thus, guarantee independent sources of blood supply for the nail

matrix, supporting normal nail growth even in the presence of temporary perfusion

deficits (FLECKMAN et al. 1997).

Tab. 2.4 Human nail apparatus – basic data (PAUS et al. 2007)

Nail plate Tightly packed cornified cells (onychocytes), arranged in lamella pattern (weakly eosin+, strongly acid-fast+. Soöver staom reveals three horizontal layers; the largest nail plate is on the first toe (covers about 50% of the digit); fingernails have a longer longitudinal axis compared to the transverse axis, while toenails usually present a greater transverse axis

Nail matrix Thick epithelium situated above the middle part of the distal phalanx of the digits; keratinizes wihtout a granular layer and gernerates the bulb of the nail plate; contains many melanocytes (up to 300/mm2) in African Americans), predominantly in suprabasal layers

Nail bed Thin epithelium consisting of 2-5 cell layers with abrupt keratinization without a granular layer; sparse or no melanocytes; contribution to nail plate fomration debated (nail bed keratinocytes supposedly contribute about on-fifth of the ventral nail plate’s thickness and mass)

Proximal nail fold Epithelium with a granular layer; its dorsal part corresponds to the skin of the dorsum of the digit and contains sweat glands is devoid of pilosebaceous units; densely innervated; the ventral part is thinner and continues into the nail matrix epithelium

Hyponychium An epithelium equivalent to the volar skin containing a granular and a thick conified layer; the hyponychial dermal-epidermal junctioin is papillary as in normal skin

Nail pigmentation Banded pigentation (longitudinal melanoncyhia) is common I Asians or African-Americans (seen in 77% of African-Americans by the age of 20, nearly 100% by the age of 50 years); rare in Caucasians

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2.3.3 Growth of the nail

In contrast to the HF, which cycles between growth, regression and resting phases,

the nail grows continuously and during adulthood (RUNNE and ORFANOS 1981).

The special arrangement of the matrix cells and the eponychium and nail bed

determine the direction of growth. The major part of nail plate formation arises from

the dorsal matrix in rodents, whereas in human the intermediate matrix contributes

the greatest mass to the nail plate (THORNDYKE 1966). Besides that, the nail bed

contributes slightly to nail growth (JOHNSON et al. 1991; DE BERKER and ANGUS

1996). The growth rate is determined by the turnover of the matrix cells; the

thickness of the nail, however, by the number of dividing matrix cells. In cases where

a part of the matrix is damaged by trauma, the nail grows upwards in an uncontrolled

manner (RUNNE 1980). On the other hand, digit tip regeneration (e.g. following

amputation at levels distal to the nail matrix) has been reported in non-primate

mammals (BORGENS 1982; MULLER et al. 1999), supporting the importance of the

nail matrix.

Variation in homeostatic control may contribute to some of the many nail

morphologies among vertebrates. Disruption of a similar balance in the epidermis

may be responsible for skin diseases, such as psoriasis. Alteration in nail

homeostasis could also result in longer, shorter, or abnormal nail plates. For

example, the HoxC13 and the nuclear repressor protein hairless (the Rhino allele,

hrrh) are independently involved in the program controlling homeostasis and nail

morphology. In the absence of either protein, long, curled, and continuously growing

nails are formed (AHMAD et al. 1998; GODWIN and CAPECCHI 1998;

MOOKHERJEE et al. 2006). Several other mutations in mice have been described,

which affect dorso/ventral patterning and disturb nail development [e.g. loss of

Wnt7a, or FOXN1, mutations in En1, LMX1B (CYGAN et al. 1997; VOLLRATH et al.

1998; KAWAKAMI et al. 2000; MECKLENBURG et al. 2004)]. In addition, several

other aspects, i.e. physiological variations like differences in the growth of toenails

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and fingernails, age or season or pathological aspects like diseases or drugs play an

important role.

2.4 Immunological background for the current study

In the following, a short overview of the two main components of normal immune

system should be given in order to better understand the special condition of

´immune privilege`, which differs from normal immune regulation.

2.4.1 Innate immune system

The innate system is thought to constitute an evolutionarily older defense strategy,

and is the dominant immune system found in plants, fungi, insects, and in primitive

multicellular organisms (JANEWAY et al. 2001). It comprises the cells and

mechanisms that defend the host from infection by other organisms, in a non-specific

manner, often but not only in the early phase. Innate immunity is present in all

individuals at all times, shows no increase in the reaction strength after repeated

exposure to a given pathogen and discriminates between groups of similar

pathogens (ALBERTS et al. 2002; JANEWAY et al. 2005). This means that, unlike

the adaptive immune system, no long-lasting or protective immunity to the host is

constituted. The main functions of the innate immune system in vertebrates include:

a) Recruitment of immune cells (i.e. neutrophils and leukocytes) to sites of

inflammation by the release of chemical factors (chemokine and cytokine interaction).

b) Activation of complement cascades and formation of antibody complexes to clear

pathogens or mark them for destruction by other cells (opsonization, cytolysis,

neutralization). c) Activation of the adaptive immune system through antigen

presentation (MHC class II), d) identification and removal of foreign substances

through phagocytosis by leukocytes.

Natural killer (NK) cells, mast cells, eosinophils, basophils; and the phagocytic cells

including macrophages, neutrophils and dendritic cells, contribute to the immune

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52

system by identifying and eliminating pathogens that might cause infection. These

cells are activated through different mechanisms, i.e., through important pattern-

recognition receptors (PRRs), expressed on macrophages and dendritic cells that

recognize pathogen-associated molecular patterns (PAMPs), Toll-like receptors,

activating receptors (PRRs) and inhibitory receptors (KIRs) for NK cells and the

production of antimicrobial peptides (GOLDSBY et al. 2003; PLAYFAIR and CHAIN

2006).

2.4.2 Acquired immune system

The adaptive immune system is the response of antigen-specific lymphocytes to

antigen, and provides the vertebrate immune system with the ability to recognize and

remember specific pathogens (development of an immunological memory). It is

composed of highly specialized, systemic cells and processes. The responses are

generated by clonal selection of lymphocytes in contrary to the innate immune

system (JANEWAY et al. 2005). Adaptive immunity is activated in vertebrates when a

pathogen evades the innate immune system and generates a threshold level of

antigen (JANEWAY et al. 2005). The major functions of the adaptive immune system

include: a) Distinction between ´self` and ´non-self`antigens, during the process of

antigen presentation (through MHC class I and class II molecules and interaction with

T cells), b) elimination of pathogens or pathogen infected cells, c) The development

of an immunological memory, to immediately eliminate a pathogenic antigen in case

of reoccurence/reinfection.

The cells of the adaptive immune system are lymphocytes, like B cells and T cells. B-

cells are important in the humoral immune response, interacting with T helper cells

and producing antibodies, whereas T cells are involved in cell-mediated immune

responses (GOLDSBY et al. 2003; JANEWAY et al. 2005).

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Fig. 2.16 Diagram of innate and adaptive immune system (PLAYFAIR and CHAIN 2006)

2.4.3 Immune privilege: Definition and basic characteristics

The concept of immune privilege was deduced from the acceptance of allogeneic

tumor implants in defined anatomical locations, such as the eye and the brain

(GREENE 1947). Already in the nineteenth century, van Dooremaal (1873) and Zahn

(1884) noted a prolonged survival of human tumor cells and fetal cartilage xenografts

in the anterior chamber of eyes of rabbits (NIEDERKORN 2003). For the historical

perspective, however, the starting point are studies of Medawar transplanting normal

tissues into such sites (MEDAWAR 1945, 1948; BILLINGHAM et al. 1954). Because

these transplanted tissues survived longer than anticipated or were accepted

indefinitely (MUNN and MUNN 2006), the term ´immunological privilege` was

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primarily developed to illustrate transplant (e.g. corneal allograft) survival in these

sites (BILLINGHAM and BOSWELL 1953). Thus, the term immune privilege (IP)

included, originally, that a given tissue environment that hosts an allotransplant

relatively protects the transplanted cells from rejection by the host immune system as

a consequence of antigen sequestration (HEAD and BILLINGHAM 1985; BRENT

1997). Historically, it was thought that mainly passive mechanisms, notably physical

and physiologic barriers that maintained local segregation between tissues and

immune cells and antibodies, contributed most for localized immune privilege.

However, with the recognition that the establishment of IP in the fetomaternal

placentar unit is vital for avoiding fetal rejection (MELLOR and MUNN 2000;

EHRLEBACHER 2001) and that ocular IP is indispensable for normal eye function

(NIEDERKORN 2002), this narrow definition of IP was extended to tissue sites where

the local establishment of an immunosuppressive/tolerogenic environment exerts

biologically important functions to suppress a cytotoxic immune attack on the cells

and antigens harbored within these sites (PAUS et al. 2003; ITO et al. 2008b).

Correspondingly, the collapse of eye IP may result in autoimmune uveitis that is

potentially blinding ocular inflammatory diseases (ITO et al. 2008b).

Today, the term IP has become associated with a small list of defined anatomical

compartments that are protected by IP from excessive inflammatory activity and from

the normal immune system, such as the anterior chamber of the eye, the

fetotrophoblast, the testis, the central nervous system behind the blood-brain barrier,

the anagen hair follicle epithelium, the proximal nail matrix, and the hamster cheek

pouch, (NIEDERKORN 2002); (HEAD and BILLINGHAM 1985; STREILEIN 1993;

BRENT 1997; JANEWAY et al. 2001; PAUS et al. 2003, 2005; ITO et al. 2005c).

Collapse of IP is now appreciated to play a major role in different autoimmune

diseases such as multiple sclerosis (BRUNO et al. 2002), mumps orchitis (FILIPPINI

et al. 2001), and autoimmune chronic active hepatitis (LOBO-YEO et al. 1990;

FILIPPINI et al. 2001).

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In general, IP is established and maintained, e.g., by (STREILEIN 1993; BRENT

1997; MELLOR and MUNN 2000; FUZZI et al. 2002; NIEDERKORN 2002; PAUS et

al. 2005):

� Absence of lymphatics.

� Establishment of extracellular matrix barriers to hinder immune cell trafficking,

such as blood-retinal barrier, blood-brain barrier.

� Downregulation or absence of classical MHC class I expression, which

sequesters (auto)antigens in tissue sites and hinders their presentation to

CD8+ T cells with a matching T cell receptor.

� Expression of non-classical MHC class I molecules (such as the MHC class Ib

molecules HLA-G in humans and Qa-2 in mice or HLA-E as a ligand of the NK

cell inhibitory receptor CD94/NKG2A. The interaction of HLA-E with this

receptor for example may result in inhibition of NK cell- and cytotoxic T cell-

dependent lysis (GAO et al. 1997; BRAUD et al. 1998; PACASOVA et al.

1999; TRIPATHI et al. 2006).

� Functional impairment of antigen-presenting cells, for example, by

downregulation of MHC class II expression.

� Local production of potent immunosuppressants such as TGF-β1, TGF-β2, IL-

10, and α-MSH, macrophage migration inhibitory factor (MIF) and other

factors.

� Expression of Fas Ligand (FasL) in order to delete autoreactive, Fas-

expressing T cells.

In addition to these mechanisms of IP maintenance, new conditions and factors have

been explored that may also contribute to the generation and maintenance of relative

IP, such as IDO, MIF and others which will be discussed later.

IP is a relative, not an absolute, state and only some of these mechanisms, the

composition and relative importance of which vary between tissue sites, may be

present in a recognized IP compartment (HOBBS et al. 2002; JANEWAY et al. 2005).

Thus, it is an ongoing debate, which and how many mechanisms have to be existent

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to call a tissue site immunoprivileged. For example, even though fetal cells

expressing paternal antigens can leave the fetomaternal placentar unit and do evoke

a maternal response (TAFURI et al. 1995), and even though paternal DNA is found in

the lesions of one of the classical dermatoses associated with pregnancy, fetal

rejection is usually not a consequence. This suggests that the critical issue is which a

certain extent a state of tolerance to the alloantigen-bearing cells is established, and

with which quality immunotolerance is maintained throughout the persistence of

these antigens.

2.5 Immune privilege in skin appendages

Skin with its appendages is the largest organ of the mammalian body and is

continuously facing exposure to various kinds of antigens, including viruses, bacteria,

fungi and parasites as well as a large variety of chemical substances and allergens.

The skin immune system responses to most of these agents and has to find the

balance between proper immune defenses, the limitation of damaging defenses,

maintenance of immune tolerance against autoantigens and avoidance of deleterious

autoimmunity and destruction. At least two skin epithelia are now recognized to meet

key characteristics of IP sites, i.e. the anagen hair bulb of the human HF and the

proximal nail matrix (ITO et al 2005c; PAUS et al. 2005). This fact is noteworthy

because the mammalian skin and its IP sites have so far been largely ignored by

mainstream IP. Therefore, one can at best sketch outline of the potential significance

of skin immune privilege, drawing primarily upon the limited available information that

has been published so far on the IP of HF and nail.

HFs are one of the defining skin features of mammals. The HFs undergo life-long

cycles of growth (anagen), regression (catagen) and relative quiescence (telogen),

the so-called HF cycle, which result from complex bi-directional interactions between

the HF epithelium and its specialized, inductive mesenchyme. During anagen

development, a new hair shaft (HS) factory - the anagen hair bulb - is reconstructed

and the HF recapitulates in part key morphological events of its own morphogenesis.

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Then the HFs generate pigmented keratin fibres systems (hair shafts), which require

precisely coordinated interactions between hair follicle keratinocytes, melanocytes

and fibroblasts (PAUS and PEKER 2003). Since melanocyte-associated antigens

frequently are the target of cutaneous autoimmunity (e.g. in the context of vitiligo,

halo nevi or malignant melanoma regression) (COTSARELIS and MILLAR 2001), it is

reasonable to expect that HF melanocytes and their pigmentary activities may also

be targeted by autoimmune responses (ITO et al. 2008b). Indeed, it is now believed

that HF melanocyte autoantigens play a key role as potential immune targets in one

of the most frequent autoimmune diseases of man, alopecia areata, where, e.g., the

presence of autoantibodies against melanocyte-associated autoantibodies has long

been appreciated (GILHAR et al. 2005, 2007; PAUS et al. 2005; GILHAR and

KALISH 2006).

More than three decades ago, Billingham had already reported, that HFs provide a

special milieu which enables transplanted allogeneic melanocytes to escape

detection and elimination by the host immune system. In Billingham’s experiments

genetically incompatible homografts of black skin epidermis were transplanted onto

white skin beds (BILLINGHAM 1971) (Fig. 2.17). The result was that the donor

epidermis immediately lost its pigmentation, reflecting rejection of the foreign

melanocytes. However, shortly thereafter, black hair shafts began to pierce the (now

white) epidermis, which could only be explained by the possibility, that at least some

epidermal donor melanocyte allotransplants must have survived or escaped rejection

in the host hair bulbs, and that they had resumed their transfer of melanosomes to

precortical hair matrix keratinocytes (BILLINGHAM and SILVERS 1971; GILHAR and

KALISH 2006).

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Fig. 2.17 Billingham’s experiment: Survival of epidermal melanocyte allotransplants in the host anagen HF. When black ear skin epidermis is transplanted onto skin bed of genetically incompatible white guinea pigs, the transplant rapidly loses its pigmentation. This indicates that the allogeneic melanocytes are rejected. But surprisingly, shortly therafter, some black hair shafts pierce the (now white) epidermis – indicating that at least some donor melanocytes have escaped the host immune system and have begun to transfer melanosomes to precortical hair matrix keratinocytes with the result of generating black hair shafts (BILLINGHAM 1971; BILLINGHAM and SILVERS 1971).

Despite these exciting early findings indicating that the anagen hair bulb is a site of

IP, the concept of HF IP still awaits systematic exploration by mainstream

immunology (ITO et al. 2008b) and functional evidence. This has become even more

urgent, since the proximal human nail matrix (ITO et al. 2005c) recently has also

been found to be a site of relative IP, and since the gene and protein profiling data

(MORRIS et al. 2004; TUMBAR et al. 2004; GILHAR et al. 2005; OHYAMA et al.

2006) suggest that the bulge region is a strong candidate as intracutaneous

immunoprivileged site.

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2.5.1 The anagen hair bulb as an immunoprivileged site

Fig. 2.18 The anagen hair bulb HE staining shows an human anagen VI hair bulb. CTS: Connective tissue sheath, DP: Dermal papilla, IRS: Inner root sheath, MK: Matrix keratinocytes, ORS: Outer root sheath. Bar 100µm.

More than a decade after Billingham’s discovery, HARRIST et al. (1983) reported the

distribution of major histocompatibility (MHC) antigens in normal human skin

including human terminal HFs. They found that MHC class I molecules were present

on the surface of epidermal basal and spinous layer keratinocytes, and on the ORS

epithelium in the infundibulum of the HF. In contrast, DP, proximal ORS and IRS

showed negative MHC class I expression. Ia-like antigen positive dendritic cells were

also rarely observed in the deep portion (around the proximal hair follicles). On the

other hand, distal ORS showed strong positive expression of MHC class I and many

Ia-like antigen positive dendritic cells. This striking down-regulation of MHC class I

expression in the proximal epithelium of anagen hair bulbs (Fig. 2.18) was confirmed

to exist in human (HARRIST et al. 1983), rat (BROECKER et al. 1987) and mouse

HFs (WESTGATE et al. 1991), and then re-analyzed in greater detail in stage VI

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human anagen scalp HFs (PAUS et al. 1994b). In these studies, the question

whether the human hair follicle bulge, i.e. the area of stem cell, is also relatively

immunoprivileged has been left uncontrolled so far.

Besides MHC class I negativity, additional features of HF biology strongly supported

the concept that anagen hair bulbs in all mammalian species belong to a site with

relative IP (PAUS et al. 2005):

Fig. 2.19 Distribution of peri- and intrafollicular CD4+, CD8+ T cells and CD1a+ cells. The distribution of immune cells in and around human HF differs from that in and below the interfollicular epidermis. Around the human anagen hair bulb, CD4+, CD8+ and CD1a+ cells are only rarely found. (ITO et al. 2008b)

• In the proximal portion of the HF, there is a sharply reduced number of

apparently non-functional, MHC class II antigen-negative, CD1a+ Langerhans cells,

(PAUS et al. 1994b; CHRISTOPH et al. 2000) as compared with the distal part of hair

follicles (upper portion of HFs) (Fig. 2.19).

• In contrast to the ORS distal of the infundibulum of the sebaceous gland, the

anagen hair bulb displays almost none intraepithelial T cells; and in mice, γ/δ-TCR+

lymphocytes are not detected below the bulge region (CHRISTOPH et al. 2000).

Human anagen-VI scalp HFs extremely rarely show CD4+ T cells, and a CD8+

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lymphocyte is almost never caught trafficking through the proximal follicle epithelium

(PAUS et al. 1994b) (Fig. 2.19).

• Like the other well defined immune privileged tissues (HEAD and

BILLINGHAM 1985; STREILEIN 1993), the hair bulb is characterized by the absence

of lymphatic drainage pathway and the presence of a special extracellular matrix

barrier around the hair follicle, both of these conditions may contribute to hinder

immune cell trafficking (BROECKER et al. 1987; STENN and PAUS 2001).

• Importantly, anagen hair bulbs in mice and humans express potent

immunosuppressants, such as TGF-β1 (TOKURA et al. 1997; STENN and PAUS

2001), ACTH (SLOMINSKI et al. 1993), and α-MSH (SLOMINSKI et al. 1993, 2000;

BOTCHKAREV et al. 1999; PAUS et al. 1999a; ITO et al. 2005a).

2.5.2 Downregulation of MHC class Ia and NK cells

NK cells are large granular, non-T, non-B lymphocytes, which destroy certain tumor

cells. NK cells are important in innate immunity against viruses and other intracellular

pathogens, as well as in antibody-dependent cell-mediated cytotoxicity (ADCC).

Normally, NK cells attack autologous (e.g. virally infected or malignantly transformed)

cells, with both the allogeneic MHC class I type or absent or low MHC class I

expression. In sites of IP, where weak or absent MHC class Ia expression is the main

characteristic, this becomes a dilemma, because NK cells are attracted, which are

programmed to destroy MHC class Ia-negative cells. In human anagen HFs and

adjacent tissue only very few perifollicular NK cells have been found (CHRISTOPH et

al. 2000). The same is true for the nail apparatus, where only very rarely CD56+ cells

were detected in the mesenchyme adjacent to the PNM (ITO et al. 2005c).

Nevertheless, a small number of T cells and NK cells also exists in sites of IP (i.e.

HF, nail apparatus) (CHRISTOPH et al. 2000; ITO et al. 2007), so there have to be at

least some compensatory mechanisms available against NK cell effects.

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NK cells express two types of surface receptors of opposing kinds that control their

cytotoxic activity and help to distinguish infected/foreign from uninfected/self cells.

One type is an ´activating receptor`, the second type are inhibitory receptors, Killer

cell immunoglobulin (Ig)-like receptors (KIR) and heterodimer CD94/NKG2A (LONG

1999; BOEHM 2006), which are specific for MHC class Ia alleles. If target cells

express MHC class I molecules, NK cell activation is prevented through interaction

with KIR by phosphorylation of immunoreceptor tyrosine inhibitory motif (ITIM)

followed by binding to phosphatases (LONG 1999; VIVIER et al. 2002; JANEWAY et

al. 2005; LANIER 2005). NKG2D is expressed not only in NK cells but also in CD8+

T cells and recognizes the MHC class I chain-related A (MICA) molecule on target

cells, which stimulates these immune cells to attack the target cells (BAUER et al.

1999; WU et al. 1999; MIDDLETON et al. 2002).

Fig. 2.20 Activation of NK cell activity In some circumstances, inhibitory receptors recognizing ligands other than MHC class I proteins may suppress NK cell responses. When interacting with target cells expressing ligands for both inhibitory and activating receptors, the outcome is determined by the summation of the strength of signals. The amount of activating and inhibitory receptors on the NK cells and the amount of ligands on the target cell, as well as the qualitative differences in the signals transduced, determine the extent of the NK cell response. (LANIER 2005)

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MICA is induced in response to heat or cellular stress. NK cells are therefore active

only against target cells which have lost MHC expression (e.g. after viral infection or

malignant transformation) because then the interaction with KIR is not possible, and

because in these cells NKG2D is expressed on NK cells recognizing the MHC class I

chain-related A gene (MICA) on target cells (BAUER et al. 1999; WU et al. 1999)

(Fig. 2.20).

In order to investigate NK cell activity in the context of HF IP, ITO et al. (2008a)

studied an organ-specific, cell-mediated autoimmune disease on skin from patients

with AA, thought to result from a collapse of HF IP (GILHAR et al. 2005; PAUS et al.

2005; GILHAR and KALISH 2006). Indeed, in skin of AA patients both, a marked up-

regulation of NK cell numbers with a defect in inhibiting perifollicular NK cell

activation was observed (ITO et al. 2008a) and an upregulation of the activating

NKG2D molecule and MICA. The normal anagen HFs may escape from NK cell

attack by combining active suppression of NK cell (e.g. by MIF and KIR up-

regulation) with reducing the chance of NK cells to receive stimulatory signals (e.g.

by down-regulation of NKG2D expression on NK cells and of its ligand, MICA, on the

potential target - HF epithelium). These recently revealed defects must now be taken

into account in AA pathogenesis, and in the development of more effective treatment

scenarios for AA and related autoimmune diseases that are also characterized by IP

collapse (ITO et al. 2005d).

MHC class Ib molecules such as HLA-G and E inhibit cell killing by NK cells (see

chapter 3.10.4). In IP sites, i.e. the fetotrophoblast, HLA-G is recognized by an

inhibitory receptor on the NK cell, which prevents the NK cell from killing placental

cells (JANEWAY et al. 2005). HLA-E binds a very restricted subset of peptides,

derived from the leader peptide HLA-G and by forming a complex with this HLA-G,

HLA-E can bind to the receptor NKG2a on NK cells. This interaction will lead to an

inhibition of the NK cell dependent cytotoxicity (JANEWAY et al. 2005).

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2.5.3 Infantile human nail matrix is a site of relative immune

privilege

ITO et al. (2005a) recently discovered that the HF is not the only intracutaneous

compartment with an IP. The nail apparatus, which has many stem cells, is often

attacked by chronic inflammatory diseases (e.g. chronic eczema, lichen planus,

psoriasis, alopecia areata, lupus erythematosus, scleroderma, bullous dermatoses),

which may result in substantial, often irreversible changes to this functionally

important skin appendage (TOSTI and PIRACCHINI 1999; HANNO 2000; DE

BERKER et al. 2001; TOSTI et al. 2001). Like the HF, the nail apparatus is

constantly exposed to environmental damage, and thus requires a well-functioning

and balanced immune response against infectious attack and tissue damage

(HANNO 2000). At the

same time, not unlike the

eye, a careful balance must

be struck here between

sufficient and undesired,

autodestructive immune

responses, if severe nail

apparatus damage has to

be avoided.

Fig. 2.21 The similarity of anatomical structure between HF and nail Nail apparatus and HF share some anatomical features. Hair matrix, ORS and hair shaft are mirrored to PNM, nail bed and nail plate, respectively (ITO et al. 2008b).

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On the basis of the substantial architectural similarities between hair follicle and nail

apparatus (e.g. nail and hair matrix) (Fig. 2.21), it seemed expectable that the –

previously only ill-defined – nail apparatus immune system shows striking similarities

with the HF immune system (ITO et al. 2005c). Immunohistological analysis, in

particular, suggested that nail tissue also harbors a distinct area of IP. ITO et al.

(2005c) showed that HLA-A/B/C expression is prominently down-regulated on both

keratinocytes and melanocytes of the proximal nail matrix (PNM), compared to other

regions of the nail epithelium (Fig. 2.22).

The PNM also shows moderate HLA-G immunoreactivity and strong IR for locally

generated immunosuppressants such as TGF-β1, α-MSH and ACTH, as well as for

macrophage migration inhibitory factor (MIF) as prominent inhibitor of NK cell activity.

Around the PNM, there are only very few CD1a+, CD4+ or CD8+ cells, compared to

nail fold and hyponychium (Fig. 3.5). CD1a+ cells in and around the PNM show

reduced MHC class II and CD209

expression, indicating diminished antigen-

presenting capacity (ITO et al. 2005c).

Fig. 2.22 Distribution of HLA-ABC, CD4+ and CD8+ T cells in the murine nail apparatus (ITO et al. 2008b)

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Taken together, this suggests that the nail immune system strikingly differs from the

skin immune system, but shows intriguing similarities to the HF immune system,

including the establishment of an area of relative immune privilege in the PNM. The

functional advantages of establishing IP in the nail matrix are still uncertain (e.g.

stringent containment of inflammation in a skin appendage that is notoriously under

the attack of infectious agents and is prone to trauma-induced inflammatory tissue

damage?). Also, it is unknown yet which key autoantigens are shared between nail

apparatus and HFs, and whether the collapse of IP in the nail matrix may also

underlie the frequent involvement of the nail in AA. However, it is already clear that

our – as yet, poor - understanding of the immunopathogenesis of nail growth

disorders necessitates systemic dissection of the nail immune system and its IP.

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2.6 The immune privilege collapse model of AA pathogenesis

Fig. 2.23 ´Immune privilege collapse model` of alopecia areata pathogenesis Some endo-/exogenous factors induce IFN-γ production that upregulate MHC class I in hair bulb. Then, AA-associated follicular autoantigens are presented via MHC class I to autoreactive T cells that result in secondary autoimmune phenomena (ITO et al. 2008b).

More than ten years ago, PAUS and co-workers claimed the hypothesis, which was

the first to apply Billingham’s concept of HF IP to the pathogenesis of AA (PAUS et

al. 1993; PAUS et al. 1994a; PAUS et al. 1999b) (Fig. 2.23). Although this concept

was over years ignored, it is widely accepted today in AA research. While the

pathogenesis of AA still has not been fully elucidated, recent consensus appears that

AA reflects an organ-restricted, T cell-mediated autoimmune disease (ITO et al.

2008b). The most important results in this respect were reported by GILHAR and

KALISH (2006, 2007), and colleagues who demonstrated that AA lesions can be

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induced by the transfer of MHC class I-restricted CD8+ T cells alone, that anagen HF

antigens are needed to stimulate T cells for effective triggering of AA lesions after cell

transfer, and that these antigens can be substituted for by HF melanocyte antigens

(WHITING 2003; GILHAR et al. 2005). The key histological feature of AA is a

lymphocytic infiltrate around the lower HF, which may show a characteristic ´swarm

of bees` pattern (SPERLING and LUPTON 1995), but - in chronic AA - can also be

much more discreet than widely assumed (WHITING 2003). CD4+ T-cells

predominate in the infiltrate surrounding the HF, while T-cells within the follicular

epithelium are predominantly of the CD8+ type (TODES-TAYLOR et al. 1984).

In AA lesions, antigen presenting cells including Langerhans cells and macrophages,

infiltrate the dystrophic HFs, and often melanin deposition is observed around these

HFs (HORDINGSKY 2003). Trigger for the pathogenesis could be infectious foci,

bacterial superantigens, psychoemotional stressors, skin microtrauma, or other

damage to the HF, which are possibly aided by predisposing immunogenetic factors.

The result is a peri- and/or intrafollicular rise in AA of IFN-γ secretion, ectopically up-

regulating MHC class Ia expression in the proximal HF epithelium (PAUS et al. 2003;

ITO et al. 2008b). This MHC class I up-regulation occurs in the normally MHC class I-

negative hair matrix of anagen hair bulbs, where immunogenic melanogenesis-

associated antigens are massively generated. Once the HF enters anagen and its

pigmentary unit engages in active melanogenesis [i.e., during anagen III/VI

(SLOMINSKI and PAUS 1993b)], these anagen- and/or melanogenesis-associated

autoantigens are no longer sequestered but can be ectopically presented in the

normally MHC class I-negative epithelial hair bulb and, thus, seriously endangering

maintenance of the HF IP. If an individual has pre-existing autoreactive CD8+ cells,

which in addition receive appropriate co-stimulatory signals and help from CD4+ T

cells (and, possibly, additional signals via CD4 as well), a cytotoxic T cell attack on

the hair matrix is the result. This attack activates a vicious circle of secondary,

follicle-damaging autoimmune phenomena, whose quality and magnitude largely

determine the resulting degree of HF damage (dystrophy) and thus the actual clinical

manifestation, progression, and course of AA (ITO et al. 2008b).

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This basic hypothesis was later extended to account for a key role of locally

generated immunosuppressants [e.g. α-MSH, TGF-β1 and IGF-1 (ITO et al. 2004)]

and NK cell-suppressing activities [e.g. MIF (ITO et al. 2005d)] as ´guardians of HF

immune privilege`, the insufficient activity/function of which was thought to

predispose individuals towards AA development, while immune privilege repair via

these agents was suggested to underlie spontaneous AA remission and hair

regrowth (PAUS et al. 2005).

Collapse and restoration of IP in the anagen hair bulb in vitro

As described before, IFN-γ plays an important role in the pathogenesis of AA.

Compared to other cytokines such as IL-1 and TNF-α, IFN-γ offers the most potent

cytokine stimulus for ectopic MHC class I expression in murine pelage HFs in vivo

(RUECKERT et al. 1998). In addition, recent studies have demonstrated that IFN-γ

deficient mice are resistant to the development of alopecia areata (FREYSCHMIDT-

PAUL et al. 2006). ITO et al. have recently developed a standardized and highly

reproducible in vitro assay that recreates the key feature of IP collapse postulated

above in the human system: the ectopic upregulation of HLA-A/B/C expression in the

matrix of normal human anagen scalp HFs. Using this new in vitro assay and very

sensitive immunostaining techniques, confirmed by in situ hybridization and RT-PCR,

they could show that IFN-γ is indeed a very potent stimulator of ectopic MHC class I

expression in microdissected, organ-cultured human scalp HFs from healthy donors

in anagen VI (ITO et al. 2004). Low-dose IFN-γ (75 IU/ml) can be exploited

experimentally to induce IP collapse of normal human anagen HFs in vitro, likely via

an IRF-1-mediated mechanism (ITO et al. 2004). Higher doses of IFN- γ (500 IU/ml)

also act as potent catagen inducer in human scalp HFs (ITO et al. 2005b). The ability

of IFN-γ to induce follicular MHC class I and II was used to test the hypothesis that

AA results from loss of immune privilege. C3H/HeJ female mice were injected

intravenously with IFN-γ to induce follicular MHC. These injected mice demonstrated

an increased rate of development of alopecia areata (GILHAR et al. 2005).

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Thus, this in vitro model allows also screening for candidate agents that restore IP by

down-regulating the IFN-γ induced, ectopic MHC class I expression in human

anagen HFs. So far, three immunomodulators, which are known to be locally

produced in the anagen hair bulb, α-MSH, TGF-β1, and IGF-1 (SLOMINSKI et al.

1993, 2000; BOTCHKAREV et al. 1999; PAUS et al. 1999a), demonstrated the

capacity of down-regulating ectopic MHC class Ia expression, on both the protein

and the mRNA level, when added to the culture medium after IFN-γ administration

(ITO et al. 2004). The fact, that natural and by the HF itself generated

immunomodulators might be used for IP restoration in AA is encouraging. These and

other related agents also promise to carry with them minimal risks of toxicity, which

may be further reduced by their topical application (e.g., via HF-targeted liposome

preparations). Today, AA treatment focuses on targeting the secondary autoimmune

phenomena associated with AA, but it is still not convincing or satisfying. Instead, it

would be much more promising to concentrate on the restoration of the HFs lost or

compromised IP - both for preventing the progression of AA lesions and for inducing

hair regrowth. This IP restoration therapy does not require any prior knowledge of the

relevant key autoantigens or the specific autoreactive T cells, and it can resort to the

administration of well-known nonspecific immunomodulators that chiefly down-

regulate ectopic MHC class I expression in the anagen hair bulb.

These reasons explain the need of further evaluating and testing different substances

as possible IP restorer, in order to find a suitable candidate with minimal side effects.

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2.7 The human bulge region as a site of relative immune privilege?

Recent gene and protein expression data obtained from mice have raised the

possibility that follicular IP is not purely limited to the anagen hair bulb (COTSARELIS

2006a). By microarray analysis of keratin 15 promoter-driven-GFP labelled ORS cells

of murine pelage HFs, which included bulge stem cells. MORRIS et al. (2004)

identified downregulated transcript levels for several histocompatibility genes (e.g. β2-

microglobulin, H2-Q8, H-2K2, H-2D). Independently, transcriptional profiling of

murine and human bulge label-retaining cells revealed an increase of TGF-β2 mRNA

(TUMBAR et al. 2004; OHYAMA et al. 2006), a molecule that is known to help

maintain the immune-privileged milieu in the eye and brain (NIEDERKORN 2006;

SIGLIENTI et al. 2007).

The concept of bulge IP is further supported by the observation that CD200 IR

preferentially occurs in the bulge region of mice and humans. The cell surface

glycoprotein attenuates inflammatory responses, while CD200-deficient mice develop

an inflammatory, cicatricial alopecia (ROSENBLUM et al. 2004). This has invited the

proposal that CD200 may function as an immunosuppressive, ´no danger` signal for

the HF (ROSENBLUM et al. 2006). OHYAMA et al. (2006) also demonstrated that

the bulge region of normal human scalp HFs prominently expresses CD200 at the

gene and protein level, along with other stem cell markers. Since protection of these

epithelial stem cells from immune destruction is essential for preserving the

regenerative and cycling capacity of HFs (PAUS and COTSARELIS 1999;

COTSARELIS 2006a; TIEDE et al. 2007a), it seems logical that the bulge region

should also have an established relative IP (Fig. 2.24).

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Fig. 2.24 The human HF bulge region HE staining shows the human HF bulge with the insertion point of the arrector pili muscle. APM: Arrector pili muscle, BM: Basement membrane, CTS: Connective tissue sheath, HS: Hair shaft, IRS: Inner root sheath, ORS: Outer root sheath, SG: Sebaceous gland. Bar 100µm.

However, previous immunohistological analyses have provided contradictory

evidence on this matter. The first study published on MHC class Ia antigen

expression in normal terminal human HFs reported prominent IR in the infundibular

region of the ORS, with diminished expression of HLA-A, B, C and β2-microglobulin

in the isthmus region, although the bulge region had not been specifically analysed

(HARRIST et al. 1983). Subsequent immunohistological studies of MHC class Ia

expression in murine pelage HFs (PAUS et al. 1994b, 1998) did not identify a

prominent downregulation of MHC class Ia antigens in the bulge. However, the

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murine MHC class Ia analogue, H-2Db antigen, was slightly downregulated in both

the isthmus and bulge (PAUS et al. 1994b). In a study of normal human skin, the

isthmus region of the ORS in terminal anagen VI HFs clearly expressed HLA-A/B/C

antigens, though possibly at a somewhat reduced level of IR compared to the distal

ORS (CHRISTOPH et al. 2000). In addition, IR for β2-microglobulin (CHRISTOPH et

al. 2000), which stabilizes MHC class Ia molecules allowing proper (self-) peptide

presentation, was also identified (JANEWAY et al. 2005). Furthermore, CD1a+

and/or MHC class II+ Langerhans cells were detected in this region of the ORS,

although their numbers were reduced compared with the distal follicle (CHRISTOPH

et al. 2000). On superficial analysis, these findings supported the concept that HF IP

is restricted to the anagen hair bulb (PAUS et al. 2003, 2005).

Thus, although the murine gene profiling data derived from isolated cultured cells

suggests the existence of an IP within the bulge, convincing protein evidence for this

aspect is still missing. Since IP is a phenomenon that refers to entire tissue

compartments, we aimed to provide in situ protein IR evidence that would support or

refute the hypothesis of bulge IP in human HFs.

2.8 Bulge IP collapse and the pathogenesis of PCA

Cicatricial alopecias (PCA; scarring alopecias, permanent alopecias) are an

uncommon, but clinically important group of inflammatory disorders that result in

permanent loss of human scalp hair. Histological examination reveals the

characteristic replacement of HF with scar-like fibrous tissue (WHITING 2001) and

perifollicular lymphocytic infiltrates that lead to hydrophic degeneration of the basal

layer of the outer root sheath, besides other skin architecture changes.

In contrast to reversible forms of inflammation-induced, autoimmune hair loss (e.g.

alopecia areata) that characteristically attack the actual hair shaft factory (i.e. the

anagen hair bulb), the infiltrate in cicatricial lesions is centered round the eHFSC

located in the bulge (Fig. 2.25 A,B) (PAUS and COTSARELIS 1999; COTSARELIS

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2006a). This typical peri-bulge location already suggests that immuno-destruction of

eHFSC is a likely key event in its pathogenesis. It has been demonstrated that

Keratin 15 immunostaining, which highlights the bulge region of the ORS (but is not

limited to it) (KLOEPPER et al. 2008), is reduced especially in patients with a dense

peri-follicular inflammatory infiltrate around the bulge (AL-REFU et al. 2008;

POZDNYAKOVA and MAHALINGAM 2008). Together with the reported reduction of

proliferation rate these findings suggest the important role of eHFSC loss in the

bulge, although not the only component in cicatricial alopecia pathogenesis (MOBINI

et al. 2005). On the other hand, the occurrence of perifollicular inflammatory cell

infiltrates around the bulge region is quite often (JAWORSKY et al. 1992;

EICHMULLER et al. 1998) and causes only rarely irreversible hair loss. Therefore,

(immuno-) protective mechanisms normally in place must be severely defective or

disrupted.

Fig. 2.25 Hypothesized model of the pathogenesis of cicatricial alopecia CCLE: cutaneous cicatricial lupus erythematosus (HARRIES et al. in press)

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On the basis of the recent new microarray data and our current study it was

hypothesized, that loss of relative bulge IP, in combination with the exposure of

eHFSC to a directly cytotoxic immune attack, may therefore play a key role in the

pathogenesis of cicatricial alopecia (HARRIES et al. 2008) (Fig. 2.25). Preliminary

work in patients with cicatricial alopecia indicate that MHC class Ia, β2-microblobulin

and MHC class II IR is significantly up-regulated in the bulge region of lesional skin,

compared with un-involved skin (HARRIES and PAUS, unpublished observations).

While loss of bulge IP offers a plausible explanation for how eHFSC can become

exposed to destructive immune attack, it is still unknown, which intrafollicular

target(s) or stimulators [e.g. IFN-γ (TORO et al. 2000; WENZEL and TUTING 2007;

WENZEL et al. 2007)] of such auto-aggressive immune responses remain to be

identified. It is also unclear whether IP collapse occurs early in the disease process

(i.e. before inflammation develops), or only later on, as a secondary phenomenon.

Since of the irreversible nature of this disease and unsatisfactory therapeutic options

(HARRIES et al. 2008), it is important to further explore the immuno pathogenesis of

cicatrical alopecias and to search for future therapeutic strategies in immuno

protection of eHFSC and restitution of their IP.

2.9 Function of immunoprivileged sites

While it is now clear that IP is indispensable for normal eye function to avoid

inflammatory reactions, and that IP in the fetomaternal placentar unit is vital for the

suppression of fetal rejection (STREILEIN 1993; BRENT 1997; MELLOR and MUNN

2000; EHRLEBACHER 2001; NIEDERKORN 2002, 2003), it still remains to be

satisfactorily demonstrated by convincing functional evidence what exactly HF IP

might be good for. With regard to ocular IP, NIEDERKORN concludes that the

existence of ocular IP is necessary for the normal function of the visual axis, the

preservation of which requires that ocular inflammation is regulated very stringently

and innocent bystander immune damage that may result in irreversible eye damage

is scrupulously avoided (NIEDERKORN 2002; STREILEIN 2003). Along the same

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lines, establishment of an IP milieu in the HF may be needed to ensure a safe

environment for HF cycling, the process of which depends on a constitutively active

safeguarding system to protect it against immune injury.

In this context it is noteworthy that, apart from epidermal melanocytes (in vitiligo),

thyroid epithelium (in autoimmune thyroiditis) and the synovium (in rheumatoid or

psoriatic arthritis) (PAUL 1999; JANEWAY et al. 2001), the HF represents one of the

most frequent targets of immune-mediated disease, resulting in the development of

alopecia areata (AA), or even in permanent hair loss (due to immune destruction of

epithelial HF stem cells) as it is seen in the scarring alopecia associated with lichen

planopilaris, lupus erythematodes, scleroderma, and folliculitis decalvans (HERMES

and PAUS 1998; PAUS and COTSARELIS 1999). Therefore, one reasonable

general hypothesis is that HF IP serves to reduce the chances of autoimmune HF

damage (PAUS et al. 2005).

Skin melanocytes are prone to be the target of immune-mediated injury (e.g., in

vitiligo, halo nevi, regressing malignant melanoma, and during immunotherapy of

metastasizing melanoma). Notably, in AA lesions, the characteristic inflammatory cell

attack on lesional HFs almost exclusively targets anagen hair bulbs, which are in the

process of active pigment production (i.e. anagen III-VI HFs). In addition, recovering

HFs in AA patients typically generate white hair shafts (PAUS et al. 1994a; DAWBER

and FENTON 1997). Thus, a more specific hypothetical function of HF-IP is to

sequester melanocyte-/melanogenesis-associated autoantigens from immune

recognition and to protect the hair bulb from potentially deleterious autoaggressive

immune responses. Immunogenetically distinct individuals may differ in their relative

level of protection from, and relative risk of, anti-HF autoimmunity. In case of

constitutively insufficient IP capacity or functional collapse of the HF IP, this would

result in a greatly enhanced risk of autoimmune attack on the follicle (PAUS et al.

1994a, 1999b, 2005).

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It is interesting to note that during neonatal HF morphogenesis in mice, in contrast to

the epidermis, distal follicular keratinocytes begin to express MHC class I only at the

late stage of development when almost all skin cells already express MHC class I

molecules, paralleling the timing of maturation of the skin immune system (PAUS et

al. 1998, 1999b; OHYAMA et al. 2006). This observation raises the question if IP is

also needed for the proper development of HF epithelium. Perhaps IP needs to be

established by the time, when the full complement of immunocytes (intraepithelial T

cells and Langerhans cells), immunoregulatory surface molecules, secreted

immunomodulators, and HF melanocytes has been assembled and expressed in

their designated locations.

In any case, the currently available – largely phenomenological – evidence, taken

together, suggests that the HF IP is generated and maintained during each anagen

phase (and then disassembled again during catagen and telogen phases) in order to

sequester potentially deleterious, anagen- and/or melanogenesis-associated

autoantigens from immune recognition by appropriately sensitized CD8+ T cells with

cognate receptors, primarily via down-regulation of MHC class I and by the local

production and secretion of potent immunosuppressants (PAUS et al. 1993, 1999b;

HARRIES et al. 2008; HARRIES et al. in press).

2.10 Immune privilege markers

IP is a relative, not an absolute state, which reflects the net result of multiple

interconnected active and passive mechanisms (FERGUSON and GRIFFITH 2006;

SIMPSON 2006). But it is an ongoing debate, how many and which of these

mechanisms have to be established in a given tissue location to justify the term ´IP`

for this site (FERGUSON and GRIFFITH 2006). Since no universally accepted

criteria have as yet been defined that apply to all IP tissues, ultimate proof for the

human bulge, the murine vibrissal follicle or nail apparatus as sites of relative IP can

only arise from functional studies, such as Billingham’s famous (as yet never

repeated) melanocyte allotransplant experiment in guinea pigs which still represents

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the only existing functional evidence of bulb IP (BILLINGHAM 1971; PAUS et al.

2005). Also, in order to demonstrate that a putative ´IP` status of any tissue

compartment is to truly relevant to organ or system homeostasis, it needs to be

shown that loss of IP actually can lead to pathology (FERGUSON and GRIFFITH

2006). Nonetheless, in the following part, we discuss some of the as important

agreed IP markers with their expression patterns and supposed mechanisms.

2.10.1 MHC class I molecules

The major histocompatibility complex (MHC) is a cluster of genes on human

chromosome 6 or mouse chromosome 17. It encodes a set of membrane

glycoproteins called the MHC molecules (JANEWAY et al. 2005) which are grouped

into three classes (I-III). MHC class I is separated to classical (MHC Ia) and non-

classical (MHC Ib). The classical MHC class I gene, is subdivided into HLA-A, HLA-B

and HLA-C in human and Qa, Tla and M in mice. These molecules present peptides

generated in the cytosol in particular to CD8+ T cells. The MHC class I molecule

consists of a single three domain α chain, which is non-covalently associated with β2-

microglobulin. MHC class I is basically expressed on almost all nucleated cells in the

body (LE GAL et al. 1999; JANEWAY et al. 2005), except those in areas of an

established IP (PAUS et al. 2005; NIEDERKORN 2006), which often do have little or

no capacity for regeneration (LAMPSON and FISHER 1984; ABI-HANNA et al. 1988;

LE BOUTEILLER 1994). The lack or down-regulation of classical MHC class I

expression is probably the most characteristic feature of immunoprivileged sites and

tissues.

On the one hand, limited or absent MHC class I expression hinders (auto-)antigen

presentation to autoreactive CD8+ T cells (PAUS et al. 2005) and protects them from

lysis by these cells, contributing to IP. But on the other hand, the reduced or absent

expression of MHC class I molecules creates an immunological dilemma, as it

attracts the attention of NK cells, which are programmed to destroy both cells, with

incompatible MHC class I type and MHC class I-negative cells (LJUNGGREN 1991).

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This is especially important for the allogeneic fetus with maternal and paternal

antigens, as NK cells account for 70% of the lymphocytes found in the pregnant

human uterus (NIEDERKORN 2006). Thus, other mechanisms have to compensate

for the paucity or frank absence of MHC class Ia, such as non-classical MHC class Ib

molecules, TGF-β or MIF.

Both skin appendages, the proximal nail matrix and the proximal epithelial anagen

hair bulb of the normal mammalian HF - e.g. in human (BROECKER et al. 1987),

mice (PAUS et al. 1994a) and rat HFs (WESTGATE et al. 1991) - demonstrate virtual

absence of MHC class I-antigen expression.

Interestingly, IR for classical MHC class Ia antigen in the HF is hair cycle dependent,

both in mice and human (PAUS et al. 1994a, 2005). These details have been first

investigated using the murine hair cycle as a model, exhibiting significant differences

between various follicle compartments (Fig. 2.26) (PAUS et al. 1994a). The cycle-

dependency of murine HF MHC class I antigen were illustrated in experiments in

which anagen was induced in the back skin of the mouse (e.g. C57BL/6) by

depilation of the HFs in telogen (PAUS et al. 1990). During the entire hair cycle, the

distal part of the HF continuously showed strong classical MHC class I IR. In the

telogen stage, the entire HF compartments had a strong classical MHC class I IR

except for the dermal papilla. Shortly after anagen induction, this IR pattern changed.

In anagen stages II-III, anagen hair matrix and dermal papilla showed negative

classical MHC class I IR. In anagen stage VI, the IRS, in addition to the hair matrix,

had no classical MHC class I IR, which the MHC class Ia IR in dermal papilla

reappeared. In spontaneously developed catagen HFs, only the slowly receding IRS

keratinocytes and dermal papilla remained MHC class Ia negative (PAUS et al.

1994a; ITO et al. 2008b).

In the human HF cycle corresponding expression changes remain to be elucidated in

greater detail, but MHC class I expression in human anagen VI scalp has also been

investigated (CHRISTOPH et al. 2000; ITO et al. 2004). So far, MHC class I IR was

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found on all interfollicular cutaneous cells. In comparison with the epidermis and the

distal ORS, the isthmus region of the ORS showed substantially reduced MHC class

I IR, and a striking restriction of MHC class I IR to basal keratinocytes. The proximal

ORS, IRS, all hair bulb keratinocytes and most of the DP displayed no detectable

MHC class I IR. Sporadically, single MHC class I+ cells were found within the DP. In

contrast, perifollicular fibroblasts and immune cells of the perifollicular CTS were

MHC class I+. In the murine vibrissal follicle or nail apparatus, MHC class I IR has

not been studied yet, although MHC class Ia is one of the most important immune

parameters, and although whisker hairs are often used in experiments on hair

growth.

Fig. 2.26 The cycle-dependency of murine HF MHC class I antigen During the entire hair cycle, the distal part of the HF continuously shows strong classical MHC class I IR, but IR in the proximal or lower HF changes. (PAUS et al. 2005)

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2.10.2 β2-microglobulin

β2-microglobulin is a smaller non-covalently associated chain of MHC class Ia

(TOSTI and PIRACCHINI 2008, JANEWAY et al. 2005), is not polymorphic and is not

encoded in the MHC locus. It often serves as an additional verification marker of

MHC class Ia expression. As a result, β2-microglobulin is expressed on almost every

nucleated cell, except for those of IP (CHRISTOPH et al. 2000; PAUS et al. 2005;

NIEDERKORN 2006) demonstrated in the human anagen HF a prominent

expression pattern in the epidermis and distal ORS, whereas IR decreases towards

the lower HF part and was absent in the proximal ORS, IRS, and hair bulb. So far, a

detailed analysis of the bulge region has not been done. In contrast, the

immunoprivileged PNM did not display reduced β2-microglobulin (ITO et al. 2005c).

In addition, β2-microglobulin interacts with the so-called MHC class I pathway-

associated molecules, which are likely to contribute to the compensation work of

MHC class I negativity, and currently available information suggests that they are

intimately involved in maintaining HF IP (ITO et al. 2008b). Briefly, antigen

presentation in the context for the MHC class I molecules to CD8+ T cells requires

the ATP-dependent transporter in antigen presentation (TAP), consisting of the

subunits TAP1 and TAP2 (PAMER and CRESSWELL 1998). TAP1 and TAP2 genes

are in the MHC class II region that must be expressed for MHC class I molecules to

be assembled efficiency (MOMBURG et al. 1994). Peptides generated in the cytosol

by the proteasome are translocated by TAP1 and TAP2 into rough endoplasmatic

reticulum (RER) by a process that requires the hydrolysis of ATP. Within the RER

membrane, newly synthesized class I α chain associates with calnexin until β2-

microglobulin binds to the α chain. The class I α chain-β2 microglobulin heterodimer

then binds to calreticulin and the TAP-associated protein tapasin. When a peptide

delivered by TAP is bound to the class I molecules, folding of MHC class I is

complete and it is released from the RER and transported through the Golgi to the

surface.

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The expression of these MHC class I pathway molecules, such as TAP and β2

microglobulin was analysed in human and murine HFs. ITO et al. (2004) showed that

β2-microglobulin and TAP2 IR mirrored the low or absent expression level of HLA-

A/B/C molecules in the proximal ORS and hair matrix of anagen hair bulbs compared

to distal ORS and epidermis in the normal human scalp skin sections (Fig. 2.27)

Therefore, this immunoprivileged region of the skin epithelium is characterized by a

down-regulation not only of HLA-A/B/C and β2 microglobulin but also of major MHC

class I pathway molecules. This reflects the situation in murine HFs in vivo and

underscores the relative defect of these follicular tissue compartments in the

presentation of self-peptides to CD8+ T cells (RUECKERT et al. 1998).

Fig. 2.27 The expression of MHC class I pathway molecules on the murine anagen HF. The expression of the MHC class I pathway molecules TAP and β2-microglobulin is mirroring the IR of HLA-A/B/C molecules. (ITO et al. 2008b)

2.10.3 MHC class II molecules

MHC class II antigens are encoded by the class II region of the MHC complex,

expressed mainly on B cells, macrophages (CD11b on mice) and dendritic cells, and

are involved in the interaction of these with CD4+ T cells. MHC class II molecules are

composed of two non-covalently associated transmembrane glycoprotein chains, α

and β, which also have two domains each. In IP sites it is considered that the

capacity of APCs (i.e. LC and macrophages) may be impaired by a downregulation of

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MHC class II (NIEDERKORN 2003; PAUS et al. 2003, 2005). As a consequence, no

or less activation of naïve T cells occurs, because activation and antigen

presentation requires the simultaneous delivery of a co-stimulatory signal, together

with the expression of both MHC molecules (MHC class I and II) (FITZPATRICK

2008; JANEWAY et al. 2005).

In eye IP it was found, that the single corneal endothelial cell layer that lines the

anterior chamber expresses little or no detectable class II MHC (and MHC class I)

determinants (NIEDERKORN 2002). In addition, other factors like TGF-β2 alter the

behavior of APCs, downregulate their MHC class II molecules and their production of

the TH1-inducing cytokine IL-12, while stimulating their synthesis of IL-10 (which

activates TH2 response) (D`ORAZIO and NIEDERKORN 1998; NIEDERKORN

2002). Since IP is composed of several different mechanisms it is unclear, which

effect was first and which are induced by others.

MHC class II+ cells or NLDC-145+ Langerhans cells are essentially absent (PAUS et

al. 1998, 1999b) in the murine anagen hair bulb. Very few CD1a+ or ultrastructurally

identified LCs must be also functionally impaired because they do not express MHC

class II (MOSELEY 1997; CHRISTOPH et al. 2000). In the human HF, CHRISTOPH

et al (2000) reported that HLA-DR+ cells representing LC were prominently

expressed in the epidermis, but were particularly densely distributed in the ORS

surrounding the follicular canal and around the sebaceous gland. The isthmus region

and the proximal ORS showed sharply reduced numbers of MHC II+ cells compared

with the distal ORS and the bulb displayed only very rarely positive cell, the DP none.

In the CTS MHC class II+ cells were, in particular, rare in the isthmus region,

whereas in the distal CTS and around the hair bulb the number was higher. Although

the work of CHRISTOPH et al. (2000) was detailed, they had not focused on the

bulge region or distinguished between isthmus, bulge and proximal ORS.

In the nail apparatus, LC and macrophages in or around the PNF showed the

expected strong HLA-DP/DQ/DR IR, suggesting that these CD1a+ or CD68+ cells

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normally perform their well-recognized ´professional` antigen-presentation functions

via MHC class II. In contrast, intraepithelial LC in dorsal nail matrix and dermal

macrophages surrounding the entire nail matrix revealed no or very low IR for HLA-

DP/DQ/DR (ITO et al. 2005c).

2.10.4 MHC class Ib molecules

The non-classical MHC class Ib molecules in humans are HLA-E, -F, -G and –H, in

mice they are called H-2Qa1, to Qa10, H-2T1 to T24, and H-2M1 to M7. These

molecules are encoded within the MHC gene but not highly polymorphic like the

MHC class Ia and MHC class II molecules, and present only a restricted set of

antigens. Non-classical MHC class Ib molecules such as HLA-G and HLA-E in

humans or Qa-2 in mice are expressed in the immunoprivileged fetotrophoblast

(KOWATS 1990; JANEWAY et al. 2005; MOSCOSO et al. 2006; YIP et al. 2006), the

eye (NIEDERKORN et al. 1999; LE DISCORDE et al. 2003), and the proximal nail

matrix (ITO et al. 2005c), and are therefore thought to help maintaining IP

(JANEWAY et al. 2001; FUZZI et al. 2002).

HLA-G and HLA-E impair specific cytolytic T cell functions and have the capacity to

engage the NK cell inhibitory receptor CD94/NKG2 preventing lysis by NK cells

(KOWATS 1990; BRAUD et al. 1998; LEE 1998; LE GAL et al. 1999; ROUAS-

FREISS et al. 2000; TRIPATHI et al. 2006). Originally it was proposed that HLA-G

functions as a ligand with CD94/NKG2A receptor inducing the inhibitory effect on NK

cells. Today, it seems as if HLA-G only acts as a leader peptide, which brings the

mesenchymal-facing intracellular located HLA-E to the cell surface by forming a

complex, and that the following interaction of HLA-E with the CD94/NKG2A receptor

may result in inhibition of NK cell- and cytotoxic T cell-dependent lysis. HLA-E also

might interact with CD8+ T cells directly (GAO et al. 1997), and it is reported that

trophoblast cells also elaborate a soluble isoform of HLA-G that induces apoptosis of

activated CD8+ T cells (CAROSELLA et al. 2000; NIEDERKORN 2006).

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In fact, expression of a mouse non-classical MHC class I equivalent, Qa-2, could be

detected in the periinfundibular region of the murine outer root sheath throughout the

entire hair cycle (PAUS et al. 1994a). The hair cycle-independent expression of Qa-2

raises the possibility that these MHC class Ib molecules are involved in the regulation

of follicular IP, rather than in the intriguing, non-specific anti-infection defense of the

HF immune system (HIS) (PAUS et al. 1994a; PAUS et al. 1998). It also encourages

the hypothesis that MHC class Ib expression may play a similar T and NK cell-

inhibitory role in the context of the HF-IP as it does in the fetomaternal placentar unit

(LE GAL et al. 1999). In addition, ITO et al. (2005c) demonstrated a strong up-

regulation of HLA-G expression in the proximal nail matrix compared to the proximal

nail fold. They also concluded that HLA-G expression may serve to inhibit an attack

of NK cells on this MHC class I negative tissue.

2.10.5 CD4+ and CD8+ T cells

CD4 (cluster of differentiation 4) and CD8 are transmembrane glycoproteins. CD4 is

expressed on the surface of T helper cells, regulatory T cells, monocytes,

macrophages, and dendritic cells (JANEWAY et al. 2005). On T cells, CD4 is a co-

receptor that assists the T cell receptor (TCR) to activate its T cell following an

interaction with an antigen presenting cell. CD4 amplifies the signal generated by the

TCR by recruiting a tyrosinase kinase, which is essential for activating many

molecules involved in the signaling cascade of an activated T cell. In addition, CD4

also interacts directly with MHC class II molecules on the surface of the APC. CD8

also serves as a co-receptor for the TCR. Like the TCR, CD8 binds to MHC class I

protein and attacks the cell, if it has no or foreign MHC class I protein on its surface.

T is predominantly expressed on the surface of cytotoxic T cells, but can also be

foundon NK cells. To function, CD8 forms a dimer, consisting of a pair of CD8 chains.

It was demonstrated, that contrary to the epidermis, the sebaceous gland and the

rest of the hair follicle epithelium, the anagen hair bulb is almost devoid of

intraepithelial T cells; in humans, CD4+ or CD8 T cells here are extremely rare in the

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proximal HF epithelium (CHRISTOPH et al. 2000), whereas in mice no γδ-TCR

lymphocytes are found at all here (PAUS et al. 1994 ; PAUS et al. 1998).

In the human nail apparatus ITO et al. (2004) demonstrated a high density of CD4+ T

cells in PNF, accumulated CD4+ T cells in epithelium and mesenchyme, that were

not observed in normal human skin, and very rarely detected CD4+ T cells in

adjacent proximal nail matrix (PNM) resembling the hair bulb. Essentially, the same

observation was made for CD8+ T cells, even though their total number was

2.10.6 α-MSH and ACTH

α-melanocyte-stimulating hormone is a tricapeptide derived from a precursor

hormone called propiomelanocortin (EIPPER and MAINS 1980). This POMC

molecule is source for several bioactive peptides including adrenocorticotropin

(ACTH), melanocyte-stimulating hormone (MSH) and the endogenous opioid β-

endorphin (LUGER et al. 2000). Although POMC peptides were originally considered

as neuropeptides, it is now well established that many peripheral tissues including

the skin (BOEHM et al. 2000; SLOMINSKI et al. 2000) autonomously express POMC

and process it via expression of distinct enzymes to POMC-derived peptides. For

example, human HFs are an important extrapituitary site of melanocortin expression

and show prominent α-MSH expression (ITO et al. 2005a; KAUSER et al. 2005). It is

important to note that the tridecapeptide sequence of α-MSH is contained within

ACTH, so that sometimes both molecules are detected by immunohistochemistry and

are expected to display similar functions.

Many studies over the last years revealed a lot of effects on different levels. Besides

its pigmentary effects, α-MSH also has potent protective and anti-inflammatory

effects on central cells and on peripheral non-immune cells, that express

melanocortin receptors (MC-R). Five MC-Rs exist, which belong to the superfamily of

G-coupled receptors with 7 transmembrane domains (CONE et al. 1996; LUGER et

al. 1999; ROOSTERMAN et al. 2006). In many animal models for different diseases,

these anti-inflammatory effects have been validated and it was shown that α-MSH

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affects various pathways, i.e. NF-κB activation, expression of adhesion molecules

and chemokine receptors, production of pro-inflammatory cytokines and mediators,

IL-10 synthesis, cell proliferation and activity, inflammatory cell migration and

expression of anti-oxidative enzymes and apoptosis (LUGER and BRZOSKA 2007;

TAYLOR 2007) (Fig. 2.28).

Fig. 2.28 The different roles of α-MSH as immunomodulator (LUGER et al. 2000)

In addition or as a result, it is thought that these molecules provide the special

environment in immunoprivileged sites. TAYLOR and co-workers detected α-MSH in

pM levels in the aqueous humor of the eye and found that such naturally occurring

doses of the peptide had suppressive effects on the production of IFN-γ by antigen-

stimulated primed murine lymph node cells (TAYLOR et al. 1992, 1994; TAYLOR

2007). Further studies investigated the effect of α-MSH on T cells and showed that

aqueous humor treated T cells were capable of suppressing inflammation induced by

delayed-type hypersensitivity T cells and that CD25+CD4+ Tregs were induced,

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which suppressed the in vitro production of IFN-γ by other inflammatory T cells

(TAYLOR et al. 2000; NAMBA et al. 2002). In the meantime, at least four different

aqueous humor factors have been identified, suppressing DTH responses in the eye,

namely α-MSH, TGF-β, vasoactive intestinal peptide (VIP), and calcitonin gene-

related peptide (CGRP) (TAYLOR 1999).

In astrocytes and microglia of the CNS α-MSH suppressed TNF-α production

presumably via the MC1R (WRONG et al. 1997), as also shown for other cell types

from other tissues (TAHERZADEH et al. 1999). Moreover, recent studies

demonstrated that α-MSH suppresses experimental autoimmune encephalomyelitis

(EAE), and can promote re-establishment of immune tolerance to autoantigens in the

brain (TAYLOR and KITAICHI 2007) and in the human anagen hair bulb (ITO et al.

2004; LUGER and BRZOSKA 2007). Since α-MSH also blocks accessory signals

such as CD86 and CD40 and additionally induces suppressor factors such as IL-10,

it may be one of the signals required for the downregulation of an immune response

and possibly the induction of tolerance (LUGER et al. 1999).

Earlier studies have also demonstrated that the POMC-derived peptides α-MSH and

ACTH are principal inducers of human epidermal pigmentation via their action at the

melanocortin-1 receptor (MC-1R) (ROUSSEAU et al. 2007). α-MSH also promotes

human HF pigmentation by up-regulation of intrafollicular melanogenesis,

melanocyte dendricity, and melanocyte proliferation (KAUSER et al. 2005).

POMC gene transcription and translation is again hair cycle-dependent in the skin of

C57BL/6 mice and increases significantly in the active growth stage of the hair cycle

(anagen) (SLOMINSKI et al. 1992). Since these molecules have immunosuppressive

effects they may provide the special environment for HF IP. On normal skin, ACTH

immunoreactivity is localized on the ORS cells of anagen HFs that is not detected in

the epidermis and dermis of corporal skin (SLOMINSKI et al. 1993). During anagen,

keratinocytes of the ORS and hair matrix express α-MSH (PAUS et al. 1999a). The

concentration of ACTH significantly increases during depilation-induced anagen

measured by radioimmunoassay (SLOMINSKI et al. 1998). Together with the finding

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that the epithelium of human anagen VI HFs in scalp skin also prominently expresses

α-MSH and ACTH, it became obvious that ACTH stimulates intrafollicular cortisol

generation (ITO et al. 2005a), and α-MSH is a potent general immunosuppressant

(COOPER et al. 2005). These results indicate that both melanocortins may contribute

the maintenance of IP milieu in anagen stage.

2.10.7 TGF-β

The transforming growth factor β belongs to a family of multifunctional cytokines that

have wide-ranging and opposing effects on many cellular processes, including

development, cell growth, cell differentiation, carcinogenesis, wound healing, fibrosis,

inflammation and host defense (LETTERIO and ROBERTS 1998). Three isoforms of

TGF-β are described in mammals (TGF-β1, TGF-β2, TGF-β3), which are all encoded

by a different gene and have a different expression, regulation and function

(DERYNCK et al. 1985; DERYNCK et al. 1988; SIGLIENTI et al. 2007). The

signalling occurs through transmembrane serin/threonine kinase receptors (KHALIL

1999; OKUNIEFF et al. 2005). Knockout mice for TGF-β have nonoverlapping

phenotypes, indicating diverse noncompensated functions of TGF family members

(SHULL et al. 1992; KULKARNI et al. 1993).

In addition, pathways mediated by TGF-β may obscure immune surveillance

mechanisms, resulting in failure to recognize or respond adequately to self, foreign,

or tumor-associated antigens. This is why TGF-β is also considered to play an

important role for improvising niches of IP, by recruitment of a cohort of powerful

immunosuppressive cells (COBBOLD et al. 2006; WAHL et al. 2006). The role of

TGF-β2 in IP has been demonstrated in the brain (FABRI et al. 1995; SIGLIENTI et

al. 2007) and the anterior chamber of the eye (STREILEIN et al. 2002;

NIEDERKORN 2003, 2006; CAPSI 2006; TAYLOR and KITAICHI 2007). In the brain

for example, it was shown that downregulation of TGF-β2 facilitates inflammation in

the CNS by astrocyte/microglia interactions (SIGLIENTI et al. 2007). In the eye

chamber, TGF-β is present in the aqueous humor at concentrations that produce

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profound inhibition of NK cell-mediated cytoloysis (APTE and NIEDERKORN 1996;

APTE 1997; APTE et al. 1998; NIEDERKORN 2003, 2006).

TGF-β influences the behaviour of APCs in promoting differentiation of Ag-specific T

cells into CD4þCD25þFoxp3þ regulatory T cells (Tregs), which are found in infiltrating

tumors and other sites of IP, where they influence CD8+ T cells and CD4+ T-helper

cells (Th1, Th2, Th17) leading to immune tolerance (D`ORAZIO and NIEDERKORN

1998; TAKEUCHI et al. 1998; KEZUKA and STREILEIN 2000; STREILEIN et al.

2002). Through a similar mechanism Tregs also impair NK cell activity to block

immune surveillance. Moreover, TGF-β inhibits IL-2 dependent proliferation of Ag-

specific T cells (WAHL et al. 2006).

TGF-β1 and TGF-β2 are also considered to contribute to the HF IP (PAUS et al.

2003; PAUS et al. 2005; NIEDERKORN 2006). TGF-β1 is prominently expressed in

the HF epithelium of mice and man, and is strongest during late anagen and the

onset of catagen in cells of the ORS and epithelial strand (FOITZIK et al. 1999,

2000). TGF-βRII positive cells were also found in the proximal and central region of

the ORS during the late anagen and catagen induced hair growth in mice (WELKER

et al. 1997; FOITZIK et al. 2000; NIEDERKORN 2003; ITO et al. 2008b). In addition,

TGF-β1 was shown to have the capacity to restore IP after an IFN-γ induced collapse

(ITO et al. 2004). Besides that, in the nail apparatus and, in particular, in the PNM

prominent expression of TGF-β1 besides other immunosuppressants (ACTH, α-MSH,

IGF-1, MIF) was found (ITO et al. 2005c). In addition, several other effects of TGF-β1

have been reported, such as an elusive endogenous regulator of catagen induction in

vivo in mice (FOITZIK et al. 2000).

In HF biology it is not known yet, whether the anagen HF recruits all these factors to

maintain and restore its IP (PAUS et al. 2005; ITO et al. 2008b). In addition, the

production/secretion of α-MSH, TGF-β, IGF-1, IL-10, and neutrophins (with the latter

putatively being capable of deleting autoreactive CD8+ T cells via stimulation of the

p75 neurotrophin receptor) may be up-regulated whenever the HF becomes the

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target of immune injury and/or is threatened with a collapse of its IP. On the other

hand, HF-derived α-MSH, TGF-β, IGF-1, IL-10, well-recognized natural

immunosuppressants (LUGER et al. 1998; PAUL 1999; JANEWAY et al. 2001), may

efficiently dampen the secondary autoimmune phenomena that we envisage as

driving force behind the specific clinical characteristics and the progression of AA in

any given patient (SLOMINSKI et al. 1992). Therefore, together with ACTH, α-MSH,

and cortisol, TGF-β1 likely constitutes an integral part of an entire system of

intrafollicularly generated, secreted ´IP maintenance/protective factors` (PAUS et al.

2005), which remains to be dissected comprehensively.

2.10.8 MIF

MIF is a highly conserved protein, with homologues found in evolutionary divergent

organisms including vertebrates, insects, worms and plants (VIGANO et al. 2007).

Originally, it was described as a factor able to inhibit the random migration of

macrophages (RICH and LEWIS 1932) produced by activated T cells (GEORGE and

VAUGHAN 1962), and has been shown to regulate the immune response by

affecting a number of macrophage and lymphocyte features including cytokine

synthesis, cell activation, and phagocytosis. In addition, it has been implicated to play

a role in several immune and inflammatory conditions, including sepsis (BOZZA et al.

1999), delayed type hypersensitivity (BLOOM and BENNET 1966; BERNHAGEN et

al. 1996), and rheumatoid arthritis (LEECH et al. 1999).

MIF protein is ubiquitously expressed by a variety of cells (although primarily by T

cells and macrophages) indicating a more far-reaching non-immunological

involvement in a variety of pathologic states (SHIMIZU 2005). In normal human skin

(SHIMIZU et al. 1996) localized cytoplasmic MIF IR in keratinocytes, especially in the

epidermal basal layer, in endothelial cells, eccrine sweat ductal cells and

myoepithelial cells of eccrine sweat glands, whereas in lesional psoriatic human skin

IR was up-regulated (STEINHOFF et al. 1999).

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MIF is also expressed in the classical IP sites as the fetotrophoblast (VIGANO et al.

2007), eye (APTE et al. 1998), brain (GALAT et al. 1993), human proximal nail matrix

(ITO et al. 2005c), and human anagen scalp HFs (ITO et al. 2007), and is considered

to play a pivotal role in IP maintenance by suppression of NK cell activity (APTE et

al., 1998; TAYLOR 2003; CASPI 2006; PAUS et al. 2005). MIF may inhibit NK-cell

mediated cytotoxicity against MHC class I-negative cells by preventing the release of

cytolytic perforin granules from NK cells (APTE and NIEDERKORN 1996; APTE et al.

1998; ARCURI et al. 2006; NIEDERKORN 2006). This would explain why immune-

privileged tissues that are highly vulnerable to lysis by NK cells, due to their lack of

MHC class I expression, are not under constant NK cell attack. So far, MIF IR was

shown to be present throughout most of the epithelium of normal anagen scalp HFs,

particularly in the proximal IRS and ORS, whereas the epithelium of lesional AA HFs

displayed greatly reduced or absent MIF IR. This suggests that HFs in established

AA exhibit a decreased capacity for suppressing undesired NK cell functions. In the

human nail apparatus, MIF IR is upregulated in the PNM compared with the PNF and

the epidermis (ITO et al. 2005c).

2.10.9 IDO

IDO is a nonomeric heme-containing and rate-limiting enzyme for tryptophan

degradation in extrahepatic tissues (HAINZ et al. 2007; MUNN and MELLOR 2007).

It catabolizes tryptophan into N-formylkynurenine which is converted into 3-

hydroxyanthranilic acid (HIGUCHI and HAYAISHI 1967; KWIDZINSKI and

BECHMANN 2007), ultimately leading to the biosynthesis of the cellular co-factor

nicotinamide adenine dinucleotide (NAD+). Tryptophan is an essential amino acid,

and is required for normal T-cell proliferation, but also for protein synthesis and for

the generation of the neurotransmitter serotonin (HAINZ et al. 2007).

IDO expression is inducible in response to pro-inflammatory cytokines such as IFN-γ,

LPS and IFN-β (CARLIN et al. 1989; CURRIER et al. 2000) in many tissues and cell

types including macrophages, dendritic cells, tumor cells, bone marrow stromal cells,

astrocytes, endothelial cells (HAINZ et al. 2007; KWIDZINSKI and BECHMANN

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2007), but most prominently in APCs (HAINZ et al. 2007). Previous observations

made from other immunoprivileged sites, e.g. lung or allografts (SWANSON et al.

2004; LI et al. 2006) skin (VON BUBNOFF et al. 2004), anterior chamber of the eye

(CHEN et al. 2007), fetus (MELLOR and MUNN 2008) and pancreatic islet cells

(JALILI et al. 2007) indicated, that IDO has a yet un-known important role in the

maintenance of IP.

The catabolism of tryptophan by IDO expression in tissues or APCs limits T cell

proliferation by creating a tryptophan-depleted environment and also affects NK cell

function, reduces inflammation and enhances Fas-mediated T cell apoptosis via their

tryptophan degradation products (MUNN et al. 1998; FALLARINO et al. 2002;

MELLOR and MUNN 2008). For example, the number of allogeneic concepti in

female mice treated with IDO inhibitor (1-methyl tryptophan) was reduced

significantly compared to control mice (COBBOLD et al. 2006; ITO et al. 2008b).

Thus, IP may be maintained as a result of the enzymatic activity IDO (MELLOR and

MUNN 2000), which downregulates T cell function by tryptophan depletion (MUNN et

al. 1998). In addition, it was found, that murine regulatory T cells can condition

dendritic cells to express IDO functional activity and to, thus, exercise

immunosuppressive functions in the context of IP (XU et al. 2000; FALLARINO et al.

2003). Downregulation of MHC class I levels on the surface of IDO-expressing

keratinocytes (LI et al. 2004), inhibition of complement activation (XU et al. 2000) and

a placenta-specific, β2-microglobulin-dependent process linked to MHC class I

antigen presentation (HOBBS et al. 2002) are other examples for additional, newly

recognized mechanisms that favour maternal immunotolerance of pregnancy; uterine

natural killer cells may also be involved (EHRLEBACHER 2001). But so far, no IDO

protein expression in normal skin has been shown.

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Fig. 2.29 Molecular mechanisms of IDO-induced immunosuppression (A) IDO initiates the rate-limiting step in the degradation of tryptophan along the kynurenine pathway. (B) IDO enzymatic activity results in the local depletion of tryptophan and a local increase in the concentration of downstream metabolites. The decrease in tryptophan can cause a rise in the level of uncharged transfer RNA (tRNA) in neighboring T cells, resulting in activation of the amino acid-sensitive GCN2 stress-kinase pathway. In turn, GCN2 signaling can cause cell cycle arrest and energy induction in responding T cells. The local increase in tryptophan metabolites can cause cell cycle arrest, apoptosis, and differentiation of new Tregs from uncommitted CD4+ T cells. (MUNN and MELLOR 2007)

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2.10.10 CD200

The type-1 transmembrane cell surface glycoprotein CD200 (formerly Ox-2) is

expressed in a diversity of tissues on cells of both hemopoietic and non-hemopoietic

origin, including thymocytes, neurons of the central nervous system and the retina,

cells of the glomeruli, cells of degenerating ovarian follicles, vascular endothelium,

some T cells, B cells and dendritic cells in both peripheral blood and lymphoid tissue

(BUKOVSKY et al. 1983; CLARK et al. 1985, 2003). This pattern of CD200

expression is largely conserved in mice, rats and humans (WRIGHT et al. 2001).

Recently, it has been demonstrated that the normal HF epithelium in mice and man

also prominently expresses CD200, a prototypic ´no-danger` signal (OHYAMA et al.

2006; ITO et al. 2007; MEYER et al. 2008a). The expression of CD200 has

previously been shown in the mouse HF, in which it was predominantly detected in

the outermost layer of ORS throughout the length of the mouse HF and could not be

localized in the bulge area. But gene expression studies have identified CD200

transcripts as being up-regulated in the hair bulge region of humans and mice, and

CD200 has become a useful bulge marker for the selection of epithelial stem cells

(TUMBAR et al. 2004; KLOEPPER et al. 2008). In the human anagen HF

homogenously CD200 IR was found in the outermost layer of the ORS, in the bulge,

in the proximal part of the isthmus, in the DP (including blood vessels), and in the

companion layer. The arrector pili muscle and the sweat gland mesenchyme

displayed most prominently IR (KLOEPPER et al. 2008).

When CD200 interacts with the CD200 receptor (CD200R), it induces intracellular

signalling within receptor bearing cells (WRIGHT et al. 2001, 2003) and delivers a

immune-inhibitory signal, which reduces inflammation and promotes tolerance

(PAUS et al. 2003; MORRIS et al. 2004). In vitro, CD200+ dendritic cells inhibit the

secretion of pro-inflammatory cytokines by activated T-cells (MORRIS et al. 2004),

and CD200R binding on APC down-regulates APC activity (ROSENBLUM et al.

2004). Therefore, CD200-CD200R interactions are thought to act as a “no danger”

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signal that links innate and adaptive immunity in a given tissue location so as to

dampen the chance of undesired immune responses (MORRIS et al. 2004). In

addition, CD200 is thought to influence the IDO production (FALLARINO et al. 2002),

that could play a yet undefined role in the maintenance of relative IP (see 3.10.9).

Loss of this ´no danger` signal could be important in the pathogenesis of autoimmune

primary cicatricial alopecia (HARRIES et al 2008). The concept that CD200-CD200R

interactions are important for protecting the HF epithelium from immune-mediated

attack and eHFSC damage comes from the CD200 knock-out mouse model

(MORRIS et al. 2004). When skin was grafted from CD200-/- mice onto gender-

matched wild-type hosts (CD200+/+) inflammation, hair loss and follicular scarring in

the grafted skin was caused. Of note was that the grafted skin survived long-term

and the epidermis remained normal throughout. Histologically, a peri- and intra-

follicular mononuclear inflammatory cell infiltrate was seen with intra-follicular

oedema and increased levels of apoptosis. Inflammation eventually resolved after all

hairs were lost and replaced with tracks of scar tissue. This supports that CD200 is a

key component in protecting eHFSC from immune attack and also plays a central

role in maintaining bulge IP.

2.10.11 Mast cells

Mast cells arise from pluripotential stem cells, reside and mature in tissues including

the skin and mucosal membranes, particularly at the interface of the host and its

environment, but also next to nerves, blood vessels and glandular structures

(BOTCHKAREV et al. 1997b; WEBER et al. 2003; BROWN et al. 2007). Mast cells

are characterized by their content of cytoplasmic granules, which contain pro-

inflammatory mediators, that are released upon mast cell activation (i.e. histamine,

leukotriens, prostaglandins, proteases, cytokines) (YOUNG 1997; MEKORI 2004;

THEOHARIDES and KALOGEROMITROS 2006; CHRISTY and BROWN 2007;

METZ et al. 2007).

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Mast cells have long been only recognized for their role in the genesis of allergic

inflammation [antigen-specific, IgE-mediated degranulation and secretory activities in

type 1 hypersensitivity reactions (BOS 1997)]. This changed since recently an

alternative activation pathway in addition to those elicited by FcεRI cross-linking was

found. Mast cells are equipped with many different receptors or their surface, through

these can be stimulated by complement components, neuropeptides, stress

hormones and TLR (bacteria and viruses), indicating a participation in innate and

acquired immune responses (GALLI et al. 2005; GILFILLAN and TKACZYK 2006).

Mast cell activation and expression on the cell surface can, besides other effects,

modulate T cell and DC function (METZ et al. 2008b; SAYED et al. 2008). For

example, MHC class II and T cell co-stimulatory molecule expression by mast cells

provides potential antigen-presenting cell capability. Mast cells also express most

cytokines (i.e. TNF-α) that control T cell differentiation pathways and directly regulate

T cell fate decision through processing and presenting antigen to T cells through

class I and class II MHC routes (MCLACHLAN et al. 2003; SUTO et al. 2006;

JAHANYAR et al. 2008; SAYED et al. 2008). For example, it has been demonstrated,

that mast cell-deficient mice do not get disease in models of rheumatoid arthritis or

bullous pemphigoid, indicating an essential role in these Ab-mediated, organ-specific

autoimmune diseases. In contrast, in Ab-mediated systemic lupus erythematosus

mast cell-deficient mice have normal or enhanced disease progression, since they

appear to be protected against multi-organ autoimmunity (CHRISTY and BROWN

2007) and it has been demonstrated that mast cells are required for normal

cutaneous wound healing (WELLER et al. 2006).

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Fig. 2.30 Stimulatory and immunosuppressive functions of mast cells. (CHRISTY and BROWN 2007)

Interestingly, recent reports indicate, that MCs may also be critical in suppressing

skin immune responses and in maintaining IP (Fig. 2.30). DEPINAY et al. (2006)

explained the reduced delayed-type hypersensitivity in the skin of MC knock-in mice,

that have been bitten by Anopheles mosquitoes, by MC degranulation and induction

of the immunosuppressive IL-10 (DEMEURE et al. 2005; DEPINAY et al. 2006). And

recently LU et al. (2006) suggested that MCs are crucial for optimal expression of

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99

peripheral tolerance to skin allografts. In addition, MCs can also dampen an immune

response directly through expression of certain cytokines such as IL-10 or TGF-β or

indirectly by facilitating T regulatory cell (Treg) activity (SAYED et al. 2008). In

summary, mast cells are now regarded to be ´good and bad guys`, just like every

other cell (MAURER et al. 2003b; MAURER and METZ 2005).

Mast cells show hair-cycle dependent functional and numerical changes in rats

(MORETTI et al. 1963) and mice (MAURER et al. 1994, 1997; PAUS et al. 1994c).

Furthermore, the ratio of degranulated mast cells increases substantially in the

transition periods (between anagen-catagen, catagen-telogen and telogen-anagen)

(PAUS et al. 1994c; BOS 1997). In human HFs, mast cells were shown to be

localized homogenously in the whole CTS (CHRISTOPH et al. 2000), although, a

detailed analysis remains to be done. There are reports from HF organ cultures that

the number and activation status of mast cells change in response to treatment (VAN

BEEK et al. 2008; MEYER et al. unpublished data). However, one has to keep in

mind, that mast cells in mice and man are very similar regarding their distribution,

responses to stimulating signals, and mediators, but that they also differ in several

respects. For example, human MCs do not contain serotonin, some proteases and

cytokines such as IL-3 and IL-4 can differ in their effects (MAURER and METZ 2005).

So there is need to clarify the role of MCs.

In the human nail matrix, dermal mast cells were demonstrated to be reduced in

vicinity of the nail matrix, compared to the PNF (ITO et al. 2005c). This low number of

periunguinal mast cells is in contrast to high density of mast cells found in

mesenchyme surrounding the human hair matrix (CHRISTOPH et al. 2000). Given

that mast cells are important cellular elements of innate immunity (MAURER et al.

2003b), this low mast cell number may compromise the defense capacity of this

region of nail mesenchyme. In contrast, in the more distally located, terminally

differentiated nail epithelium, production of antimicrobial peptides was found

(DORSCHNER et al. 2004), which may compensate and supply the innate immune

defense.

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100

2.10.12 ICAM-1

Intercellular adhesion molecules belong to the immunoglobulin superfamily. They are

cell-surface ligands for the leukocyte integrins and are crucial in the binding of

lymphocytes and other leukocytes to certain cells, including APCs and endothelial

cells. ICAM-1 (CD54) is the most prominent ligand for the integrin CD11a:CD18 or

LFA-1. It is rapidly inducible on endothelial cells by infection, and plays a major role

in local inflammatory responses (CAMELI et al. 1994; BOS 1997; GOLDSBY et al.

2003; JANEWAY et al. 2005).

Many studies have demonstrated ICAM-1 effects in normal inflammation (facilitation

of lymphocytes to migrate to the location of inflammation). ICAM-1 is considered to

be one of the components in uninflamed tissue, which endangers the status of IP. In

mice, the peri- and infrainfundibular ORS constitutively expresses ICAM-1 and thus

attract LFA-1 expressing immunocytes (e.g. macrophages) (MUELLER-ROEVER et

al. 2000). This is not surprising because the ORS and the bulge region are the

preferred sites for the occurrence of dense perifollicular inflammatory cell infiltrates

under stress (ARCK et al. 2001, 2003). In addition, it has been shown that in normal,

uninflamed mouse skin, a very small, but notable percentage of HFs are permanently

deleted by an inflammatory cell attack on the ICAM-1+ bulge region of anagen and

catagen pelage HFs, without any macroscopic evidence for this striking phenomenon

of ´programmed organ deletion (POD)` (EICHMUELLER et al. 1998). ICAM-1 IR in

normal human skin was found on most CTS and dermal fibroblasts and immune

cells, but the strongest ICAM-1 IR was seen on perifollicular inflammatory cell

clusters (PICC) (JAWORSKY et al. 1992). In addition, ICAM-1 expression was found

on HF keratinocytes in the close vicinity of perifollicular inflammatory cell clusters

(CHRISTOPH et al. 2000).

Whether such physiological POD of isolated (or possibly malfunctioning) HFs also

occurs in human skin, and whether at least some forms of cicatricial alopecias only

represent an excessive form of POD (PAUS et al. 1999) remains to be investigated.

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101

Furthermore, it was reported that human nail epithelium does not express ICAM-1

and that nail matrix cells express a low level of ICAM-1 in vitro (PICARDO et al.

1994). But in situ studies in the infantile human nail apparatus did not demonstrate

ICAM-1 IR in any epithelial compartment; only the expected IR in blood vessels was

found (ITO et al. 2005c).

2.10.13 β-defensin 2

β-defensins (also designated BD, and HBD in human) belong to the group of the

cationic antimicrobial peptides (CAPs or AMPs), that represent a highly conserved

element of innate immune defense from the evolutionary point of view, because they

are also present in plants, insects and lower vertebrates. The substances are

involved in the protection of epithelial surfaces, such as the skin or airway surfaces,

against microbial colonization (BOMAN 1995; NISSEN-MEYER and NES 1997;

HARDER et al. 2001; GLAESER et al. 2005; MOOKHERJEE and HANCOCK 2007;

ABTIN et al. 2008). Since the 1980s many peptides with antimicrobial activity were

also found and investigated in humans (HARDER et al. 1997, 2001; BRAFF et al.

2005a, 2005b; GLAESER et al. 2005; ABTIN et al. 2008), like ß defensins,

cathelicidin (LL-37) (LARRICK et al. 1995; FROHM et al. 1997) and the S100 protein

psoriasin.

In general, CAPs or AMPs not only act as an antibiotic, but also in the context of

wound healing (CARRETERO et al. 2008; GALLO 2008), stimulation of the migration

and proliferation of epidermal keratinocytes (NIYONSABA et al. 2007) angiogenesis

(KOCZULLA et al. 2003) and, furthermore, of modulating the immune response [e.g.

influencing the migration of macrophages by modulating their chemokine and

chemokine receptor expression and increasing the production of various cytokines

such as IL-6, IL-8, and GMCSF (NIYONSABA et al. 2005; GALLO 2008)]. For

cathelicidin (LL-37) it was even shown to affect the antimicrobial activity of mast cells

(DI NARDO et al. 2003).

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102

β-defensins are small cationic peptides with broad-spectrum antimicrobial activity.

Human β-defensin 2 is locally regulated by inflammation and is the first member of

the β-defensin family that is locally inducible by inflammation. The murine homolog of

human β-defensin 2, which is called β-defensin 3, is present in the respiratory system

and in low levels in the epithelial cells of the intestine and lung (BALS et al. 1999).

The unique murine β-defensin 2 (Defβ2) is not expressed in airways of untreated

mice (MORRISON et al. 1999), but is upregulated in the airways by

lipopolysaccharide and may contribute to host defense at the mucosal surface of the

airways. Despite the fact that β-defensins are looked upon as classic antimicrobial

peptides, it has recently been demonstrated that they have also immunomodulatory

activity and link between innate and adaptive immunity, being chemotactic for CD4+

T cells and immature dendritic cells (YANG et al. 1999).

Although the HF ostium of skin and the nail hyponychium in the nail apparatus

represent a major potential port of microbial entry into the skin, clinical signs of

bacterial infection are exceptionally rare. This suggests the presence of a highly

effective AMP defense system. The ORS and sebaceous gland of human

pilosebaceous units express prominent IR for human β defensin-1 and 2 (HBD1 and

2), which appear to be up-regulated in acne vulgaris lesions (CHRONNELL et al.

2001). Toll-like receptors (TLRs) -2, -4 and -5 are expressed in murine pelage HFs

and their activation by microbial stimuli induced the production of β defensin-2

(SELLERI et al. 2007). In addition, it was reported, that human, porcine, and murine

nails contain AMPs, and that the human cathelicidin LL-37 can destroy Candida

albicans (DORSCHNER et al. 2004).

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2.11 K(D)PT – a candidate as hair growth modulator and IP restorer

in anagen hair bulbs?

α-Melanocyte-stimulating hormone (α-MSH) is a cleavage product of the

neuropeptide pro-hormone pro-opiomelanocortin (POMC), which plays an important

role, e.g., in immune and pigmentary homeostasis (LIPTON and CATANIA 1997;

LUGER et al. 2000; BOEHM et al. 2006; WOOD et al. 2006). Earlier studies have

demonstrated that the POMC-derived peptides α-MSH and ACTH are principal

inducers of human epidermal pigmentation via their action at the MC-1R (LIN and

FISHER 2007; ROUSSEAU et al. 2007). α-MSH also promotes human HF

pigmentation by up-regulation of intrafollicular melanogenesis, melanocyte dendricity,

and melanocyte proliferation (KAUSER et al. 2005). In addition, numerous studies

over the last few years have provided plenty of evidence that α-MSH is a well

tolerated immuno modulator with cytoprotective and antiinflammatory effects

(LUGER et al. 1999, 2000, 2003; LUGER and BRZOSKA 2007). α-MSH is supposed

to be a mediator of tolerance induction in the brain (TAYLOR and KITAICHI 2007)

and eye (NIEDERKORN 2006), affecting several pathways with the capacity to re-

establish IP in anagen human hair bulbs (ITO et al. 2004). Such a collapse is

hypothesized as the reason for the development of the autoimmune disease alopecia

areata (AA).

Synthetic, α-MSH related tripeptides can imitate these pigmentary and

immunomodulatory effects, and promise minimal side effects upon clinical use, since

they are closely related to a well-tolerated endogenous neuropeptide (HADLEY and

DORR 2006). Most of the anti-inflammatory activities of α-MSH can be exerted by its

C-terminal tripeptide KPV. In this study, K(D)PT, a derivative of KPV, is of particular

interest. In K(D)PT, the hydrophobic amino acid valine of KPV is substituted by the

more polar amino acid threonine. K(D)PT also corresponds to amino acid 193-195 of

IL-1β, one of the major pro-inflammatory cytokines which also inhibits human HF

growth in vitro (PHILPOTT et al. 1996; HOFFMANN et al. 1997).

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Recent studies indicated that K(D)PT itself exerts anti-inflammatory effects in human

sebocytes (MASTROFRANCESCO et al. 2007) (suppression of IL-1β- and LPS

induced NFκB activation, IL-6 and IL-8 expression) but so far, it is unknown which

effect K(D)PT has on HF biology. On the other hand and in contrast to α-MSH, C-

and N-terminal fragments of α-MSH were shown to have no melanotropic effect in

frog and lizard skin bioassays (HASKELL-LUEVANO et al. 1996), so that it was

expected that K(D)PT has no melanogenic effect. Therefore we used our organ-

cultured human anagen HF model, comparing normal and pro-inflammatory

conditions.

The physiochemical properties, expected low costs of production of K(D)PT, its

combined anti-inflammatory and anti-microbial effects and the small molecular size,

would make this substance suitable for the future local treatment of immune-

mediated inflammatory skin disease, and as an IP restorer or as a hair growth

modulator (BRZOSKA et al. 2008).

Fig. 2.31 Biosynthesis of POMC peptides an natural melanocortins. Posttranslational processing of POMC by enzymes at specific cleavage sites, yields peptide hormones such as ACTH, α-MSH, γ-MSH and β-endorphin peptides. (B) Peptide sequences of the natural melanocortins, the central melanotropic pharmacophor of α-MSH and the α-MSH-related C-terminal tripeptides. Structural homologies are labeled in red (BRZOSKA 2008).

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2.12 Questions addressed in this study

1. Does the human HF bulge region belong to a site of relative immune privilege?

2. Do any differences exist between the IP in the anagen hair bulb and the bulge

region?

3. Which effects does the α-MSH related tripeptide K(D)PT have on organ cultured

HF biology?

4. Can K(D)PT influence the constitutive and IFN-γ induced MHC class-I and MHC

class II expression in isolated organ-cultured HFs and restore IP?

5. Which effects has K(D)PT on mast cells and degranulation?

6. Does K(D)PT effect the human HF cycle or does it have effects on hair matrix

proliferation and –apoptosis?

7. Is the murine vibrissal follicle a site of relative immune privilege?

8. Is the murine proximal nail matrix a site of relative immune privilege?

2.13 Experimental design

For this purpose, human uninflamed scalp skin from a total of 16 different patients

was randomly collected during routine face-lift surgery, and, additionally, murine

snout skin and nail apparatus from a total of 18 control mice was used. Both routine

and increased-sensitivity immunohistochemical staining techniques were employed,

and the corresponding IR patterns were evaluated by quantitative

immunohistochemistry. For functional evidence, we performed full thickness human

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scalp skin organ cultures to investigate whether IFN-γ, a key inducer of IP collapse in

hair bulbs, has a similar effect on the putative bulge IP.

In addition, we investigated the effects of the new synthetic α-MSH related tripeptide

K(D)PT onto HF IP and biology in our organ-culture HFs model under pro-

inflammatory conditions, complemented by immunohistochemistry.

For staining controls, internal positive controls of the reproduction of published

follicular IR patterns were chosen, if available. Otherwise appropriate positive

controls (e.g. lymphnodes, spleen, brain) were used. Only specific IR patterns that

were well reproducible between at least (3-)5 different individuals were

photodocumented and are reported here.

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3 Materials and methods

3.1 Tissue collection

3.1.1 Human tissue collection

Fig. 3.1 Human scalp skin specimen

Fronto-temporal and occipital human skin scalp biopsies were obtained from 16

females undergoing routine face-lift surgery after informed consent. The donors were

selected randomely (aged 45-67 years, mean age 52.5 years) and all experiments

were performed according to the Helsinki guidelines.

Scalp specimens were placed immediately after surgery into William’s E serum-free

medium supplemented with 2.5 fold concentrated penicillin (250 IU/ml), streptomycin

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(250mg/ml) and fungazone (12.5mg/ml amphotericin B), and transported overnight to

the labaratory at 4°C (all cell culture reagents were obtained from Gibco BRL,

Paisley, Scotland.

The skin samples were cut into thin strips (approximately 0.5 cm x 1 cm) (Fig. 3.1)

with a scalpel, embedded in Cryomatrix Shandon (Pittburg, PA, USA), snap-frozen in

liquid nitrogen, and stored at –80°C until use.

For immunohistochemistry, 5-7 µm thick cryosections were processed. The common

way of processing cutaneous tissue is by using vertically oriented sections of the

skin, with the epidermis at one pole and the hypodermis at the other. One difficulty is

that vertically orientated sections of the skin very rarely match the slanting of the

HFs, because they do not grow uniformely in a 90° angle to the epidermis. As a

consequence, complete longitudinal sections of the HF from the bulb to the epidermis

can be rarely produced and the HFs almost always appear incomplete. For every

analyzed antigen, scalp skin cryosections of at least 3-5 different individuals were

examined (3-10 cryosections per patient).

3.1.2 Murine tissue collection

Mouse tissue samples were gained from 18 six to 14 weeks old female C57BL/6 and

Balb/c mice of control groups from different experiments, that were housed in

community cages under standardized conditions (12 hours light/dark-cycle, water

and mouse chow ad libitum) in the animal facility of the University Hospital

Schleswig-Holstein. The mice were kindly provided from a cooperation with Prof. Dr.

D. Zillikens from the Department of Dermatology, Venerology and Allergology,

University of Lübeck. Samples that had been fixated in Bouin’s solution were kindly

collected by Prof. Dr. W. Meyer (Institute of Anatomy, University of Veterinary

Medicine Hannover) in cooperation with the Institute of Pharmacology, of the same

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university. The mice were anaesthesized and euthanized by cervical dislocation.

Immediately after death, samples were taken as described below.

For dissecting the nail samples, a nail was gripped with a forceps and cut with a

scissors proximal to the joint of the first and second phalanx. Every single nail of the

fore- and hindlimb was taken. One or two specimens each were placed horizontally

into a little plastic tablet form, embedded in Cryomatrix Shandon (Pittburg, PA, USA),

and snap frozen in liquid nitrogen. This embedding technique allows sagittal

cryosections of the whole nail apparatus.

In order to macrodissect the whisker pad, first, the hair in this location was trimed

with clippers or carefully removed with a scalpel. Second, the skin was cut with a

scalpel over the bridge of the snout posterior to the whisker pad. Then the incision

was extended medially along both sides of the mouth, paralleling the upper lips to the

nose. It was necessary to cut the deeper tendinous tissues in order to free the snout

skin and to prevent cutting of the large vibrissal follicles. Using a sterile forceps, the

whisker pad was pulled forward of the face, separating the tissue from the nasal

bridge. These tissue samples were placed in a vertical orientation (epidermis left,

dermis right) in a plastic tablet, embedded with Cryomatrix Shandon (Pittsburg, PA,

USA), snap frozen in liquid nitrogen and stored at –80°C until use. The samples were

cut into ~6-8 µm thick cryosections and then processed for immunohistochemistry (or

stored at –80°C until use). Vertically orientated sections of the whisker pad were

performed, showing the epidermis at one pole and the hypodermis at the other in

order to get complete longitudinal sections of the vibrissal follicle from the bulb to the

epidermis. The nail apparatus was processed for sagittal sections. Per antigen,

whisker pad and nail cryosections deriving from at least 5 different mice were

examined (3-8 cryosections per mouse).

Specimens, which were collected in the Institute of Pharmacology in Hannover, were

immersed immediately after removal in Bouin’s solution (BOECK 1989) [for

preparation 1500 ml picric acid, 500 ml formaldehyde (37-40%) and 100 ml glacial

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110

acetic acid were mixed) and fixed for at least 48 hours. The cut samples were then

placed in labeled plastic tablets and run through different fixing steps with

intermediate changes of the solution: 70 % ethanol (>1 day), 80% ethanol (> 1 day),

96 % ethanol (1 ½ h), isopropanol (1 1/2 h), xyelene I and II (each 1 ½ h), and

paraffine I (over night), paraffine II (2 h), paraffine III (1 ½ h). In the end, the samples

were positioned into a metallic form, effused with paraffin, and were then placed for

hardening in the fridge. After performing sections by using a microtome (5µm thin),

the slides were dried overnight at 37°C.

3.2 Human hair follicle isolation

Human anagen VI HFs were microdissected from frontotemporal and occipital human

scalp skin obtained from female donors (age range: 46-65 years) undergoing face-lift

surgery with informed consent and ethics committee as previously described

(PHILPOTT et al. 1990; MAGERL et al. 2002; MAGERL et al. 2004).

Before and during isolation, the skin samples were maintained in isolation medium

(William’s E medium, Biochrom, Cambridge UK) with 1% antibiotic/antimycotic

mixture (100x, Gibcom Germany, Karlsruhe) containing penicillin G, streptomycin

and amphotericin B to reduce the number of infection.

First, human scalp specimens were divided into 0.5 cm x 1 cm thin strips. Then the

skin strips were cut under sterile conditions at the dermo-hypodermal fat interface

using a scalpel blade (Fig. 3.2). The epidermis was removed and discarded. Under a

binocular dissecting microscope, the dermis was fixed with a blunt forceps to partially

extrude the upper portion of the HFs from the hypodermis. Simultaneously, the

CTS/ORS of the HF was gently gripped with watchmaker’s forceps and the intact HF

pulled out. This technique resulted in the isolation of intact HFs without any visible

damage (the HF was surrounded completely by the intact CTS). Otherwise, HFs had

not survived during the following culture period.

MATERIAL AND METHODS

111

Fig. 3.2 Isolation of human HFs Isolation of human HFs. A,B) Cut through the skin at the dermo-hypodermal fat interface. C,D) Parts of the dermis are removed. E) The skin is compressed, so that the HF steps out. F) The ORS of the follicle is gripped in the forceps and pulled out. G) Isolated pigmented HFs.

3.3 Human HF organ culture

Normally pigmented human anagen VI HFs were microdissected from temporal and

occipital human scalp skin obtained from female donors (age range: 46-65 years);

grey and white HFs were excluded from study. The isolated HFs were maintained

and cultured in serum-free William’s E medium (Biochrom AG, Berlin, Germany),

which was supplemented with 2 mmol/L L- glutamine (Invitrogen, Paisley, UK), 10

µg/ml insulin (Sigma-Aldrich, Taufkirchen, Germany), 10 ng/ml hydrocortisone

(Sigma-Aldrich), and 1% antibiotic/antimycotic mixture (100x, Gibco, Germany,

A

C

E

D

B

F G

MATERIAL AND METHODS

112

Karlsruhe). HFs were placed in a threesome in a 24-well plate with 500 µl complete

medium (Figs. 3.3, 3.4) and incubated free-floating in the wells in an atmosphere of

5% CO2 and 95% air at 37°C. On day 1, medium was changed and treatment began.

Fig. 3.3 Hair follicles in a 24-well plate

Fig. 3.4 Isolated hair follicles

MATERIAL AND METHODS

113

Six independent HF organ cultures with two different set-ups were used (three

HFs/well, three test groups and three controls) and compared.

In the first assay (Restoration assay) (Fig. 3.5), HFs were pre-treated with 75IU/ml

IFN-γ (Peprotech, Rocky Hill, NJ) for 4 days, followed by addition of 10-8, 10-9 or 10-

10M K(D)PT on day 3 (Assay 1). Control HFs were cultured for 4 days with K(D)PT

(10-8 M), IFN-γ (75 IU/ml), or with the vehicle (distilled water).

Alternatively (Assay 2, protection assay) (Fig. 3.5), HFs were first treated with

K(D)PT (10-8, 10-9 or 10-10M) for 48 hours (control groups were cultured in complete

medium only), followed by addition of 75 IU/ml IFN-γ (treatment for 4 days). Control

HFs were exclusively treated with K(D)PT, IFN-γ, or in the vehicle from day 3 on. We

chose low-dose 75IU/ml IFN-γ on the basis of our previous studies (ITO et al. 2004;

ITO et al. 2005b) in which we had demonstrated that this low dose of IFN-γ sufficed

to induce immune privilege collapse in the human anagen hair bulb (i.e. induction of

ectopic MHC class I immunoreactivity) without inducing premature catagen

development (the latter had to be avoided since premature catagen-induction shuts

off HF pigmentation) (SLOMINSKI et al. 1994). Every second day, hair shaft

elongation was measured using an inverted microscope and medium and substances

were renewed. 5 (assay 1) or 7 days (assay 2) after microdissection, HFs were

frozen in liquid nitrogen, stored at -80°C and processed for cryosectioning.

Fig. 3.5 Experimental design of the HF organ culture

MATERIAL AND METHODS

114

3.4 Full thickness human scalp skin organ culture

Two independent full-thickness organ cultures with adult human scalp skin were

performed. Human scalp skin was transferred and washed in William’s E medium

(Biochrom, Cambridge, UK) supplemented with 100 IU/ml penicillin G (Sigma, St

Louis, MO, USA), 10 lg/ml streptomycin (Sigma), 0.25 lg/ml amphotericin B (Gibco,

Karlsruhe, Germany) for the preparation. The outgrowing hair shafts were shaved

down to the level of the epidermis. Then 3-4 mm biopsies of intact scalp skin were

punched out parallel to the direction of hair growth, using an Acu-puncher (STIEFEL,

Offenbach am Main, Germany).

Human scalp tissue pieces were carefully placed randomised into William’s E

medium, which was supplemented with 100 IU/ml penicillin/10 lg/ml streptomycin, 10

lg/ml of insulin (Sigma), 10 ng/ml of hydrocortisone (Sigma) and 2 mmol/l of l-

glutamine (Invitrogen, Paisley, UK). Each treatment group consisted of three to four

punch biopsies and was placed in a 6-well-multi well plate with 3 ml complete

medium (Fig. 3.6).

Fig. 3.6 Full thickness human scalp skin punch biopsies in 6-well multi well plate

MATERIAL AND METHODS

115

The skin fragments were left to float freely in the medium, with the epidermis up at

the air/liquid interface and the dermis/hypodermis down. The cultures were

maintained at 37°C in a gassed incubator with 95% air and 5% CO2. After the first

over-night incubation, medium was changed and treatment began.

Control punch biopsies were treated only with PBS; the other five groups were

treated with five different IFN-γ (Peprotech, Rocky Hill, NJ) concentrations (100

IU/ml, 500 IU/ml, 1000 IU/ml). The treatment was always combined with a medium

change on day 1 and 3. After 5 days of cultivation, the specimens were embedded,

snap-frozen in liquid nitrogen and stored at-80°C until use.

3.5 Histological stainings

3.5.1 Hematoxylin-Eosin staining

The hematoxylin-eosin staining is a dichromatic staining, based on the basic dye

hematoxylin and the ethanol-based acidic dye eosin (BOECK 1989).

Cryosections (stored at -80°C) were fixed in acetone for 10 min at -20°C followed by

air drying for 30 min at room temperature and two times washing in Tris-buffered

saline (TBS) and 10 min in distilled water.

For the preparation of TBS buffer, 6.1 g Tris-base and 8.8 g sodium chloride (NaCl)

were diluted in 800 ml distilled water. Then the pH was adjusted with 1N hydrochloric

acid (HCL) to 7.6, and the solution was dropped up to 1000 ml with distilled water.

The sections were stained in Mayers hemalum (Merck, #1.04302) for 10 min and

rinsed under tap water approximately 15 min. The counterstaining was performed

with 0.1% eosin (Sigma, #E4382) for approximately 1 min. The final differentiation

was as follows via an ascending ethanol series: one time in 70%, two times in 96%,

two times in 100% ethanol and two times in xylene, all steps with carefully dipping 10

to 15 times. The slides were mounted with synthetic resin (Eukitt, O.Kindler &Co,

Freiburg, Germany).

MATERIAL AND METHODS

116

3.5.2 Trichromatic staining

This method was used for slides fixed in Bouin’s solution; with cryosections the

staining results were not successful.

The method was performed according to Masson-Goldner (BOECK 1989) and used

mainly as general map for the detection of connective tissue (collagen fibres, green),

that can well be distinguished from, i.e., muscles (red). For deparaffinization, the

slides were treated as follows: two times washing in xylene for 10 min each, one time

washing for 2 min each in isopropanol, 96 % ethanol, 80% ethanol, 70 % ethanol,

and clearing in distilled water.

Then the sections were stained in hemalum according to Delafield for 8 min, dipped

carefully in 0.1 % HCl in distilled water and rinsed under tap water approximately 15

min. For the following staining solution, 0.2 g Ponceau de xyline and 0.1 g acid

fuchsin were solved in 300 ml distilled water and 0.6 ml glacial acetic acid was

added. The slides were stained for 5 min in acid fuchsin-Ponceau, followed by 5 min

washing in 0.1 % acetic acid, 10 min washing in a solution of 10 g phosphortungstic

acid and 5 g orange G in 250 ml distilled water and a second time of 5 min. washing

in 0.1 % acetic acid. The counterstaining was performed using light green (0.5 g light

green + 250 ml distilled water + 0.5 ml acetic acid) for 5 min followed again by a 5

min washing step in 0.1 % acetic acid, and dehydration via the ascending ethanol

series as follows: three min washing each in 80 % ethanol, in 96 % ethanol and 96%

isopropanol, and two times clearing in xylene substitute (Merck, Darmstadt,

Germany) for 5 min each. The slides were mounted with synthetic resin (Eukitt, O.

Kindler GmbH Co., Freiburg, Germany).

3.5.3 Toluidine blue staining

This staining method is simple and widely used as a technique for the detection of

mast cells. Mast cells are often present in connective tissue, and their cytoplasm

MATERIAL AND METHODS

117

contains granules composed of heparin and histamine. Toluidine blue should colour

mast cells violet or red purple (metachromatic staining) and the background blue

(orthochromatic staining).

Cryosections (6-8 µm thick) which had been stored at -80°C were air dried for 10

min. Then the slides were washed for 2 min in distilled water at room temperature

followed by staining for 1 min in toluidine blue according to RICHARDSON et al.

(1960) (working solution: 1g toluidine blue 0,1g di-sodiumtetraborate-10-hydrate,

0,2g paraformaldehyde in 100ml distilled water). The staining step was stopped by

three times washing in distilled water, and the slides were then differentiated and

dehydrated via an ascending ethanol series as follows: three times dipping in 70%

ethanol, three times dipping in 96% ethanol, 3 min washing in 96% ethanol, two

times washing 3 min in 100% ethanol and two times clearing in xylene substitute

(Merck, Darmstadt, Germany) for 10 min each. The slides were coversliped with a

synthetic resin mounting medium (Eukitt, O. Kindler GmbH Co., Freiburg, Germany).

3.5.4 Leder`s esterase staining

Leder`s esterase staining (LEDER 1964) uses the enzyme chloroacetate esterase as

a marker for cells of the neutrophil series, monocytes, and mast cells in tissues, and

produces a strong red to deep pink cytoplasmic staining of mast cells, whereas the

nuclei appear in green (light green) or blue (hematoxylin). We performed this method

mainly to detect mast cells in the CTS of isolated HFs.

Cryosections of cultured HFs which have been stored at -80°C were air dried for 10

min at room temperature. The slides were fixed in 1% paraformaldehyde (in PBS) for

10 min and then washed three times in distilled water for 5 min each.

For the preparation of the incubation medium several different working buffers were

prepared: The Sörensen working buffer consisted of 32.8 ml of the Sörensen stock

solution A (2.73 g Na2HPO4 in 250 ml distilled water) and 7.2 ml Sörensen stock

solution B (2.27 g KH2PO4 in 250 ml distilled water). A pararosanilin solution was

MATERIAL AND METHODS

118

made of 0.5 g pararosanilin powder (Merck, Germany), 20 ml of distilled water and

2,5 ml concentrated hydrochloric acid, which was heated carefully, filtered and stored

in the refridgerator. Immediately before staining, the nitrosylated pararosanilin was

prepared by mixing 150 µl of the pararosanilin solution with 150 µl 4% sodium nitirite

in distilled water. In the end the incubating medium was prepared by mixing and

filtering of 10 mg naphtol-ASD chloroacetate (Merck, Germany), 1 ml N,N-

dimethylformamide, 35 ml of Sörensen working buffer and 200 µl of the nitrosylated

pararosanilin The slides were stained in this incubating medium for 40 min, followed

by washing in running tap water for 5 min. The counterstaining was performed either

using light green or hematoxylin for approximately 1 to 2 min, followed by another 5

min wash in running tap water. Afterwards the slides were air dried and dehydrated

via an ascending ethanol series as follows: three times dipping in 70% ethanol, three

times dipping in 96% ethanol, 3 min washing in 96% ethanol, two times washing 3

min in 100% ethanol and two times clearing in xylene substitute (Merck, Darmstadt,

Germany) for 10 min each. The slides were coversliped with synthetic resin mounting

medium (Eukitt, O. Kindler GmbH Co., Freiburg, Germany).

3.5.5 Ki-67 / TUNEL

To evaluate apoptotic cells in co-localization with the proliferation marker Ki-67, a Ki-

67 / TUNEL (terminal dUTP nickendlabeling) double-staining method was used.

The Ki-67 antigen is a nuclear protein, which is identified by its reactivity with

monoclonal antibody from Ki-67 clone (GERDES et al. 1984); two isoforms of 345

and 395 kDa have been identified (GERDES et al. 1991). The Ki-67 antigen is

preferentially expressed during all active phases of the cell cycle (G1, S, G2 and M-

phase), but it is absent in resting cells (G0-phase) (GERDES et al. 1984). During

interphase, the antigen can be exclusively detected within the nucleus, whereas in

mitosis most of the protein is relocated to the surface of the chromosomes. The

antigen is rapidly degraded as the cell enters the non-proliferative state (SCHOLZEN

MATERIAL AND METHODS

119

and GERDES 2000), and there appears to be no expression of Ki-67 during DNA

repair process (KEY et al. 1994).

Apoptosis is a form of cell death that eliminates compromised or superfluous cells.

During this process, the DNA is cut into fragments via endonuclease activity. The

DNA strand breaks are detected by enzymatically labeling the free 3’-OH termini with

modified nucleotides. These nucleotides are enzymatically added to the DNA by the

terminal deoxynucleotidyltransferase (TdT), catalysing a template-independent

addition of nucleotide triphosphates to the 3’-OH ends of double-stranded or single-

stranded DNA. DNA fragments which have been labelled with the

digoxigenin-nucleotide are then allowed to bind an anti-digoxigenin antibody that is

conjugated to FITC.

Cryostat sections of treated (with 12G10 (+), TS2/16 (+) and mAb13 (-) β1 integrin

antibodies) and untreated cultured HFs were fixed in paraformaldehyde (10 min,

room temperature) and ethanol-acetic acid (2:1; 5 min, -20°C), using washing steps

with PBS in-between (three times for 5 min). Then the slides were labelled with a

digoxigenin-deoxyUTP (ApopTag Fluorescein In Situ Apoptosis detection kit;

Intergen, Purchase, USA) in the presence of TdT (60 min, 37°C), followed by

incubation with a mouse anti-Ki-67 antiserum (DAKO, Glostrup, Denmark) overnight

at 4°C.

Using washing steps with PBS, TUNEL-positive cells were visualized by an

anti-digoxigenin FITC-conjugated antibody (ApopTag kit) (30 min, room

temperature), whereas Ki-67 was detected by a rhodamine-labelled goat anti-mouse

antibody (Jackson ImmunoResearch, West Grove, USA) (45 min, room temperature).

Finally the cryosections were counterstained with DAPI (Boehringer Mannheim,

Germany) for 1 min and mounted with Fluoromount-G (Southern Biotechnologies,

Birmingham, USA).

Negative controls were performed by omitting TdT and the Ki-67antibody.

MATERIAL AND METHODS

120

3.6 Immunohistochemistry

3.6.1 Primary antibodies

Tab. 3.1 Primary antibodies and secondary detection systems Anitbodies used for immunohistological stainings are listed and described in detail. ABC-AP: Avidin-biotin complex, alkaline phosphatase, ABC-HRP: Avidin-biotin complex, horseradish peroxidase, FITC: Fluorescein isothiocyanate, IF: Immunofluorescence, Ig: Immuno globulin, NA: Not applicable, Rh: Rhodamine, TSA: Tyramide signal amplification conjugated.

Anti-human antibodies Primary

antibody

Origin Clone Ig

class

Vendor Dilu-

tion

Secon-

dary

system

References

HLA-ABC mouse W6/32 IgG2akappa

DAKO, Glostrup, Denmark

1:50 1:50 1:1500

EnVision® IF-FITC/Rh TSA

ITO et al. (2004); ITO et al. (2005c)

HLA-DP/ DQ/ DR

mouse CR3/43 IgG1, kappa

DAKO, Glostrup, Denmark

1:50 1:50 1:1500

EnVision® IF-FITC/Rh TSA

ITO et al. (2004)

CD200 mouse MRC OX 104

IgG1 Serotec, Oxford, UK

1:400 TSA OHYAMA et al. (2006); TIEDE et al. (2007a)

β2-micro-globulin

mouse Tü99 IgM, kappa

BD Pharmingen, Heidelberg, Germany

1:100 1:100

EnVision® IF-FITC/Rh

HARRIST et al. (1983); CHRISTOPH et al. (2000); BLASCHITZ et al. (2001)

α-MSH rabbit - IgG Sigma-Aldrich, München, Germany

1:2000 TSA KONO et al. (2001)

TGF-β2 rabbit - - Santa Cruz, Heidelberg, Germany

1:4000 TSA DE KOZAK et al. (2007)

IDO mouse 10.1 - AbD Serotec, Oxford, UK

1:50 ABC-HRP SCHELER et al. (2007)

MIF mouse 12302 IgG1 R&D Systems, Minneapolis, USA

1:50 ABC-HRP ITO et al. (2007)

HLA-E mouse - - kindly produced and provided by Daniel Geraghty, Fred Hutchinson, Re-search Centre, Seattle, USA

1:100 IF-FITC -

MATERIAL AND METHODS

121

Tab. 3.2 Primary anti-mouse antibodies and secondary detection systems Anitbodies used for immunohistological stainings on mouse tissue are listed and described in detail. ABC-AP: Avidin-biotin complex, alkaline phosphatase, ABC-HRP: Avidin-biotin complex, horseradish peroxidase, FITC: Fluorescein isothiocyanate, IF: Immunofluorescence, Ig: Immuno globulin, NA: Not applicable, Rh: Rhodamine, TSA: Tyramide signal amplification conjugated.

Anti-mouse antibodies Primary

antibody

Origin Clone Ig

class

Vendor Dilution Secon-

dary

system

References

MHC I (H-2d, H-2b,p,q)

rat ER-HR52

IgG2a BMA Biomedical,

Augst, Switzerland

1:50 1:150

ABC-HRP RUECKERT et al. (1998)

MHC II rat ER-TR3 IgG2b BMA Biomedical,

Augst, Switzerland

1:200 1.200

ABC-HRP IF-

FITC/Rh

PAUS et al. (1998); EICHMUELLER et

al (1998)

CD4 (L3T4)

rat RM4-5 IgG2a BD Pharmingen, Heidelberg, Germany

1:100 ABC-HRP PAUS et al. (1998); EICHMUELLER et

al (1998)

CD8a (Ly-2)

rat 53-6.7 IgG2a kappa

BD Pharmingen, Heidelberg, Germany

1:10 ABC-HRP PAUS et al. (1998); EICHMUELLER et

al (1998) CD11b (Ly-40)

rat M1/70 IgG2b BMA Biomedicals,

Augst, Switzerland

1:8000 ABC-HRP PAUS et al. (1998)

ICAM-1 (CD54)

hamster 3E2 IgG BD Pharmingen, Heidelberg, Germany

1:50 ABC-HRP EICHMUELLER et al (1998)

TGF-β1 rabbit - IgG Santa Cruz, Heidelberg, Germany

1:750 ABC-HRP CHUNN et al (2006)

β-defen-sin 2

goat - IgG Santa Cruz, Heidelberg, Germany

1:10 ABC-HRP -

3.6.2 Avidin Biotin Complex-Peroxidase

Cryosections (6-7 µm thick) which had been stored at -80°C were air dried for 10 min

and fixed in acetone at -20°C for 10 min. Then the slides were washed tree times for

5 min each in TBS buffer (for preparation see chapter 3.5.1) followed by a blocking

step against the endogenous peroxidase in 3 % H2O2 in TBS for 15 min at room

temperature. After three times of 5 min washing the samples, sections were circled

MATERIAL AND METHODS

122

with a fat marker and then preincubated with normal serum 10% (DAKO, Glostrup,

Denmark) in TBS for 20 min in a moist chamber. The normal serum was of the same

origin as the secondary antibody (i.e. goat, rabbit, hamster).

After preincubation the slides were not washed but the normal serum was only

dropped, and the primary antibody (see Tabs. 3.1, 3.2) was applicated with its

appropriate dilution in TBS together with 2 % of normal serum overnight at 4 °C.

After three times washing for 5 min each in TBS, the slides were stained either with

goat anti-mouse or goat anti-rabbit (1:200 in TBS, Jackson ImmunoResearch,

Cambridgeshire, UK), goat anti-rat (1:200 in TBS, Beckman Coulter, Marseille,

France) or rabbit anti-goat (1:200 in TBS, Santa Cruz, Heidelberg, Germany)

biotinylated secondary antibodies. After 45 min incubation at room temperature,

slides were washed three times for 5 min in TBS. A 30 min application of ABC-

peroxidase solution (avidin-biotin-complex, peroxidase conjugated, Vector

Laboratories, Burlingame, CA, USA) followed at room temperature. It is important to

note is, that the solution had to be prepared 30 min before usage.

The sections were labelled with the peroxidase-chromogen 3,3’-diaminobenzidine

(DAB) (Vector Laboratories, Burlingame, CA, USA) or 3-amino-9-ethylcarbazole

(AEC) (DAKO, Burlingame, CA, USA) for 2-10 min (see Tabs. 3.1, 3.2). Both

substrate solutions DAB and AEC were prepared according to their protocols

immediately before use as follows: For preparation, 2 drops of Buffer stock solution,

5 drops of DAB stock solution, i.e. 3 drops of the AEC stock solution and 2 drops of

the hydrogen peroxide solution, were added to 5 ml of distilled water, and mixed well

in between. If necessary or desired, the sections were counterstained with Mayer’s

hemalum (Merck, Darmstadt, Germany) using washing steps in between. Finally the

slides were mounted with DAKO Faramount (DAKO, Glostrup, Denmark).

As negative controls, the primary antibodies were omitted, performing the same

staining steps as described before.

MATERIAL AND METHODS

123

3.6.3 EnVision®-alkaline phosphatase

Cryosections (6-8 µm thick) which had been stored at -80°C were air dried for 10 min

and then fixed in acetone at -20°C for 10 min. After a three times washing step for 5

min each in TBS (as described before, see 3.5.1), the section samples were

preincubated with normal serum 10%, that was of the same origin as the secondary

antibody (i.e. goat, rabbit, hamster; DAKO, Glostrup, Denmark), in TBS for 20 min.

The slides were washed three times for 5 min in TBS and then preincubated with

10% normal goat serum (DAKO, Glostrup, Denmark) in TBS for 20 min. The

application of the primary antibodies (see Tabs. 3.1, 3.2) followed in their appropriate

dilution at 4 ˚C in TBS overnight.

A three times washing step in TBS for 5 min each was followed by the incubation

with EnVision® solution (alkaline phosphatase, anti rabbit / mouse, IgG-labelled

polymer, DAKO, Glostrup, Denmark) for 30 min and another three times washing

step. Afterwards Fast Red AP-chromogen (Sigma, Saint Louis, USA) was employed

for 3 to 10 min to visualize the immunosignals, and finally, sections were

counterstained with Mayer’s hemalum (Merck, Darmstadt, Germany) and mounted

with DAKO Faramount (DAKO, Glostrup, Denmark).

3.6.4 Immunofluorescence

To identify the IR in the bulge region of human HFs via immunofluorescence staining,

we used primary antibodies (see Tabs. 3.1, 3.2) and the secondary antibodies goat

anti-mouse, anti-rat or anti-rabbit IgG conjugated with fluorescein isothiocyanate

(FITC) or rhodamine (1:200 in TBS, Jackson ImmunoResearch, Cambridgeshire,

UK).

For Immunofluorescence staining, cryosections (6-8 µm thick) which had been stored

at -80°C were first air dried for 10 min and then fixed in acetone at -20°C for 10 min.

A three times washing step in TBS (produced as described before, see 4.5) for 5 min

each followed, and then the section samples were preincubated for 20 min with

MATERIAL AND METHODS

124

normal serum 10% (DAKO, Glostrup, Denmark) in TBS at room temperature. Then

primary antibodies (see Tabs. 3.1, 3.2) in their appropriate dilution in TBS were

applicated and the sections were incubated overnight at 4°C. After washing three

times for 5 min in TBS, a 45 min incubation at room temperature with the appropriate

secondary antibody in TBS was arranged. Then the slides were washed three times

for 5 min in TBS again, counterstained with DAPI (4’,6-diamidin-2’-phenylindol-

dihydrochlorid, Boehringer Mannheim, Germany) for 1 min and mounted with

Fluoromount-G (Southern Biotechnologies, Birmingham, USA).

The fluorescent dye DAPI binds selectively to DNA (deoxyribonucleic acid) and forms

strongly fluorescent DNA-DAPI complexes with high specificity. On adding DAPI to

the tissue it is rapidly taken up into cellular DNA yielding highly fluorescent nuclei and

no detectable cytoplasmatic fluorescence.

3.6.5 Tyramide signal amplification (TSA)

After standard fixation for 10 min in acetone at -20°C, the cryosections (6-7µm thick)

were washed three times for 5 min using TNT buffer (0.1 mol/l Tris-HCl, pH 7.5;

containing 0.15 mol/l NaCl and 0.05 % Tween 20). Then endogenous horseradish

peroxidase was blocked by washing with 3% H2O2 in PBS (phosphate buffered

saline) for 15 min.

For the preparation of PBS, 1.8 g NaH2PO4 x H2O sodium dihydrophosphate

monohydrate and 8.0 g sodium chloride (NaCl) were diluted in 800 ml distilled water.

The pH was adjusted to 7.2 with 1N sodium hydroxide (NaOH). At the end the

solution had to be dropped up to 1000 ml with distilled water.

Preincubation was performed with the incubation of avidin and biotin for 15 min each,

with three times 5 min washing steps in TNT in between followed by another

preincubation step with 5% normal goat serum (DAKO, Glostrup, Denmark) in TNT

for 30 min. Afterwards the primary antibodies (see Tabs. 3.1. 3.2) diluted in TNT

were added and the sections were incubated overnight at 4˚C. Next day, the

MATERIAL AND METHODS

125

application of the appropriate biotinylated secondary antibody (goat anti-mouse

(1:200 in TNT, Jackson ImmunoResearch, Cambridgeshire, UK) followed with an

incubation for 45 min at room temperature. After a three times washing step,

streptavidin horseradish peroxidase (TSA kit; Perkin-Elmer, Boston, USA) was

administrated (1:100 in TNT) for 30 min at room temperature, and the reaction was

amplified by tetramethylrhodamine- or FITC-tyramide amplification reagent at room

temperature for 5 min (1:50 in amplification diluent provided with the TSA kit). The

cryosections were counterstained with DAPI (Boehringer Mannheim, Germany) for 1

min and mounted with Fluoromount-G (Southern Biotechnologies, Birmingham,

USA).

For all immunostaining assays, primary antibodies were omitted as negative control.

3.7 Histomorphometry

3.7.1 Assessment of hair cycle stages

In order to observe spontaneous or induced catagen development, immunostained

HF slides were used for HF cycle staging according to previously well-defined

morphological criteria (MUELLER-ROEVER et al. 2001; STENN and PAUS 2001),

and the percentage of HFs in anagen, early, mid and late catagen were determined.

3.7.2 Assessment of proliferating matrix keratinocytes

Cryosections of organ-cultured HFs treated with different concentrations of the test α-

MSH related tripeptide K(D)PT, with or without a combination with IFN-γ were then

stained for Ki-67 / TUNEL and investigated at 400x magnification. All Ki-67-positive

(red) and all TUNEL-positive cells (green) below Auber’s line (AUBER 1952) were

counted as well as the total number of keratinocytes, stained with DAPI (blue) (Fig.

3.7). The amount of Ki-67-positive cells, and, TUNEL-positive cells respectively

relating to DAPI-positive cells was shown in per cent.

MATERIAL AND METHODS

126

Fig. 3.7 Auber’s line marked in the human HF The Auber’s line runs through the widest part of the hair bulb at the level of the maximal diameter of the dermal papilla; Below this line every Ki67+ cell, TUNEL+ cell and DAPI+ cell is counted and the percentage for proliferation and apoptosis calculated; dotted white line: Auber’s line (AUBER 1952).

3.7.3 Assessment of immunostaining intensity

The immunostaining intensity for selected antigens examined was compared by

quantitative immunohistochemistry as previously described (ITO et al. 2004b, 2005b,

2005c), using NIH image software (National Institute of Health, Bethesda, Maryland).

In regard to the special tissue, different reference areas were defined and analyzed.

In general, the human scalp HF was divided anatomically into five reference

compartments: (1) epidermis, (2) distal ORS: middle of the isthmus, (3) bulge:

insertion point of the APM, (4) proximal ORS, (5) hair matrix, and analyzed deriving

from 3-5 different individuals.

The vibrissal follicle was divided according to the human HFs into five compartments

1) isthmus, (2) distal ORS: superior swelling of the ORS, (3) bulge: inferior swelling of

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the ORS, (4) proximal ORS, (5) hair matrix, and analyzed, deriving from 5 different

mice.

The nail apparatus was analyzed within two reference areas (1) the proximal nail

matrix and (2) the proximal nail fold as shown.

3.7.4 Assessment of MHC class I IR in isolated HFs

Immunostaining intensity was compared by quantitative immunohistochemistry as

previously described (ITO et al. 2004), using NIH image software (NIH, Bethesda,

Maryland). In the run-up to analyse, reference areas were defined according to the

objective of the study. The mean intensity was measured at five previously defined

reference areas in the hair matrix, proximal ORS and CTS/DP (Fig. 3.8) and the

average was calculated.

Fig. 3.8 Reference areas for the quantitative analysis of MHC class I IR CTS: Connective tissue sheath, DP: Dermal papillam IRS: Inner root sheath, MK: Matrix keratinocytes, ORS: Outer root sheath. Original magnification: 200x.

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3.7.5 Assessment of mast cells

By evaluating the toluidine blue and with Leder`s esterase method stained HFs, the

total number of mast cells was counted in the defined reference areas. In isolated

HFs, all mast cells located in the CTS were counted, whereas in the human and

murine tissue in situ mast cells were counted at a 400x magnification of the defined

compartments (see 3.7.3) and the mean value was calculated. In addition, non-

degranulated and degranulated mast cells were distinguished. Mast cells with more

then six granules outside of the cell membrane were regarded as ´degranulated`.

3.7.6 Assessment of MHC class II, CD4, CD54, CD11b, mast cells

and c-kit positive cells

Positively stained cells were counted by quantitative immmuno histomorphometry

following our previously described morphometrical techniques (PAUS et al. 1998;

CHRISTOPH et al. 2000; ITO et al. 2004).

Again, the human scalp HF was divided anatomically into five reference

compartments: (1) epidermis, (2) distal ORS: middle of the isthmus, (3) bulge:

insertion point of the APM, (4) proximal ORS, (5) hair matrix. In these compartments

HLA-DP/DQ/DR, CD54 positive cells and mast cells were counted at a 400x

magnification and analyzed on deriving from 3-5 different individuals.

In the same way, the vibrissal follicle was divided into (1) isthmus, (2) distal ORS:

superior swelling of the ORS, (3) bulge: inferior swelling of the ORS, (4) proximal

ORS, (5) hair matrix, and analyzed at 400x magnification for MHC class II, CD4,

CD11, CD54 positive cells and mast cells (N= 5 mice).

The murine nail was divided into the nail matrix and the PNF as shown anatomically,

and MHC class II, CD4, CD11b, CD54, and mast cells (toluidine blue staining) in

these compartments cells were counted at 400x magnification per macroscopic field

from one nail (N=5-7 individuals).

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3.7.7 Microscopical equipment

For fluorescence and light microscopy, a Keyence Biozero-8000 microscope

(Keyence Corporation, Biozero-8000, Higashi-Nakajima, Osaka, Japan) was used, in

combination with Nikon lenses (Japan).

3.7.8 Statistical analysis

For the evaluation of statistical significance, all data obtained from the experiments

were pooled and analyzed with the Mann-Whitney-U-Test for unpaired samples

(GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego,

California, USA). The results were expressed as standard error of the mean (mean

±SEM) and values of p<0.05 were regarded as significant.

The IR for MHC class I in human scalp skin in situ and in the full thickness punch

biopsies was analysed by quantitative immuno histomorphometry, using NIH image

software. The reference area for ‘distal ORS’ was on the level of the insertion of the

sebaceous duct, the ´bulge` at the insertion point of the arrector pili muscle and the

proximal ORS point was situated in the middle between the bulge and the hair bulb.

(N=5, *p<0.001, mean+/-SEM), p value was calculated with the Mann-Whitney-Test

for unpaired samples.

The mean immunostaining intensity for MHC class I in isolated HFs was measured

for four previously defined reference areas in the hair matrix of anagen hair bulbs

(Fig. 3.8) by NIH image, and the average staining intensity was calculated (N= 6-36

HFs from 6 patients/group).

The percentage of HFs in the different hair cycle stages treated with the K(D)PT was

calculated as compared to the control (N=8-12 follicles / group, six representative

experiments), p value was calculated with the Mann-Whitney-U-Test for unpaired

samples.

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Ki-67+ / TUNEL+ cells were counted below Auber’s line were counted as well as the

total number of keratinocytes, stained with DAPI. The amount of Ki-67+ cells, and,

TUNEL+ cells respectively relating to DAPI-positive cells was shown in per cent.

N=6-18 follicles / group, *p<0.05, mean ± SEM, p value was calculated with the

Mann-Whitney-U-Test for unpaired samples.

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4 Results

4.1 Immune privilege and the human hair follicle bulge

4.1.1 Demonstration of MHC class Ia and β2-microglobulin

expression on CD200+ cells

In order to accurately define MHC class Ia protein expression in the bulge region, we

used the broad-specificity antibody HLA-A/B/C against all MHC class Ia antigens,

which are expressed on all nucleated cells, except in areas of IP (NIEDERKORN

2006). Compared to the distal ORS or the epidermis, immunohistochemistry clearly

showed that the ORS bulge region indeed expressed substantially lower or absent

MHC class Ia IR (Fig. 4.1 A,B). This was evident both by light microscopy (Fig. 4.1)

and Immunofluorescence (Fig. 4.4), complemented by quantitative analysis of MHC

class I IR (***p<0.001) (Fig. 4.3). In the distal ORS, especially in the infundibulum

and upper parts of the isthmus both basal and suprabasal staining was observed,

whereas IR in the bulge ORS was mainly limited to the basal layer of the ORS.

The proximal ORS, IRS and all matrix keratinocytes of the immunoprivileged anagen

hair bulb displayed extremely weak or absent MHC class Ia IR (Fig. 4.1 C).

Interestingly, compared to the bulge, the proximal ORS showed lower, although not

significant MHC class I IR (Fig. 4.3) whereas the level of HLA-A/B/C associated IR in

the anagen hair matrix compared to the bulge was significantly reduced (*p<0.05)

(Figs. 4.2, 4.3).

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Fig. 4.1 IR is downregulated in the human bulge region for MHC class I (A-C) Immunohistological identification (EnVision®) of MHC class I expression. (A,B) HLA-A/B/C IR is prominently downregulated in the bulge region compared to the distal ORS and the epidermis. (C) The anagen hair matrix shows no MHC class I expression. APM: Arrector pili muscle, B: Bulge, CTS: Connective tissue sheath, E: Epidermis; HS: Hair shaft, MK: Matrix keratinocytes, ORS: Outer root sheath, SG: Sebaceous gland, Bars: (A): 200µm; (B,C): 100µm.

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Fig. 4.2 Schematic drawing of MHC class I IR pattern Schematic drawing of the MHC class I IR pattern in the pilosebaceous unit and in the epidermis. APM: Arrector pili muscle, B/FT: Bulge/Follicular trochanter; BM: Basement membrane; CL: Companion layer; CTS: Connective tissue sheath, DP: Dermal papilla, E: Epidermis, HS: Hair shaft, IRS: Inner root sheath, ORS: Outer root sheath, SG: Seba-ceous gland; IR: Immunoreactivity

Fig. 4.3 Quantitative immunohistochemistry for MHC class I The immunostaining intensity for MHC class I was analysed by quantitative immunohistochemistry using NIH image software. The reference area for ‘distal ORS’ is on the level of the insertion of the sebaceous duct, the ‘bulge’ at the insertion point of the arrector pili muscle, the ´proximal ORS` point is situated in the middle between the bulge and the hair bulb and the fourth reference area are the hair matrix keratinocytes of the hair bulb. N=5, *p<0.05, ***p<0.001, mean ± SEM, p value was calculated by Mann-Whitney-U-Test for unpaired samples.

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Since the immunoregulatory molecule CD200 belongs to the so-called bulge marker

(although it is not restricted to it), we performed a double TSA staining with MHC

class I molecule. We found prominent CD200 IR in the outermost layer of the ORS

between the insertion of the APM and the insertion of the SG duct (parts of the

isthmus and the bulge region) besides some staining in the DP including blood

vessels, the APM, the homogeneously stained companion layer and SW

mesenchyme and epithelium (data not shown). Most importantly, the intensity of

MHC class Ia-like IR was inversely correlated with that of CD200 (Fig. 4.4 A,B):

Again, MHC class I protein expression was only prominent in the epidermis, and in

the ORS of infundibulum and isthmus, but absent or extremely weak in the basal

bulge ORS (Figs. 4.4 B, 4.5).

Fig. 4.4 MHC class I IR is downregulated in CD200+ bulge ORS cells Double Immunofluorescence staining (TSA) shows the expression pattern of CD200 molecule (FITC, green signal) and MHC class I (Rhodamine, red signal) (counterstained with DAPI, blue). CD200 is upregulated in the bulge whereas MHC class I protein expression is downregulated compared to the distal ORS and epidermis. (A) High magnification of the bulge ORS displays very strong CD200 expression and almost none MHC class I expression. APM: Arrector pili muscle, HS: Hair shaft, ORS: Outer root sheath, SG: Sebaceous gland, Bars: (A): 100µm; (B): 20µm.

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Fig. 4.5 Schematic drawing of CD200 IR pattern Schematic drawing of the CD200 IR pattern of the employed antibody in the pilosebaceous unit and in the epidermis. APM: Arrector pili muscle, B/FT: Bulge/Follicular trochanter; BM: Basement membrane; CL: Companion layer; CTS: Connective tissue sheath, DP: Dermal papilla, E: Epidermis, HS: Hair shaft, IRS: Inner root sheath, ORS: Outer root sheath, SG: Sebaceous gland; IR: Immunoreactivity.

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As β2-microglobulin is non-covalently associated with MHC class Ia molecules, it was

used as an additional verification for the MHC class Ia expression pattern. Using

immunofluorescence and the EnVision® staining method (data not shown) we

demonstrated that β2-microglobulin IR exactly

paralleled that of MHC class Ia (Figs. 4.6, 4.7) and

confirmed the results by immuno histomorphometry

(Fig. 4.8) (**p<0.01).

Fig. 4.6 IR in the human hair follicle for β2-microglobulin Expression pattern of β2-microglobulin in normal human scalp skin (Immunofluorescence). The staining intensity of β2-microglobulin is very strong in the epidermis (A) and isthmus (B), it decreases in the infundibulum (C), whereas the bulge region (D) and the hair bulb (E) of human anagen VI HFs show no IR (green signal). Bars (A-E): 50µm.

Fig. 4.7Schematic drawing of β2-microglobulin IR pattern

Schematic drawing of the CD200 IR pattern of the employed antibody in the pilosebaceous unit and in the epidermis. APM: Arrector pili muscle, B/FT: Bulge/Follicular trochanter; BM: Basement membrane; CL: Companion layer; CTS: Connective tissue sheath, DP: Dermal papilla, E: Epidermis, HS: Hair shaft, IRS: Inner root sheath, ORS: Outer root sheath, SG: Sebaceous gland; IR: Immunoreactivity.

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Fig. 4.8 Quantitative immunohistochemistry for β2-microglobulin The immunostaining intensitiy for β2-microglobulin was analysed by quantitative immunohistochemistry using NIH image software. The reference area for ‘distal ORS’ is on the level of the insertion of the sebaceous duct and for the ‘bulge’ at the insertion point of the arrector pili muscle. N=5, **p<0.01, mean ± SEM, p value was calculated by Mann-Whitney-U-Test for unpaired samples.

4.1.2 Demonstration of MHC class II+ cells in the bulge

A monoclonal antibody specific against HLA-DP/DQ/DR was used for the localization

of intraepithelial MHC class II+ antigen-presenting cells such as Langerhans cells

and macrophages (SMITH et al. 1987). The most prominent expression of MHC

class II was epidermal (data not shown) and expression was particularly dense in the

infundibulum and upper parts of the isthmus (Fig. 4.9 A,B). Here we show, that the

bulge region, as the proximal ORS, exhibited a significantly reduced expression of

MHC class II compared to the upper parts of the HF (Figs. 4.9 C,D). As expected,

the bulb region only rarely showed single MHC class+ cells (Fig. 4.9 D) that were not

found in the DP. This was confirmed by quantitative analysis using NIH image (Figs.

4.10, 4.11).

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While the distal perifollicular CTS displayed a high density of MHC class II+ cells,

most of which may represent macrophages, the lowest density was observed in the

isthmus region of the CTS. The CTS around the proximal HF and the bulb showed

more MHC class II+ cells than the CTS at the level of the isthmus region.

Fig. 4.9 IR in the human bulge region for MHC class II Immunohistochemical identification of MHC class II protein expression (HLA-DP/-DQ/DR) in normal human scalp skin (EnVision®) (A-D) /red). (A) MHC class II+ cells can be found in high numbers in the epidermis (A) and distal ORS of isthmus and infundibulum (B) whereas the bulge region and the hair bulb show only rare MHC class+ cells. APM: Arrector pili muscle, B: Bulge, CTS: Connective tissue sheath, E: Epidermis; HS: Hair shaft, MK: Matrix keratinocytes, ORS: Outer root sheath, SG: Sebaceous gland, Bars (A-D): 100µm.

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Fig. 4.10 MHC class II+ cells in the human hair follicle and human bulge region Positively stained cells are counted by quantitative immuno histomorphometry following previously described morphometry techniques (PAUS et al, 1998; CHRISTOPH et al, 2000; ITO et al, 2004). The human scalp HF was divided anatomically into five reference compartments: distal ORS=middle of the isthmus, (3) bulge= insertion point of the APM, (4) proximal ORS, (5) hair matrix of the hair bulb. Then HLA-DP/DQ/DR positive cells were counted at 400 times magnification and analyzed. N=4 .*p<0.05, ***p<0.001, mean ± SEM, p value was calculated by Mann-Whitney-U-Test for unpaired samples.

Fig. 4.11 Schematic drawing of MHC class II IR pattern Schematic drawing of the MHC class II pattern of the employed antibody in the pilosebaceous unit and in the epidermis. APM: Arrector pili muscle, B/FT: Bulge/Follicular trochanter; BM: Basement membrane; CL: Companion layer; CTS: Connective tissue sheath, DP: Dermal papilla, E: Epidermis, HS: Hair shaft, IRS: Inner root sheath, ORS: Outer root sheath, SG: Sebaceous gland; IR: Immunoreactivity.

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4.1.3 Demonstration of HLA-E expression in CD200+ bulge cells

By using double Immunofluorescence staining, we found that HLA-E protein

expression was limited to the CD200 positive cells in the basal layer of the follicular

bulge (Fig. 4.12 A-B) and yet appeared to occupy an intracellular position close to

the mesenchyme-facing side of the cell. There was no cell surface expression of

HLA-E by these cells.

Fig. 4.12 HLA-E expression by CD200+ cells in the human bulge, double Immunofluorescence (A) The bulge region demonstrates double staining with HLA-E (Rhodamine, red) and CD200 (FITC, green), compared with the distal ORS. (B) The confocal microscopic view of the outer most layer of the ORS of the bulge demonstrates that HLA-E protein expression (rhodamine red, arrowhead) is limited to within the CD200+ positive cells of the bulge. APM: Arrector pili muscle, arrows: HLA-E+ cells. Bars: (A): 100µm; (B): 20µm.

Fig. 4.13 Schematic drawing of MHC class II IR pattern Schematic drawing of the MHC class II pattern of the employed antibody in the pilosebaceous unit and in the epidermis. APM: Arrector pili muscle, B/FT: Bulge/Follicular trochanter; BM: Basement membrane; CL: Companion layer; CTS: Connective tissue sheath, DP: Dermal papilla, E: Epidermis, HS: Hair shaft, IRS: Inner root sheath, ORS: Outer root sheath, SG: Sebaceous gland; IR: Immunoreactivity.

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4.1.4 Locally generated immunosuppressants complement the

bulge IP

Since immunosuppressants like TGF-β and α-MSH play a major role in IP, it is

important that we could show a substantial increase of α-MSH IR in the bulge and

proximal ORS - in particular in the suprabasal layers - (Fig. 4.14 A-C) compared to

the epidermis and the distal ORS (Figs. 4.14 A, 4.16).

Fig. 4.14 IR in the human bulge region for α-MSH α-MSH (green) shows upregulated IR in the ORS of the bulge region and proximal ORS compared to the upper part of the HF and the epidermis. APM: Arrector pili muscle, E: Epidermis; FT: Follicular trochanter, ORS: Outer root sheath, SG: Sebaceous gland, Bars (A): 200µm; (B,C): 20µm.

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However, though TGF-ß2 was also prominently expressed in the bulge, the staining

intensity was not found to be significantly upregulated here compared to basal layer

epidermis (Fig. 4.15 A-C) or other HF compartments like the human HF bulb (Figs.

4.15 C, 4.17), even when using the highly sensitive TSA-staining method.

Quantitative immunohistochemistry did also not reveal significant differences in the

IR intensity between the bulge region and other parts of the ORS (data not shown).

Fig. 4.15 TGF-β2 is expressed in the bulge ORS Immunostaining for the immunosuppressive α-MSH (by using TSA method) (FITC, green signal, counterstained with DAPI, blue) shows an upregulation of IR in the bulge, and in the proximal ORS compared with the epidermis and distal ORS. APM: Arrector pili muscle, E: Epidermis, ORS: Outer root sheath, SG: Sebaceous gland, dotted white line indicates the APM; Bars (A) 100µm, (B,C) 20µm.

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Fig. 4.16 Schematic drawing of α-MSH IR pattern Schematic drawing of the α-MSH IR pattern of the employed antibody in the pilosebaceous unit and in the epidermis. APM: Arrector pili muscle, B/FT: Bulge/Follicular trochanter; BM: Basement membrane; CL: Companion layer; CTS: Connective tissue sheath, DP: Dermal papilla, E: Epidermis, HS: Hair shaft, IRS: Inner root sheath, ORS: Outer root sheath, SG: Sebaceous gland; IR: Immunoreactivity.

Fig. 4.17 Schematic drawing of TGF-β2 IR pattern Schematic drawing of the TGF-β2 IR pattern of the employed antibody in the pilosebaceous unit and in the epidermis. APM: Arrector pili muscle, B/FT: Bulge/Follicular trochanter; BM: Basement membrane; CL: Companion layer; CTS: Connective tissue sheath, DP: Dermal papilla, E: Epidermis, HS: Hair shaft, IRS: Inner root sheath, ORS: Outer root sheath, SG: Sebaceous gland; IR: Immunoreactivity.

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4.1.5 Demonstration of macrophage migration inhibitory factor and

indoleamine-2,3-dioxygenase

Next, we investigated the local expression of the immunosuppressant factor MIF.

Compared to the epidermis and the distal ORS (Fig. 4.18 A,B), MIF-IR was

upregulated in the bulge and proximal ORS (Fig. 4.18 A), where it was limited to the

suprabasal layer. Quantitative analysis of MIF-IR in the bulge confirmed this

significant upregulation compared to the distal ORS and to the epidermis (Figs. 4.19,

4.23) (*p<0.01).

Fig. 4.18 IR is upregulated in the human bulge region for MIF MIF (red) IR can be found throughout the whole ORS, with an upregulation in the bulge region and proximal parts of the ORS. The epidermis and APM are weakly stained for MIF. APM: Arrector pili muscle, BM: Basement membrane, HS: Hair shaft, ORS: Outer root sheath, SG: Sebaceous gland, dotted white line indicates the APM; Bars: (A) 200µm; (B) 50µm.

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Fig. 4.19 Quantitative immunohistochemistry for MIF The immunostaining intensity for MIF was analysed by quantitative immunohistochemistry using NIH image software. The reference area for ‘distal ORS’ is on the level of the insertion of the sebaceous duct, the ‘bulge’ at the insertion point of the arrector pili muscle, the ´proximal ORS` point was situated in the middle between the bulge. N=3, *p<0.05, ***p<0.001, mean ± SEM, p value was calculated by Mann-Whitney-U-Test for unpaired samples.

IDO blocks T cell responses via withdrawal of essential amino acids 33-36. We

examined its IR pattern, using the avidin-biotin complex method and provide the first

immunohistochemical in situ evidence of IDO in human HFs.

The lower parts of the isthmus, the outermost layer of the ORS in the bulge region,

and segments of the basal layer in the proximal ORS revealed substantially

increased IDO IR (Fig. 4.20 A), whereas the basal layers of the epidermis and distal

ORS showed only weak intracellular IR (Fig. 4.20 B).

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Fig. 4.20 IR is upregulated in the human bulge region for IDO IDO IR (brown, DAB substrate) is upregulated in the basal layers of the bulge ORS (A) compared to the distal ORS and epidermis (B). The APM is weakly stained positive. APM: Arrector pili muscle, BM: Basement membrane, E: epidermis, HS: Hair shaft, SG: Sebaceous gland; Bars: (A) 50µm; (B) 200µm.

Interestingly, the staining intensity decreased in the proximal ORS with a sparse

distribution of IDO+ keratinocytes (Fig. 4.21 B) and no IR was found in the hair bulb

(data not shown) (Fig. 4.23). Quantitative analysis corroborated a significantly

increased IR in the bulge, compared to distal ORS and epidermis (Fig. 4.22)

(***p<0.001).

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Fig. 4.21 IR in different parts of the proximal ORS for IDO A heterogenous staining intensity of IDO is seen in the proximal ORS (red, AEC substrate). Compared to the suprabulge ORS (A), IR decreased in lower parts of the proximal ORS with a sparse distribution of IDO+ keratinocytes (B). No IR was found in the IRS or the hair bulb (data not shown). APM: Arrector pili muscle, BM: Basement membrane, HS: Hair shaft; IRS: Inner root sheath; ORS: Outer root sheath; Bars: (A, B) 50µm.

Fig. 4.22 Quantitative immunohistochemistry for IDO The immunostaining intensity for IDO was analysed by quantitative immunohistochemistry using NIH image software. The reference area for ‘distal ORS’ is on the level of the insertion of the sebaceous duct, the ‘bulge’ at the insertion point of the arrector pili muscle. N=7, ***p<0.001, mean ± SEM, p value was calculated by Mann-Whitney-U-Test for unpaired samples.

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Fig. 4.23 Schematic drawings of MIF and IDO IR pattern Schematic drawing of the MIF and IDO IR pattern of the employed antibody in the pilosebaceous unit and in the epidermis. APM: Arrector pili muscle, B/FT: Bulge/Follicular trochanter; BM: Basement membrane; CL: Companion layer; CTS: Connective tissue sheath, DP: Dermal papilla, E: Epidermis, HS: Hair shaft, IRS: Inner root sheath, ORS: Outer root sheath, SG: Sebaceous gland; IR: Immunoreactivity.

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4.1.6 Influence of IFN-γ on ectopic MHC class I protein expression

in the bulge ORS

In order to check whether IFN-γ also causes ectopic MHC class I expression in the

bulge epithelium such as in the hair matrix, we performed organ culture of full-

thickness human scalp skin fragments under serum-free conditions and treated them

with IFN-γ or vehicle, and evaluated MHC class I IR by quantitative immuno

histomorphometry. The cultured human anagen VI HFs in situ showed a consistent

tendency towards ectopic MHC class I expression in the bulge region (Fig. 4.24).

However, only high-dose IFN-γ treatment (1000 IU/ml) reached the level of

significance (Fig. 4.25) (*p<0.05). Thus, just as the anagen hair bulb IP, the MHC

class I-based IP of the human bulge collapses after exposure to sufficient amounts of

IFN-γ.

Fig. 4.24 Influence of IFN-γ on MHC class I expression of human HFs in full thickness organ culture Full-thickness human scalp skin punch biopsies were organ cultured and groups were treated with IFN-γ (100, 500, 1000 IU/ml) or PBS. Then, Immunofluorescence staining for MHC class I (green) was performed and IR was compared. (A) PBS control group, (B) 1000 IU/ml IFN-γ treated sample. APM: Arrector pili muscle, HS: Hair shaft, ORS: Outer root sheath, SG: Sebaceous gland, Bars: (A,B) 50µm.

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Fig. 4.25 Quantitative analysis of MHC class I staining intensity in the bulge Full-thickness human scalp skin punch biopsies were organ cultured and groups were treated with IFN-γ (100, 500, 1000 IU/ml) or PBS. Then, IF staining for MHC class I (green) was performed and IR in the bulge region of anagen VI HFs was compared. The group treated with 1000 IU/ml IFN-γ revealed a significant increase in MHC class I IR. N=2, *p<0.05, mean ± SEM, p value was calculated by Mann-Whitney-U-Test for unpaired samples.

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4.2 Influence of the α-MSH related tripeptide K(D)PT on human hair

follicle biology in situ under pro-inflammatory conditions

4.2.1 Influence of K(D)PT on IFN-γ induced MHC class I and II

protein expression

IFN-γ significantly upregulated MHC class Ia IR in the hair bulb of human anagen VI

HFs, whereas the control HFs treated with K(D)PT 10-8M alone displayed no

differences compared to vehicle treated HFs (Fig. 4.2 C). In the majority of

experiments none of the K(D)PT doses could significantly and reliably reduce the

IFN-γ-induced MHC class I IR in the hair matrix and proximal ORS (Fig. 4.27 A,B),

although K(D)PT 10-8 often showed the tendency to and evoked in one patient a

significant downregulation of MHC class Ia IR (Figs. 4.26, 4.27 C).

Fig. 4.26 Isolated HFs stained for MHC class I EnVision® staining of isolated and cultured HFs for MHC class I (red).

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Fig. 4.27 Quantitative immunohistochemistry for MHC class I in isolated treated HFs HFs were isolated and treated with different doses of the test substance K(D)PT with or without combination of IFN-γ and appropriate controls. In the restoration assay HFs were pre-treated with IFN-γ, in the protection assay culture period started with a pre-treatement with K(D)PT first and IFN-γ second (Fig. 7.3). Then the immunostaining intensity for MHC class I was analysed by quantitative immunohistochemistry using NIH image software. The mean of following reference areas were compared (see Fig. ): Two reference areas were in the hair matrix of the hair bulb, two in the ORS and one in the CTS of the DP. (A) pooled data of two experiments, restoration assay; (B) pooled data of two experiments, protection assay; (C) individual case shows significant downregulation. N= 6, *p<0.05, ***p<0.001, mean ± SEM, p value was calculated by Mann-Whitney-U-Test for unpaired samples.

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Similarly, the expected significant upregulation of MHC class II+ cells in the hair bulb

of IFN-γ treated control HFs (Fig. 4.28) could not be significantly and reliably

downregulated by any K(D)PT dose, neither in the number of MHC class II+ cells

(Fig. 4.28), nor in the staining intensity (Fig. 4.29). However, K(D)PT showed the

tendency for downregulation and we also observed interindividual differences and

susceptibility (data not shown).

Fig. 4.28 Quantitative analysis of the mean number of MHC class II+ cells Quantitative immuno histomorphometry using NIH Image of MHC class II+ cells (A) Assay 1: pre-treatment with IFN-γ, pooled data of two experiments, N=10-30 HFs/group, (B) Assay 2: pre-treatment with K(D)PT, pooled data of two experiments, N=10-30 HFs/group, N= 6-18 HFs/group, *p<0.05, **p<0.01, ***p<0.001, mean ± SEM, p value was calculated by Mann-Whitney-U-Test for unpaired samples. Original magnification 630x.

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Fig. 4.29 Staining intensity of MHC class II+ cells Immunostaining for MHC class II+ cells in K(D)PT treated HFs (A) versus control HFs (B) [using the tyramide signal amplification (TSA) method, green signal] did not reveal any marked differences in the IR level (no formal quantification done).

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4.2.2 Reaction of K(D)PT on the IFN-γ induced upregulation of total

mast cell numbers and mast cell degranulation

Following treatment, cryosections of organ-cultured HFs were stained with the Leder

esterase staining (Fig. 4.30 A) and for c-kit (Fig. 4.30 B), in order to investigate how

K(D)PT influenced the number of mast cells and their activation status.

Fig. 4.30 Staining for mast cells (A) Representative photodocument of a Leder esterase stained HF, using the standard leder esterase protocol, cytoplasmic mast cell granules stained red, green counterstain, original magnification 400x. (B) Representative photodocument of c-Kit staining (green signal/FITC), original magnification 200x.

Stimulation with IFN-γ induced both a significant upregulation of mast cell numbers

and an increased degranulation compared to K(D)PT– or vehicle-treated control

groups (Fig. 4.31). High dose K(D)PT 10-8M always showed a strong reduction of this

increase in mast cell numbers (Fig. 4.31), although significance was only reached in

the restoration assay (assay 2) with IFN-γ pre-treatment. This was confirmed by c-Kit

immunofluorescence staining in the CTS of the HF (Fig. 4.30).

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Fig. 4.31 Quantitative analysis of the number of mast cells and their degranulation ratio Every Leder estease stained HF was analysed quantitatively regarding its number of mast cells (non-degranulated/degranulated) and the mean was calculated. Representative experiment of assay 2, n=10-18 hair follicles/group. Mean ± SEM, *p<0.05. p value was calculated by Mann-Whitney-U-Test for unpaired samples.

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4.2.3 Influence of K(D)PT on the hair cycle

Microdissected and cultured HFs were assessed with respect to their hair cycle

status. As shown in Fig. 4.32, this procedure revealed significant differences in the

ratio of HFs in distinct hair cycle stages: In the vehicle and the K(D)PT control groups

most of the HFs were still in anagen (~ 75%) and only a small part in early catagen

stage. The treatment with the pro-inflammatory cytokine IFN-γ reduced significantly

the percentage of HFs, which were in anagen (~ 50%) and displayed more HFs in

mid catagen. We could demonstrate, that supplementation with high dose K(D)PT

(10–8M, 10-9M) significantly counteracted against the effects of IFN-γ, both in assay 1

and assay 2. In addition, the effects seemed to inhibit even more catagen

transformation than in the vehicle treated control groups (~ 90% of the HFs in anagen

stage) (Fig. 4.32).

Fig. 4.32 Influence of K(D)PT on the hair cycle stage The total number (%) of HFs in the different hair cycle stages is shown. High-dose K(D)PT treated HFs under pro-inflammatory stimulus counteract the IFN-γ induced catagen transformation, and display a similar or even higher percentage of HFs being in anagen. N=8-12 follicles/group, pooled data of four experiments, Mean ± SEM, *p<0.05, **p<0.01. p value was calculated by Mann-Whitney-U-Test for unpaired samples.

* *

* *

RESULTS

158

4.2.4 Influence of K(D)PT on hair matrix keratinocyte proliferation

and apoptosis

The finding of the effects on the hair cycle was further supported by quantitative

immunohistochemistry of Ki-67 / TUNEL-double-stained HF sections (see Fig. 3.7).

In microdissected, organ-cultured human scalp HFs, K(D)PT stimulated hair matrix

proliferation under pro-inflammatory conditions, thus counteracted against the IFN-γ

induced reduction of matrix keratinocye proliferation and led to the increase of

apoptotic cells (Fig. 4.33). Nonetheless, interindividual differences were observed

and these results can only be regarded as preliminary.

Fig. 4.33 Ki-67 / TUNEL staining on human HFs The Ki-67 / TUNEL staining demonstrates the influence of K(D)PT on hair MK proliferation and apoptosis under pro-inflammatory condition. K(D)PT alone had no relevant effect compared to vehicle treated control HFs. In combination with IFN-γ high-dose K(D)PT counteracts against INF-γ induced proliferation reduction and apoptosis stimulation. N=6-18 follicles/group. Mean ± SEM, *p<0.05, **p<0.01, ***p<0.001. p value was calculated by Mann-Whitney-U-Test for unpaired samples.

RESULTS

159

4.3 Immune privilege and murine sinus hair follicles

4.3.1 Demonstration of MHC class I molecules

Staining for MHC class I by avidin-biotin-complex immunohistochemistry showed

homogenous IR in the epidermis and in the distal ORS

of the isthmus and infundibulum (Figs. 4.34, 4.36). At

the level of superior swelling IR started to decrease

(Fig. 4.34 B) and was even more reduced in the bulge

region or the inferior swelling of the ORS (Fig. 4.34 C).

Interestingly, in these hourglass shaped parts, the basal

layers of the ORS were almost devoid of IR. And almost

none staining intensity was found in the proximal ORS

or the follicle bulb (Fig. 4.34 E). Quantitative

immunohistochemistry revealed significant differences

in IR intensity between the bulge region and the

superior swelling of the ORS (Fig. 4.35). Notably, we

also found significant differences between the bulge

region and the proximal ORS (Fig 4.35).

Fig. 4.34 IR in the murine vibrissal follicle for MHC class I MHC class I (red) shows strong IR in the epidermis and distal ORS, reduced staining intensity in the bulge region (inferior swelling of the ORS) and almost none IR in the proximal ORS and bulb. HS: Hair shaft, DP: Dermal papilla, SO: Superior swelling of the ORS. Bars: (A-E) 50µm.

RESULTS

160

Fig. 4.35 Quantitative immunohistochemistry for MHC class I The immunostaining intensity for MHC class I was analysed by quantitative immunohistochemistry using NIH image software. The vibrissal follicle was divided according to the human HFs into five compartments 1) isthmus, (2) distal ORS=superior swelling of the ORS, (3) bulge= inferior swelling of the ORS, (4) proximal ORS, (5) hair matrix, and analyzed. deriving from 5-10 different mice. N=5, *p<0.05, **p<0.01, ***p<0.001, mean ± SEM, p value was calculated by Mann-Whitney-U-Test for unpaired samples.

Fig. 4.36 Schematic drawing of MHC class I IR pattern Schematic drawing of the MHC class I IR pattern in the murine vibrissal follicle. C: Capsule, CS: Cavernous sinus, CV: club vibrissae, DP: Dermal papilla, Epi: Epidermis, GM: glass membrane, HM: Hair matrix, HS: Hair shaft, IO: Inferior swelling of the ORS, IRS: Inner root sheath, ORS: Outer root sheat, RS: Ring sinus, RW: Ring wulst, SG: Sebaceous gland, SO: Superior swelling of the ORS. IR: Immunoreactivity.

RESULTS

161

4.3.2 Demonstration of MHC class II molecules

A monoclonal antibody specific against the murine Ia region of the H-2 complex,

corresponding to the human HLA-DR region, was used for the localization of

intraepithelial MHC class II+ antigen-presenting cells such as Langerhans cells and

macrophages. MHC class II expression was particularly dense in the epidermis, in

the infundibulum and upper parts of the isthmus (Figs 4.37, 4.38). The superior and,

in particular, the inferior swelling of the ORS exerted significantly reduced MHC class

II+ cells compared to upper parts of the HF, whereas lower parts revealed an even

decreased number of MHC class II+ cells towards the hair bulb. The bulb region

showed only rare MHC class II+ (data not shown) and only single MHC class+ cells

were found in the DP cells (Fig. 4.38 D).

Fig. 4.37 Schematic drawing of MHC class II IR pattern Schematic drawing of the MHC class II IR pattern in the murine vibrissal follicle. C: Capsule, CS: Cavernous sinus, CV: club vibrissae, DP: Dermal papilla, Epi: Epidermis, GM: glass membrane, HM: Hair matrix, HS: Hair shaft, IO: Inferior swelling of the ORS, IRS: Inner root sheath, ORS: Outer root sheat, RS: Ring sinus, RW: Ring wulst, SG: Sebaceous gland, SO: Superior swelling of the ORS. IR: Immunoreactivity:.

RESULTS

162

Fig. 4.38 MHC class II positive cells in the murine vibrissal follicle Immunohistochemical identification of MHC class II+ cells (brown) in normal murine snout skin (ABC method). MHC class II+ cells can be found in a relative high number in the ORS of upper parts of the vibrissal follicle and isthmus, whereas the inferior swelling of the ORS and lower parts of the follicle show only single or no positive cells in the ORS. (A) epidermis, isthmus, superior swelling and inferior swelling of the ORS; (B) lower parts of the vibrissal follilce: hair shaft and hair bulb; (b) superior swelling of the ORS. Bars: (A-B) 100µm, (b) original magnification 400x.

RESULTS

163

HS

4.3.3 Demonstration of CD4+ T cells

CD4+ T cells were predominantly expressed in upper parts of the vibrissal follicle, in

the isthmus, whereas in the superior swelling of the ORS and even more in parts of

the inferior swelling of the ORS, a significantly reduced number of this cell type was

found (Fig. 4.39). CD4+ T cells were also found in lower parts of the vibrissal follicle,

although most of them were localized in the sinus. The bulb region showed only

single CD4+ T cells (Figs 4.39, 4.41) and no CD4+ T cell could be detected in the

dermal papilla. Quantitative analysis corroborated the signigicant differences

between the compartments (Fig. 4.40).

Fig. 4.39 IR in the murine vibrissal follicle for CD4+ T cells Immunohistochemical identification of CD4+ T cells (brown) in normal murine snout skin (ABC method). CD4+ T cells can be found in high numbers in upper parts of the vibrissal follicle and isthmus, whereas the inferior swelling of the ORS and lower parts of the follicle show only rare CD4+ T cells. HS: Hair shaft, DP: Dermal papilla, SO: Superior swelling of the ORS. Bars: (A-E) 50µm.

RESULTS

164

Fig. 4.40 Quantitative immunohistochemistry for CD4 The immunostaining intensity for CD4 was analysed by quantitative immunohistochemistry using NIH image software. The vibrissal follicle was divided according to the human HFs into five compartments 1) isthmus, (2) distal ORS=superior swelling of the ORS, (3) bulge= inferior swelling of the ORS, (4) proximal ORS, (5) hair matrix, and analysed. deriving from 5-10 different mice. N=5, *p<0.05, **p<0.01, mean ± SEM, p value was calculated by Mann-Whitney-U-Test for unpaired samples.

Fig. 4.41 Schematic drawing of CD4 pattern Schematic drawing of the CD4 IR pattern in the murine vibrissal follicle. C: Capsule, CS: Cavernous sinus, CV: club vibrissae, DP: Dermal papilla, Epi: Epidermis, GM: glass membrane, HM: Hair matrix, HS: Hair shaft, IO: Inferior swelling of the ORS, IRS: Inner root sheath, ORS: Outer root sheat, RS: Ring sinus, RW: Ring wulst, SG: Sebaceous gland, SO: Superior swelling of the ORS. IR: Immunoreactivity.

Isthmus SO IO prox. ORS Hair bulb0

1020

25

30

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***

**

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mb

er

of

CD

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lls

RESULTS

165

4.3.4 Demonstration of CD11b molecules

In order to detect CD11b on myeloid cells, we used again the avidin-biotin-complex

staining method. CD11b+ cells were often visible particularly in the epidermis, the

infundibulum and upper parts of the isthmus (Fig. 4.42). The superior and inferior

swelling of the ORS, as well as lower parts

of the vibrissal follicle, demonstrated a

reduced number of CD11+ cells.

The bulb region and DP showed only

single CD11b+ cells (Figs. 4.42, 4.44).

Interestingly, many CD11b+ cells occurred

in the surroundings of the vibrissal capsule.

This was confirmed by quantitative analysis

(Fig. 4.43).

Fig. 4.42 IR in the murine vibrissal follicle for CD11b Immunohistochemical identification of CD11b cells (brown) in normal murine snout skin (ABC method). CD11b+ cells can be found in high numbers in upper parts of the vibrissal follicle and isthmus, whereas the inferior swelling of the ORS and lower parts of the follicle show only rare CD11b+ cells. DP: Dermal pailla, IO: Inferior swelling of the ORS, SO: Suprior swelling of the ORS, 100x maginification

RESULTS

166

Fig. 4.43 Quantitative immunohistochemistry for CD11b The immunostaining intensity for CD11b was analysed by quantitative immunohistochemistry using NIH image software. The vibrissal follicle was divided according to the human HFs into five compartments 1) isthmus, (2) distal ORS=superior swelling of the ORS, (3) bulge= inferior swelling of the ORS, (4) proximal ORS, (5) hair matrix, and analysed. deriving from 5-10 different mice. N=5, *p<0.05, **p<0.01, mean ± SEM, p value was calculated by Mann-Whitney-U-Test for unpaired samples

Fig. 4.44 Schematic drawing of CD11b IR pattern Schematic drawing of the CD11b IR pattern in the murine vibrissal follicle. C: Capsule, CS: Cavernous sinus, CV: club vibrissae, DP: Dermal papilla, Epi: Epidermis, GM: glass membrane, HM: Hair matrix, HS: Hair shaft, IO: Inferior swelling of the ORS, IRS: Inner root sheath, ORS: Outer root sheat, RS: Ring sinus, RW: Ring wulst, SG: Sebaceous gland, SO: Superior swelling of the ORS. IR: Immunoreactivity.

RESULTS

167

4.3.5 Demonstration of TGF-β1 molecules

A polyclonal antibody against TGF-β1 was used for the localization of precursor and

mature TGF-β1+ and TGF-β2+ cells. The most prominent expression of TGF-β was

epidermal (Fig. 4.45 A) and in the ORS of the infundibulum - showing both a basal

and suprabasal cytoplasmic staining. In the

superior swelling of the ORS

immunohistochemistry decreased and was

almost exclusively evident in the basal

layers of the ORS. No staining was detected

in the IRS (Fig. 4.45 B). In contrast, the

bulge region (namely the inferior swelling of

the ORS) exerts a highly increased

immunostaining intensity compared with the

SO parts (Fig. 4.45 C). Interestingly, the

ORS of the bulge region did not

demonstrate a uniform expression pattern,

but an increased IR in the basal layers with

a sparse distribution of more intense stained

TGF-β1+ keratinocytes. Staining intensity

was reduced in the lower vibrissal follicle

and in the hair bulb (Fig. 4.45 D).

Fig. 4.45 IR in the murine vibrissal follicle for TGF-β1 on cryosections Immunohistochemical identification of TGF-β1 (brown) in normal murine sinus follicle cryosections (ABC method). TGF-β1 IR can be observed in high numbers in upper parts of the vibrissal follicle and isthmus, whereas the inferior swelling of the ORS and lower parts of the follicle show only rare CD4+ T cells. E: HS: Hair shaft, DP: Dermal papilla, SO: Superior swelling of the ORS. Bars: (A-E) 50µm.

RESULTS

168

Fig. 4.46 IR in the murine vibrissal follicle for TGF-β1 on Bouin fixated specimens Immunohistochemical identification of TGF-β1 (brown) in Bouin fixated normal murine sinus follicle sections (ABC method). The ORS is stained homogenously, the IRS shows no Immunoreactivity, whereas cells of the sinus (A) and ring wulst (B) were also positive for TGF-β1 expression pattern. Bars: (A-E) 20µm.

Fig. 4.47 Schematic drawing of TGF-β1 pattern Schematic drawing of the TGF-β1 pattern in the murine vibrissal follicle. C: Capsule, CS: Cavernous sinus, CV: club vibrissae, DP: Dermal papilla, Epi: Epidermis, GM: glass membrane, HM: Hair matrix, HS: Hair shaft, IO: Inferior swelling of the ORS, IRS: Inner root sheath, ORS: Outer root sheat, RS: Ring sinus, RW: Ring wulst, SG: Sebaceous gland, SO: Superior swelling of the ORS. IR: Immunoreactivity.

RESULTS

169

4.3.6 Demonstration of mast cells

In order to detect mast cells, a toluidine blue staining was performed. We could

show, that the number of mast cells was especially high in the upper part of the

vibrissal follicle, i.e. the isthmus (Fig. 4.48) and decreased continuously towards the

lower part (Fig. 4.48 A,B). No significant difference in mast cell numbers could be

shown within the hourglass shaped parts and their surrounding tissue (Fig. 4.49).

Interestingly, in almost every vibrissal follicle, mast cells could be detected (Fig.

4.50).

Fig. 4.48 Demonstration of mast cells in the murine vibrissal follicle Immunohistochemical identification of mast cells (purple) in murine sinus follicle sections (Toluidine blue staining). The ORS is stained homogenously, the IRS shows no Immunoreactivity, whereas cells of the sinus (A) and ring wulst (B) were also positive for TGF-β1 expression pattern. Bars: (A-E) 20µm.

RESULTS

170

Fig. 4.49 Quantitative immunohistochemistry for mast cells Positively stained mast cells are counted by quantitative immunohistomorphometry. The vibrissal follicle was divided according to the human HFs into five compartments 1) isthmus, (2) distal ORS=superior swelling of the ORS, (3) bulge= inferior swelling of the ORS, (4) proximal ORS, (5) hair bulb, and analysed deriving from 5-10 different mice. N=5, *p<0.05, **p<0.01, mean ± SEM, p value was calculated by Mann-Whitney-U-Test for unpaired samples.

Fig. 4.50 Schematic drawing of mast cell expression pattern Schematic drawing of the mast cell expression pattern in the murine vibrissal follicle. C: Capsule, CS: Cavernous sinus, CV: club vibrissae, DP: Dermal papilla, Epi: Epidermis, GM: glass membrane, HM: Hair matrix, HS: Hair shaft, IO: Inferior swelling of the ORS, IRS: Inner root sheath, ORS: Outer root sheat, RS: Ring sinus, RW: Ring wulst, SG: Sebaceous gland, SO: Superior swelling of the ORS. IR: Immunoreactivity.

RESULTS

171

4.4 Immune privilege and the murine mouse nail apparatus

4.4.1 Demonstration of MHC class I molecules

Fig. 4.51 IR in the murine nail apparatus for MHC class I Expression pattern of MHC class I in normal murine nail apparatus (brown signal). The staining is very strong in the PNF and decreases towards the PNM. Bars: (A) 100µm, (B,C) 20µm.

By using avidin biotin-complex staining method with a monoclonal antibody against

H-2Dd, H-2b,p,q, we found that MHC class I IR in the murine nail apparatus changed

interindiviudally and also in regard on the fixation method applied (cryosections

versus Bouin-fixated tissue).

In most of the mice cryosections samples we found that the nail matrix epithelium,

and very prominently the PNM, displayed significantly downregulated MHC class I

Immunoreactivity (Fig. 4.51 A,C), compared with PNF (Fig. 4.51 B), nail bed, and

hyponychium (Fig. 4.51 A). This low or absent MHC class I expression in nail matrix

epithelium was mirrored in the immediately adjacent nail mesenchyme (Figs. 4.51-

4.53). The pad epithelium was only weak or not stained.

RESULTS

172

Fig. 4.52 Quantitative immunohistochemistry for MHC class I Positively stained MHC class I are counted by quantitative immunohistomorphometry. Analysis deriving from 5-10 different mice. N=5, *p<0.05, mean ± SEM, p value was calculated by Mann-Whitney-U-Test for unpaired samples.

Fig. 4.53 Schematic drawing of MHC class I expression pattern Schematic drawing of the MHC class I pattern in the murine nail apparatus. Hyp: Hyponychium, NB: Nail bed, NP: Nail plate, P: Pad, PNF: Proximal nail fold, PNM: Proximal nail matrix, PH: Phalanx, SW: Sweat gland, IR: Immunoreactivity.

RESULTS

173

4.4.2 Demonstration of MHC class II molecules

In order to detect MHC class II+ cells we used again avidin-biotin-complex method.

MHC class II+ cell were found in high numbers in the epidermis and in the vicinity of

the PNF (Fig. 4.54 A), whereas no MHC class II+ cells were detectable in the PNM

(*p<0.05) and in its mesenchymal vicinity (***p<0.001) (Fig. 4.54 B). The nail bed

only rarely showed MHC class II+ cells. In the foot pad epithelium, the greatest

number of intraepithelial MHC class II+ cells could be demonstrated (Fig. 4.54 D),

whereas the dermis showed a reduced number compared to the PNF and PNM

dermis (Fig. 4.56). This was confirmed by quantitative analysis using NIH imaging

(Fig. 4.55).

Fig. 4.54 IR in the murine nail apparatus for MHC class II Expression pattern of MHC class II in normal murine nail apparatus (brown signal). (A) MHC class II+ cells can be found in high numbers in the epidermis and in the vicinity of the PNF, whereas the NB shows only single (B) and the PNM show no MHC class II+ cells (C). In pad epithelium most of the intraepithelial MHC class II+ cells can be demonstrated. Bars: (A-D) 200µm.

RESULTS

174

Fig. 4.55 Quantitative immunohistochemistry for MHC class II Positively stained MHC class II cells are counted by quantitative immunohistomorphometry. Analysis deriving from 5-10 different mice. N=5, *p<0.05, mean ± SEM, p value was calculated by Mann-Whitney-U-Test for unpaired samples.

Fig. 4.56 Schematic drawing of MHC class II expression pattern Schematic drawing of the MHC class II pattern in the murine nail apparatus. Hyp: Hyponychium, NB: Nail bed, NP: Nail plate, P: Pad, PNF: Proximal nail fold, PNM: Proximal nail matrix, PH: Phalanx, SW: Sweat gland, IR: Immunoreactivity.

RESULTS

175

4.4.3 Demonstration of CD4+ T cells

CD4+ T cell localization was identified by avidin-biotin-complex staining. We could

show, that the number of CD4+ T cells was especially high in the vicinity of the PNF

(Fig. 4.57 A) and decreased continuously towards the PNM (Fig. 4.57 B).

Intraepithelial CD4+ T cells were only found in the PNF and foot pad, but not in the

PNM; quantification was not done (Fig. 4.58).

Fig. 4.57 IR in the murine nail apparatus for CD4+ T cells Expression pattern of CD4 in normal human scalp skin (brown). NP: Nail plate, PNF: Proximal nail fold, PNM: Proximal nail matrix, Bars: (A,B) 100µm.

Fig. 4.58 Schematic drawing of CD4 expression pattern Schematic drawing of the CD4 expression pattern in the murine nail apparatus. Hyp: Hyponychium, NB: Nail bed, NP: Nail plate, P: Pad, PNF: Proximal nail fold, PNM: Proximal nail matrix, PH: Phalanx, SW: Sweat gland, IR: Immunoreactivity.

RESULTS

176

4.4.4 Demonstration of TGF-β1 molecules

A polyclonal antibody against TGF-β1 was used for the localization of precursor and

mature TGF-β1+ and TGF-β2+ cells. The most prominent expression of TGF-β was

visible in the epidermal basal layer (Fig. 4.59 A) and in the ORS of HFs (Fig. 4.59

B). In addition, other epithelial parts also showed homogenously TGF-β1 IR like the

suprabasal layers of the epidermis, the epidermis in the foot pad and the PNM.

Moreover, we found IR, although with less intensity, in sweat glands and single other

dermal cells.

Fig. 4.59 IR in the murine nail apparatus for TGF-β1 Expression pattern of TGF-β1 in normal murine nail apparatus (brown). (A) The epidermal basal layers (A) and

Outer root sheath of the HFs are intensely stained for TGF-β1. HF: Hair follicle, NP: Nail plate, PNF: Proximal nail fold, PNM: Proximal nail matrix, Bars: (A-D) 200µm.

Fig. 4.60 Schematic drawing of TGF-β1 expression pattern Schematic drawing of the TGF-β1 expression pattern in the murine nail apparatus. Hyp: Hyponychium, NB: Nail bed, NP: Nail plate, P: Pad, PNF: Proximal nail fold, PNM: Proximal nail matrix, PH: Phalanx, SW: Sweat gland, IR: Immunoreactivity.

RESULTS

177

4.4.5 Demonstration of CD54 molecules

By using avidin-biotin-complex staining, we observed that cells positive for

intracellular adhesion molecule 1 (ICAM-1) were limited to the mesenchyme (Fig.

4.61 A), except for single positive ICAM-1+ cells in the hair follicle ORS (data not

shown). Most of the ICAM-1 positive cells could easily be detected in endothelial

cells (Fig. 4.61 B) of small blood vessels near the epidermis of the PNF, whereas the

number of positive cells was slightly reduced in the vicinity of the PNM and the foot

pad. The nail bed showed only rarely contained ICAM-1+ cells (Figs. 4.61, 4.62).

Fig. 4.61 IR in the murine nail apparatus for CD54 Immunohistological identification of CD54 expression (brown). NP: Nail plate, PNF: Proximal nail fold, PNM: Proximal nail matrix, Bars: (A) 100µm, (B) 20µm.

Fig. 4.62 Schematic drawing of CD54 expression pattern Schematic drawing of the CD54 expression pattern in the murine nail apparatus. Hyp: Hyponychium, NB: Nail bed, NP: Nail plate, P: Pad, PNF: Proximal nail fold, PNM: Proximal nail matrix, PH: Phalanx, SW: Sweat gland, IR: Immunoreactivity.

RESULTS

178

4.4.6 Demonstration of mast cells

In order to detect mast cells, again, toluidine blue staining was performed. We found

no significant differences of mast cell numbers in the vicinity of the PNM, compared

to the PNF (Fig. 4.63 A) or the foot pad. This was confirmed by quantitative

immunohistochemistry. Interestingly, the nail bed displayed a significantly reduced

number of mast cells than the PNF (*p<0.05) (Fig.4.64).

Fig. 4.63 IR in the murine nail apparatus for mast cells Immunohistochemical identification of mast cells in normal murine nail apparatus (purple). (A) Mast cells are uniformly distributed in the vicinity of PNF and PNM and foot pad. NP: Nail plate, PNF: Proximal nail fold, PNM: Proximal nail matrix, Bars: (A) 100µm, (B) 20µm.

NP

PNM

PNF

A

B

RESULTS

179

Fig. 4.64 Quantitative immunohistochemistry for MHC class I Positively stained mast cells are counted by quantitative immunohistomorphometry. Therefore, the murine nail is divided into the proximal nail matrix, proximal nail fold, nail bed and pad and mast cells (toluidine blue staining) are counted at 400 times magnification per macroscopic field from one nail (N=5-7 individuals). Analysis deriving from 5-10 different mice. N=5, *p<0.05, mean ± SEM, p value was calculated by Mann-Whitney-U-Test for unpaired samples.

Fig. 4.65 Schematic drawing of mast cell expression pattern Schematic drawing of the mast cell expression pattern in the murine nail apparatus. Hyp: Hyponychium, NB: Nail bed, NP: Nail plate, P: Pad, PNF: Proximal nail fold, PNM: Proximal nail matrix, PH: Phalanx, SW: Sweat gland, IR: Immunoreactivity.

RESULTS

180

4.4.7 Demonstration of β-defensin 2

We investigated the local expression of precursor and mature β-defensin 2 producing

cells in the murine nail apparatus. A strong up-regulation of staining intensity

appeared in parts of the

PNM close to the nail plate

compared to the PNF and

parts of the foot pad (Fig.

4.66). The staining was

cytoplasmic and a descreet

IR could also be shown in

the epidermal basal layers of

the PNF and pad (Figs. 4.66,

4.67).

Fig. 4.66 IR in the murine nail apparatus for β-defensin 2 Immunohistochemical identification of β-defensin 2 in normal murine nail apparatus (brown). (A) Staining intensitiy is in the PNF reduced compared to dorsal parts of the PNM (B). NP: Nail plate, PNF: Proximal nail fold, PNM: Proximal nail matrix, Bars: (A,B) 100µm.

Fig. 4.67 Schematic drawing of β-defensin 2 expression pattern Schematic drawing of β-defensin 2 expression pattern in the murine nail apparatus. Hyp: Hyponychium, NB: Nail bed, NP: Nail plate, P: Pad, PNF: Proximal nail fold, PNM: Proximal nail matrix, PH: Phalanx, SW: Sweat gland, IR: Immunoreactivity.

DISCUSSION

181

5 Discussion

5.1 Introductory remarks

Recent gene profiling data in mice have raised the possibility, that follicular IP is not

purely limited to the anagen hair bulb, but that the epithelial stem cell region in the

ORS of HFs, termed the bulge, may also represent an area of relative IP. Since

protection of these epithelial stem cells from immune destruction is essential for

preserving the regenerative and cycling capacity of the follicle, it seems logical that

the bulge region should also have an established relative IP. However, convincing

protein evidence for this idea is still missing. In the current study, we aimed to

support or refute the hypothesis of bulge IP in human HFs.

By using highly sensitive immunohistochemistry, including Immunofluorescence, we

provide the first comprehensive in situ-protein expression evidence, which follows-up

leads from gene profiling data that suggests, contrary to previous assumptions, that

the HF bulge fully meets classical criteria for immune privileged tissues: MHC class

Ia, β2-microglobulin and MHC class II immunoreactivity (IR) were found to be down-

regulated in the human bulge. The immuno supressants α-melanocyte stimulating

hormone (α-MSH), transforming growth factor-β2 (TGF-β2), macrophage migration

inhibitory factor (MIF) and indoleamine-2,3-dioxygenase (IDO) are upregulated in the

CD200+, stem cell-rich bulge region. In addition, these CD200+ cells also co-express

HLA-E.

Our systematic bulge immuno phenotyping experiments were complemented with a

functional study, which should determine, whether IFN-γ, a recognized key inducer of

IP collapse in human anagen hair bulbs, had a similar effect on the putative bulge IP.

We showed a significant ectopic upregulation of MHC class Ia in bulge cells of organ-

cultured human scalp skin. By this skin organ culture assay other IP markers can be

evaluated in future experiments, particularly substances to restore IP after IFN-γ

induced collapse.

DISCUSSION

182

In the current study, the effects of the α-MSH related tripeptide K(D)PT were

explored with our organ-culture HF model, and it was demonstrated that it failed to

act as an agent to protect IP or to re-establish IP. Nevertheless, it revealed many

other interesting effects on HF biology (e.g. melanogenesis, mast cells, hair cycle),

suggesting further investigation.

Moreover, we contributed substantially to the immunological characterization of the

murine vibrissal follicle and murine nail apparatus and showed that the vibrissal bulb,

and in a less extent the bulge and proximal nail matrix, can be considered to be also

immunoprivileged to a certain. However, many gaps in the immunobiology of the

murine vibrissal follicle and nail apparatus remain to be closed in future studies.

5.2 Methods employed

Tissue specimens:

In the current study, normal human fronto-temporal and occipital uninflamed scalp

skin was obtained from females only, undergoing routine face-lift surgery after

informed consent. By choosing samples from the ´female` sex only, the influence of

hormonal differences was prevented. The patients were supposed to have no pre-

treatment and did not take any drugs to exclude any non-biological influences,

although a guarantee did not exist. The donors were selected randomly (aged 45-67

years, mean age 52.5 years) and all experiments were performed according to the

Helsinki guidelines.

For the in situ protein immunohistochemistry of the bulge, scalp skin of 16 patients

was investigated, which was obtained from both regions of the skin (occipital and

fronto-temporal). However, no region-related or age-dependent differences in the IR

pattern of the markers were obvious. Per antigen, 3-10 cryosections per patient of at

least 5 different individuals were examined. This representative amount of patients

substantially limited the possibility of inter-individual differences, which always also

occur within a population. For the isolated HF organ culture, only occipital human

DISCUSSION

183

scalp skin from six donors (age range: 46-65 years) was used. Per experiment the

isolated HFs from one donor were randomly and uniformly distributed into the six

different test- and control groups, so that interindividual differences between the test

groups could be excluded. Despite this aspect, we found interindividual differences

when comparing the experiments. Per test group normally 18 HFs were cultured,

taking into account losses associated with the treatment or with the cutting, so that a

statistical evaluation was possible. Two experiments started with a number of 12 HFs

per group, due to a limited availability of HFs in the sample. For the full-thickness

skin organ culture, scalp skin of different regions from two patients was cultivated

(age 53 and 63 years). Again, no age- or patient-related difference, as well as no

region-related difference in the IR pattern of the staining could be observed.

Important for this culture was the appropriate size of the skin sample, i.e. in case that

the size was too large, apoptosis in the skin specimens occurred, because the

medium could not penetrate into the centre of a sample.

In the cooperating hospitals, scalp specimens were placed immediately after surgery

into supplemented William’s E serum-free medium, and transported overnight to the

laboratory at a temperature of 4°C. One critical point of specimen collection was the

time period between tissue removal and its immersion into tempered William’s E

medium. The optimal procedure is to place the sample into the medium immediately

after removal, but since the hospitals were widely distributed over Germany, it was

not possible to take the samples oneself. A second critical point was to keep the

specimen refrigerated at a temperature of 4°C during the transport overnight (only

one hospital in Lübeck delivered scalp skin on the same day of surgery). High

outdoor temperatures might have influenced tissue quality, although the samples

were transported on ice packs, it could not be checked whether the material was sent

well tempered during transport. After arriving in our laboratory, nevertheless, the skin

was immediately processed for the in situ immunohistochemistry experiments, for HF

organ culture or for full-thickness skin culture. Despite all critical aspects that could

have affected tissue quality, we ensured that no negative influence on tissue and

reaction quality was observable light microscopically during the course of our study.

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184

Mouse tissue samples were taken from 18 control C57/BL6 and Balb/c mice from

other cooperating working groups on the campus. Specimens were collected

immediately after cervical dislocation to minimize autolysis. For obtaining a

representative result, 3-10 cryosections per patient of at least 5 different individuals

were examined per antigen. Although we collected samples of different mouse

strains (C57BL6 or Balb/c), which had a different immunological background and

susceptibility for autoimmune diseases, we could not detect any difference in the

pattern of the IR staining. The same observation was made for possible age-related

differences or anatomical differences between the fore- and the hind limb.

Fixation:

The routine fixation method was to immerse the sample into Shandon Cryomatrix,

followed by snap-freezing in liquid nitrogen and storing at -80°C until use. In contrast

to fixation in Bouin’s solution, this method is supposed to be less efficient. Bouin’s

fixation has the advantage that the fluid penetrates faster into the tissue and

therefore produces good protein conservation and tissue morphology excluding also

shrinkage artefacts. Most experiments were based on cryosections, because the

experimental laboratory was specialized in such methodical approach, including the

fact that there was much experience available for the use of anti-mouse antibodies

on cryosections. Furthermore it is important to note, that the freezing technique had

the advantage in the Cryomatrix embedding step compared to Bouin fixation with

paraffin embedding: All specimens could be accurately placed in an optimal angle

into the cups, so that longitudinal or sagittal cuts became possible. Furthermore, the

possibility of adjusting the cutting level (angle) facilitates the production of

longitudinal sections. For future research it should be considered to use the more

rapid freezing technique at lower temperatures.

Performing cryosections:

DISCUSSION

185

We took the rather common way of processing cutaneous tissue and carried out

vertical cuts, showing the epidermis at one pole and the hypodermis at the other. The

aim was to obtain as many longitudinal scalp HF and vibrissal sections, respectively

sagittal sections of the murine nail apparatus, as possible. Since the follicles do not

grow in the same angle or parallel, it was difficult to receive longitudinal sections from

the bulb to the epidermis. Cutting the organ cultured punch biopsies was particularly

difficult, since they were of such a small size (4 mm diameter) and often contained

two or three HFs only. If a section on a the isthmus level was complete in one HF,

and another HF of the follicular unit next to it showed a complete lower HF, the skin

sample was taken into evaluation. An additional aspect which had to be taken into

account, was the fact that IR of several antigens were shown to be hair cycle

dependent. Since IP in the bulge or hair bulb is only present in anagen VI, it was

important to ensure, that only HFs and vibrissal follicles in anagen were evaluated.

Therefore, the best way to differentiate was to investigate complete longitudinal

follicles, or, at least, to obtain the lower HF to examine the shape and structure of the

hair bulb and DP (STENN and PAUS 2001; WHITING 2004) to exclude HFs of a

different cycling stage from evaluation.

Despite the fact, that it was not sure at the beginning of the experiments, whether the

bone tissue of the phalanges could be cut with a normal cryostat blade, we learned

that it was possible to obtain sections of good quality without decalcification and with

an optimal cutting technique. Sometimes the bone structure was slightly disrupted,

but this fact did not affect the staining quality or evaluation. In contrast, in Bouin-

fixated samples a step of decalcification with 25% EDTA was included.

Immunohistochemistry and evaluation:

In this work not only standard immunohistochemistry, including Immunofluorescence

was employed, but also highly sensitive visualization methods (EnVision®, TSA

immunofluorescence) (see e.g. ROTH et al. 1999; ITO et al. 2004b, 2005c; BODO et

al. 2005; GAISER and BERNHARDS 2007). Since previous studies had shown

contrary results compared to the recent gene profiling data, these special sensitive

DISCUSSION

186

methods should provide more reliable results. Regarding the nail apparatus, we

observed that ABC methods based on alkaline phosphatases cross-reacted

unspecifically with the bone tissue. For this reason, all stainings based on avidin-

biotin-complex method were performed with horseradish peroxidase. In general, it

was also found, that the substrate AEC could not be reliably used on murine tissue.

Sometimes no staining was seen, even though DAB provided good results. Whether

this observation is due to the staining kit (although different ones were used) or to the

special tissue, could not be clarified. In addition, Masson Goldner staining could not

be performed on cryosections, due to the fixation method, which leads to a reduced

staining intensity of epithelial tissue. As a consequence most overview stainings were

performed with HE.

Furthermore, MHC class Ia staining on murine nail apparatus specimens revealed

contradictory results, although positive controls worked. On cryosections, sometimes

parts of the epidermis in the PNF were stained, sometimes not. The PNM has not

produced any positive staining and the epithelium of the foot pad was often negative

for MHC class I IR, although dermal cells were stained. In contrast, Bouin-fixed tissue

showed a homogenous staining all over the epithelium (including the PNM, PNF and

foot pad). However, region-related, age-or sex- or breed-related differences in the IR

pattern of the staining could not be detected.

5.3 Immune privilege in the human hair follicle bulge, murine nail

and sinus hair follicle

5.3.1 The human HF bulge

The aim of the current study was to show by immunohistochemistry and organ

culture, whether the human anagen hair bulb fully meets the classical criteria for

immune privileged tissues, as recently shown in gene profiling data (MORRIS et al.

DISCUSSION

187

2004; TUMBAR et al. 2004; OHYAMA et al. 2006), or whether previous contradictory

assumptions on the protein level are correct.

Since downregulation of MHC class Ia IR is one hallmark of IP, it was very important

to verify its exact and distinctive expression pattern in the human HF. HARRIST et al.

(1983) published the first MHC class Ia antigen expression data in normal terminal

human HFs, reporting prominent IR in the infundibular region of the ORS with

diminished expression of HLA-A, B, C and β2-microglobulin in the isthmus region,

whereas the bulge was not referred to. Later CHRISTOPH et al. (2000)

demonstrated strong HLA-A/B/C IR and β2-microglobulin IR in the ORS isthmus

region of the ORS, though possibly at a somewhat reduced level of IR compared to

the distal ORS (CHRISTOPH et al. 2000). They concluded, that the anagen hair bulb

was the only site of IP within the HF (PAUS et al. 2003), which became generally

accepted. However, with the recent gene and profiling data, this theory had to be

abandoned. We demonstrated a convincing and significant downregulation of MHC

class Ia and β2-microglobulin in the bulge region, compared to the isthmus and

epidermis (Figs. 4.1, 4.6), by using high sensitive immunohistochemistry. Intriguingly,

we could also present a difference in MHC class Ia IR between the bulge and the

bulb.

There are several aspects which might explain why a bulge IP had not been

suggested before: 1) At that time, object of interest was to investigate the

pathogenesis of AA, with the IP collapse model in the pathogenesis of AA (PAUS et

al. 2003, 2005), other HF disorders were not discussed. 2) The bulge was ignored as

a prominent and important structure or as a reservoir of eSCs in the HF. Originally, it

was thought that stem cells mainly reside in the hair bulb and have to be protected

(PAUS et al. 1999b; PAUS and COTSARELIS 1999). It was not considered that an

attack of the bulge eSCs could cause irreversible HF damage. 3) The bulge is not

easily detected as a part of the HF, so that detailed analyses remained inconclusive.

Since the characterization of the bulge by KLOEPPER et al. (2008), with the finding

DISCUSSION

188

of the leading structure ´follicular trochanter` and the insertion point of the APM, this

structure became more distinctive.

In addition, we confirmed a significant downregulation of antigen-presenting functions

of intraepithelial dendritic cells (seen as downregulation of MHC class II expression)

(Figs. 4.9-4.11). These finding was expected, since CHRISTOPH et al. (2000) had

already shown significantly reduced numbers of MHC class II+ cells in the isthmus

and proximal ORS compared with the distal ORS, although again, no detailed

analysis of the bulge had been carried out. It is important to note, that this

downregulation much more severely hampered antigen presentation in the bulb

compared to the bulge, as also true for the MHC class II-dependent antigen. Here,

very few CD1a positive or ultrastructurally detectable Langerhans cells were clearly

MHC class II-negative, while the number of dendritic cells in the bulge region still

displayed some MHC class II expression, albeit the fact that their number was

substantially reduced.

We could detect an upregulation of non-classical MHC class Ib molecules, which has

previously been shown to be important in the IP maintenance of other systems, like

the fetotrophoblast (MOSCOSO et al. 2006; YIP et al. 2006), the eye (NIEDERKORN

et al. 1999; LE DISCORDE et al. 2003), and the proximal nail matrix (ITO et al.

2005c). We focused on HLA-E, since its expression in human skin has not been

extensively studied until now. In addition, its effect is more directly than, for example,

the one of HLA-G. HLA-E is a ligand of the NK cell inhibitory receptor CD94/NKG2A

and may result in inhibition of NK cell- and cytotoxic T cell-dependent lysis (GAO et

al. 1997; BRAUD et al. 1998; PACASOVA et al. 1999; TRIPATHI et al. 2006),

although HLA-E is also regarded to interact with CD8+ T cells directly (GAO et al.

1997). In our study, we observed a mesenchymal-facing intracellular location of HLA-

E protein in CD200+ keratinocytes within the bulge (Figs. 4.12., 4.13), which

corresponds to a previous report of HLA-E sequestration to the endoplasmic

reticulum in other tissues (ULBRECHT et al. 1992), and suggests that HLA-E is

stored in the bulge ORS, ready to be transported to the cell surface upon recruitment.

DISCUSSION

189

The role of the immunosuppressants has long been underestimated, although recent

studies showed that α-MSH suppresses experimental autoimmune

encephalomyelitis, and that α-MSH and TGF-β can promote re-establishment of

immune tolerance to autoantigens in the brain (TAYLOR and KITAICHI 2007) and

the human anagen hair bulb (PAUS et al. 2003; PAUS et al. 2005; NIEDERKORN

2006). TGF-β1 for example, is prominently expressed in the HF epithelium of mice

and humans, and shows its highest IR during late anagen and the onset of catagen

in cells of the ORS and epithelial strand (FOITZIK et al. 2000).

Our finding of the upregulation of the potent, locally generated “IP guardian” α-MSH

in the bulge (Fig. 4.14), along with strong IR of the immunosuppressant TGF-β2 (Fig.

4.15), probably contributes to the maintenance of bulge IP in a manner similar to that

recognized in the eye, brain and fetal IP (WAHL et al. 2006; SIGLIENTI et al. 2007).

TGF-β is thought to inhibit NK cell-mediated cytoloysis, contributing to the

mechanism of IDO (APTE and NIEDERKORN 1996; APTE 1997; APTE et al. 1998;

NIEDERKORN 2003, 2006), and influences the behavior of APCs in promoting

differentiation of Ag-specific T cells into CD4þCD25þFoxp3þ regulatory T cells

(Tregs) (D`ORAZIO and NIEDERKORN 1998; TAKEUCHI et al. 1998; KEZUKA and

STREILEIN 2000; STREILEIN et al. 2002).

We also present the first in situ immunohistochemical evidence of IDO expression in

human HFs and demonstrated an upregulation of IDO in the bulge region (Figs.

4.20-4.22), that underscores previous observations made from other

immunoprivileged sites [(e.g. lung or allografts (SWANSON et al. 2004; LI et al.

2006); skin (VON BUBNOFF et al. 2004), anterior chamber of the eye (CHEN et al.

2007) and fetus (MELLOR and MUNN 2008)]. IDO is the rate limiting tryptophan-

catabolizing enzyme, which limits T cell proliferation by creating a tryptophan-

depleted environment, and also affects NK cell function, reduces inflammation and

enhances Fas-mediated T cell apoptosis through their tryptophan degradation

products (MUNN et al. 1998; FALLARINO et al. 2002; MELLOR and MUNN 2008).

Even though T cells (CD4+ or CD8+ cells) are predominantly distributed in the distal

HF epithelium, a small number of T cells and NK cells exists in and around the

DISCUSSION

190

human HF bulge (CHRISTOPH et al. 2000; ITO et al. 2007). In addition, since every

CD8+ and NK cell could endanger or attack the ORS bulge cells upon their MHC

class Ia negativity, IDO might be vital in preserving HF immune integrity and keeping

these cells inactivated.

In addition, in the light of the presented upregulation of MIF in the human HF bulge,

which is expressed in several IP sites such as the fetotrophoblast (VIGANO et al.

2007), eye (APTE et al. 1998), brain, human proximal nail matrix (ITO et al. 2005c),

and human anagen scalp HFs (ITO et al. 2007), the bulge IP concepts also gains

importance. We confirmed the previously reported MIF IR pattern in human

epidermis (SHIMIZU et al. 1996; ITO et al. 2007), and were able to show an

increased MIF IR in the bulge region, proximal of the ORS and IRS of human HFs

(Figs. 4.18, 4.19). MIF may inhibit NK-cell mediated cytotoxicity against MHC class I-

negative cells by preventing the release of cytolytic perforin granules from NK cells

(APTE et al. 1998; ARCURI et al. 2006). This would explain why immune-privileged

tissues, that are highly vulnerable to lysis by NK cells due to their lack of MHC class I

expression, are not under constant NK cell attack. By comparative studies of human

scalp skin from healthy donors and patients with AA, it has been already shown that

the epithelium of lesional AA HFs displayed a greatly reduced or absent MIF IR. This

observation suggests that HFs in established AA have a decreased capacity for

suppressing undesired NK cell functions.

We could confirm the previously demonstrated strong upregulation of the ´no

danger`-signal CD200 (ROSENBLUM et al. 2004; OHYAMA et al. 2006; KLOEPPER

et al. 2008), that inhibits pro-inflammatory cytokine release of CD200 receptor+ mast

cells, downregulates APC activity (ROSENBLUM et al. 2006), and is thought to

influence IDO production (FALLARINO et al. 2002). As recently reported (OHYAMA

et al. 2006; KLOEPPER et al. 2008), CD200 is present in the outermost ORS layer,

between the insertion of the APM and the insertion of the SG duct [note that this

region is classically defined as isthmus (WHITING 2004), while only its proximal end

includes the bulge region]. This cell surface protein is upregulated in the human

DISCUSSION

191

bulge, although not restricted to it, and therefore does not qualify as a specific human

epithelial HF stem cell marker (KLOEPPER et al. 2008). But for our purpose the

expression pattern was distinct enough, and because of its immunomodulatory

functions contributing to IP, we used CD200 for double Immunofluorescence

stainings with MHC class I and HLA-E to confirm the IR of both antigens within the

bulge.

In order to obtain functional evidence, some experiments were run with our

established full-thickness human skin culture. It was shown that the putative bulge IP

is subject to the same IP collapse-inducing pro-inflammatory stimulus (IFN-γ) (Fig.

4.25) as the bulb IP in mice and humans (RUECKERT et al. 1998; ITO et al. 2004),

providing the strongest evidence available so far, that the human bulge also

represents an area of relative IP. Interestingly, more than ten times higher doses of

IFN-γ were required to induce a statistically significant collapse in the bulge than in

the bulb (1000 IU/ml vs. 75 IU/ml). This could be a result of a different experimental

design: ITO et al. (2005) used the isolated HF organ culture and 75 IU/ml IFN-γ to

induce an ectopic upregulation of MHC class Ia IR in hair matrix keratinocytes,

whereas higher doses like 500 or 1000 IU/ml IFN-γ induced catagen transformation

in the HFs. In contrast, to explore the bulge region regarding its effects on IFN-γ, it

was necessary to use the full thickness organ culture model (LU et al. 2007) (for the

isolation of HFs, the epidermis and bulge region is cut off). Since the specimens were

much larger than isolated HFs, including a longer penetration distance, it could be

expected that much higher doses of agent had to be added.

However, our immunohistochemical study also suggests that the bulge and bulb IP in

human scalp HFs differ in several important aspects: The level of constitutive MHC

class Ia, as associated with β2-microglobulin and MHC class II protein expression in

the bulge, is much higher than in the bulb (Figs. 4.3, 4.8, 4.10). Since β2-

microglobulin protein expression is virtually absent in the human anagen bulb

(CHRISTOPH et al. 2000; ITO et al. 2004), but not in the bulge, in theory, leaving

unclear how the rest of HLA-A/B/C molecules can be stabilized here (and thus

DISCUSSION

192

rendered fully functional by association with β2-microglobulin). This suggests that the

presentation of MHC class I-dependent autoantigens, virally encoded or tumor

antigens, is still possible in the bulge region of the ORS (albeit its significant

reduction compared to other ORS regions), while this presentation is almost

impossible under physiological conditions in the anagen bulb. Whether such

observation indicates a more labile relative IP in the bulge than in the bulb or not,

cannot be assessed easily. The state of IP is a complex of different mechanisms

(e.g. IDO, TGF-β or MIF), which are upregulated in the bulge and might reduce the

risk of IP collapse. Furthermore, for such an important site as the bulge with its eSCs,

it might be necessary to have a capacity of defense via the immune system in case

of inflammation.

Taken together, even though this concept of bulge IP remains to be supported by

additional functional evidence, the in situ protein expression data presented here

emphasize that the bulge in fact has a strong - and perhaps stronger - immune

protection than that of the anagen bulb. IDO and MIF showed higher IR levels around

the bulge compared to the proximal follicle, and CD200 and HLA-E were solely

expressed in the bulge, having both marked immunomodulatory functions. These are

likely to be the key aspect in protecting the bulge and the stem cells from immune

attacks, not only in anagen but throughout the complete hair cycle. Supportive

evidence for this finding is provided by ROSENBLUM et al. (2006), who showed that

CD200-/-skin grafts quickly develop permanent alopecia when grafted onto wild type

mice.

On the other hand, MHC I, MHC II and β2-microglobulin IR levels were not as low as

those in the hair bulb, which may indicate that the bulge IP is constitutively more

prone to inciting an immune response than the bulb, resulting in an inflammatory cell

attack on bulge epithelial stem cells (COTSARELIS and MILLAR 2001). The latter

aspect is the most characteristic histological feature of cicatricial alopecia. However,

from a clinical point of view, alopecia areata (AA) (the prototypical bulb IP collapse

model) is far more common than any of the primary cicatricial alopecias (PCA). It

seems logical that, if PCA were the ´clinically visible consequence of bulge IP failure`

DISCUSSION

193

and the bulge more susceptible to IP collapse, we should see far more cases of PCA

than actually done. Furthermore, the required IFN-γ dose to induce IP collapse within

the organ culture was much higher than in the bulb (ITO et al. 2004), suggesting that

the bulge IP is less susceptible to IP collapse. This theory is corroborated by

evidence from molecular studies that show a down-regulation of INF-γ receptors in

the bulge (MORRIS et al. 2004).

5.3.2 The murine sinus hair follicle and nail apparatus

In order to immunologically characterize murine vibrissal follicles and nail apparatus,

and to get a basic understanding if parts of these tissues also might have the benefit

of relative IP, we focused on a small amount of IP marker and some general

immunological parameters. Although MHC class Ib, MIF, IDO and CD200 are

important actors in IP, we waived investigating these antigen IR on murine tissue due

to temporary and economical reasons. In addition, in the case of MIF and IDO for

example, cross reaction of the anti-human Abs with the mouse tissue impeded its

usage. Moreover, concerning the murine specimens, we focused on MHC class Ia

expression only and did not analyze the β2-microglobulin expression pattern,

because it was likely to parallel the findings for MHC class Ia, with which it is

associated. Inspection of human HFs was more important, since a lot of data on this

matter have been published before (HARRIST et al. 1983; CHRISTOPH et al. 2000)

and recent microarray data have claimed a β2-downregulation (MORRIS et al. 2004;

TUMBAR et al. 2004).

Since MHC class Ia IR is one of the striking features of IP (JANEWAY et al. 2005;

NIEDERKORN and WANG 2005; PAUS et al. 2005), we analyzed the MHC class Ia

expression pattern. Staining for MHC class I showed homogenous IR in the

epidermis and in the distal ORS of the isthmus and infundibulum (Figs. 4.35, 4.37).

At the level of superior swelling, IR started to decrease (Fig. 4.35 B) and was even

more and significantly reduced in the bulge region or the inferior swelling of the ORS

DISCUSSION

194

(Fig. 4.34 C). Interestingly, in these hourglass shaped parts, the basal layers of the

ORS were almost devoid of IR. And almost no staining intensity was found in the

proximal ORS or the follicle bulb (Fig. 4.34 E). Thus, in the vibrissal follicle the

expression pattern was similar to the human HF, apart from structural differences.

The negativity in the basal layers of the bulge is different from the human HF and

could be important to reduce immune regulation, because the basal layers are facing

to the blood sinus and environment. But since a residual level of MHC class Ia+ cells

is still presented in the other layers of the ORS, they could be activated in case of

need. Otherwise, MHC class I negativity may sequester (auto) antigens from

presentation to CD8+ T cells and NK cells.

We also demonstrated that the nail matrix epithelium, and most prominently the

PNM, displayed substantially downregulated MHC class I IR, compared with PNF,

nail bed, and hyponychium (Fig. 4.51), as it was also shown for the human nail

apparatus (ITO et al. 2005c). This low or absent MHC class I expression in nail

matrix epithelium was mirrored in the immediately adjacent nail mesenchyme (Fig.

5.52). The pad epithelium was only weak or not stained. The absence of MHC class I

in the PNM compared to the PNF, suggested the PNM to be also immunoprivileged.

On the one hand, it is not fully comprehensible, why the epithelium of the foot pad

also displayed a reduced IR. One possible explanation could be that the foot pad

epithelium is the part, on which the mice weigh themselves down, thus, the stratum

corneum is more distinctive and acts as the main barrier against pathogens, water,

and pressure. On the other hand, another explanation could be that our staining

problems influenced the result. As mentioned before, we obtained varying results,

although the positive controls (or the vibrissal follicle) worked. On cryosections, parts

of the epidermis in the PNF sometimes were stained and sometimes not. The PNM

has never demonstrated positive staining, and the epithelium of the foot pad was

often negative for MHC class I IR, although fibroblasts / fibrocytes in the dermis were

stained. In contrast, Bouin-fixed tissue showed a homogenously staining of the entire

epithelium (including the PNM, PNF and foot pad). It would be easy to suggest that

staining of the cryosections did not work at all. But the fact that we obtained

convincing staining results in normal murine back skin and snout / vibrissal follicles

DISCUSSION

195

contradicts this hypothesis. Because of the lack of data in the literature and despite

the exchange of information with international experts in this field, it was difficult to

decide which IR pattern was reliable. Given that the PNM is indeed negative for MHC

class Ia IR, it could be possible that the PNM is another site of IP.

In accordance with the findings in the human bulge (MEYER et al. 2008a), we could

demonstrate a significant downregulation of antigen-presenting functions of

intraepithelial dendritic cells (seen as downregulation of MHC class II expression) in

the vibrissal bulb, bulge and PNM. These findings are in line with the theory of IP

analogue to the HF (MEYER et al. 2008a) or human nail apparatus (ITO et al.

2005c). The MHC class II expression was particularly dense in the epidermis, in the

infundibulum and upper parts of the isthmus (Fig. 4.38), whereas the superior and

inferior swelling of the ORS exerted significantly reduced MHC class II+ cells. Similar

to the human HF, lower parts revealed an even decreased number of MHC class II+

cells towards the hair bulb, and only single MHC class+ cells were present in the DP.

In the murine nail matrix, MHC class II+ cells were found in high numbers in the

epidermis and in adjacent PNF (Fig. 4.54 A), whereas no MHC class II+ cells were

detectable in the PNM and in its mesenchymal vicinity (Fig. 4.54 B). The nail bed

only rarely showed MHC class II+ cells (Fig. 4.54 B). In the foot pad epithelium, the

greatest number of intraepithelial MHC class II+ cells could be identified (Fig. 4.54

D), whereas the dermis showed a reduced number compared to the PNF and PNM

dermis (Figs. 4.55, 4.56). We focused on the detection of surface MHC class II

molecules, since their downregulation correlates with the suppressing of the APC

capacity, independent of the cell type (LC, macrophages, DC). The results of the

CD11b IR in the vibrissal follicle and the nail apparatus underlines the MHC class II

findings and reveals that most of the MHC class II positive cells were macrophages.

Nonetheless, in future experiments it would be interesting to perform double

stainings, as ITO et al. (2005) have done to distinguish APCs in detail, and also to

evaluate how many APC are localized in the different parts and which percentage is

impaired of their antigen-presenting capacity (seen as downregulation of MHC class

II+ cells on the surface).

DISCUSSION

196

As demonstrated for the human HF (CHRISTOPH et al. 2000), we also detected that

CD4+ T cells and CD8+ T cells (data not shown) were predominantly expressed in

the epidermis and upper parts of the vibrissal follicle (in particular in the isthmus and

around the sebaceous gland), whereas in the superior swelling of the ORS and even

more in the bulge, a significantly reduced number of this cell type was obvious (Fig.

4.39). The bulb region showed only single CD4+ T cells (Fig. 4.39) and no CD4+ T

cells in the dermal papilla. The same observation was made for CD8+ T cells, even

though their total number was lower (data not shown). This similarity is also true for

the murine nail apparatus compared to the human system. As known from the human

nail apparatus (ITO et al. 2005c), we could identify a high density of CD4+ T cells in

the vicinity of the PNF, but very rarely in adjacent PNM. Intraepithelial CD4+ T cells

were only present in the PNF and foot pad. This absence or downregulation of CD4+

T cells may contribute to an immunoprivileged environment since the mechanism of

antigen presenting is also dependent on CD4+ T cells and co-stimulatory signals. In

addition, a low number of CD8+ T cells could reduce the risk of an attack on cells

without MHC class I surface molecules to protect the stem cells.

We demonstrated a similar expression pattern of the immunosuppressant TGF-β1, in

both the vibrissal follicle (Fig. 4.45) and the nail apparatus (Fig. 4.59) compared to

the human HF. In the vibrissal follicle, the bulge exerts, when compared to the SO

and lower parts of the follicle, an upregulated TGF-β1 IR, indicating a special function

of TGF-β1. In the nail apparatus, we found homogenously TGF-β1 IR (i.e. the

suprabasal layers of the epidermis, the epidermis in the foot pad and the PNM),

besides prominent expression of TGF-β in epidermal basal layers and in the ORS of

HFs (Fig. 4.59 B). As already described recently (WAHL et al. 2006; SIGLIENTI et al.

2007), TGF-β1 is considered to play an important role in the maintenance of IP by

inhibiting NK cell-mediated cytolysis and CD8+ T cell attacks, in particular, in areas of

low or absent MHC class Ia expression. The aforementioned findings suggest that

besides these and other IP-associated effects, TGF-β1 somehow influences and

supplies cells and tissues in another way, because it is so widely expressed.

DISCUSSION

197

Interestingly, according to the findings in the human HF, the matrix keratinocytes in

the bulb revealed a reduced staining intensity. This indicates that the assumed IP

within the bulb either is not as strong as in the bulge, or that the role of TGF-β1 in the

bulb is not really essential, because MHC class I negativity persists.

In addition to these antigens directly associated with IP, we also investigated some

other key immunological parameters on the mouse specimens: Mast cells, CD54 and

β-defensin 2: Mast cells are multifunctional and involved in many different

interactions of innate and adaptive immunity (YOUNG 1997; MCLACHLAN et al.

2003; MEKORI 2004; SUTO et al. 2006; THEOHARIDES and KALOGEROMITROS

2006; CHRISTY and BROWN 2007; METZ et al. 2007; JAHANYAR et al. 2008;

SAYED et al. 2008). They are also considered to contribute to IP (DEPINAY et al.

2006; SAYED et al. 2008), and we aimed to define the expression of mast cells

pattern in murine specimens.

Contrary to previous findings in the human HF (CHRISTOPH et al. 2000), where

mast cells were distributed homogenously all over the CTS of the human HF, we

could show a compartment-dependent distribution of mast cells in the vibrissal

follicle. Here we found that the number of mast cells was especially high in the upper

part and the isthmus, and decreased continuously towards the lower part, whereas

almost no mast cell was evident around the vibrissal bulb (Figs. 4.48 A,B, Fig. 4.49).

However, no significant difference in mast cell numbers could be demonstrated within

the hourglass shaped parts and their surrounding tissue, whereas many mast cells

were localized outside the capsule around the vibrissal follicle.

On the one hand, such decreased mast cell numbers towards the bulb suggest a

contribution of mast cell interaction to the maintenance of IP in the vibrissal follicle,

although the mechanisms are still unclear. Since it is supposed that mast cells play a

role in the pathogenesis of AA, which is based on hair bulb IP collapse, this idea

would underline our findings in mice. On the other hand, it cannot be fully explained,

why the mast cell results strongly differ between the murine vibrissal follicle and the

human HF. An explanation would be that the vibrissal capsule is the cause of the

difference and acts as a strong barrier against the invasion of mast cells from the

DISCUSSION

198

surrounding. Furthermore, it is unknown so far under which circumstances mast cells

reveal pro-inflammatory or immunosuppressive functions, or when they act as ´good`

or ´bad guys` (MAURER et al. 2003b; MAURER and METZ 2005). For this reason, it

is also important to keep in mind that not only the mast cell numbers but also the

activation status or ratio of degranulated mast cells compared to non-degranulated

have to be considered. Either the previous findings of CHRISTOPH et al (2000) were

not distinctive enough, or the ratio of degranulation is the main limiting or important

factor.

In the murine nail apparatus, we found a homogenous distribution of mast cells

without any significant differences in mast cell numbers in the compartments of the

murine nail apparatus (Fig. 4.64). The only exception were the surroundings of the

nail bed, where significantly less mast cells were detectable than in the PNF. Since

this part has a sterile environment and is lying between two important hard tissue

structures, namely the nail plate and the bone where much pressure occurs, it might

be that a low number of mast cells should reduce the risk of unnecessary

inflammation induced by trauma. However, according to the human nail system, the

expected reduced numbers of dermal mast cells in the vicinity of the proximal nail

matrix was not confirmed. The explanation of ITO et al. (2005) for these results

emphasized that low mast cell number may compromise the defense capacity of this

region of nail mesenchyme, whereas in the terminally differentiated nail epithelium

locally produced AMPs (DORSCHNER et al. 2004) may compensate and supply the

innate immune defense. However, the authors did not investigate the mast cell

activation status. Since we did not find such differences, the explanation suggested is

not convincing and it remains to be controlled by future experiments whether the

activation status of mast cells differs in the defined tissue compartments, and plays a

more important role in IP than mast cell numbers.

ICAM-1 is rapidly inducible on endothelial cells by infection, playing a major role in

local inflammatory responses (CAMELI et al. 1994; BOS 1997; GOLDSBY et al.

2003; JANEWAY et al. 2005). Since it is may be involved in IP maintenance or,

DISCUSSION

199

collapse respectively, we also examined this molecule. In this connection, it is

noteworthy to recognize that ICAM-1 expression pattern obviously differs

substantially between the follicle and the nail apparatus immune system. ITO et al

(2005) reported in their in situ studies that the infantile human nail apparatus did not

demonstrate ICAM-1 IR in any epithelial compartment, except for the expected IR in

blood vessels. In the current study, we could confirm this expression pattern also for

the murine nail apparatus. We identified ICAM-1+ positive cells as limited to the

mesenchyme (Fig. 4.61 A), except for endothelial cells of small blood vessels near

the epidermis of the PNF. The number of positive cells was slightly reduced in the

vicinity of the PNM and the foot pad. In contrast, studies of the HFs in mice and

humans have demonstrated constitutively expression of ICAM-1 in the peri- and

infrainfundibular ORS, thus attracting LFA-1 expressing immunocytes (MUELLER-

ROEVER et al. 2000). This is not surprising, because the ORS and the bulge region

has been shown to be the preferred site for the occurrence of dense perifollicular

inflammatory cell infiltrates under stress (ARCK et al. 2001, 2003). In addition, it was

reported that in normal, uninflamed mouse skin, a very small, but notable percentage

of HFs are permanently deleted by an inflammatory cell attack on the ICAM-1+ bulge

region of anagen and catagen pelage HFs, without any macroscopic evidence for this

striking phenomenon of ´programmed organ deletion (POD)` (EICHMULLER et al.

1998). ICAM-1 IR in normal human skin was found on most CTS and dermal

fibroblasts and immune cells, but the strongest ICAM-1 IR was seen on perifollicular

inflammatory cell clusters (PICC) (JAWORSKY et al. 1992). In addition, ICAM-1

expression was found on HF keratinocytes in the close vicinity of perifollicular

inflammatory cell clusters (CHRISTOPH et al. 2000), in particular in the perifollicular

bulge region (MEYER et al. 2008c). Whether such physiological POD of isolated (or

possibly malfunctioning) HFs also occurs in human skin, and if at least some forms of

cicatricial alopecias only represent an excessive form of POD (PAUS et al. 1999),

remains to be investigated.

Furthermore, in the current study a strong up-regulation of the staining intensity of β-

defensin 2 was found in parts of the PNM close to the nail plate, as compared to the

DISCUSSION

200

PNF and parts of the foot pad (Figs. 4.66, 4.67). The staining was cytoplasmic and a

discreet IR could also be shown in other epithelial parts. These results are slightly

contradictive and unexpected, because APMs are expected to be secreted in

epithelial parts facing to the environment (DOERFL 1982; DORSCHNER et al. 2004).

However, results from wild mammals on the production of antimicropial peptides

have demonstrated that not only the epidermis, but also the complete hair follicle

complex, i.e. including the entire ORS, the apocrine tubular glands and the

sebaceous glands can express CAPs or ß-glucan receptors (MEYER et al. 2003,

2008b, MEYER and SEEGERS 2004).

Taken together, our results suggest that the nail immune system shows similarities to

the HF immune system, including the possible establishment of an area of relative IP

in the PNM. The functional advantages of establishing IP in the nail matrix are still

uncertain. Also, it is unknown yet, which key autoantigens are shared between nail

apparatus and HFs, and whether the collapse of IP in the nail matrix may also

underlie the frequent involvement of the nail in AA.

5.3.3 Comparison of the human HF bulge, murine sinus hair follicle

and nail apparatus

The human HF, the murine vibrissal follicle and the nail apparatus show one common

aspect: They all belong to skin appendages and are relatives from the evolutionary

point of view. They consist of epithelial and mesenchymal structures and are in direct

contact with the environment. In addition, many structures and their functions of the

human HF and the vibrissal follicle are exactly the same ones.

Despite the above mentioned similarities, there are several aspects in which these

tissues differ, and which make a direct comparison difficult, if they belong to sites of

IP or whether the supposed function of IP could be similar. In the following, only the

most relevant aspects will be emphasized: Human HFs (as well as murine pelage

DISCUSSION

201

HFs) undergo life-long cycles of growth (anagen), regression (catagen) and relative

quiescence (telogen), the so-called HF cycle. During anagen development, i.e. when

a new hair shaft (HS) factory - the anagen hair bulb - is reconstructed, the HF

recapitulates in part key morphological events of its own morphogenesis. This

astounding phenomenon of cyclic organ regeneration during adult life is only possible

because this ´miniorgan` is richly endowed with eSCs. Terminal HFs for example,

function as a protection against the environment, act as a heat balance and

mechanoreceptor, or contribute to the individual and characteristic appearance.

In contrast, vibrissal follicles show also life-long cycling, but termination of hair growth

in the previous hair cycle and initiation of hair regeneration in the following cycle

partially overlaps. Thus, telogen is not obvious; otherwise the specific sensory

function of the tactile organ would be interrupted. The nail apparatus, however, is a

permanent structure without cycling. The nail plate is continuously produced by the

nail matrix and nail bed. In conclusion, the signals for eSCs are considered to differ

considerably from each other, which might affect also their respective defense

mechanisms.

Nonetheless, apart from such differences, the suggested function of bulge IP and IP

in the PNM is the protection of epithelial stem cells. Since IP is a phenomenon, which

is described in many tissues (SIMPSON 2006), independent of the fact that these

often strongly differ from each other (brain, fetus, anterior chamber of the eye, testes,

hamster cheek pouch), so comparisons are possible, regarding to the widely

accepted markers. In this study, we presented a down-regulation of MHC class Ia -

one of the key IP mechanism - and MHC class II molecules in all three tissues

investigated. In addition, the expression of important immunosuppressive molecules

like TGF-β could be demonstrated. The distribution pattern of CD4+ and CD8+ T

cells in the vibrissal follicle and nail apparatus also resemble the one of human HF.

Taken together, although we observed only a small amount of IP marker in the

murine specimens, available data suggest that the vibrissal bulge and bulb, as well

as the PNM also belong to a site of relative IP.

DISCUSSION

202

5.4 Effects of K(D)PT on the hair follicle immune system

α-MSH is known to have melanotropic, immunomodulatory and restoration capacity

in anagen hair bulbs (ITO et al. 2004), and acts as a mediator of tolerance induction

in the brain (TAYLOR and KITAICHI 2007) and eye (NIEDERKORN 2006).

One of the key questions investigated in this study was whether the synthetic α-MSH

related tripeptide K(D)PT can restore HF IP after IFN-γ induced collapse, or whether

it has protective functions, respectively, as it was shown for other immuno

suppressants (ITO et al. 2004). Since low or absent expression of MHC class I

protein in hair matrix keratinocytes is the hallmark of HF IP (ITO et al. 2004), we

imitated an IP collapse by application of IFN-γ (ITO et al. 2004). We chose a dose of

75 IU/ml IFN-γ to prevent a transition of the anagen VI HFs into catagen, because

hair bulb IP is hair cycle dependent and occurs in anagen VI only.

Our MHC class I results (Fig. 4.27) show that K(D)PT in the concentrations tested

failed to act as an agent for either re-establishing IP or for protecting against its

collapse. As shown previously (ITO et al. 2004), 75 IU/ml IFN-γ significantly

upregulated MHC class Ia IR in the hair bulb of human anagen VI HFs, but none of

the applicated K(D)PT doses could significantly and reliably reduce the IFN-γ I-

induced MHC class I IR in the hair matrix and proximal ORS. This is corroborated by

the observed, comparably limited effects on the downregulation of MHC class II+

cells (Fig. 4.28). However, high-dose K(D)PT (10-8M) may well down-regulate the

number of MHC class I and II+ cells (and/or their expression level!) in susceptible

individuals. So far, it is unclear, which factors make the individual susceptible or not.

In accordance to previous studies, the tested dose range of K(D)PT was chosen in

relation to the concentration of α-MSH, which was detected as an IP restorer (ITO et

al. 2007).

Intriguingly, K(D)PT in dose-dependent manner reduced both, the number of mature

mast cells and their degranulation / activation status and counteracted IFN-γ pro-

inflammatory mast cell-directed stimuli (Fig. 4.31). Since α-MSH mast cell interaction

probably takes place via the MC-1R (LUGER et al. 2000), this might be also relevant

DISCUSSION

203

for K(D)PT. Mast cells not only elicit the first and fastest innate immune response

after recognition through Toll-like receptors and cytokine release (JAHANYAR et al.

2008), but are also material to the modulation of adaptive immune response

(secretion of pro-inflammatory and anti-inflammatory mediators, acting as antigen-

presenting cells and expression of co-stimulatory molecules) (JAHANYAR et al.

2008). This functions could lead into excessive inflammation and are required for

cutaneous wound healing (WELLER et al. 2006). In addition, recent studies showed

that inhibition of mast cell activation confers to the maintenance of immune privilege

(FIJAK and MEINHARDT 2006), i.e. the optimal expression of peripheral tolerance to

skin allografts (MAURER et al. 2003a; METZ et al. 2008a). Thus, K(D)PT obviously

exerts immunomodulatory capacity, and deserves full exploration as a mast cell

inhibitor in all inflammatory skin conditions where mast cells play a major role (e.g.

allergy, atopic eczema, urticara).

It should also be noted, that K(D)PT inhibits inflammation-induced premature catagen

(Fig. 4.32) development, which also matches with the Ki67/TUNEL results (Fig.

4.33). Such unexpected effect of an α-MSH-related peptide on HF cycling suggests

that K(D)PT might become exploitable for counteracting telogen effluvium induced by

various pro-inflammatory scalp skin processes that induce premature catagen

development (e.g. seborrhoic dermatitis). In addition, we found that K(D)PT does not

appear to greatly influence hair shaft formation (data not shown) or to stimulate hair

matrix keratinocyte proliferation indicating that K(D)PT is unlikely to have major or

general hair growth-stimulatory properties. On the contrary, the observation that

K(D)PT tended to reduce apoptosis in IFN-γ treated HFs raises the possibility, that

K(D)PT under inflammatory conditions may operate as a general ´epithelial

proliferation protector` in the HF and elsewhere in human skin.

In conclusion, despite its failure to impress as a reliable HF IP protectant / restorer,

K(D)PT may yet prove to be of importance in the management of inflammatory skin

diseases in which mast cells play a major role. Specifically, topical application of

K(D)PT, allowing the administration of high peptide doses with a favourable benefit /

DISCUSSION

204

risk ratio, may become exploitable for the treatment of post-inflammatory poliosis, or

for treating the recalcitrant poliosis that is characteristically seen during the recovery

phase of alopecia areata, an organ-specific autoimmune hair growth disorder, in the

pathogenesis of which IFN-γ is recognized to play a key role (GILHAR et al. 2007).

Beyond these effects there may be plenty other so far unknown functions useful for

the treatment of skin diseases.

5.5 Conclusions

On the basis of current and generally accepted knowledge, we present suggestive

phenomenological and functional evidence that the bulge of human anagen VI HFs

represents another site of relative IP within human skin, as predicted by previous

gene expression profiling data. However, our in situ-protein expression results also

suggest that this IP site, the most vital part of the follicle with its stem cell reservoir,

displays a number of features that distinguish it from the bulb IP. It remains to be

clarified, whether these differences are signs of different IP strength, development or

importance. In any case, the full thickness scalp skin organ culture offers good

opportunities to further explore bulge IP collapse and restoration in regard to other

parameters.

The immunophenotyping results obtained in the course of this study shed further light

on the in situ-expression patterns of some immunological and IP associated markers

in the murine vibrissal follicle and nail apparatus. The results suggest that the

vibrissal bulb, in particular, but also the bulge and to a lesser extent the proximal nail

matrix, probably meet the criteria to name these sites as immunoprivileged.

Nonetheless, some parameters remain to be studied, and functional evidence should

be provided before a definite statement regarding IP establishment can be made.

Irrespective of whatever function the IP of the mammalian vibrissal follicle and nail

apparatus has, these findings contribute to immuno biology in general and fill

scientific gaps, facilitating further research in the field of IP.

DISCUSSION

205

When interpreting the current data, it has to be pointed out that IP is a relative, not an

absolute state, reflecting the net result of multiple interconnected active and passive

mechanisms (FERGUSON and GRIFFITH 2006; SIMPSON 2006). In addition, it is

an ongoing debate, how many and which of these mechanisms have to be

established in a given tissue location to justify the term ´IP` (FERGUSON and

GRIFFITH 2006). Since no universally accepted criteria have as yet been defined

that apply to all IP tissues, ultimate proof for the human bulge, the vibrissal follicle or

the proximal nail matrix as sites of relative IP can only arise from functional studies,

such as the famous (as yet never repeated) melanocyte allotransplant experiment

(BILLINGHAM 1971; PAUS et al. 2005).

Furthermore, the physiologically relevant HF organ culture assay employed here,

simulating IP collapse via IFN-γ treatment, offers a highly instructive experimental

tool for the research of substances as IP restorer, like K(D)PT, and ideally

complements human cell culture models available (BELL et al. 1979). Despite its

failure to act as a reliable HF IP protectant / restorer, K(D)PT may yet prove to be

very useful in the management of inflammatory skin diseases in which mast cells

play a major role.

5.6 Perspectives

In order to demonstrate that a putative ´IP` status of any tissue compartment is truly

relevant to organ or system homeostasis, it needs to be shown that loss of IP actually

can lead to pathology (FERGUSON and GRIFFITH 2006). While it may be argued

that primary cicatricial alopecias (PCA), such as lichen planopilaris and CCLE-

associated scarring alopecia, represent just such examples for pathology as a result

of bulge IP loss, where an autoaggressive inflammatory cell attack on the HF‘s stem

cell region damages and destroys its stem cell-based regenerative capacity.

Definitive evidence that this is indeed the case has not yet been published.

In cooperation with Dr. M. Harries from the University of Manchester a current study

follows-up this lead and investigates, whether loss of bulge IP causes primary

DISCUSSION

206

cicatricial alopecias (HARRIES et al. 2008; HARRIES et al. submitted). Therefore,

punch scalp biopsies from healthy donors, and patients with PCA (unaffected vs.

affected scalp skin) are collected and compared immunohistochemically in respect of

IP parameters.

In a second step, full thickness organ culture experiments should be performed, to

evaluate substances, which are capable of restoring bulge IP. Another optimal test

system, in which this missing proof of pathology may be generated, would be to use

xenotransplants of hair-bearing human skin onto SCID mice (GILHAR et al. 1998), as

ideally after reconstitution with a human immune system upon transfer of appropriate

human stem cells.

In regard to the murine vibrissal follicles and nail apparatus, some parameters remain

to be investigated with respect to the in situ expression pattern, e.g. MHC class Ib

molecules, the ´no danger signal` CD200, IDO, MIF, and other immunosuppressants.

In addition, it would be helpful to perform also full-thickness skin organ culture, to

prove whether or not the suspected IP site could be induced to collapse.

With regard to the common autoimmune disease AA, which is considered to be

caused by the loss of IP within the human anagen hair bulb, it would be promising to

concentrate on the restoration of the HFs lost or compromised IP, both for preventing

the progression of AA lesions and for inducing hair regrowth. This IP restoration

therapy does not require any prior knowledge of the relevant key autoantigens or the

specific autoreactive T cells, and it can resort to the administration of well-known

nonspecific immunomodulators that drastically down-regulate ectopic MHC class I

expression in the anagen hair bulb. Although K(D)PT failed to restore IP, it exerts

several other interesting functions, which should be explored systematically.

SUMMARY

207

6 Summary

Katja C. Meyer

Detailed analysis of immunoprivileged tissues in skin appendages

Recent gene profiling data in mice have raised the possibility, that follicular IP is not only

limited to the anagen hair bulb, but that the epithelial stem cell region in the ORS of HFs,

termed the bulge, may also represent an area of relative IP. Since protection of these

epithelial stem cells from immune destruction is essential for preserving the regenerative and

cycling capacity of the follicle, it seems logical that the bulge region should also have an

established relative IP.

The current study on the immune status of human HFs and murine sinus hair follicles and

nail apparatus, is based on both routine and increased-sensitivity immunohistochemical

(including Immunofluorescence) staining techniques, and evaluation of these IR patterns by

quantitative immunohistochemistry, histochemical and histomorphometric assessments.

Additionally, full thickness human scalp skin organ culture and the HF organ culture model

were used for functional evidence.

The in situ protein expression data presented here provide evidence, that the bulge region of

anagen VI HFs meets all criteria of an IP site, and suggests that the bulge has a perhaps

stronger immune protection than that of the anagen bulb. Contrary to previous reports, we

demonstrated that MHC class Ia, β2-microglobulin and MHC class II IR were down-regulated

in the human bulge relative to the distal ORS. The locally secreted immunosuppressants α-

MSH and TGF-β were prominently expressed in the bulge. In addition, IDO and MIF showed

higher IR levels around the bulge compared to the proximal follicle and CD200 and HLA-E

were solely expressed in the stem cell-rich bulge region. Besides these systematic bulge

immuno phenotyping experiments, we obtained functional evidence for an established IP in

the bulge, by showing that IFN-γ can induces significant ectopic MHC class Ia expression in

bulge cells of organ-cultured human scalp skin, as it had been shown for the anagen hair

bulb. However, our results also revealed that the bulge and bulb IP in human scalp HFs differ

in several important aspects: The level of constitutive MHC I, MHC II and β2-microglobulin IR

was not as low as in the hair bulb, which may indicate that the bulge IP is constitutively more

prone to inciting an immune response than the bulb, resulting in an inflammatory cell attack

on bulge epithelial stem cells.

SUMMARY

208

This thesis also contributes considerably towards the immunological characterization of the

murine vibrissal follicle and murine nail apparatus, and showed that the vibrissal bulb, and to

a lesser extent, the bulge as well as the proximal nail matrix can also be considered to be

sites of relative IP. Both tissues showed a substantial downregulation of MHC class I and II in

the examined IP sites. Prominent IR of the immunosuppressant TGF-β1 was demonstrated

in the PNM, bulge and weaker in the bulb, exerting also an important role in IP. Furthermore,

the presented distribution pattern of CD4+ and CD8+ T cells was similar to the human HF.

Besides these IP-associated findings, we could observe that mast cell numbers decreased in

vibrissal follicles towards the vibrissal bulb, whereas no relevant differences in the

distribution could be shown in the murine nail apparatus. In addition, we identified ICAM-1+

positive cells to be limited to the mesenchyme of the nail apparatus. The number of these

cells was slightly reduced in the vicinity of the PNM and the foot pad, but no intraepithelial

clusters were observed, in accordance with previous studies on the human nail apparatus.

Unexpected results were obtained for β-defensin 2, showing substantially higher levels of IR

in the PNM and in the entire ORS of the vibrissal follicle, and only weak IR in epidermal

layers. Nonetheless, whether mast cells, ICAM-1, and β-defensin 2 contribute to the

mechanisms for or against IP, remains to be further explored.

The analyses of this study were concluded by exploring the effects of the α-MSH related

tripeptide K(D)PT by using an organ-culture HF model. K(D)PT failed to act as a protection or

restoration agent of IP. However, K(D)PT counteracted IFN-γ-induced upregulation of mast

cell numbers and degranulation. In addition, we demonstrated that K(D)PT inhibited

inflammation-induced premature catagen development, whereas it did not considerably

influence hair shaft formation in vitro or stimulate the proliferation of hair matrix keratinocytes.

In summary, the current thesis has provided evidence for a second epithelial IP within the

human HF, which could even be stronger than that of the bulge. Moreover, the presented

results indicate that the murine PNM, and the bulge and bulb of vibrissal follicles also enjoy

IP, so that some gaps in basic immunological research could be filled. This was

complemented with the exploration of the new candidate K(D)PT for IP-restoration or

protection and the demonstration of several effects, which may involve that K(D)PT may be

used pharmacologically as a hair growth modulator.

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7 Zusammenfassung

Katja C. Meyer

Detaillierte Analyse von immunpriviligierten Geweben in Hautanhangsorganen

Neueste Daten auf Genebene von Mäusen haben die Vermutung nahegelegt, dass nicht nur

die anagene Haarwurzel zum follikulären Immunprivileg (IP) zählt, sondern auch die

sogenannte Wulstregion in der äußeren Wurzelscheide des Haarfollikels, die dessen

epitheliale Stammzellregion darstellt.

Methodische Grundlagen für die vorliegende Studie an Haarfollikeln des Menschen und an

Sinushaarfollikeln und dem Nagelapparat der Maus waren primär hochspezifische

immunhistochemische Färbungen (inklusive Immunfluoreszenz), die qualitativ und quantitativ

ausgewertet wurden. Weiterhin wurden für die Erarbeitung funktioneller Daten

Vollhautorgankulturen von humanen Kopfhautproben sowie Kulturen mit mikrodissezierten

humanen Haarfollikeln verwendet.

Die hier präsentierten in situ Protein Reaktivitätsverteilungsmuster erbringen den Beweis,

dass die Wulstregion des Anagen VI Haarfollikels alle Kriterien eines immunprivilegierten

Gewebes erfüllt. Sie lassen sogar vermuten, dass die Wulstregion einen stärkeren

Immunschutz aufweist als die anagene Haarwurzel. Im Gegensatz zu früheren

Veröffentlichungen zeigen wir in unserer Arbeit, dass die Immunreaktivität von MHC Klasse

Ia, β2-Mikroglobulin und MHC Klasse II Molekülen in der Wulstregion, verglichen mit der

distalen äußeren Wurzelscheide, stark herabgesetzt ist. Die lokal sezernierten

Immunosuppressoren α-MSH und TGF-β waren ebenfalls in der Wulstregion prominent.

Zusätzlich zeigten IDO und MIF höhere Immunintensitäten im Bereich der Wulstregion als im

proximalen Haarfollikel und die Moleküle CD200 und HLA-E waren alleinig in der

stammzellreichen Wulstregion vertreten. Neben dieser systematischen,

immunphänotypischen Analyse, konnten wir zusätzlich einen funktionellen Beweis für das

Bestehen eines IP in der Wulstregion erbringen: Ähnlich den Beobachtungen vorheriger

Experimente an der immunprivilegierten anagenen Haarwurzel, konnten wir demonstrieren,

dass eine Behandlung mit IFN-γ in organ-kultivierten humanen Skalp-Vollhautstanzen eine

Hochregulation von MHC Klasse Ia IR in der Wulstregion bewirkte. Gleichzeitig zeigen die

Ergebnisse jedoch auch, dass sich die Wulstregion und die anagene Haarwurzel hinsichtlich

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verschiedener Aspekte unterscheiden: Die Intensitätsstärke von MHC Klasse I, Klasse II und

β2-Mikroglobulin ist in der Wulstregion höher als in der Haarwurzel, was darauf hin deutet,

dass das IP der Wulstregion unmittelbarer eine Immunreaktion zulässt als die Haarwurzel

und die Folge eine entzündliche Zellattacke auf die epithelialen Stammzellen in der

Wulstregion und Zerstörung sein könnte.

Mit dieser Arbeit wurde auch ein Beitrag zur immunologischen Charakterisierung von

Sinushaarfollikel und dem Nagelapparat der Maus geleistet. Es konnte gezeigt werden, dass

die Haarwurzel des Sinushaarfollikels, und zu einem geringeren Maße dessen Wulstregion

sowie die proximale Nagelmatrix, ebenfalls zu immunologisch privilegierten Regionen

zählen. Beide Gewebetypen in den genannten Regionen wiesen eine stark reduzierte

Färbungsintensität für MHC Klasse I und II Moleküle auf. Ferner war TGF-β1 stark präsent in

der proximalen Nagelmatrix, in der Wulstregion und, weniger intensiv, in der Haarwurzel, so

dass eine wichtige Funktion für diese Substanz zu unterstellen ist. Das Verteilungsmuster

von CD4+ und CD8+ T Zellen war ebenfalls ähnlich den Verhältnissen im humanen

Haarfollikel. Neben diesen IP-assoziierten Ergebnissen konnten wir zeigen, dass die Zahl an

Mastzellen innerhalb des Sinushaarfollikels von der Epidermis zur Haarwurzel kontinuierlich

abnimmt, wohingegen kein relevanter Unterschied im Verteilungsmuster innerhalb des

Maus-Nagelapparates zu verzeichnen war. Zusätzlich wurden ICAM-1 positive Zellen

identifiziert, die auf das Mesenchym des Nagelapparates begrenzt waren. In direkter Nähe

zur proximalen Nagelmatrix und zum Fußballen waren etwas weniger ICAM-1+ Zellen zu

beobachten. Intraepitheliale Anhäufungen dieser Zellen wurden, entgegen vorheriger

Berichte, im humanen Nagelsystem nicht gefunden. Unerwartete Resultate wurden ebenfalls

für β-Defensin 2 erhoben, da die proximale Nagelmatrix und die gesamte ORS des

Sinushaarfollikels substantiell höhere Reaktionsintensitäten aufwiesen, als in den

epidermalen Zellschichten. Ob und welche Rolle Mastzellen, ICAM-1+ Zellen und das

antimikrobielle Peptid β-Defensin 2 zur Erhaltung des IP spielen, sollte in zukünftigen

Versuchen weiter erforscht werden.

Die Analysen dieser Studie wurden ergänzt mit der Erforschung von K(D)PT hinsichtlich

seiner Effekte auf den Haarfollikel durch den Einsatz unseres HF-Organ-Kulturmodels. Diese

Substanz, ein neues synthetisches, mit α-MSH verwandtes Tripeptid, war jedoch als

Wirkstoff zur Protektion oder Wiederherstellung des IP nicht überzeugend. Wir konnten

allerdings demonstrieren, dass K(D)PT der IFN-γ induzierten Zunahme von Mastzellen und

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211

der Rate an Degranulation entgegenwirkte; zusätzlich inhibierte K(D)PT die

entzündungsinduzierte vorzeitige Catagenentwicklung.

Zusammenfassend gesehen, erbringt die vorliegende Dissertation den Beweis, dass die

Wulstregion ein zweites epitheliales IP innerhalb des HFs darstellt. Dieses IP ist

möglicherweise stärker oder bedeutender, als dasjenige der Haarwurzel. Zusätzlich weisen

die hier vorgestellten Ergebnisse darauf hin, dass die murine PNM des Nagelapparates,

sowie die Wulstregion und Haarwurzel des Sinushaarfollikels ebenfalls immunologisch

privilegiert sein könnten; zudem war es möglich, einige Lücken in der immunologischen

Grundlagenforschung zu schließen. Ergänzt wurde diese Studie durch die Erforschung eines

neuen Haarmodulator-Kandidatens, dem K(D)PT, das zwar nicht als IP-Modulator

einzusetzen ist, jedoch zur Behandlung von Haarausfall und gegen Vergrauung genutzt

werden könnte.

ANNEX

212

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ANNEX

9 Annex

Parts of this thesis were published as follows:

Articles: ITO, T., MEYER, K.C., ITO, N., PAUS, R. (2008): Immune Privilege and the Skin. Curr. Dir. Autoimmun. 10, pp 27–52 MEYER, K.C., KLATTE, J.E., REITHMAYER, K., DINH, H.D., HARRIES, M.J., MEYER, W., SINCLAIR, R., PAUS, R. (2008) Evidence that the bulge region is a site of relative immune privilege in human hair follicles Br. J. Dermatol. Br J Dermatol. 2008 Sep 15. [Epub ahead of print], PMID: 18795933 HARRIES, M.J., MEYER, K.C., PAUS, R. (2009)

Hair loss as a result of cutaneous autoimmunity: Frontiers in the immunopathogenesis of primary cicatricial alopecia Autoimmun. Rev. 2008 Oct 13. [Epub ahead of print] PMID: 18926937 Meyer, K.C., BRZOSKA, T., ABELS, C., PAUS, R. The role of α-MSH related tripeptide K(D)PT in human hair follicle immunology and biology in situ under pro-inflammatory conditions Br. J. Dermatol. 2008 Oct 25. [Epub ahead of print] PMID: 19016700 Abstracts: MEYER, K.C., KLOEPPER, J.E., TIEDE, S., PAUS, R. (2007): The bulge – a second site of epithelial immune privilege within the human anagen hair follicle? Exp. Dermatol., 16, 3, P087, 221-222 KLOEPPER, J.E., MEYER, K. C., TIEDE, S., PAUS, R. (2007): The human hair follicle bulge: Immunohistological examination of potential stem cell-niche elements, and further indications that it displays relative immune privilege J. Invest. Dermatol. 127, S198 MEYER, K. C., KLOEPPER, J.E., DINH, H.V., SINCLAIR, R., PAUS, R. (2008): The immunoprivileged bulge: Regeneration pool and immunological “Achilles’ heel” of the hair follicle? Exp. Dermatol., 17, Number 3, March 2008 P134, Page 264

ANNEX

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REITHMAYER, K., MEYER, K.C., GLAESER, R., HARDER, H., SCHROEDER, J.M., PAUS, R. (2008): Antimicrobial defense of human hair follicle epithelium: inducible expression of RNase 7 and psoriasin and constitutive expression of hornerin Dermatol., 17, 3, P51, Page 264 VAN BEEK, N., BODÓ, E., KROMMINGA, A., GÁSPÁR, E., MEYER, K.C., ZMIJEWSKI, M.A., SLOMINSKI, A., WENZEL, B.E., PAUS, R. (2008): Human hair follicles are direct targets for thyroid hormones: involvement in anagen prolongation, hair matrix proliferation, hair pigmentation and metabolism J. Invest. Dermatol. 128, S156-S156

Additional publications generated during this thesis project: MEYER, K.C., BRZOSKA, T., ABELS, C., PAUS, R. (2008) The α-MSH related tripeptide K(D)PT stimulates hair follicle pigmentation in situ under pro-inflammatory conditions Br. J. Dermatol. (in press) VAN BEEK, N., BODÓ, E., KROMMINGA, A., GÁSPÁR, E., MEYER, K.C., ZMIJEWSKI, M.A., SLOMINSKI, A., WENZEL, B.E., PAUS, R. (2008) Thyroid hormones directly alter human hair follicle functions: Anagen prolongation, and stimulation of both hair matrix keratinocyte proliferation and hair pigmentation. J. Clin. Endocrinol. Metab. 2008 Nov;93(11):4381-8. Epub 2008 Aug 26. REITHMAYER, K., MEYER, K.C., GLAESER, R., HARDER, H., SCHROEDER, J.M., PAUS, R. Antimicrobial defense of human hair follicle epithelium: inducible expression of RNase 7 and psoriasin and constitutive expression of hornerin Br. J. Dermatol. (submitted) HARRIES, M.J., MEYER, K.C., CHAUDHRY, I., GRIFFITHS, C., PAUS, R.

Does collapse of the hair follicle bulge immune privilege play an important role in the pathogen? Br. J. Dermatol. (submitted)

260

Mein besonderer Dank gilt…

… meinem lieben Doktorvater, Herrn Prof. Dr. Wilfried Meyer (Anatomisches

Institut, Stiftung Tierärztliche Hochschule Hannover), für die Vermittlung meines

Dissertationsthemas an der Universität zu Lübeck, für die hervorragende und

verantwortungsbewusste Betreuung aus der Ferne und bei meinen Aufenthalt in

Hannover, für die zahlreichen hilfreichen Hinweise und Anregungen und vor allem für

die zu jedem Zeitpunkt gewährte fachliche und menschliche Unterstützung und sein

klassisch-motivierenden Zuspruch „Ach, das schaffen wir schon…!“.

… meinem lieben Betreuer und hervorragenden Chef vor Ort, Herrn Prof. Dr. Ralf

Paus (Klinik für Dermatologie, Allergologie und Venerologie, Universität zu Lübeck),

für die Vergabe des Themas, für die ständigen fachlichen Anregungen und

Hilfestellungen, für das Vertrauen in mich und meine Arbeit, das jederzeit offene Ohr

und seinen ständigen Optimismus und Enthusiasmus bei all meinen Vorhaben.

… dem Direktor der Hautklinik der Universität zu Lübeck, Herrn Prof. Dr. D.

Zillikens, für die Möglichkeit, in seiner Klinik als Doktorandin zu arbeiten, für den

fachlichen Austausch im Rahmen des Autoimmunitätsschwerpunktes der

Medizinischen Fakultät der Universität zu Lübeck und für das gute Arbeitsklima

innerhalb seines Hauses. Ebenso gilt mein Dank Herrn PD. Dr. Ralf Ludwig für den

inhaltlichen Austausch und die Bereitstellung meiner Maushautproben.

… unseren kollaborierenden plastischen Chirurgen, Herrn Dr. Dr. W. Funk (Klinik Dr.

Koslowski, München), Herrn W. Moser (Moser-Klinik, Augsburg) und Herrn J. Levy

(Atriumklinik in Holzkirchen, Holzkirchen), ohne deren wertvolle Lieferungen von

menschlicher Skalphaut aus elektiven Eingriffen diese Arbeit niemals hätte

durchgeführt werden können. Dieser Dank gilt natürlich auch den anonymen

Spendern, deren großzügige Bereitschaft, ihre Hautexzidate zu Forschungszwecken

zur Verfügung zu stellen, die Grundvorausetzung zur Durchführung dieser Arbeit

war.

... Herrn Dr. Christoph Abels und Dr. Thomas Brzoska von der Firma Wolff,

Bielefeld Arzneimittel für die Möglichkeit, die erzielten Ergebnisse über K(D)PT in

meine Dissertation zu integrieren .

… meinen Kooperationspartnern Dr. Matthew J. Harries, Prof. Dr. Taisuke Ito,

Prof. Dr. Rodney Sinclair und Hope V. Dinh für die Bereitstellung diverser

Antikörper und sonstige professionelle Hilfestellungen.

… der AG Wollenberg (Leiter: PD Dr. Ralph Pries) und der AG Wenzel (Leiter: Dr.

Björn Wenzel) an der Universität zu Lübeck für die Erlaubnis, diverse Instrumente

mitnutzen zu dürfen und für die immerwährend gute Zusammenarbeit. Besonderer

Dank gilt hier unmittelbar meinem Tischnachbarn Dr. Björn Wenzel und Frau Silvia

Grammersdorf, für jegliche Unterstützung und interessanten Austausch.

… meiner treuesten Mitdoktorandin, Dr. Jennifer Klatte, die mir den Einstieg

besonders erleichterte, ihr Wissen mit mir teilte, mir in jeglicher Hinsicht und zu jeder

Zeit beiseite stand und steht, und am allerbesten wusste, in welcher emotionalen

Lage der Promotionsphase ich mich gerade befand.

... meinem Laborchef Herrn Dr. Stephan Tiede, der mit seiner besonderen Art und

seinem trockenem Humor den Alltag mitbestimmt hat und auf den ich in

unwegsamen Situationen zählen konnte.

… Frau Dr. Enikö Bodó und Frau Katrin Reithmayer für deren Anteilnahme, Rat

und Tat im Labor und auf allen Ebenen der Dissertation.

… der Chef-MTA Frau Astrid Becker, Frau Nadine Dörwald, Frau Gaby Scheel

und Frau Antje Winter-Keil für ihre menschliche und fachliche Unterstützung, und

die sehr gute Organisation des Labors.

262

… allen bisher nicht genannten Mitgliedern der AG Paus für die tolle

Arbeitsatmosphäre, für die geduldige und engagierte Unterstützung und alle

gemeinsamen Aktivitäten: Nina van Beek, Nina Biedermann, Nancy Ernst, Tobias

Fischer, Lütfiye Hastedt, Elisabeth Gáspár, Céline Hardenbicker, Sybille Hasse,

Benedikt Kany, Patrick Kleditzsch, Dorothee Langan, Viktoria Naumann, Burkhard

Poggeler, Christian Plate, Yuval Ramot, Katrin Reithmayer, Anne Spiegelberger, Koji

Sugawara, Sai Kailash Uppalapati, Ronny Wachtel, Friederike Wiersma, Annika

Hanning.

… meinen lieben Freunden und meiner gesamten Familie für das offene Ohr, den

Rückhalt, das Vertrauen und die Zuversicht

... meinem Freund Robert, für die Geduld, die Hilfestellung und den Stolz, den er für

mich und mein Vorhaben empfand.

… meiner lieben Mutter, Ernst und meinem lieben Bruder für ihre guten Gedanken

und die liebevolle Unterstützung.