BASEMENT MEMBRANE ZONE PROTEINS, EPITHELIAL INTEGRINS AND TGF-� SYSTEM IN REEPITHELIALIZATION, DERMATITIS HERPETIFORMIS AND PSORIASIS Modulation by isotretinoin, betamethasone andcalcipotriol

TOMILEIVO

Department of Dermatology andVenereology, University of Oulu

Department of Anatomy and Institute ofBiomedicine, University of Helsinki

OULU 2000

OULUN YLIOPISTO, OULU 2000

BASEMENT MEMBRANE ZONE PROTEINS, EPITHELIAL INTEGRINS AND TGF-� SYSTEM IN REEPITHELIALIZATION, DERMATITIS HERPETIFORMIS AND PSORIASIS Modulation by isotretinoin, betamethasone and calcipotriol

TOMI LEIVO

Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in Auditorium 3 of the University Hospital of Oulu, on September 1st, 2000, at 12 noon.

Copyright © 2000Oulu University Library, 2000

OULU UNIVERSITY LIBRARYOULU 2000

ALSO AVAILABLE IN PRINTED FORMAT

Manuscript received 27 June 2000Accepted 10 July 2000

Communicated by Docent Ilkka Harvima Docent Matti Kallioinen

ISBN 951-42-5712-X

ISBN 951-42-5711-1ISSN 0355-3221 (URL: http://herkules.oulu.fi/issn03553221/)

In memory of my motherPirkko Leivo, MD

1933-1996

Leivo, Tomi, Basement membrane zone proteins, epithelial integrins and TGF-�system in reepithelialization, dermatitis herpetiformis and psoriasis. Modulation byisotretinoin, betamethasone and calcipotriolDepartment of Dermatology, University of Oulu, FIN-90220 Oulu, FinlandDepartment of Anatomy, Institute of Biomedicine, University of Helsinki, FIN-00014Helsinki, Finland2000Oulu, Finland(Manuscript received 27 June 2000)

Abstract

TGF-�s are cytokines that signal through the receptor complex of type I and type II receptors.Hemidesmosome (BP180, BP230, plectin/HD1, �6�4 integrin), anchoring filaments (laminin 5),and anchoring fibrils (collagen VII) form a hemidesmosomal adhesion complex that provides stableadherence of keratinocytes to the epidermal basement membrane. Nidogen, collagen IV, andlaminins are components of the basement membrane, integrins are cell adhesion molecules, andtenascin-C is a matrix protein.

The expression of TGF-� receptors I and II was studied in normal and psoriatic epidermis byimmunohistochemistry. TGF-�1 and TGF-�2 in suction blister fluid and serum were determined byELISA. Suction blister fluid and serum samples were obtained from acne patients before and afteroral isotretinoin treatment. Suction blister fluid samples were also obtained from healthy volunteersfrom a control site and a betamethasone-pretreated site. The expression of basement membranezone proteins was studied in uninvolved dermatitis herpetiformis skin by the immunofluorescencetechnique. The ultrastructure of the hemidesmosomal inner plaque was studied in uninvolveddermatitis herpetiformis skin by electron microscopy. The suction blister method was used to studyintact blisters, open wounds (=blister roofs removed right after blister induction) and calcipotriol-pretreated open wounds in healthy volunteers. The reepithelialization rate and the expression ofbasement membrane zone proteins and integrins during reepithelialization were studied byhaematoxylin and eosin stainings and the immunofluorescence technique.

In normal epidermis, TGF-� receptors I and II were detected in the basal epidermis. Diffusioncalculations suggest that circulation is likely to be a major source of TGF-� for TGF-� receptors inthe basal epidermis. Downregulation of TGF-� receptors I and II was seen in lesional psoriaticepidermis, suggesting that hyperproliferating lesional epidermis may have lost TGF-�-mediatedgrowth inhibition. Isotretinoin caused a 19% local increase in suction blister fluid TGF-�1.Betamethasone caused a 17% decrease in suction blister fluid TGF-�1, presumably due toglucocorticoid-induced vasoconstriction. Immunoreactivity for BP230 and plectin/HD1 wasdecreased in the basement membrane zone in uninvolved dermatitis herpetiformis skin in asignificant proportion of the patients, suggesting distinct molecular changes in BP230 and plectin/HD1. This may be a factor contributing to blister formation. Reepithelialization rate wasconsiderably slower in intact blisters than in open wounds and was not affected by calcipotriol. Thismay suggest that, in some bullous diseases, removal of the blister roof could accelerate blisterhealing and that calcipotriol treatment does not delay wound epithelialization. BP230 and plectin/HD1 appeared earlier in intact blisters than in open wounds. Reepithelialization took place on acontinuous laminin sheath in intact blisters, but the laminin sheath in open wounds was partiallydiscontinuous. The present results suggest, that a continuous laminin sheath may inhibitreepithelialization, and that the formation of the hemidesmosomal inner plaque at the leading edgetakes place earlier in the more slowly reepithelializing intact blisters than in open wounds. Integrin�v�5 and integrin �5 antibodies showed divergent distributions in regenerating epidermis.

Keywords: suction blister, hemidesmosome

Acknowledgements

First of all, I wish to express my deepest gratitude to my supervisor, Professor AarneOikarinen, M.D., Ph.D., Head of the Department of Dermatology in the University ofOulu, for his friendly support and guidance during this study and for providing financialresources.

I express my respectful gratitude to Professor Ismo Virtanen, M.D., Ph.D., Head of theInstitute of Biomedicine in the University of Helsinki, for providing laboratory facilitiesand a wide variety of antibodies to carry out this work and for his advice during thisstudy.

I thank Docent Ilmo Leivo, M.D., Ph.D., for assisting me with dermatologicalmorphology and immunohistochemistry and for advice on scientific writing.

I thank Docent Urpo Kiistala, M.D., Ph.D., former Head of the Department ofDermatology in the Central Military Hospital, for providing an opportunity to performclinical experiments at his department and for introducing to me the suction blistermethod.

I thank Docent Ilkka Harvima, M.D., Ph.D., and Docent Matti Kallioinen, M.D.,Ph.D., the reviewers of my study, for their constructive criticism.

I thank Docent Arja-Leena Kariniemi, M.D., Ph.D., for providing biopsy material forthis study.

I thank Professor Jorma Keski-Oja, M.D., Ph.D., for providing antibodies and for hisadvice.

I thank my closest colleagues Jouni Lohi, M.D., Ph.D., and Jan Rissanen, Cand.Med.,for collaboration and friendship. Their support has been of considerable value during thisstudy.

I thank Pirkko Arjomaa, M.Sc. (Chem.), Head of the Department of ClinicalChemistry in the Central Military Hospital, for collaboration and for placing the facilitiesof her laboratory at my disposal.

I thank Professor Robert Burgeson, Ph.D., Professor Katsushi Owaribe, Ph.D., DocentPekka Autio, M.D., Ph.D., Gerd Molander, M.D., Csaba Kiraly, M.D., Ph.D., Marja-LiisaKotovirta, M.D., and Jouko Lohi, M.D., Ph.D., for collaboration.

At the Department of Anatomy, I thank Professor Yrjö Konttinen, M.D., Ph.D., TaneliTani, M.D., Ph.D., and Kim Fröjdman, Ph.D., for encouragement and a pleasentatmosphere. I also thank Ms Paula Hasenson, Ms Marja-Leena Piironen, Ms AnneReijula, Ms Aili Takkinen, Mr Reijo Karppinen and Mr Hannu Kamppinen for excellenttechnical assistance and Ms Outi Rauanheimo for secretarial assistance.

At the Department of Dermatology in the Central Military Hospital, I thank the headnurse of the department, Marjatta Nieminen, and her entire nursing personnel forassistance in the clinical studies of this thesis, and medical researcher Harry M. Larni forencouragement.

I thank department secretary Liisa Laine for providing information on biopsy materialfrom hospital archives.

I thank all the volunteers who participated in this study.I thank Sirkka-Liisa Leinonen, Lic. Phil., for revising the English of this thesis.I thank MATINE, the Research Institute of Military Medicine, the Finnish Medical

Society Duodecim, the Oulu University Hospital, Oulun Yliopiston Tukisäätiö, OrionPharma, Labsystems, Schering-Plough, UCB Pharma Finland, Leo Pharma, Roche,Novartis Finland and Pharmacon/Tamro for financial support.

I express my warmest gratitude to my brother Timo Leivo, LL.M. (Helsinki), LL.M.(Brügge), my sister Tiina Leivo, M.D., M.Sc (Econ.), and my parents Pirkko Leivo �,M.D., and Professor Veikko Leivo, D.Sc. (Tech.), for their support and encouragementduring this study. I thank my parents for their encouraging attitude towards education.

I express my warmest appreciation to Miss Niina Vesterinen whose support andencouragement has been very essential for the completion of this study.

Helsinki 2000

Tomi Leivo

Abbreviations

BMZ basement membrane zoneBPA bullous pemphigoid autoantiseraDNA deoxyribonucleic acidFITC fluorescein isothiocyanateKDa kilodaltonLAP latency-associated proteinLTBP latent transforming growth factor-�-binding proteinmRNA messenger ribonucleic acidPBS phosphate-buffered salineT�R-I transforming growth factor-� type I receptorT�R-II transforming growth factor-� type II receptorTGF-� transforming growth factor-�UEA-I ulex europaeus I agglutinin

List of original articles

This thesis is based on the following articles, which are referred to in the text by theirRoman numerals:

I Leivo T, Leivo I, Kariniemi A-L, Keski-Oja J & Virtanen I (1998) Downregulationof transforming growth factor-� receptors I and II is seen in lesional but not non-lesional psoriatic epidermis. Br J Dermatol 138: 57-62.

II Leivo T, Arjomaa P, Oivula J, Vesterinen M, Kiistala U, Autio P & Oikarinen A(2000) Differential modulation of TGF-� by betamethasone-17-valerate andisotretinoin; corticosteroid decreases and isotretinoin increases the level of TGF-� insuction blister fluid. Skin Pharmacol Appl Skin Physiol 13: 150-156.

III Leivo T, Lohi J, Kariniemi A-L, Molander G, Kiraly CL, Kotovirta M-L, OwaribeK, Burgeson RE & Leivo I (1999) Hemidesmosomal molecular changes in dermati-tis herpetiformis; decreased expression of BP230 and plectin/HD1 in uninvolvedskin. Histochem J 31: 109-116.

IV Leivo T, Kiistala U, Vesterinen M, Owaribe K, Burgeson RE, Virtanen I & Oika-rinen A (2000) Reepithelialization rate and protein expression in suction-inducedwound models; comparison between intact blisters, open wounds and calcipotriol-pretreated open wounds. Br J Dermatol 142: 991-1002.

V Leivo T, Virtanen I & Oikarinen A. Increased immunoreactivity for integrin �5 sub-unit in suprabasal cell layers in regenerating epidermis. Submitted.

Contents

Abstract AcknowledgementsAbbreviations List of original articles 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172. Review of the literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2.1. Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182.1.1. Epidermis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182.1.2. Dermis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192.1.3. Basement membrane zone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

2.2. Molecular components of epidermal BMZ . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202.2.1. Hemidesmosomal adhesion complex . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

2.2.1.1. BP230, plectin/HD1, BP180 and a6�4 integrin(hemidesmosome) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

2.2.1.2. Laminin 5 (anchoring filaments) and other lamininswithin the BMZ 23

2.2.1.3. Type VII collagen (anchoring fibrils) . . . . . . . . . . . . . . . . . . . 242.2.2. Nidogen and collagen IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

2.3. Epithelial integrins (�v�5, �5, �9) and tenascin-C . . . . . . . . . . . . . . . . . . . . . . 242.4. TGF-� system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

2.4.1. Structure and activation of TGF-���������������������������������������������������������������2.4.2. TGF-� receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262.4.3. Biological effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262.4.4. Expression in circulation and in skin . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

2.5. Dermatitis herpetiformis skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292.5.1. Epidermal BMZ in dermatitis herpetiformis skin . . . . . . . . . . . . . . . . . 30

2.6. Cutaneous reepithelialization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312.6.1. Reepithelialization rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312.6.2. BMZ components, integrins and tenascin-C in reepithelialization . . . . 32

2.7. Psoriasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342.7.1. TGF-� system and psoriasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

2.8. Pathophysiology of acne . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352.9. Isotretinoin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

2.9.1. TGF-� and isotretinoin action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362.10.Topical glucocorticoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

2.10.1. TGF-� and glucocorticoid action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382.11.Calcipotriol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

3. Aims of the study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394. Methods and materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

4.1. Suction blister method (II, IV, V) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404.2. Immunohistochemistry (I, III, IV, V) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

4.2.1. Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414.2.2. Immunoperoxidase method (I) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424.2.3. Indirect immunofluorescence method (III, IV, V) . . . . . . . . . . . . . . . . . 424.2.4. Direct immunofluorescence method (III) . . . . . . . . . . . . . . . . . . . . . . . 43

4.3. Lectinhistochemistry (III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434.4. Electron microscopy (III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434.5. Quantitative sandwich enzyme immunoassay technique (II) . . . . . . . . . . . . . . . 43

4.5.1. Human TGF-�1 immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434.5.2. Human TGF-�2 immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

4.6. Suction blister fluid samples in conjunction with topical glucocorticoid treatment (II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

4.7. Suction blister fluid and serum samples in conjunctionwith oral isotretinoin treatment (II) 45

4.8. Tissue samples (I, III, IV, V) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454.8.1. Normal skin controls (I, III), psoriasis (I) and

dermatitis herpetiformis (III) skin samples . . . . . . . . . . . . . . . . . . . . . . 454.8.2. Specimens of normal skin by body site (IV) . . . . . . . . . . . . . . . . . . . . . 464.8.3. Intact blisters and open wounds (IV, V) . . . . . . . . . . . . . . . . . . . . . . . . 464.8.4. Calcipotriol-pretreated and vehicle-pretreated open wounds (IV) . . . . 47

4.9. Statistical methods (II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

5.1. Distribution of TGF-� receptors I and II in normal epidermisand in psoriatic epidermis (I) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

5.2. TGF-�1 in healthy skin and modulation by topical glucocorticoid treatment and ageing (II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

5.3. Modulation of TGF-�1 and TGF-�2 in suction blister fluid and serum by oral isotretinoin in acne patients (II) 51

5.4. BMZ in uninvolved dermatitis herpetiformis skin (III) . . . . . . . . . . . . . . . . . . . 515.4.1. Expression of BMZ components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

5.4.1.1. BP230 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525.4.1.2. Plectin/HD1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525.4.1.3. BP180, �6 integrin, �4 integrin, nidogen, laminin 5,

laminin �3, laminin �1�1, collagen �1�2 (IV)and type VII collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

5.4.2. Ultrastructure of inner hemidesmosomal plaque . . . . . . . . . . . . . . . . . . 535.5. Reepithelialization rate in intact blisters, open wounds and

calcipotriol-pretreated open wounds (IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

5.6. Expression of BMZ components and epithelial integrins duringreepithelialization in intact blisters and open wounds (IV, V) . . . . . . . . . . . . . . 545.6.1. BP180, BP230, and plectin/HD1 (IV) . . . . . . . . . . . . . . . . . . . . . . . . . . 545.6.2. �4, �5, �v�5, �5, and �9 integrins, type VII collagen

and tenascin-C (IV, V) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555.6.3. Laminin 5, laminin �5, and laminin �1 (IV) . . . . . . . . . . . . . . . . . . . . . 565.6.4. BP180, BP230, and plectin/HD1 in different body sites (IV) . . . . . . . . 56

5.7. Effect of calcipotriol pretreatment on BP180, BP230, plectin/HD1, �4 integrin, laminin 5 and tenascin-C expression in regenerating and normal skin (IV) . . . . . . . . . . . . . . . . . . . . . . . . 57

6. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 586.1. TGF-�s and TGF-� receptors I and II in normal skin,

serum, and psoriatic skin (I, II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 586.2. Effect of oral isotretinoin and topical betamethasone

on TGF-� in suction blister fluid and serum (II) . . . . . . . . . . . . . . . . . . . . . . . . 616.3. Integrity of BMZ in uninvolved dermatitis

herpetiformis skin (III, IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626.4. Reepithelialization rate in intact blisters and open wounds

and modulation by calcipotriol (IV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 646.5. BMZ components and epithelial integrins in reepithelialization

in intact blisters and open wounds (IV, V) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 657. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 688. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

1. Introduction

Transforming growth factor-�s (TGF-�s) are cytokines that show a multitude of effectson the cellular differentiation and growth of almost all cell types. TGF-�s exert theireffects via transmembrane protein receptors, TGF-� type I (T�R-I) and TGF-� type II(T�R-II) receptors, both of which are required for TGF-� signalling through the receptorcomplex. (Taipale et al. 1998.) Hemidesmosome (BP180, BP230, plectin/HD1, �6�4integrin), anchoring filaments (laminin 5), and anchoring fibrils (collagen VII) constitutea functional unit called �hemidesmosomal adhesion complex�, which provides stableadherence of keratinocytes to the underlying epidermal basement membrane (Borradori& Sonnenberg 1999). Collagen IV, laminins, and nidogen are components of theepidermal basement membrane, integrins function as cell adhesion molecules, andtenascin-C is an extracellular matrix protein (Burgeson & Christiano 1997, Eble 1997,Vollmer 1997). Psoriasis, dermatitis herpetiformis, and reepithelialization all showcharacteristic epidermal changes, i.e. hyperproliferation and loss of differentiation, blisterformation, migration and hyperproliferation, respectively (van de Kerkhof & van Erp1996, Garrett 1997, Wojnarowska et al. 1998). Topical betamethasone, oral isotretinoin,and topical calcipotriol are drugs that are extensively used in clinical practice (Orfanos etal. 1997, Griffiths & Wilkinson 1998, van de Kerkhof 1998).

The purpose of the present studies was to examine the role of the TGF-� system,basement membrane zone (BMZ) proteins and integrins in the above epidermal changes.The effects of betamethasone and isotretinoin on the expression of TGF-� in skin andserum were also investigated, as were the effects of calcipotriol on the reepithelializationrate and on the expression of BMZ proteins during reepithelialization.Immunohistochemical techniques, electron microscopy, enzyme-linked immunoassaysand in vivo experiments were combined to elicit novel information on these issues.

2. Review of the literature

2.1. Skin

Skin can be divided into an outer epithelial component, epidermis, and an underlyingconnective tissue, dermis. The two are separated by the basement membrane zone. (Chan1997.)

2.1.1. Epidermis

Epidermis is a stratified squamous epithelium, and consists predominantly of keratinocytelayers. It also contains melanocytes, Langerhans cells and Merkel cells. Epidermis can bedivided into four layers: the basal cell layer, squamous cell layer, granular cell layer andstratum corneum. Basal cells are columnar in shape and form a single layer on thebasement membrane. Squamous cells are polygonal and form a mosaic pattern of cells,usually five to ten layers thick. They become flattened toward the surface, with their longaxes arranged parallel to the skin surface. Granular cells are diamond-shaped or flattened.Their cytoplasm is filled with keratohyaline granules. The granular layer is only one tothree cell layers thick in areas where the stratum corneum is thin, but measures up to tenlayers in areas with a thick stratum corneum. The cells of stratum corneum are anuclearand arranged in orderly vertical stacks. Desquamation of corneocytes takes place in theupper portion of stratum corneum. (Lever & Schaumburg-Lever 1990a.)

The proliferative cell compartment of normal epidermis includes all cells of the basalcell layer and approximately 2/3 of the cells in the first suprabasal cell layer. Thedifferentiated cell compartment consists of all viable cells above the first suprabasal celllayer plus approximately 1/3 of the cells in the first suprabasal cell layer. The meanepidermal turnover time is 39 days, consisting of 13 days for the proliferativecompartment, 12 days for the differentiated compartment, and 14 days for stratumcorneum. (Weinstein et al. 1984.) Epithelial stem cells are cells that remain in the tissueand retain their ability to proliferate throughout life. The division of stem cells generatesstem cells and transit-amplifying cells. Transit-amplifying cells undergo several rounds of

19

division, after which all of their daughters become committed to terminal differentiationand leave the basal cell layer. In the hair-bearing skin, which covers most body sites, stemcells lie where the epidermis is thinnest, overlying the dermal papillae. Stem cells divideless frequently than transit-amplifying cells in vivo. Regions of normal epidermis with ahigh proportion of basal cells in mitosis alternate with areas with a much lower mitoticindex. (Jones 1997.)

Epidermis has a multiplicity of functions: It prevents the inward or outward passage ofwater and electrolytes and arrests the penetration of micro-organisms and destructivechemicals. It plays a role in immunological host defense, absorbs ultraviolet radiation,and exerts sensory functions. (Archer 1998.)

2.1.2. Dermis

Dermis largely consists of ground substance, in which polysaccharides and protein formproteoglycan macromolecules, which attract and retain water. Running through andattached to this matrix are protein fibers of several kinds, such as interstitial collagenfibers, and elastic fibers, which are composed of elastin and microfibrils. The size andarrangement of collagen fibers distinguishes two main regions in dermis. Papillary der-mis includes subepidermal papillae between the rete ridges and the subpapillary layerforming a narrow ribbon between the rete ridges and the subpapillary blood vessels. Theremaining part of dermis is referred to as reticular dermis. The collagen fibril diameterincreases progressively from papillary to reticular dermis. The predominant cellular ele-ments are fibroblasts, which secrete extracellular matrix proteins. The other cells includemast cells, macrophages, lymphocytes and other leukocytes, and melanocytes. Dermisalso contains blood and lymph vessels, nerves, nerve endorgans and epidermal appendag-es. (Lever & Schaumburg-Lever 1990a, Eady et al. 1998.)

Embryonal stratum germinativum, or the basal cell layer, gives rise to keratinizing epi-dermis but also to epidermal appendages, i.e. hair, sweat and sebaceous glands, all ofwhich are situated in dermis. Sebaceous glands are usually found in association with hairstructures. Pilosebaceous units involving follicles that produce small hairs but are associ-ated with large sebaceous glands are called sebaceous follicles. Sebaceous follicles have afollicular canal lined by keratinizing stratified squamous epithelium, a fine vellus-likehair and multilobar sebaceous glands. The glands are connected to the follicular canalthrough short sebaceous ducts lined by keratinizing stratified squamous epithelium. Seba-ceous glands are composed of undifferentiated, differentiated, and mature cells. Undiffer-entiated cells are located at the periphery of the gland adjacent to the basement mem-brane. As cell division takes place at the periphery, undifferentiated cells are pushedtoward the sebaceous duct and develop into differentiated lipid-containing cells. Theincreased concentration of lipids in mature cells leads to cell disintegration and the forma-tion of sebum. (Lever & Schaumburg-Lever 1990a,b, Leyden 1995, Cunliffe & Simpson1998.)

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2.1.3. Basement membrane zone

The cutaneous BMZ is a 0.5- to 1.0-�m-thick band-like ultrastructurally defined area. Itcan be divided into four distinct areas: hemidesmosome, lamina lucida, lamina densa, andsub-lamina densa. Hemidesmosomes are multiprotein complexes at the undersurface ofbasal keratinocytes. Lamina lucida is an electron-lucent area between thehemidesmosome and the electron-dense lamina densa layer of the basement membrane.Fine filamentous structures, known as anchoring filaments, traverse lamina lucida andconnect hemidesmosomes to lamina densa. Sub-lamina densa contains fibrillar structures,known as anchoring fibrils, which connect lamina densa to the upper papillary dermis.Both keratinocytes and fibroblasts synthesize protein components of BMZ. The majorfunction of cutaneous BMZ is to serve as an adherent connection between epidermis anddermis. It restricts the transit of molecules between epidermis and dermis on the basis ofsize and charge, but permits the passage of migrating cells under normal (i.e. melanocytesand Langerhans cells) or pathological (i.e. lymphocytes, neutrophils and tumor cells)conditions. It also influences the behaviour of keratinocytes by modulating cell polarity,proliferation, migration, and differentiation. (Burgeson & Christiano 1997, Chan 1997.)

2.2. Molecular components of epidermal BMZ

2.2.1. Hemidesmosomal adhesion complex

Hemidesmosome, anchoring filaments and anchoring fibrils constitute a functional unitcalled �hemidesmosomal adhesion complex�, which provides stable adherence ofkeratinocytes to the underlying epidermal basement membrane (Fig. 1) (Borradori &Sonnenberg 1999).

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Fig. 1. Hemidesmosomal adhesion complex. Modified from Borradori & Sonnenberg 1999.

2.2.1.1. BP230, plectin/HD1, BP180 and �6�4 integrin(hemidesmosome)

Under an electron microscope, hemidesmosomes are distinguishable by their electron-dense cytoplasmic inner and outer plaque and a sub-basal dense plate, which occurs in theupper lamina lucida of the basement membrane. Five major components ofhemidesmosomes have been identified: cytoplasmic plaque proteins plectin/HD1 andBP230 (bullous pemphigoid antigen 1), which link keratin intermediate filaments tohemidesmosome, and transmembrane integrin �6�4 heterodimer and BP180 (bullouspemphigoid antigen 2, collagen XVII), which serve as cell receptors connecting the cellinterior to the extracellular matrix. Recent findings suggest that HD1 and plectin are thesame protein (Okumura et al. 1999). (Burgeson & Christiano 1997, Hirako & Owaribe1998, Jones et al. 1998, Moll & Moll 1998, Borradori & Sonnenberg 1999.)

BP230 is located at the inner hemidesmosomal plaque and is involved in the linkage ofthe keratin cytoskeleton to the inner plaque. In BP230 knockout mice, skin blistering dueto basal cell rupturing parallel to and just above the cell base was detected. The innerplate was lacking, and the mutant hemidesmosomes did not associate with keratinfilaments. Keratinocyte growth and the attachment of hemidesmosomes to the basementmembrane appeared unperturbed, while retardation of reepithelialization was noted. (Guo

22

et al. 1995.) In bullous pemphigoid, where blister formation takes place in lamina lucida,autoantibodies occur against BP230. The question of whether they are a secondarymanifestation or the cause of bullous pemphigoid, however, remains unclear. BP230interacts with BP180 and probably also with �4 integrin. (Jones et al. 1998, Moll & Moll1998, Borradori & Sonnenberg 1999.)

Plectin/HD1 is located at the inner hemidesmosomal plaque and is involved in thelinkage of the keratin cytoskeleton to the inner plaque. Plectin/HD1 gene mutations,which cause tissue separation within basal keratinocytes at the level of the innerhemidesmosomal plaque in epidermolysis bullosa patients, demonstrate the importance ofthis protein in providing integrity within basal keratinocytes. In these patients, aconsiderable reduction in the number of inner plaques and impaired keratin filamentattachment have been detected by electron microscopy (McMillan et al. 1998). Plectin/HD1 interacts with �4 integrin and possibly with BP180. (Uitto & Pulkkinen 1996,Burgeson & Christiano 1997, Hirako & Owaribe 1998, Borradori & Sonnenberg 1999.)

Transmembrane BP180 consists of extracellular and cytoplasmic domains. Theextracellular domain extends to the lamina densa and may partly constitute the anchoringfilaments. Its ligand(s) remain(s) to be identified. The extracellular domain may undergoproteolytic processing resulting in the formation of a 120 kilodalton (kDa) fragment,which is incorporated into the basement membrane and may have cell adhesion properties(Tasanen et al. 2000). BP180 is a target molecule in a subtype of cicatricial pemphigoid,in which blistering skin shows separation within the lamina densa. BP180 is one of thetwo main target molecules in bullous pemphigoid, in which blister formation takes placewithin lamina lucida (Schumann et al. 2000). The serum level of anti-BP180autoantibodies correlates with disease severity. BP180 gene defects in epidermolysisbullosa patients cause dermo-epidermal cleavage within lamina lucida. However, in short-term adhesion assays the initial adhesion of BP180-deficient keratinocytes, which werederived from an epidermolysis bullosa patient, to extracellular matrix proteins (laminin 1,laminin 5, fibronectin, type IV and V collagens) was not substantially impaired(Borradori et al. 1998). BP180 can associate with �6�4 integrin and interacts with BP230and probably also with plectin/HD1. In a previous study, where biopsies from freshsuction blisters from three volunteers were immunostained with bullous pemphigoidautoantisera (BPA), positive staining was detected only in the blister roof, while theblister floor remained negative (Woodley et al. 1983). Regional variation has beendescribed in the expression of bullous pemphigoid antigen with patient sera (Goldberg etal. 1984). (Hirako & Owaribe 1998, Moll & Moll 1998, Shimizu 1998, Borradori &Sonnenberg 1999.)

In contrast to most integrins associated with the actin cytoskeleton, the �6�4 integrinis found to be concentrated in hemidesmosomes at sites where keratin filaments attach.The cytoplasmic domain of the �4 subunit interacts with plectin/HD1 and BP180, andpossibly with BP230. The extracellular domain of �6 is thought to interact with theextracellular domain of BP180. It has been shown that cells which overexpress the tailless�4 molecule do not show defective interaction with laminins, e.g. laminin 5, in short-termadhesion assays, although the hemidesmosome assembly is inhibited and the cells haveless adhesive morphology compared to control cells (Spinardi et al. 1995). This suggeststhat the main function of hemidesmosomes is to stabilize the �6�4-mediated adhesion tothe basement membrane. Accumulation of �6�4 integrin at points called stable anchoring

23

contacts, where BPA labeling is usually codistributed with �6�4, was only detected innonmotile keratinocytes in culture (Carter et al. 1990). Mutations in the �6 and �4 genesin epidermolysis bullosa patients cause dermo-epidermal cleavage within lamina lucida.Integrin �6�4 binds with high affinity to laminin-5, which is enriched in the cutaneousbasement membrane. (Giancotti 1996, Jones et al. 1998, Borradori & Sonnenberg 1999.)

2.2.1.2. Laminin 5 (anchoring filaments) and other lamininswithin the BMZ

Laminins are heterotrimers constituted by the association of three different gene products,the �, � and � chains. Laminin 5 (�3�3�2) is concentrated below the hemidesmosomalplaques. It traverses lamina lucida and connects hemidesmosomal �6�4 integrin to typeVII collagen, the major constituent of anchoring fibrils. Laminin 5 is involved in theassembly of hemidesmosomes. Laminin 5 gene mutations, which cause dermo-epidermalcleavage within lamina lucida, demonstrate the importance of laminin 5 for keratinocyteanchorage to the epidermal basement membrane. The anchoring function of laminin 5 ispreferentially mediated by hemidesmosomal �6�4 integrin. As a subunit of the laminin5-laminin 6 (�3�1�1) complex, laminin 5 is also a strong ligand forinterhemidesmosomal �3�1 integrin (Champliaud et al. 1996). The �1 chain of laminin 6connects the laminin 5-laminin 6 complex to the collagen IV network via nidogen.Integrin �3�1 is situated along the basolateral surface of the basal keratinocytes andassociated with actin-containing focal contacts. The migration properties of laminin 5 aremediated preferentially by �3�1 integrin. Exogenous and endogenous laminin 5 can bothinhibit and promote keratinocyte migration (Zhang & Kramer 1996, O�Toole et al. 1997).After secretion, laminin 5 undergoes complex extracellular enzymatic processing of its�3 and �2 chains. This processing results in laminin 5 subunit polypeptides of differentforms, a fact that may partly explain the multifunctional nature of laminin 5. It has beenshown that laminin 5, which contains a �3 subunit of 160 kDa, induces the formation ofhemidesmosomes in epithelial cells and retards their motility, whereas laminin 5 proteincomplexes, which contain a 190 kDa unprocessed �3 laminin subunit, promote cellmotility (Goldfinger et al. 1998). Cleavage of the laminin �2 chain has been suggested totrigger epithelial cell migration on laminin 5 (Koshikawa et al. 2000). There is ampleevidence for the existence of laminins other than laminins 5 and 6 within the dermo-epidermal junction. There is increasing evidence for the presence of an �5-chaincontaining laminin, probably laminin 10 (�5�1�1). (Miner et al. 1997, Virtanen et al.2000.) The integrins �3�1 and �6�4 are potential receptors for laminin 10 (Eble et al.1998, Kikkawa et al. 1998, Kikkawa et al. 2000). The recently described laminin �3chain is seen within the basement membrane of the dermo-epidermal junction at points ofnerve penetration (Koch et al. 1999). (Burgeson & Christiano 1997, Aumailley & Smyth1998, Jones et al. 1998, Borradori & Sonnenberg 1999.)

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2.2.1.3. Type VII collagen (anchoring fibrils)

Anchoring fibrils are composed predominantly, if not exclusively, of type VII collagen.Type VII collagen is composed of three identical �1 (VII) polypeptides. Anchoring fibrilsoriginate and terminate in lamina densa, forming individual semicircular loops in upperpapillary dermis, which constitute a network of anchoring fibrils that stabilize the BMZ.In the basement membrane, type VII collagen binds to type IV collagen within laminadensa and to laminin 5 in lamina lucida. Type VII collagen gene mutations cause tissueseparation below lamina densa in dystrophic epidermolysis bullosa patients, anddemonstrate the importance of type VII collagen for BMZ stability. In epidermolysisbullosa acquisita, autoantibodies against type VII collagen lead to skin blistering.(Burgeson & Christiano 1997, Shimizu 1998.)

2.2.2. Nidogen and collagen IV

The major constituents of all basement membranes are the laminin and collagen IVnetworks. These two networks apparently have only weak affinity for each other. Theyare connected and stabilized by nidogen, which also binds to other basement membraneproteins. High-affinity binding of nidogen to laminins involves a single binding site onthe laminin �1 chain, which is why all laminins that contain �1 chain are potential ligandsfor nidogen. The most widely expressed form of collagen IV trimer is composed of two�1(IV) chains and one �2(IV) chain. The collagen IV network is formed by self-assembly and is localized predominantly to lamina densa. It is considered to beresponsible for the mechanical stability of basement membranes. In addition to the�1(IV) and �2(IV) chains, �5(IV) and �6(IV) chains have also been detected in thebasement membrane of normal skin (Ninomiya et al. 1995, Tanaka et al. 1997). (Timpl &Brown 1996, Burgeson & Christiano 1997, Mayer et al. 1998).

2.3. Epithelial integrins (�v�5, �5, �9) and tenascin-C

Integrins are a family of transmembrane receptors, which mediate cell-matrix and cell-cell adhesion in various cell types, including epithelial cells. Each integrin consists of aheterodimer of an � and � subunit. The subunits are transmembrane glycoproteins thatrange in size from 95 kDa to 210 kDa. The extracellular domain binds to various ligands,including extracellular matrix proteins, and to other cell surface receptors. Thecytoplasmic domain interacts with cytoskeletal proteins. The association of the twosubunits is necessary for the expression of integrin on the cell surface. In addition to theirfunction as adhesion receptors, integrins are also known to function as signalingreceptors, participating in a diverse array of cellular effects, including spreading,migration, proliferation, differentiation, and cell survival. Integrins have been shown toregulate the expression of a number of genes, including those encodingmetalloproteinases and cytokines. (Meredith et al. 1996, Sheppard 1996, Eble 1997.)

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Vitronectin, a soluble serum factor, has been shown to serve as a ligand for �v�5integrin and to use �v�5 integrin on human keratinocyte cell surfaces in mediating skinkeratinocyte migration (Kim et al. 1994). Restoration of �v�5 expression in neoplasticoral keratinocytes has been shown to result in increased capacity for terminaldifferentiation and suppression of anchorage-independent growth (Jones et al. 1996). Thecytoplasmic domain of the �5 subunit has been shown to increase cell migration(Pasqualini & Hemler 1994). The �5 subunit is known to pair up only with the �vsubunit, whereas the �v subunit is able to associate with several � subunits (Berman &Kozlova 2000). In immunohistochemical studies of normal human epidermis, no ornegligible positivity has been detected for the �v�5 complex using P1F6 antibody (Clarket al. 1996, Haapasalmi et al. 1996), and no or weak positivity in basal keratinocytes hasbeen detected for the �v subunit (Hertle et al. 1992, Cavani et al. 1993, Haapasalmi et al.1996), while distinct positivity has been detected for the �5 subunit in the basal cell layer(Pasqualini et al. 1993). The �5 subunit associates only with the �1 subunit. Fibronectinserves as a ligand for �5�1 integrin. In immunohistochemical studies no positivity(Cavani et al. 1993, using P1D6 antibody, Pellegrini et al. 1992), weak positivity in thebasal cell layer (Hertle et al. 1992), and distinct positivity in the basal cell layer have beendetected for �5 integrin in normal human epidermis (Clark et al. 1996). The �9 subunitassociates with the �1 subunit to form �9�1 integrin. Its specific in vivo functions are stillunknown. Tenascin-C is a well characterized ligand for �9�1 integrin (Yokosaki et al.1998). In immunohistochemical studies, �9 integrin has been described to be abundantlyexpressed in the basal cell layer in normal mouse epidermis (Palmer et al. 1993, Stepp etal. 1995). (Eble 1997).

Tenascin-C is a large extracellular matrix glycoprotein expressed in multiple isoformsproduced by alternative splicing of FN type III repeats. The role of tenascin-C in vivo isstill a matter of debate. Tenascin-C has been suggested to have anti-adhesive or adhesion-modulating effects, especially in conjunction with fibronectin. Tenascin-C appears to bindto the cell surface through both nonintegrin and integrin receptors, e.g. �v�6 and �9�1integrins (Prieto et al. 1993, Yokosaki et al. 1994, Haapasalmi et al. 1996). Theexpression of tenascin-C is stimulated by several growth factors, including TGF-�1.Mechanical force has been shown to induce tenascin-C production in chick embryofibroblasts (Chiquet-Ehrismann et al. 1994). Both keratinocytes and dermal fibroblastsare considered to be able to produce tenascin-C. In normal skin, tenascin-C is detected inpapillary dermis, where it is sparsely distributed immediately beneath the basementmembrane (Lightner et al. 1989). Tenascin-C expression is upregulated in papillarydermis in a number of skin diseases involving epidermal hyperproliferation, such aspsoriasis and epidermal tumors, in wound healing, and in perilesional skin inepidermolysis bullosa patients (Stamp 1989, Schalkwijk et al. 1991, Schenk et al. 1995).(Vollmer 1997, Latijnhouwers et al. 1997, Tuominen et al. 1997).

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2.4. TGF-� system

2.4.1. Structure and activation of TGF-�

Three mammalian TGF-� isoforms (TGF-�1-3) have been discovered, of which TGF-�1has been most extensively studied. TGF-�s are secreted from cells in either small (100kDa) or large (220 kDa) latent complexes. Small complexes contain the active TGF-� (25kDa) and its prodomain, the latency-associated protein (LAP). Large complexes containthe active TGF-�, LAP and the latent TGF-�-binding protein (LTBP). Most cell linessecrete large latent TGF-� complexes. Association of small latent TGF-� with LTBPresults in rapid secretion of the complex. LTBPs are important in targeting TGF-� to theextracellular matrix, although more than 90% of LTBP is not bound to TGF-� andprobably serves a structural role in the extracellular matrix. The regulation of TGF-�activity in tissues is still poorly understood. Proteases are thought to be in a key role inreleasing TGF-� from the matrix and in the activation of latent TGF-� by dissociation ofLAP from active TGF-�. Integrin �v�6 has been shown to bind to TGF-�1-LAP and isconsidered to be able to activate endogenous latent TGF-�1 (Munger et al. 1999). (Clark& Coker 1998, Roberts 1998, Taipale et al. 1998.)

2.4.2. TGF-� receptors

TGF-� signalling is mediated by the transmembrane receptors T�R-I and T�R-II. T�R-II,to which TGF-� is bound, associates with T�R-I to form a signalling complex. T�R-I isnot expressed in cells defective of T�R-II. Endoglin is a non-signalling receptor which isexpressed particularly on endothelial cells. T�R-I, T�R-II and endoglin bind to TGF-�1and TGF-�3 with higher affinity than they show in binding to TGF-�2. (Bassing et al.1994, Taipale et al. 1998, Wrana 1998.)

2.4.3. Biological effects

TGF-�s exert potent regulatory effects during embryogenesis and show a multitude ofeffects on cellular differentiation and growth in the adult. Human TGF-� isoforms exertsimilar, but not identical, biological activities. The three major biological effects of TGF-� are: 1) inhibition of the growth of epithelial, endothelial, and hematopoietic cells, 2)stimulation of extracellular matrix formation, and 3) immunosuppression. TGF-�1reversibly inhibits the growth of human keratinocytes in culture (Shipley et al. 1986).TGF-�1 either enhances or inhibits the differentiation of human keratinocytes in culture,depending on the culture medium. TGF-� may either stimulate or inhibit the proliferationof fibroblasts, and stimulates the synthesis of multiple extracellular matrix components,e.g. collagens, fibronectin, vitronectin, tenascin and laminin 5. TGF-� suppresses matrixdegradation by downregulating the expression of proteinases and by inducing proteaseinhibitors. TGF-�s stimulate new bone formation (Bostrom & Asnis 1998). In growing

27

keratinocyte colonies, TGF-�1 upregulates and downregulates integrin receptors andinduces hemidesmosomal �6�4 integrin to lose its basal topography and becomepericellular (Zambruno et al. 1995). Ectopic expression of �5 integrin has been shown toincrease the expression of T�R-II messenger ribonucleic acid (mRNA) and protein(Wang et al. 1999). (Reiss & Sartorelli 1987, Matsumoto et al. 1990, Clark & Coker1998, Roberts 1998, Taipale et al. 1998.)

Transgenic and knockout mice give valuable information on the in vivo functions ofTGF-�s and their receptors in skin. In TGF-�1-null mice, massive inflammatory lesionswere detected in many organs (Kulkarni et al. 1993), while skin alterations were limitedto epidermal hyperproliferation without significant histological changes (Glick et al.1993). In transgenic mice which overexpressed TGF-�1 in epidermis, the proliferativecapacity of epidermal basal cells was almost nonexistent, and a reduced number of hairfollicles were detected (Sellheyer et al. 1993). Unexpectedly, in another study wheretransgenic mice overexpressed TGF-�1 in epidermis suprabasally, an increase in theproliferative rate was detected without significant histological changes (Cui et al. 1995).In transgenic mice that overexpressed a dominant negative-T�R-II in the epidermis,hyperplastic and hyperkeratotic epidermis and an increased rate of proliferation andexpansion of the proliferative compartment were detected (Wang et al. 1997). In anotherstudy, where transgenic mice overexpressed a dominant negative-T�R-II in the basal cellcompartment and in the follicular cells of the skin, histology as well as both proliferationand differentiation were normal, but an increase in carcinoma incidence was seen(Amendt et al. 1998).

2.4.4. Expression in circulation and in skin

Platelets and bone are the major sources of human TGF-�1, while bone is the majorsource of human TGF-�2. Human platelets contain TGF-�1 in a latent form, but do notcontain TGF-�2 (Cheifetz et al. 1987, Grainger et al. 1995). In a previous study, normalhuman subjects had 4.1 2.0 ng/ml total (i.e. latent + active) TGF-�1, <0.2 ng/ml totalTGF-�2, and <0.1 ng/ml total TGF-�3 in their plasma, and 84.1 32.4 ng/ml total TGF-�1 in their serum. Plasma TGF-�1 levels showed no significant changes related to age orhormonal status, but any given individual showed up to 3-fold fluctuations during the 2-year study period. (Wakefield et al. 1995.) In another previous study, normal humansubjects were described to have 56 ng/ml total TGF-�1 in their serum (Bonifati et al.1996). In a recent study using an enzyme-linked immunosorbent assay kit from the samemanufacturer as was used in article II, a surprisingly low serum TGF-�1 concentration(0.04 ng/ml) was reported, while suction blister fluid TGF-�1 remained below thesensitivity level of 5.0 pg/ml. The authors of the above study did not specify whether ornot TGF-�1 activation had been done or how the serum samples had been collected.(Giacalone et al. 1998.) Plasma TGF-�1 may be in the form of a small or large latentcomplex, or possibly bound to a carrier molecule. TGF-�1 is transferred transplacentallyto fetuses and the transferred protein appears to play a critical role in embryogenesis

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(Letterio et al. 1994). For total TGF-�2, a 150 pg/ml serum concentration and a 1300 pg/ml plasma concentration have been reported in healthy controls (Stoiser et al. 1998,Grainger et al. 1999). (Roberts 1998.)

In situ hybridization and immunohistochemical studies of normal human skin giveinformation on the in vivo role of TGF-�s and their receptors in maintaining thehomeostasis of healthy skin. In several studies, negligible TGF-�1 mRNA expression hasbeen detected in epidermis and dermis (Schmid et al. 1993a, Zhang et al. 1995, Schmid etal. 1996), while one study reported TGF-�1 mRNA positivity in the cytoplasm ofepithelial cells of all skin adnexa, especially in the outer root sheath (Gruschwitz et al.1990). Immunohistochemical studies show controversial results on the tissue distributionof TGF-�s in skin. In several immunohistochemical studies, epidermis has been describedto be negative for the TGF-�1 protein (Falanga et al. 1992, Wataya-Kaneda et al. 1994,Ciernik et al. 1995, Zhang et al. 1995, Karonen et al. 1997). In two studies, however,immunoreactivity for intracellular TGF-�1 was detected in the suprabasal keratinocytelayers, while epidermis remained negative for separately studied extracellular TGF-�1(Kane et al. 1990, Stamp et al. 1993). In one study, TGF-�1 was detected intracellularlyin basal and suprabasal keratinocytes (Raghunath et al. 1998), while in another study itwas seen in epidermis without specifying the location in detail (Rudnicka et al. 1994). Inseveral studies, dermis has been described to be negative for the TGF-�1 protein (Kane etal. 1990, Rudnicka et al. 1994, Wataya-Kaneda et al. 1994, Zhang et al. 1995). In a fewother studies, the TGF-�1 protein has been detected in the dermal matrix to variableextents (Falanga et al. 1992, Stamp et al. 1993, Raghunath et al. 1998). In these studies,TGF-�1 has also been detected in dermal structures other than the extracellular matrix,such as vascular cells (Falanga et al. 1992, Stamp et al. 1993), the outer root sheath andnerve fibres (Stamp et al. 1993), sweat ducts (Falanga et al. 1992), and smooth musclecells (Raghunath et al. 1998).

Negligible TGF-�2 mRNA expression has been detected in epidermis and dermis(Schmid et al. 1993a, Schmid et al. 1996), although the epithelial cells of skin adnexahave shown TGF-�2 mRNA positivity (Gruschwitz et al. 1990, Schmid et al. 1996). In afew immunohistochemical studies TGF-�2 was not detectable in epidermis (Falanga et al.1992, Ciernik et al. 1995, Zhang et al. 1995), whereas in some other studies it was(Rudnicka et al. 1994, Wataya-Kaneda et al. 1994). Several studies have reported noimmunoreactivity for TGF-�2 in dermis (Rudnicka et al. 1994, Wataya-Kaneda et al.1994, Zhang et al. 1995), while in one study the dermal matrix and vascular cellscontained minimally detectable amounts of TGF-�2 (Falanga et al. 1992).

TGF-�3 mRNA has been clearly detectable in all epidermal keratinocytes (Schmid etal. 1993a, Schmid et al. 1996) and in hair follicle epithelia (Schmid et al. 1996), butnegligible TGF-�3 mRNA has been detected in dermis (Schmid et al. 1996). In twostudies TGF-�3 protein was clearly detectable in epidermis (Schmid et al. 1993a, Schmidet al. 1996) and in hair follicle epithelia (Schmid et al. 1996), while in one studyepidermis was negative for TGF-�3 (Wataya-Kaneda et al. 1994). In one study TGF-�3was detectable in a small number of dermal cells (Schmid et al. 1996), while in anotherstudy it was detected in the subepidermal portion of dermis diffusely, but no specific cellsreactive to TGF-�3 antibody were identified (Wataya-Kaneda et al. 1994).

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LTBP-1 immunoreactivity has been shown to co-localize with elastic fibers throughoutdermis (Karonen et al. 1997, Raghunath et al. 1998). In addition, LTBP-1 has also beendetected in the walls of some blood vessels and smooth muscle cells, while epidermisremains negative. TGF-�1 and LTBP-1 co-localized to the same individual fibrillin-containing microfibril components of elastic fibers. (Raghunath et al. 1998.)

The expression of T�R-I and T�R-II proteins and mRNAs was studied in formalin-fixed paraffin sections of human normal skin by Schmid et al. (1998). T�R-I mRNA wasvisible in epidermis and epidermal appendages. In dermis, weak hybridization signalsappeared in vascular structures and a minority of stromal fibroblasts. T�R-Iimmunostaining was detectable in epidermis, hair follicle epithelia and vascular cells.Only a subset of stromal cells within the papillary and reticular parts of dermis showedT�R-I.

T�R-II mRNA has been found in epidermis (Schmid et al. 1993, Matsuura et al. 1994,Schmid et al. 1996, Schmid et al. 1998) and in epidermal appendages of normal humanskin (Matsuura et al. 1994, Schmid et al. 1998). Weak T�R-II signals have beendescribed to appear in vascular structures and a minority of stromal fibroblasts (Schmid etal. 1998) and heterogeneously among dermal cells (Schmid et al. 1996), while nosignificant signals were seen in dermal fibroblasts or endothelial cells in one study(Matsuura et al. 1994). T�R-II immunostaining was detectable in epidermis, hair follicleepithelia, and vascular cells, while only a subset of stromal cells within the papillary andreticular dermis showed detectable immunoreactivity for T�R-II (Schmid et al. 1998).

In a previous study, endoglin was not found in any structures in normal skin (Cierniket al. 1995), while another study reported minimal staining in endothelial cells (Westphalet al. 1993).

2.5. Dermatitis herpetiformis skin

Dermatitis herpetiformis is clinically characterized by pruritic papulovesicles on extensorsurfaces and gluten-sensitive enteropathy, which may be asymptomatic. In addition to theextensor surface of the elbows and knees and the buttocks, eruptions of dermatitisherpetiformis may also be seen on the face and scalp and at sites of pressure fromclothing, e.g. tight belts. The blisters are most commonly present within urticarialplaques, but may also arise from otherwise normal-appearing skin. The blisters oftenoccur in groups and are usually small, 2-5 mm in diameter. Larger blisters areoccasionally seen, but they are usually less than 1 cm in diameter. The blisters are oftenexcoriated. The initial histologic lesion is a neutrophilic cell infiltrate and oedema indermal papillae. As microabscesses form, a separation develops between the tips of thedermal papillae and the overlying epidermis. As the microabscesses coalesce, blisterformation takes place. Neutrophils are the predominant cells in the blister, but someeosinophils are often also present. Subjacent to this, a perivascular infiltrate composed oflymphocytes, neutrophils, and eosinophils may be seen. In uninvolved skin, thecharacteristic immunopathological finding is granular deposition of IgA in the dermalpapillae beneath the epidermal basement membrane or continuous granular deposition ofIgA in the upper dermis beneath the basement membrane. IgM, IgG and C3 may

30

occasionally be found at the same location as IgA. IgA is found in uninvolved skin inpatients in spontaneous remission and in ones whose rash is controlled by drug treatment.IgA is also present in patients whose rash is controlled by a gluten-free diet, although itmay disappear in some individuals after many years of this treatment. The possiblebinding site of IgA in skin has remained unknown. A variety of circulating organ-specificautoantibodies have been detected in dermatitis herpetiformis patients, while IgAautoantibodies against skin components are notably absent. The role of deposited IgA, ifany, in the blister formation in dermatitis herpetiformis, is unknown. Increased expressionof degrading proteases has been reported in blistering skin in situ and also in blister fluid,suggesting involvement in tissue degradation and blister formation (Oikarinen et al.1983a, 1986a, Airola et al. 1995, 1997). (Smith & Zone 1993, Fry 1995, Wojnarowska etal. 1998.)

2.5.1. Epidermal BMZ in dermatitis herpetiformis skin

Immunohistochemical analyses of the cleavage plane of dermatitis herpetiformis blistersusing type IV collagen and laminin antisera and serum from bullous pemphigoid andepidermolysis bullosa acquisita patients have suggested that dermatitis herpetiformisblisters form within the lamina lucida layer of the basement membrane (Hertz et al. 1976,Klein et al. 1983, Karttunen et al. 1984, Pardo & Penneys 1990, Smith et al. 1992).Electron microscopy studies have suggested considerable variability in the location of thecleavage site. For instance, in a previous electron microscopy study of five cases ofdermatitis herpetiformis, the location of basal lamina in the blister area alternated fromthe blister roof to the blister base (Horiguchi et al. 1987). The ultrastructure ofhemidesmosomes has been reported to be normal in uninvolved and erythematousdermatitis herpetiformis skin (Riches et al. 1976). The integrity of BMZ in uninvolveddermatitis herpetiformis skin has not been studied with immunohistochemical methods.Indirect conclusions regarding the integrity of BMZ in uninvolved skin may be drawnfrom studies where BMZ proteins have been studied in blistering dermatitis herpetiformisskin and, in some cases, also in adjacent skin by immunohistochemistry. Variousantibodies recognizing laminin and collagen IV, various BPA presumably recognizingBP230 and/or BP180, one epidermolysis bullosa acquisita serum presumably recognizingcollagen VII, one antibody recognizing laminin 5, and one antibody recognizing collagenVII have been used in these studies. No findings have been reported that would suggestany major alterations in the integrity of BMZ in uninvolved skin compared to normalskin. (Hertz et al. 1976, Klein et al. 1983, Karttunen et al. 1984, Pardo & Penneys 1990,Smith et al. 1992, Airola et al. 1997.)

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2.6. Cutaneous reepithelialization

Epidermal wound healing includes keratinocyte migration, proliferation anddifferentiation. Uninjured keratinocytes along wound edges and the edges of hair folliclestumps are involved in covering the wound bed. There is a delay period, usually 18-24hours, before the onset of migration and enhanced proliferation. To be able to migrate,keratinocytes adopt an elongated morphology, reorganize the cytoskeletal filaments, andbreak free from their cell-adhering structures, e.g. desmosomes and hemidesmosomes,during the delay period. Initially, migration involves basal and suprabasal keratinocytes atthe proximal wound and hair follicle stump margins, and later on, same cells at the tip ofthe epithelial tongue. The foremost epidermal cells extend pseudopods which attach tothe substratum. The trailing portion of these cells then moves forward with epidermalcells positioned above and behind them. Subsequently, elongated extensions of overlyingflattened epidermal cells roll or slide over the basal cells and attach to the substratum.The composition of the substratum varies, mainly as far as the depth of the wound isconcerned, and is considered to have an effect on the keratinocyte phenotype, e.g.integrin expression and enzyme production. Enhanced proliferation of epidermal cellsstarts some hours after the onset of migration and occurs distally to the migrating cellsboth in the epithelial tongue and in the wound margin. After coverage of the wound floor,the enhanced proliferation still continues as the wound epithelium undergoesstratification. (Krawczyk 1971, Garlick & Taichman 1994, Coulombe 1997, Garrett 1997,Martin 1997, Pilcher et al. 1998.)

2.6.1. Reepithelialization rate

Reepithelialization has been studied in intact blisters and open wounds in 2-day-old miceusing suction-induced blisters where dermo-epidermal separation takes place withinlamina lucida. In both model systems, the pattern of movement appeared to be similar tothe above discussed �leap-frogging�. Reepithelialization was considerably faster in intactblisters than in open wounds. De novo formation of hemidesmosomes was detected alongthe basal plasma membrane of the foremost epidermal cell in intact blisters but not inopen wounds. In intact blisters, reepithelialization took place on a retained basementmembrane. Soon after removal of the blister roof in open wounds, no basementmembrane was detected anymore and the reepitheliating keratinocytes were exposed tothe dermal environment. (Krawczyk 1971.) Hemidesmosome morphogenesis occurs infour steps in the most forward migrating keratinocyte in the above discussed intactblisters: (1) extension of extracellular fine filaments from the basal plasma membrane tolamina densa; (2) appearance of a sub-basal dense plate within the extracellular filaments;(3) formation of a cytoplasmic outer plaque; (4) formation of an inner plaque andinsertion of tonofilaments (Krawczyk & Wilgram 1973). In humans with full-thicknesswounds, it has been demonstrated that occluded wounds heal faster than nonoccludedwounds (Nemeth et al. 1991).

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2.6.2. BMZ components, integrins and tenascin-C in reepithelialization

During reepithelialization, keratinocytes use integrin adhesion molecules in cell-cellinteraction and to interact with several matrix proteins, including various collagen types,fibronectin, vitronectin, tenascin-C, and laminins (Larjava et al. 1996). Integrin �4 hasbeen detected around basal and suprabasal keratinocytes at the leading edge in onesuction blister study (Kainulainen et al. 1998), while in another suction blister study, both�6 and �4 integrins were detected in pericellular distribution in the basal layer withrelative concentration at BMZ in both migrating and normal epidermis (Hertle et al.1992). No distinction between open wounds and intact blisters was made in either study.In full-thickness human cutaneous wounds, �6 and �4 labeling appeared to be clearlypolarized at the basal pole of migrating basal keratinocytes in most specimens, and thelateral and apical surfaces of basal keratinocytes and several layers of suprabasalkeratinocytes were also labeled, although to a lesser extent (Cavani et al. 1993). In an invitro wound healing model using bovine corneal explants, both �6 and �4 integrins weredetected along the entire cell surface in actively migrating epithelial cells, while BPAlabeling was only barely detectable. Antibodies to �4 integrin did not interfere withepithelial cell migration in the above explant. (Kurpakus et al. 1991.)

Laminin �2 chain and mRNA have been detected at the leading edge in healing suctionblisters. In addition, �2 immunoreactivity was detected in the suction blister floor, in thebasal keratinocytes of detached epidermis, and under regenerating epidermis, where itwas more intense than in normal skin. (Kainulainen et al. 1998.) Polyclonal laminin 1antibody, which presumably detects �1 and �1 chains in human cutaneous basementmembrane, showed positivity only in the blister base and under regenerating epidermis ina previous suction blister study (Foidart et al. 1980, Oikarinen et al. 1982). In a recentstudy, the basement membrane of normal human skin was negative for the unprocessedlaminin �3 chain (190 kDa), whereas strong positivity for this unprocessed laminin �3chain was detected in the basement membrane beneath migrating keratinocytes in healinghuman skin wounds. The authors proposed the following model to explain the role oflaminin 5 and its receptors during wound healing: Upon wounding, laminin 5 productionis upregulated and/or there is a concomitant downregulation of �3 chain proteolysis at thewound edge, resulting in an increase in the presence of unprocessed �3 laminin subunit inlaminin 5 protein complexes at the leading edge of the wound. At the leading edge �3�1integrin binds to laminin 5 containing an unprocessed �3 subunit, which promotes cellmigration over the wound bed. In the same cells, �6�4 integrin concentrates on the lateralcell surfaces. In cells some distance away from the tip of the leading edge, �6�4 integrinlocates not only at sites of cell-cell contact but also along the basal surface, where it bindsto laminin 5 containing the processed �3 subunit (160 kDa), in partially formedhemidesmosomes. (Goldfinger et al. 1999.)

Band-like BMZ positivity has been described for BP180 beneath the entire length ofthe epithelial outgrowth in oral human mucosal wounds, whereas diluted BPA, which wasthought to recognize BP230, showed no positivity in the epithelial outgrowth region(Dabelsteen et al. 1998). In wounded mouse corneas, BPA labeling has been detecteddiffusely within the cytoplasm of migrating cells at the leading edge in contrast to normalmouse corneas, where BPA labeling localizes along the basal cell membrane of basal cells(Gipson et al. 1993). In contrast to unwounded rat cornea, where plectin/HD1 appears in

33

a linear staining pattern above the basement membrane, regenerating epithelium in ratcorneas showed plectin/HD1 staining that was somewhat diffuse and appeareddiscontinuous at some locations, but still appeared to be present preferentially at the basalaspect of the basal cells (Stepp et al. 1996). In a human in vitro reepithelialization model,collagen type VII was deposited underneath the migrating tip in partial thickness wounds(Jansson et al. 1996). Disruption of type VII collagen has been observed during blisterhealing in the blister base in dermatitis herpetiformis blisters, which form within thelamina lucida (Airola et al. 1997).

The �9 subunit of �9�1 integrin has been described to be upregulated in regeneratingepithelium in mouse cornea, using a wound model where reepithelialization takes placeon intact basement membrane (Stepp & Zhu 1997). An increase in the expression of the�v�5 complex has been detected with the P1F6 antibody predominantly in basalkeratinocytes in migrating epidermis during excisional but not incisional human woundhealing (Clark et al. 1996, Haapasalmi et al. 1996). Increased immunoreactivity for the�v subunit has been detected predominantly in basal cells in regenerating epidermis ofhuman full-thickness wounds (Cavani et al. 1993, Clark et al. 1996, Haapasalmi et al.1996). In a previous suction blister study, no upregulation of �v subunit was detected inregenerating epidermis (Hertle et al. 1992). Upregulation of the �5 subunit has beendetected in regenerating human bronchial epithelium in a xenograft model (Pilewski et al.1997). In �5-deficient mice, the rate of healing of cutaneous wounds was not altered,although keratinocytes harvested from these mice demonstrated impaired migration onand adhesion to vitronectin (Huang et al. 2000). In full-thickness wounds using the P1D6antibody, �5 integrin has been shown to be upregulated in the basal keratinocytes of themigrating epidermis. Interestingly, another �5 antibody showed no reactivity in theregenerating epidermis in the same study. (Cavani et al. 1993.) In another study usingexcisional wounds and in a previous suction blister study, no upregulation of �5 integrinwas detected in regenerating epidermis (Hertle et al. 1992, Clark et al. 1996). In freshsuction blister fluid, fibronectin has been observed as intact proteins and, later duringregeneration, in the blister base as dense aggregates (Oikarinen et al. 1982, Grinnell et al.1992). In newly reepithelialized suction blisters, fibronectin was present in dermis and theBMZ in the same distribution as in unwounded epidermis (Hertle et al. 1992). Suctionblister fluid has been shown to contain intact vitronectin (Grinnell et al. 1992). In full-thickness wounds, fibronectin and vitronectin are upregulated under the tongue of thenewly forming epidermis, in addition which fibronectin is also upregulated in the plainwound bed (Cavani et al. 1993).

Tenascin-C expression was found to be increased in upper dermis at wound marginsand beneath the entire epidermal tongue in a recent study using partial thickness humanskin wounds. In the latter region, tenascin-C immunoreactivity was detected as a thindiscontinuous line. When reepithelialization was complete, tenascin-C immunostaining indermis was not only visible at the dermo-epidermal junction but also in the granulationtissue. Some immunostaining was also seen in the sheet of migrating keratinocytes and inneo-epidermis. During reepithelialization, tenascin-C mRNA was detected mostly in thebasal cells of the epidermal tongue. When reepithelialization was complete, granulationtissue was formed and the number of tenascin-C mRNA positive cells increased indermis. (Latijnhouwers et al. 1997.)

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In growing human keratinocyte colonies, TGF-�1 has been shown to upregulate theexpression of the �5�1 and �v�5 integrins and to stimulate keratinocyte migration(Nickoloff et al. 1988, Zambruno et al. 1995). TGF-�1 synthesis has been shown to occurat the leading edge in acute human skin wounds (Schmid et al. 1993).

2.7. Psoriasis

Psoriasis is a common disease with a prevalence of up to 3.0% in many populations. It isbelieved to be a multigene disease, in which expression is partly dependent on externalfactors. The clinical features of elevated, erythematous, scaly plaques reflect threeessential underlying pathophysiological processes: 1) Epidermal hyperproliferation andloss of differentiation, 2) angiogenesis and dilatation of dermal blood vessels, and 3)infiltration of inflammatory cells in both the dermal and epidermal compartments of skin.Lesional epidermis is thickened with elongation of the rete ridges and thinning of thesuprapapillary plate. The superficial layers of epidermis are characterized by focalabsence of the granular layer with parakeratotic stratum corneum characterized byremnants of nuclei. Increased activation of resting cells to become cycling epidermal cellswith normal cell cycle times is considered to account for the hyperproliferation ofepidermis in psoriatic lesions. The inflammatory infiltrate is composed of T-lymphocytes,monocytes, macrophages, mast cells and polymorphonuclear leukocytes. Vasodilatation,papillary oedema and leukocyte infiltrates appear to precede epidermal changes in early,developing lesions. Clinically uninvolved psoriatic skin has also been shown to differfrom normal skin in many respects. Among other things, slight epidermalhyperproliferation, altered keratin expression in epidermis, an increased number of T-lymphocytes in dermis, increased fibroblast proliferation, vasodilatation, abnormalpresence of plasma fibronectin in epidermis, and overexpression of fibronectin receptor�5�1 integrin in epidermis have been described (Weinstein et al. 1984, Thewes et al.1991, Bata-Csorgo et al. 1998). (van de Kerkhof & van Erp 1996, Barker 1998, Bos & deRie 1999, Nickoloff 1999.)

The enigma in the pathogenesis of psoriasis is whether psoriasis results from a primaryabnormality in epidermis or in immune system or both. Current research seems toadvocate the hypothesis that psoriasis is a T-cell-mediated disorder. T-cells are a majorconstituent of the inflammatory infiltrate in papillary dermis, and they are also present inhyperproliferating epidermis. The precise mechanism by which activated psoriatic T-cellstrigger psoriasis is unknown, but it may involve release of several cytokines which inducekeratinocyte hyperproliferation in psoriatic epidermis. Cytokines from lesional T-lymphocytes have been shown to stimulate proliferation among psoriatic uninvolved, butnot normal, stem keratinocytes (Bata-Csorgo et al. 1995). The mechanisms leading to T-cell activation and proliferation also remain speculative. (Barker 1998, Bos & de Rie1999, Nickoloff 1999.)

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2.7.1. TGF-� system and psoriasis

TGF-�1 has been shown to inhibit the proliferation of psoriatic lesional keratinocytes tothe same extent as that of normal keratinocytes (Malkani et al. 1993). In an in vitro organculture, addition of TGF-� caused thinning of both uninvolved and involved psoriaticepidermal specimens, but did not cause granular layers to appear in the involved psoriaticepidermis (Kondo et al. 1992). T-lymphocytes from a lesional psoriatic skin biopsy havebeen shown to express TGF-� mRNA (Lemster et al. 1995). TGF-�1, TGF-�2 and TGF-�3 have been shown to be able to block the lymphocyte adhesiveness of dermalmicrovascular endothelial cells isolated from normal skin, but not from psoriatic plaques,presumably due to the reduction of TGF-� receptor expression and function that wasdetected in psoriatic endothelial cells (Cai et al. 1996). Psoriatic fibroblasts fromuninvolved and involved skin have been shown to have a greater proliferative response toTGF-� compared to normal fibroblasts (Espinoza et al. 1994).

In a previous immunohistochemical study, two different antibodies were used to detectTGF-�1 protein in psoriatic epidermis. One antibody stained suprabasal keratinocytesintracellularly in normal skin, but did not stain psoriatic plaques. The other antibody didnot stain normal skin, but showed extracellular and intracellular staining in suprabasalcell layers in lesional epidermis. Both antibodies stained suprabasal keratinocytesintracellularly in uninvolved psoriatic epidermis. (Kane et al. 1991.) In anotherimmunohistochemical study, anti-TGF-�2-LAP antibody showed decreased expression,especially in the lower part of the lesional epidermis, while the immunoreactivities forTGF-�1-LAP and TGF-�3-LAP in psoriatic skin remained unchanged compared tonormal skin (Wataya-Kaneda et al. 1996). The levels of TGF-�1 mRNA have been shownto be comparable in normal, uninvolved, and lesional psoriatic epidermis (Elder et al.1989). In a previous in situ hybridization study, very weak TGF-�1 mRNA hybridizationsignals and distinct TGF-�3 mRNA hybridization signals were detected in lesional, non-lesional, and normal epidermis, while no TGF-�2 hybridization signals were detected ineither lesional, non-lesional, or normal epidermis (Schmid et al. 1993b). In another in situhybridization study, no difference was detected in the expression of T�R-II mRNAbetween normal and psoriatic lesional skin (Matsuura et al. 1994). The expression ofendoglin has been shown to be upregulated in endothelial cells in lesional, but not inuninvolved, psoriatic skin by immunoelectron microscopy (Westphal et al. 1993). In animmunohistochemical study, endoglin expression was upregulated in lesional psoriaticskin, while in uninvolved psoriatic skin it was downregulated compared to normal skin(Rulo et al. 1995). The level of serum total TGF-�1 has been shown to be increased inpsoriasis patients (91 ng/ml) compared to controls (56 ng/ml) (Bonifati et al. 1996).

2.8. Pathophysiology of acne

Acne is characterized by abnormal desquamation of sebaceous follicle epithelium(comedogenesis), dermal inflammation, excessive sebum production, and abnormality ofthe microbial flora. Comedogenesis occurs specifically in sebaceous follicles (2.1.2.).Comedones represent the retention of hyperproliferating follicular keratinocytes. Among

36

other factors, local production of cytokines in keratinocytes has been implicated in theinduction of hyperproliferation, and may also be involved in the induction of the earlydermal inflammation when the follicular epithelium has not yet ruptured. An addition oftransforming growth factor-� to the medium of an in vitro model of acne infundibulumresulted in rupturing of the infundibulum similar to that seen in the severe forms of acne.(Leyden 1995, Guy et al. 1996, Cunliffe & Simpson 1998, Guy & Kealey 1998.)

2.9. Isotretinoin

�Retinoids� is a generic term that includes both naturally occurring molecules andsynthetic compounds showing specific biological activities resembling those of vitamin A(retinol). Such compounds can exhibit their specific biological activity without beingvitamin A analogues chemically. Also, not all biologically active synthetic retinoids arecarried by cytosolic binding proteins, and binding to or activation of nuclear retinoidreceptors may not be a necessary precondition for their action. Consequently, the resultson one retinoid cannot be generalized to apply to other retinoids. Retinoic acid is a majormetabolite of vitamin A. Its two stereoisomers, isotretinoin (13-cis-retinoic acid) andtretinoin (all-trans-retinoic acid), are normal constituents of human serum. Although oralisotretinoin and topical tretinoin are both used to treat acne, their biological effects andmechanisms of action differ in several ways. For example, tretinoin shows high affinityfor certain nuclear retinoid receptors, while isotretinoin has no special affinity to anyidentified nuclear retinoid receptor. Pretreatment with tretinoin is beneficial for woundhealing following dermabrasion, while pretreatment with isotretinoin often causescomplications (Rubenstein et al. 1986, Mandy 1986). (Orfanos et al. 1997, Saurat 1998.)

Synthesized isotretinoin is extensively used to treat acne and also for chemopreventionof skin cancers. It promotes keratinocyte differentiation and directly suppresses abnormaldesquamation of sebaceous follicle epithelium, proliferation of basal sebocytes and lipidsynthesis of sebocytes. It has anti-inflammatory effects and stimulates extracellularmatrix formation. The side-effects of isotretinoin include hyperostosis and congenitalmalformations, e.g. central nervous system and craniofacial abnormalities. (Orfanos et al.1997.)

2.9.1. TGF-� and isotretinoin action

The exact mechanisms of isotretinoin-induced phenomena are unclear. Studies with otherretinoids imply that modulation of TGF-� expression may be involved. Downregulationof TGF-�2 gene expression has been reported in mouse embryos suffering from tretinoin-induced malformations (Taylor et al. 1995). Retinoic acid has been shown to induce thesecretion of TGF-�1 and TGF-�2 in normal human keratinocytes (Batova et al. 1992).Tretinoin has been shown to enhance the inhibitory effect TGF-�1 on the growth ofhuman keratinocytes (Tong et al. 1990). Treatment of human keratinocytes with tretinoinresulted in a 50% induction of TGF-�1 protein, without any change in TGF-�2. In thesame study, both topical tretinoin and topical irritant resulted in upregulation of

37

extracellular TGF-�1 in epidermis and dermis in vivo, while intracellular TGF-�1 wasdownregulated and no changes were detected in TGF-�2. (Fisher et al. 1992.) In anotherstudy, tretinoin treatment induced TGF-�1 and TGF-�2 expression in humankeratinocytes (Creek et al. 1995). Combination therapy with isotretinoin and �-interferonhas been described to increase circulating TGF-�1 in prostate cancer patients (DiPaola etal. 1997).

2.10. Topical glucocorticoids

Topical glucocorticoids are extensively used in the treatment of many types ofinflammatory dermatosis. Multiple systems are responsible for the transduction ofglucocorticoid effects at the molecular level, depending on the cell type and the activationstate. The two primary modes of action take place directly via interaction withdeoxyribonucleic acid (DNA) or indirectly through modulation of specific transcriptionfactors. Glucocorticoid molecules diffuse across the plasma membrane of the cell andbind to a specific receptor protein, the glucocorticoid receptor, which is present incytoplasm. This hormone binding to the receptor causes increased DNA-binding affinityand leads to accumulation of the steroid-receptor complex in the cell nucleus. Thesteroid-receptor complex modulates the transcription of target genes by binding tospecific DNA-binding sites or, alternatively, binds to other transcription factors andmodulates their capacity to interact with DNA. Other mechanisms of glucocorticoidaction include e.g. ability to reduce mRNA stability. (Almawi et al. 1996, Griffiths &Wilkinson 1998.)

The anti-inflammatory, immunosuppressive, and anti-mitogenic effects ofglucocorticoids are beneficial in treating inflammatory dermatosis. The detrimentaleffects of topical glucocorticoids include epidermal thinning, melanocyte inhibition,reduction in collagen synthesis and ground substance, and rebound vasodilatation, whichmay become permanent in long-term use (Oikarinen et al. 1986b, Oikarinen et al. 1998).In short-term use, topical glucocorticoids produce transient vasoconstriction of thesuperficial small vessels. Glucocorticoid-induced vasoconstriction has been shown tocorrelate well with clinical efficacy, and it has been used in ranking the glucocorticoidsfor potency. Betamethasone-17-valerate 0.1% has been classified as a �potent� topicalglucocorticoid. (Griffiths & Wilkinson 1998.) Glucocorticoid-induced vasoconstrictiondecreases the diffusion of serum proteins into suction blister fluid. The decrease in theconcentration of serum-derived proteins in suction blister fluid has been reported to be10-35% after 4-day clobetasol-17-propionate (classified as a �very potent� topicalglucocorticoid) pretreatment (Kiistala et al. 1986). A 3-day betamethasone-17-valerate0.1% pretreatment has been shown to cause a 10-20% decrease in the proteinconcentration in the suction blister fluid (Haapasaari et al. 1996). No difference wasobserved in the suction blister fluid volume between fresh control blisters and clobetasol-17-propionate-pretreated blisters (Oikarinen et al. 1983b).

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2.10.1. TGF-� and glucocorticoid action

TGF-� may be one mediator of the glucocorticoid effects. The effects of glucocorticoidson the expression of TGF-� are not uniform. Glucocorticoids may induce, suppress, orhave no effect on the expression of TGF-�. The effect of glucocorticoids on theexpression of TGF-� is dependent on the tissue, cell type, experimental conditions, andthe state of cellular activation. (Almawi et al. 1996, Almawi et al. 1998.)

2.11. Calcipotriol

Vitamin D3 analogues have an effect on nearly all cell types, including epidermal cells,fibroblasts and bone marrow-derived cells. Calcipotriol is a vitamin D3 analogue that isextensively used in the topical treatment of psoriasis. Its antiproliferative anddifferentiation-stimulating effects on keratinocytes are considered to be more importantthan its anti-inflammatory effects on dermis in healing psoriasis (Reichrath et al. 1997).Both nuclear and non-nuclear mechanisms are relevant in mediating the effects ofcalcipotriol. Calcipotriol binds to a nuclear receptor protein, the vitamin D3 receptor. Thecomplex consisting of the vitamin D3 receptor and calcipotriol modulates thetranscription of target genes by binding to specific DNA-binding sites. The vitamin D3receptor interacts with other members of the nuclear receptor superfamily, e.g. retinoicacid receptors. The non-nuclear mechanisms include e.g. regulation of intracellularcalcium concentration. (van de Kerkhof 1998.) Depending on the culture conditions,calcipotriol can either inhibit or stimulate the proliferation and differentiation of humankeratinocytes (Svendsen et al. 1997, Kragballe & Wildfang 1990). Calcipotriol has beenshown to enhance the secretion and activity of TGF-�1 and TGF-�2 in murinekeratinocytes (Koli & Keski-Oja 1993). Vitamin D has been shown to have inhibitoryeffects on cell migration (Hansen et al. 1994, Yudoh et al. 1997).

3. Aims of the study

Epidermis is a vital protective shield of the human body. Its growth control takes place tolarge extent in its basal compartment next to dermis. The purpose of this study was toinvestigate epidermal pathology and the mechanisms of clinically used drugs from twodistinct but interactive perspectives: hormonal and structural. Laboratory techniques andin vivo experiments were combined to obtain novel information on the TGF-� system,BMZ proteins and integrins in human skin.

The specific aims of this study were:1. to investigate the role of T�R-I and T�R-II in controlling the growth of normal,

lesional and nonlesional psoriatic epidermis by immunohistochemistry (I).2. to investigate the effects of topical glucocorticoid and oral isotretinoin on the

expression of TGF-�1 and TGF-�2 in suction blister fluid and circulation in vivo (II).3. to investigate the structural integrity of BMZ in uninvolved dermatitis herpetiformis

skin, in order to find out if there are structural alterations that predispose to blisterformation typical of the disease (III).

4. to investigate the reepithelialization rate and the role of BMZ proteins and integrins inreepithelialization in intact blisters and open wounds in vivo (IV, V).

5. to study the effect of calcipotriol pretreatment on the reepithelialization rate and onthe expression of BMZ proteins in reepitheliating skin in vivo (IV).

4. Methods and materials

4.1. Suction blister method (II, IV, V)

In the suction blister method, an adapter plate with five apertures ( 6 mm) covered by achamber is placed on the skin. Negative pressure is communicated into the chamber,which results in suction-induced separation of epidermis from dermis at the level of thelamina lucida layer of the basement membrane. Subsequently, interstitial fluidaccumulates from the surrounding tissue to the dermo-epidermal space. The basementmembrane forms the suction blister floor, on which reepithelialization takes place. Thebasal plasma membranes of epidermal basal cells form the suction blister roof. Suctionblister induction is painless. The lesions heal without scarring, but somehyperpigmentation may remain for up to a few months. Suction blister fluid can beregarded as representative of interstitial fluid. The blister fluid/serum concentration ratioof proteins depends on their molecular weight and follows mainly the law of diffusion.The blister fluid/serum concentration ratio of a protein decreases logarithmically as themolecular weight of the protein increases. Using the concentrations of circulating TGF-�and suction blister fluid TGF-�, it is possible to estimate the proportion of TGF-� that isderived from circulation into the suction blister fluid and the proportion released locallyinto the suction blister fluid. (Kiistala & Mustakallio 1967, Kiistala 1968, Herfst & vanRees 1978, Vermeer et al. 1979, Rossing & Worm 1981, Staberg et al. 1983.)

In this study, suction blisters were induced on topical betamethasone-pretreatedhealthy skin (II), oral isotretinoin-pretreated uninvolved acne skin (II), uninvolved acneskin (II), vehicle-pretreated healthy skin (IV), calcipotriol-pretreated healthy skin (IV),and healthy skin (II, IV, V). The test sites were located on lower abdominal skin (II, IV,V), where 200-400 mmHg negative pressure was used to induce blisters. For each suctionblister fluid sample, 5-10 intact blisters were aspirated and the fluids were pooled.

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4.2. Immunohistochemistry (I, III, IV, V)

4.2.1. Antibodies

All the antibodies used in the immunohistochemical studies are listed in Table 1 (I, III,IV, V).

Table 1. Antibodies used in the immunohistochemical studies, their specificities, andreferences or sources.

Antibody Spesificity PC/MC Reference or source

1D1 BP180 MC Owaribe et al. 1991

IE5 BP230 MC Owaribe et al. 1991

HD-121 Plectin/HD1 MC Hieda et al. 1992; Okumura et al. 1999

7A8 Plectin/HD1 MC Foisner et al. 1991

AA3 �4 integrin MC Tamura et al. 1990

GoH3 �6 integrin MC Sonnenberg et al. 1987

BIE5 �5 integrin MC Werb et al. 1989

P1D6 �5 integrin MC Chemicon, Temecula, CA, USA

Y9A2 �9 integrin MC Wang et al. 1996

P1F6 �v�5 complex MC Wayner et al. 1991

IA9 �5 integrin MC Pasqualini et al. 1993

Pc kalinin 4101 Laminin-5 (�3�3�2) PC Rousselle et al. 1991

BM-2 Laminin �3-chain MC Rousselle et al. 1991

4C7 Laminin �5-chain MC Tiger et al. 1997

114DG10 Laminin �1-chain MC Virtanen et al. 1997

1141 Laminin 1 (�1�1�1) PC Liesi et al. 1983

A9 Nidogen MC Katz et al. 1991

M3F7 Collagen �1�2 (IV) MC Foellmer et al. 1983

NP32 Collagen VII MC Sakai et al. 1986

BC-4 Tenascin-C isoforms, detects EGF-like repeats MC Siri et al. 1991; Balza et al. 1993

Anti-human IgA serum Human IgA PC Dako, Glostrup, Denmark

V-22 T�R-I PC Santa Cruz Biotechnology, Santa Cruz, CA, USA

L-21 T�R-II PC Santa Cruz Biotechnology

MC = monoclonal; PC = polyclonal

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4.2.2. Immunoperoxidase method (I)

The immunoperoxidase method was used in conjunction with the V-22 and L-21antibodies (Table 1). The frozen tissue specimens were sectioned at 6 �m and fixed inacetone precooled to -20oC for 10 min. Histostain SP reagents (Zymed Laboratories, SanFrancisco, CA) were used. In this method, the fixed specimens were first washed withphosphate-buffered saline (PBS) and then treated with 0.6 % H2O2 in methanol for 15min in order to block the endogenous peroxidase activity. After washing with PBS, thesections were incubated with non-immune sheep serum for 15 min and with the primaryantiserum for 30 min. This was followed by first washing with PBS and then incubationwith biotinylated goat anti-rabbit antibody (Zymed) for 15 min. Subsequently, thesections were once again washed with PBS, incubated with streptavidin-peroxidasecomplex (Zymed) for 15 min, and washed with PBS. The immunoreactivities weredetected by a substrate solution containing H2O2 and 3-amino-9-ethylcarbazole in 0.1mol/L acetate buffer, pH 5.2. The sections were washed with distilled water and brieflycounterstained with hematoxylin diluted with distilled water 1:5, washed with water, andembedded in water-soluble GVA-Mount embedding agent (Zymed). Control stainingsincluded staining of normal human skin and lesional psoriatic skin by replacing thespecific antisera with normal rabbit serum or by omitting the specific antiserum. In theseexperiments, no staining reactions were seen. In peptide inhibition experiments with amolar excess (>10:1) of synthetic peptides used to produce the antisera (Santa Cruz), noimmunoreaction was seen in normal human skin, which further demonstrates thespecificity of the antisera V-22 and L-21.

4.2.3. Indirect immunofluorescence method (III, IV, V)

The indirect immunofluorescence method was used in conjunction with all the otherantibodies mentioned in Table 1 except V-22, L-21 and anti-human IgA serum. Frozentissue specimens were first sectioned at 6 �m and fixed in acetone precooled to -20�C for10 min and washed with PBS. The sections were then incubated with the respectiveprimary antibody for 30 min, washed with PBS, and subsequently incubated withfluorescein isothiocyanate (FITC)-coupled goat anti-mouse IgG-antiserum, FITC-coupledgoat anti-rat IgG-antiserum or FITC-coupled goat anti-rabbit IgG-antiserum for 30 min(Jackson Immunoresearch; West Grove, PA), and once again washed with PBS. Thespecimens were embedded in sodium veronal-glycerol buffer (1:1) and examined using aLeitz Aristoplan microscope. The control stainings included staining of normal humanskin (III), uninvolved dermatitis herpetiformis skin (III) and wound samples (IV, V) byomitting the specific antiserum. No immunofluorescence reactions were seen in theseexperiments. The dermal structures in wound samples and other human tissue samplesserved as positive controls with some antibodies (IV).

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4.2.4. Direct immunofluorescence method (III)

For direct immunofluorescence testing, the frozen tissue specimens were sectioned at 5�m and dried at -20�C. The sections were incubated with FITC-coupled rabbit anti-human IgA serum, washed with PBS, dried at room temperature and embedded in FAmounting fluid (Difco Laboratories, Mich., USA) at pH 7.2.

4.3. Lectinhistochemistry (III)

Some specimens stained for indirect immunofluorescence were further incubated withtetramethyl rhodamine isothiocyanate-conjugated Ulex europaeus I agglutinin (UEA-I)(Vector Laboratories, Burlingame, CA) prior to embedding to visualize endothelia(Holthöfer et al. 1982).

4.4. Electron microscopy (III)

Fresh tissues were immediately minced into small pieces and fixed with freshly prepared5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, at 4�C. Fixed tissues were treatedwith 4% tannic acid in 0.1M phosphate buffer, pH 7.4, for 30 min at room temperature.Samples were then postfixed in 2% OsO4, dehydrated, and embedded in Epon. Thinsections were poststained with uranyl acetate and lead citrate and viewed in a JEOL 100Selectron microscope.

4.5. Quantitative sandwich enzyme immunoassay technique (II)

4.5.1. Human TGF-�1 immunoassay

Commercial immunoassay (R&D SYSTEMS) was used to detect active TGF-�1 insuction blister fluid samples and serum samples. All instructions given by themanufacturer for sample collection, storage and assay procedure were followed. Prior toTGF-�1 determinations, 1 N HCl (suction blister fluid samples) or 2.5 N Acetic acid/10M Urea (serum samples) was added to activate latent TGF-�s into an immunoreactiveactive form. The acidified samples were neutralized by adding 1.2 N NaOH/0.5 MHEPES (suction blister fluid samples) or 2.7 N NaOH/1 M HEPES (serum samples).Subsequently, standards and samples were pipetted into wells pre-coated with TGF-�1-binding T�R-IIs. After incubation and washing away of any unbound substances, anenzyme-linked polyclonal antibody specific for TGF-�1 was added to the wells tosandwich the TGF-�1 immobilized during the first incubation. Following a wash toremove any unbound antibody-enzyme reagent, a substrate solution (hydrogen peroxideand tetramethylbenzidine) was added to the wells, and color developed in proportion to

44

the amount of TGF-�1 bound at the initial step. The color development was stopped byadding sulphuric acid, and the intensity of color was measured using a microplate reader.In two non-pretreated (young males) suction blister fluid samples, TGF-�1determinations were also made prior to activation, thus giving information on theproportion of active TGF-�1 in skin.

4.5.2. Human TGF-�2 immunoassay

Commercial immunoassay (R&D SYSTEMS) was used to detect active TGF-�2 insuction blister fluid samples and serum samples. All instructions given by themanufacturer concerning sample collection, storage and assay procedure were followed.Prior to TGF-�2 determinations, all samples were acidified and neutralized in the sameway as the suction blister fluid TGF-�1 samples (see 4.5.1.) to activate latent TGF-�2 toan immunoreactive active form. Subsequently, both standards and samples were pipettedinto wells pre-coated with a monoclonal antibody specific for TGF-�2. After washingaway of any unbound substances, a peroxidase-linked polyclonal antibody specific forTGF-�2 was added to the wells. Following a wash, a substrate solution (hydrogenperoxide and tetramethylbenzidine) was added to the wells, and color developed inproportion to the amount of TGF-�2 bound at the initial step. The color development wasstopped by adding sulphuric acid, and the intensity of the color was measured using amicroplate reader.

4.6. Suction blister fluid samples in conjunction with topical glucocorticoid treatment (II)

Adult male volunteers (n = 9) in two age groups, the younger (n = 4) ranging from 21 to26 years (mean 23) and the older (n = 5) from 55 to 70 years (mean 64), were pretreatedwith 0.1% betamethasone-17-valerate (CelestodermTM, Schering-Plough) twice a day for3 days. Betamethasone was applied to a 12 x 12 cm area on the abdominal skin. The non-pretreated opposite side served as a control. Suction blisters were induced 12 h after thelast application (day 0) and on the 2nd and 7th days after the treatment on both steroid-pretreated and non-pretreated abdominal skin in each volunteer. The suction blisters wereinduced during the daytime, at approximately the same ambient temperature and with thesame amount of negative pressure in all cases. After blister induction, the blister fluidswere immediately aspirated with a micropipette and stored at -70�C until analyzed.

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4.7. Suction blister fluid and serum samples in conjunctionwith oral isotretinoin treatment (II)

All volunteers were males (age 19-24; weight 67-80 kg) free of other systemicmedications and diseases except acne. They did not use any topical treatment onabdominal skin. The isotretinoin treatment was free of charge, which ensured excellentcompliance. Paired suction blister fluid samples were collected from ten male acnepatients before and after six weeks of oral isotretinoin treatment (40 mg/day). Suctionblisters were induced on the lower abdominal skin and the blister fluids were aspiratedimmediately after blister induction and stored at -70�C until analyzed. Paired bloodsamples were drawn from a cubital vein prior to and after the isotretinoin treatment.Samples were collected from ten volunteers for serum TGF-�1 determinations and fromother nine volunteers for serum TGF-�2 determinations. The blood samples for serumTGF-�1 determinations were handled in a special way to ensure complete release ofTGF-�1 from platelets. They were allowed to clot for 1 h at room temperature andsubsequently incubated overnight at +5� C. After the samples had been centrifuged for 10min at 1000 x g, the serum was removed and stored at -70�C until analyzed. The bloodsamples for serum TGF-�2 determination were handled as follows. The samples wereallowed to clot for 30 min before centrifugation for 10 min at approximately 1000 x g.After this, the serum was removed and stored at -70�C until analyzed.

4.8. Tissue samples (I, III, IV, V)

All tissue samples for immunohistochemical experiments were snap-frozen in liquidnitrogen and stored at -70�C until used.

4.8.1. Normal skin controls (I, III), psoriasis (I) and dermatitis herpetiformis (III) skin samples

Five specimens of normal skin were obtained at surgical operations at the Jorvi Hospital(Espoo, Finland) for immunohistochemical studies (I, III). One fresh specimen of normalextensor forearm skin for electron microscopy was taken from a volunteer at theDepartment of Anatomy, University of Helsinki (Helsinki, Finland) (III). Seven lesionaland two non-lesional psoriasis skin specimens were obtained for immunohistochemicalstudies from seven voluntary patients at the Department of Dermatology, HelsinkiUniversity Central Hospital (Helsinki, Finland) (I). The histological psoriasis diagnoseswere made by an experienced dermatopathologist. Ten specimens of uninvolveddermatitis herpetiformis skin for immunofluorescence studies were obtained from thehistopathological archives at the Department of Dermatology, Helsinki University CentralHospital (III). Diagnostic direct IgA immunofluorescence testing of these ten specimenswas performed by an experienced dermatopathologist. One fresh specimen of uninvolveddermatitis herpetiformis skin for electron microscopy was obtained from South Karelia

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Central Hospital (Lappeenranta, Finland) and one from Finnish Student Health Service(Helsinki, Finland) (III). These two specimens were from patients whose specimens wereincluded in the ten above-mentioned specimens from the archives of Helsinki UniversityCentral Hospital.

4.8.2. Specimens of normal skin by body site (IV)

Nineteen biopsies of normal skin were taken for immunohistochemistry from differentbody sites in ten volunteers: proximal extensor arm (5), proximal flexor arm (4), extensorknee (2), fossa poplitea (2), scalp (3), and upper chest (3) (IV). The biopsies were takenat the Department of Dermatology, Central Military Hospital (Helsinki, Finland). Allvolunteers, aged 19 to 24 years, were healthy males with no skin disease and no systemicor topical medication.

4.8.3. Intact blisters and open wounds (IV, V)

Suction blisters were induced contralaterally on the lower abdominal skin in sixvolunteers. All volunteers, aged 19 to 24 years, were healthy males with no skin diseaseand no systemic or topical medication. In three volunteers, the blister roofs wereprotected with foamy plastic rings from mechanical trauma until the biopsies were taken(Table 2). Only intact blister samples were taken. In the other three volunteers, the blisterroofs were removed right after blister induction and the open wounds (Table 2) werecovered with dry wound dressings until the biopsies were taken. The biopsies were takenon the 2nd, 4th and 9th days after blister induction. Every biopsy was cut in half in themiddle of the wounded area. Thus, all the sections studied had a wounded areaapproximately 6 mm wide with one reepithelialization front on both sides or newepidermis on the wounded area in the older samples. The suction blister induction andbiopsies were performed in the Central Military Hospital (Helsinki, Finland).

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Table 2. Number of samples studied per wound type (IV, V).

4.8.4. Calcipotriol-pretreated and vehicle-pretreated open wounds (IV)

Three volunteers participated in a double-blind study. All volunteers were young andhealthy males, with no skin disease and no systemic or topical medication. Two areas of8x8 cm were marked contralaterally on the lower abdominal skin. Every volunteerapplied vehicle to the left-hand side and 50 �g/g calcipotriol cream (Daivonex� cream,Lövens, Denmark) to the right-hand side twice a day for 14 days. Compliance wasmeasured with a diary and ranged from 26/28 to 28/28. After two weeks� pretreatment,suction blisters were induced on both sides and the blister roofs were removedimmediately. The wound areas were covered with dry wound dressings. Biopsies weretaken on the 2nd and 4th days after blister induction (Table 2). Every biopsy was cut inhalf in the middle of the wounded area. Each volunteer served as his own control, asbiopsies were taken from both the calcipotriol- and the vehicle-pretreated sides on eachoccasion in order to exclude any interindividual variation between the two study groups.In all three cases, neither the investigator, nor the subjects were aware of the content ofthe cream used at each side. The code was opened after the findings had been analyzed.

Wound type Day HE BP180 BP230 Plectin �4 �v�5 �5 �5 �9 Ln5 Ln�5 Ln�1 VII TnBlister 2nd 3 3 3 3 3 3 3 2 1 3 2 2 2 3

Open wound 2nd 3 3 3 3 1 3 3 2 2 2 2 2 2 3Blister 4th 3 3 3 3 3 3 3 2 2 2 2 2 2 3

Open wound 4th 3 2 2 2 1 2 2 2 2 2 2 2 2 2

Blister 9th 3 3 2 3 2 3 3 2 1 3Open wound 9th 3 3 2 3 2 3 3 2 1 3

Vehicle:

Open wound 2nd 2 2 2 2 2 2 1Open wound 4th 3 3 3 3 3 1 2 3

Calcipotriol:

Open wound 2nd 2 2 2 2 2 2 1Open wound 4th 3 3 3 3 3 2 3

HE=haematoxylin-eosin; plectin=plectin/HD1; �4=�4 integrin; �v�5=�v�5 integrin; �5=�5 integrin; �5=�5integrin; �9=�9 integrin; Ln5=laminin 5; Ln�5= laminin �5 chain; Ln�1= laminin �1 chain; VII=VII collagen;Tn=tenascin-C; Vehicle=pretreatment with vehicle; Calcipotriol=pretreatment with calcipotriol.

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4.9. Statistical methods (II)

Paired-samples or independent-samples t-test was used to test the statistical significanceof the observed differences between the mean values of the different study groups. Theone-sided p-value was calculated for the difference between active and total suctionblister fluid TGF-�1, while in the other cases the two-sided p-value was used.

The value for suction blister fluid TGF-�1 before the 3-day pretreatment (day �3) inFig. 2 (see 5.2.) is the mean of all the measurements (days 0, 2 and 7) on the non-pretreated side. The other values for suction blister fluid TGF-�1 in Fig. 2 (mean, 1 SD)were calculated from the measurements made in each group (steroid or non-pretreated) ondifferent days (0, 2 and 7). The suction blister fluid TGF-�1 concentration in non-pretreated young versus non-pretreated old volunteers was determined as follows. Themean of the three TGF-�1 determinations on the non-pretreated side (day 0, 2, 7) wascalculated for each volunteer (n = 9) and served as an estimate of the suction blister fluidTGF-�1 concentration for each volunteer when the mean TGF-�1 concentration wascalculated separately for young and old volunteers. The intraindividual variability ofsuction blister fluid TGF-�1 from the non-pretreated side was determined as follows. TheSD of the three TGF-�1 determinations (day 0, 2, 7) was calculated for each volunteer (n= 9). Subsequently, the mean SD was calculated for this sample of nine volunteers.

5. Results

5.1. Distribution of TGF-� receptors I and II in normal epidermisand in psoriatic epidermis (I)

In the epidermis of normal human skin, distinct cell surface-confined and cytoplasmicimmunoreactivity for T�R-I was observed in the basal and often also the suprabasal celllayer (I, Fig. 1). In thin epidermis lacking rete ridges, this was the only reaction observed.In epidermis with well-developed rete ridges, however, a somewhat different pattern ofimmunoreactivity was observed. In these cases the basal and suprabasal cell layers of therete ridge were always immunoreactive for T�R-I, but in some cases even keratinocyteshigher up within the rete ridge showed distinct immunoreactivity. It is conceivable thatthe latter finding at least partly results from tangential sectioning along the edge of therete ridge in question. Immunoreactivity for the signalling receptor, T�R-I, however, didnot extend above the suprabasal cells in the rete ridge-free areas of adjacent epidermis. Inother areas, rete ridges did not reveal staining for T�R-I above suprabasal cells.Immunoreactions for T�R-II, which presents the ligand to T�R-I, were somewhatstronger in the epidermis of normal human skin, but showed a similar distribution as thatof T�R-I.

In lesional skin of psoriatic patients (7/7), immunoreactivity for T�R-I was distinctlyreduced or lacking compared to normal skin, and in all cases studied no or negligibleimmunoreactions were seen in the epidermis (I, Fig. 2). In psoriatic lesions,immunoreactivity for T�R-II was similar to T�R-I with no or negligible reaction. On theother hand, in nonlesional skin of psoriatic patients (2/2) distinct immunoreactions forboth T�R-I and T�R-II were seen, and they were comparable to those seen in normalskin.

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5.2. TGF-�1 in healthy skin and modulation by topical glucocorticoid treatment and ageing (II)

After the 3-day steroid pretreatment (Fig. 2), a statistically significant 17% decline (p =0,00052) from 530 pg/ml (day �3) (see 4.9.) to 440 65 pg/ml (mean SD) (day 0) wasnoted in the suction blister fluid TGF-�1 concentration, whereas the difference on the 2nd

day was not statistically significant (p = 0,21). Complete recovery was seen during the 7-day follow-up. The suction blister fluid TGF-�1 concentration was 590 100 pg/ml(mean SD) in non-pretreated young subjects (n = 4) and 490 88 pg/ml in non-pretreated old subjects (n = 5) (see 4.9.). The difference was not statistically significant, p= 0.14. The median was 580 pg/ml (range 490-710 pg/ml) for non-pretreated young and500 pg/ml (range 350-590 pg/ml) for non-pretreated old subjects.

The suction blister fluid concentration of active TGF-�1 was 44 23 pg/ml (mean, SD)(n = 2, see 4.5.1.). It accounted for approximately 5% of the total suction blister fluidTGF-�1 concentration, 840 510 pg/ml (n = 2, one-sided p = 0.13). Intraindividualvariability (see 4.9.) in suction blister fluid TGF-�1 from the non-pretreated side was 76pg/ml.

Fig. 2. TGF-�1 concentration (mean, 1 SD) in suction blister fluid in steroid-pretreated versusnon-pretreated male volunteers (n = 9).

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5.3. Modulation of TGF-�1 and TGF-�2 in suction blister fluid and serum by oral isotretinoin in acne patients (II)

The TGF-�1 level in serum was 53 9,5 ng/ml (mean SD) (n = 10) before thetreatment and 53 12 ng/ml (n = 10) after the treatment (Table 3). The TGF-�1 level insuction blister fluid was 260 45 pg/ml (mean SD) (n = 10) before the treatment and310 55 pg/ml (n = 10) after the treatment. The 19% increase in the suction blister fluidTGF-�1 concentration was statistically significant, p = 0.037. The TGF-�2 level in serumwas 340 120 pg/ml (mean SD) (n = 9) before the treatment and 370 140 pg/ml (n =9) after the treatment. The difference was not statistically significant, p = 0.20. Altogether12 of the 20 TGF-�2 determinations from suction blister fluid samples (n = 10) takenbefore the isotretinoin treatment and 3 of the 20 TGF-�2 determinations from suctionblister fluid samples (n = 10) taken after the isotretinoin treatment yielded levels lowerthan the sensitivity level of the TGF-�2 immunoassay, which corresponds to 55 pg/ml insuction blister fluid.

Table 3. Effect of isotretinoin on the total TGF-� concentration (mean SD) in suctionblister fluid (SBF) and serum.

5.4. BMZ in uninvolved dermatitis herpetiformis skin (III)

The results on polyclonal antiserum against laminin 1 (�1�1�1) have been reinterpretedto represent the localization of laminin �1�1 (Miner et al. 1997, Tiger et al. 1997,Virtanen et al. 2000).

In direct immunofluorescence, distinct granular deposits of IgA were detected underthe basement membrane in all the specimens of uninvolved dermatitis herpetiformis skin(n=10). Hematoxylin-eosin stainings of all the uninvolved dermatitis herpetiformisspecimens corresponded fully to hematoxylin-eosin stainings of normal skin controls(n=5).

SBF TGF-�1(10 patients)

Serum TGF-�1(10 patients)

Serum TGF-�2 (9 patients)

Before isotretinoin 260 � 45 pg/ml 53 � 9.5 ng/ml 340 � 120 pg/mlAfter isotretinoin 310 � 55 pg/ml 53 � 12 ng/ml 370 � 140 pg/ml

Change in concentration +19% (p = 0.037) - Statistically not significant (p = 0.20)

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5.4.1. Expression of BMZ components

5.4.1.1. BP230

Five controls of normal skin and ten specimens of uninvolved dermatitis herpetiformisskin were stained for BP230 using IE5 antibody (Table 4). In the five controls of normalskin, strong (++) band-like immunofluorescence was seen along BMZ. Four of the tenspecimens of uninvolved dermatitis herpetiformis skin showed strong (++) band-likeimmunofluorescence in BMZ. These findings were similar to those of normal skincontrols. In another four of the ten specimens of uninvolved dermatitis herpetiformisskin, weak (+) band-like immunofluorescence was seen in BMZ. In two of the tenspecimens of uninvolved dermatitis herpetiformis skin, no or negligible (+/-) staining forBP230 was observed in BMZ. Immunohistochemical stainings for BP230 were repeatedthree times in inseparate experiments giving identical results.

5.4.1.2. Plectin/HD1

Five controls of normal skin and ten specimens of uninvolved dermatitis herpetiformisskin were stained with monoclonal antibody HD-121 for plectin/HD1 (Table 4). In thefive controls of normal skin, strong (++) band-like immunofluorescence was seen alongBMZ. Seven of the ten specimens of uninvolved dermatitis herpetiformis skin showedstrong (++) band-like immunofluorescence in BMZ. These findings were similar to thoseof the normal skin controls. In three of the ten specimens of uninvolved dermatitisherpetiformis skin, weak (+) band-like immunofluorescence was detected in BMZ.

Five control specimens of normal skin and ten specimens of uninvolved dermatitisherpetiformis skin were also stained with monoclonal antibody 7A8 for plectin/HD1(Table 4). Two control specimens showed strong (++) immunofluorescence, whereasthree control specimens showed weak (+) band-like immunofluorescence in BMZ with7A8 antibody. Five uninvolved dermatitis herpetiformis skin specimens showed weak (+)immunofluorescence, whereas one uninvolved dermatitis herpetiformis skin specimenshowed strong (++) band-like immunofluorescence in BMZ. Four uninvolved dermatitisherpetiformis skin specimens showed no or negligible (+/-) immunofluorescence in BMZ.

Two control specimens and two uninvolved dermatitis herpetiformis skin specimens(case # 9U, 10U with no or negligible staining for BP230, see Table 4) were double-stained with HD-121 antibody and UEA-I lectin. The latter functioned as a marker forendothelium. Distinct immunofluorescence for plectin/HD1 was detected in theendothelium of the two control specimens, whereas weak or negligible endothelialstaining for plectin/HD1 was observed in the two uninvolved dermatitis herpetiformisskin specimens. Three normal skin controls and three uninvolved dermatitis herpetiformisskin specimens were double-stained with 7A8 antibody and UEA-I lectin. No significantdifferences in endothelial immunoreactivity were seen in these stainings (Table 4).

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5.4.1.3. BP180, �6 integrin, �4 integrin, nidogen, laminin 5,laminin �3, laminin �1�1, collagen �1�2 (IV)

and type VII collagen

Five controls and ten uninvolved dermatitis herpetiformis skin specimens were stainedfor BP180, �6 integrin, �4 integrin, nidogen, laminin 5, laminin �3, laminin �1�1,collagen �1�2 (IV) and type VII collagen. In all the uninvolved dermatitis herpetiformisspecimens studied, strong (++) band-like immunofluorescence was detected in BMZ,fully corresponding to the findings in the respective control specimens.

Table 4. Immunoreactivities for BP230 and plectin/HD1 (HD-121 and 7A8 antibodies) inthe BMZ and endothelium of normal skin and uninvolved dermatitis herpetiformis skin.

5.4.2. Ultrastructure of inner hemidesmosomal plaque

The hemidesmosomal inner plaques were studied in one normal human skin controlspecimen and in two uninvolved dermatitis herpetiformis skin specimens (case # 9U,10U, see Table 4) by electron microscopy. Both cases of dermatitis herpetiformis werenegative for BP230 and showed decreased immunoreactivity for plectin/HD1 inimmunofluorescence. In electron microscopy, the hemidesmosomal inner plaques in thetwo uninvolved dermatitis herpetiformis skin specimens appeared to be similar to theinner plaques in the control specimen (III, Fig. 4).

BP230 Plectin/HD1IE5 ab HD-121 ab 7A8 abBMZ BMZ /endothelium

Controls1C ++ ++ /D ++ /WN2C ++ ++ ++3C ++ ++ /D +4C ++ ++ + /D5C ++ ++ + /WN

Case # 1U ++ ++ +2U ++ ++ +3U ++ ++ +4U ++ ++ ++5U + ++ +/- /WN6U + ++ +7U + ++ +8U + + +/- /WN9U +/- + /WN +/-10U +/- + /WN +/- /WN

Case # 1U - 10U: uninvolved DH skin. Immunofluorescence in BMZ: ++, strong; +, weak; +/-, no or negligible. Immunofluorescence in endothelium: D, distinct; WN, weak or negligible.

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5.5. Reepithelialization rate in intact blisters, open wounds andcalcipotriol-pretreated open wounds (IV)

The normal open wound healing results include the vehicle-pretreated open woundsamples (Table 2). On the 2nd and 4th days, reepithelialization was clearly faster in theopen wounds than in the samples with intact blister roofs (IV, Fig. 1). On the 9th day, allthe wound bases in both open wounds and intact blisters were similarly covered by newacanthotic epidermis. In our double-blind study, calcipotriol pretreatment for 14 days didnot alter the reepithelialization rate on the 4th day in the open wounds compared to thevehicle-pretreated open wounds in the same individuals. The regenerating epidermis incalcipotriol- and vehicle-pretreated skin did not differ in the gross appearance ofepidermal cells and cellular differentiation.

5.6. Expression of BMZ components and epithelial integrins duringreepithelialization in intact blisters and open wounds (IV, V)

The results on normal open wound healing include the vehicle-pretreated open woundsamples. The results on immunofluorescence in 2- and 4-day-old intact blisters and openwounds are shown summarized in Table 5.

5.6.1. BP180, BP230, and plectin/HD1 (IV)

In normal-appearing skin, immunoreactivity for BP180 was detected as a strong band-like pattern in BMZ, and it was often accompanied by weak pericellular/cytoplasmicpositivity in the basal cell layer. Cytoplasmic/pericellular and band-like BMZimmunoreactivity for BP180 ranged from absent to strong at the leading edge of theregenerating epidermis in 2- and 4-day-old blisters and open wounds (IV, Fig. 2). Behindthe leading edge in the 4-day-old samples, weak/strong band-like BMZ immunoreactivitywas detected and accompanied by cytoplasmic/pericellular positivity in the basal celllayer(s) in most cases. BP180 remnants were often seen in 2-day-old blister roofs. In allbut one case, plain (i.e. not yet reepithelialized) wound bases were negative for BP180 inboth wound types.

In normal-appearing skin, strong band-like immunoreactivity for BP230 was detectedin BMZ. The tips of the reepithelialization fronts in the 2-day-old open wound samplesshowed no immunoreactivity or weak cytoplasmic immunoreactivity for BP230 (IV, Fig.2). Behind the tip of the leading edge, discontinuous weak/strong band-like positivity forBP230 was detected in the BMZ of regenerating epidermis, sometimes accompanied byweak cytoplasmic positivity in the basal cell layer. In contrast, the 2-day-old blistersamples showed a stronger and more continuous band-like staining pattern in theregenerating skin, often accompanied by some cytoplasmic positivity. In the 4-day-oldopen wounds and blisters, weak to strong band-like immunoreactivity for BP230 wasdetected in BMZ in the regenerating area.

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In normal-appearing skin, strong band-like immunoreactivity for plectin/HD1 wasdetected in the BMZ. In some cases, weak pericellular/cytoplasmic positivity for plectin/HD1 was observed in the epidermal cell layers in both regenerating and normal skin. Theleading edges of new epidermis in the 2-day-old open wounds were negative or showedvery weak discontinuous band-like positivity in BMZ (IV, Fig. 2). In contrast, the stainingfor plectin/HD1 in the 2-day-old blisters was stronger and more continuous in the BMZ ofregenerating epidermis. The 4-day-old open wounds and blisters showed distinct band-like immunoreactivity for plectin/HD1 in the BMZ of regenerating epidermis. On the 9th

day, the immunoreactivity for plectin/HD1, BP180, and BP230 was similar to that seen innormal skin.

5.6.2. �4, �5, �v�5, �5, and �9 integrins, type VII collagen and tenascin-C (IV, V)

Weak/strong immunoreactivity for �4 integrin was detected at the leading edge in thebasal cell layer and often also in the suprabasal cell layers in 2- and 4-day-old specimens(IV, Fig. 3). In addition, strong band-like reactivity for �4 integrin was detected at thebasal pole of basal keratinocytes at the leading edge in most 2-day-old samples and in all4-day-old samples. The polarized immunostaining for �4 integrin was often discontinu-ous on the 2nd day, but became continuous on the 4th day. On the 9th day, faint pericellu-lar positivity was detectable in the suprabasal cell layers in contrast to normal epidermis.The �4 integrin staining pattern was similar in intact blisters and open wounds. �5 inte-grin was also similarly expressed in both wound types. In adjacent skin, faint or weakimmunoreactivity for �5 integrin was often observed in the basal cell layer. In 2-day-oldsamples, strong or sometimes weak positivity for �5 integrin was detected in the supra-basal cell layers in regenerating epidermis, while no or weak positivity was detected inthe basal cell layer (V, Fig. 1). In 4-day-old samples, strong immunoreactivity for �5 inte-grin was detected in the suprabasal cell layers in regenerating epidermis, while weak ornegligible immunoreactivity for �5 integrin was detected in the basal cell layer. In 9-day-old samples, faint scattered immunoreactivity for �5 integrin was detected in the upperneoepidermis in most cases. The lower neoepidermis was usually negative. In one 9-day-old open wound sample, however, a similar staining pattern for �5 integrin was found asin the 4-day-old samples. No or negligible immunoreactivity was detected for the �v�5complex (IV, Fig. 3) and �5 integrin in regenerating and normal epidermis and for �9integrin in regenerating epidermis. Some specimens showed weak immunoreactivity for�9 integrin in the basal cell layer in normal epidermis.

In wounded skin, the immunoreactivity for type VII collagen corresponded fully tothat in normal skin (IV, Fig. 3). Increased strong immunoreactivity was detected fortenascin-C from day 2 to day 9 in the upper dermis both under the regenerating/regenerated epidermis and in the plain wound bases in open wound and blister samples(IV, Fig. 3). In all but one 4-day-old sample, the plain wound bases showed equally strongimmunoreactivity as the upper dermis under the regenerating/regenerated epidermis. Inmost cases, faint cytoplasmic immunoreactivity for tenascin-C was detected in the lowerepidermal cell layers in regenerating/regenerated epidermis.

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5.6.3. Laminin 5, laminin �5, and laminin �1 (IV)

Strong continuous band-like immunoreactivity for laminin 5, �5, and �1 was detected inthe plain blister floor in the 2- and 4-day-old intact blisters (IV, Fig. 4). In the openwound bases, the laminin 5, �5, and �1 immunostaining was partially discontinuous.Some open wound bases also had areas that completely lacked laminin 5, �5, and �1immunoreactivity. Under regenerating epidermis, a strong band-like BMZ immunoreac-tivity was detected for laminin 5, �5, and �1 in both wound types. No positivity wasdetected for laminin �5 or �1 in epidermal cells at the leading front, while weak to strongcytoplasmic positivity was often detected for laminin 5 at the leading front in both woundtypes. In contrast to laminin �5 and �1, strong/weak reactivity for laminin 5 was oftenfound in the blister roof in 2-day-old blisters as a discontinuous linear or intracytoplas-mic staining pattern.

5.6.4. BP180, BP230, and plectin/HD1 in different body sites (IV)

Specimens from all sites showed similar levels of immunoreactivity for BP180, BP230and plectin/HD1 in BMZ.

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Table 5. Summary of immunofluorescence results in 2- and 4-day-old blisters and openwounds (IV, V).

5.7. Effect of calcipotriol pretreatment on BP180, BP230, plectin/HD1, �4 integrin, laminin 5 and tenascin-C

expression in regenerating and normal skin (IV)

In calcipotriol-pretreated skin, the BMZ staining patterns for BP180, BP230, plectin/HD1, �4 integrin, laminin 5 and tenascin-C were similar to those in vehicle-pretreatedskin both in regenerating and healthy areas.

Molecule Localization and intensity of staining Blisters Open wounds

BP180 edge - to ++ cytoplasmic/pericellular and band-like BMZ positivity

- to ++ cytoplasmic/pericellular and band-like BMZ positivity

base - + discontinuous band-like positivity in one sample, - others

BP230 edge +/++ band-like BMZ positivity 2-day-old, -/+ cytoplasmic posit.; 4-day-old, +/++ band-like BMZ posit.

base - -Plectin/HD1 edge +/++ band-like BMZ positivity 2-day-old, -; 4-day-old, +/++ band-like

BMZ positivitybase - -

�4 integrin edge +/++ diffuse positivity, ++ polariza-tion in cells next to wound bed

+/++ diffuse positivity, ++ polarization in cells next to wound bed

base - -Laminin 5 edge - to ++ cytoplasmic positivity - to ++ cytoplasmic positivity

under regen. epid.

++ continuous band-like staining ++ continuous band-like staining

base ++ continuous band-like staining +/++ discontinuous band-like stainingroof +/++ discontinuous band-like/cyto-

plasmic stainingLaminin �5 and �1

edge - -under regen. epid.

++ continuous band-like staining ++ continuous band-like staining

base ++ continuous band-like staining +/++ discontinuous band-like stainingroof -

Tenascin-C edge -/+ cytoplasmic positivity -/+ cytoplasmic positivitybase ++ ++under regen. epid.

++ ++

Collagen VII as in normal skin as in normal skin�5 integrin ++/+ predominantly suprabasal ++/+ predominantly suprabasal�v�5 complex, �5 and �9 integrins

- staining in regenerating epidermis - staining in regenerating epidermis

Intensity of the staining: -, no or negligible; +, weak; ++, strong. Regen. epid: regenerating epidermis.

6. Discussion

6.1. TGF-�s and TGF-� receptors I and II in normal skin, serum, and psoriatic skin (I, II)

Immunohistochemical studies have shown divergent results on TGF-� expression innormal skin (see 2.4.4.). The possible reasons include, among others, formalin fixation-induced chemical changes in TGF-�s and dissolution of TGF-�s during tissue processing.In this study, suction blister fluid samples and enzyme-linked immunoassays were used toquantitate TGF-� in skin. Their advantage over immunohistochemical methods is that theabove-mentioned problems can be avoided, more accurate quantitation can be made,information on the proportions of TGF-� derived from circulation and released locallycan be obtained, as blood samples are also studied, and they are noninvasive in nature. Inaddition, the TGF-� detected in suction blister fluid gives information on the effectivefraction of TGF-� in skin, because the cytokines that are free in the interstitium ratherthan those stored in tissue are responsible for the biological effects. On the other hand,the suction blister method does not give information on the precise location of TGF-�s inskin or on the amount of TGF-�s bound to tissue or located intracellularly.

The suction blister fluid TGF-�1 level was 260 45 pg/ml in the control samples inthe isotretinoin study and 530 pg/ml in the control samples in the betamethasone study(see Fig. 2). The difference in these absolute levels may result from the fact that the TGF-� determinations and the sample collection for these two studies were made in differentlaboratories. Alternatively, differences in the study materials, e.g. uninvolved acne skinversus healthy skin, might explain the difference. However, since the primary interest wasto assess the changes in the TGF-� concentrations after medications, since the standarddeviations in the isotretinoin study and the betamethasone study were of the same relativemagnitude, and since both studies had their own controls, the difference in absolute levelsbetween these two parts does not have major significance. It has been shown that activeTGF-�1 is bound to tissue much faster than latent TGF-�1 (Wakefield et al. 1990). In thisstudy, active TGF-�1 represented approximately 5% of the total suction blister fluid TGF-�1. This suggests that activation takes place locally, with subsequent binding of theactivated cytokine. In the present study, the slight decrease (17%) in suction blister fluidTGF-�1 in old (n = 5) compared to young (n = 4) volunteers was not statistically

59

significant (p = 0.14). In view of the median, the range, and the fact that the estimatedsuction blister fluid TGF-�1 concentration for each volunteer was calculated fromdeterminations made from three separate fluid samples, it is possible that, with morevolunteers, the difference would have shown statistical significance. The suction blisterfluid TGF-�2 level remained below the sensitivity level, 55 pg/ml, in most controlsamples in the isotretinoin study. As the signaling receptors T�R-I and T�R-II have beendescribed to bind to TGF-�1 with a higher affinity than to TGF-�2, and as the suctionblister fluid TGF-�2 concentration represented only a fraction of the suction blister fluidTGF-�1 concentration, TGF-�2 may have only a minor role in maintaining thehomeostasis of healthy skin compared to TGF-�1 (Bassing et al. 1994).

Using the concentration of circulating TGF-� and suction blister fluid TGF-�, it ispossible to estimate the proportion of TGF-� that has diffused from circulation into thesuction blister fluid (Vermeer et al. 1979). The serum total TGF-�1 detected here cannotbe used in these diffusion calculations. This is due to the fact that human platelets containTGF-�1 (Cheifetz et al. 1987, Grainger et al. 1995), and that TGF-�1 was here releasedfrom the platelets during the serum TGF-�1 assay. The expected value for plasma totalTGF-�1, 2260 pg/ml, given by the manufacturer of the TGF-�1 kit, has been used here inthe diffusion calculations instead of 4100 pg/ml, which was originally used in article II.This re-estimation has been done in order to ensure that the proportion of suction blisterfluid TGF-�1 diffused from circulation will not be overestimated. This re-estimation,however, will not change the original conclusion regarding the proportion of locallyderived TGF-�1 in control suction blister fluid samples. In view of what has beenreported elsewhere for plasma total TGF-�1, 4100 pg/ml and 4800 pg/ml, the expectedvalue given by the manufacturer is not an overestimation (Wakefield et al. 1995, Graingeret al. 1999). Human platelets do not contain TGF-�2, and no major difference wasreported between the expected values for serum total TGF-�2 and plasma total TGF-�2 inthe manual of the TGF-�2 kit manufacturer (Cheifetz et al. 1987). The serum total TGF-�2 detected here, 340 120 pg/ml, has been used in the diffusion calculations.

Assuming that all circulating TGF-�1 and TGF-�2 were in a latent (100 kDa or 220kDa) form, the diffusion calculations show that a 2260 pg/ml TGF-�1 plasmaconcentration predicts a 660 pg/ml TGF-�1 suction blister fluid concentration and a 340pg/ml TGF-�2 serum concentration predicts a 100 pg/ml TGF-�2 suction blister fluidconcentration (Vermeer et al. 1979). In vivo, a small fraction of circulating TGF-�1, andperhaps TGF-�2, is in a smaller active form (25 kDa) (Roberts 1998). Consequently, thepredicted values should be somewhat higher. In the present study, the determined suctionblister fluid concentrations of total TGF-�1 and total TGF-�2 were lower than the abovepredicted values. This suggests that all the TGF-�1 and TGF-�2 detected in suctionblister fluid have diffused from systemic circulation. It also suggests that locally secretedTGF-�1 and TGF-�2 are rapidly used in an autocrine or paracrine way, or alternativelybound to tissue, and not stored in interstitial fluid.

The level of serum total TGF-�1 detected here in untreated acne patients, 53 9.5 ng/ml, is in line with the expected value given for healthy individuals, 49 ng/ml, by themanufacturer of the TGF-�1 kit and with some earlier studies, where it has been reportedto be 56 ng/ml and 84 32 ng/ml in healthy controls (Wakefield et al. 1995, Bonifati etal. 1996). Giacalone et al. have reported surprisingly low levels of TGF-�1 in suctionblister fluid (< 5.0 pg/ml) and serum (0.04 ng/ml), without specifying whether TGF-�1

60

activation had been done or how the serum samples had been collected (Giacalone et al.1998). As their level of serum TGF-�1 is considerably below those reported elsewhere,the discrepancy between their results and what has been detected here may be ignored.The level of serum total TGF-�2 detected here in untreated acne patients, 340 120 pg/ml, is in line with the expected value given for healthy individuals, 390 pg/ml, by themanufacturer of the TGF-�2 kit and with the previously reported serum total TGF-�2 inhealthy controls, 150 pg/ml (Grainger et al. 1999). Contradictory results have beenreported for the plasma total TGF-�2 concentration in healthy controls, <200 pg/ml and1300 pg/ml (Wakefield et al. 1995, Stoiser et al. 1998). Differences in these figures maybe e.g. due to differences in the used assays or the age or sex of the subjects.

In the present study, the expression of T�R-I and T�R-II was mostly, if not fully,restricted to the proliferative cell compartment in normal epidermis. The complete co-expression of the two receptors in the basal epidermis in normal skin suggests that T�R-Iand T�R-II are capable of forming functional receptor complexes for TGF-� to exert itsgrowth regulatory effects. This is in accordance with the previous studies, in which TGF-�1 has been shown to have significant antiproliferative effects on cultured keratinocytes(Shipley et al. 1986, Reiss & Sartorelli 1987, Matsumoto et al. 1990). Basal keratinocytesare likely to receive TGF-� signals in an autocrine manner, in a paracrine manner fromthe extracellular matrix where TGF-� is deposited, and, according to the present results,by diffusion from circulation. It therefore seems possible that the TGF-� receptors in thebasal epidermal area may have an important role in regulating physiological epidermalproliferation. The present results on T�R-I and T�R-II expression in normal epidermisare somewhat contradictory to Schmid et al., who detected co-expression of T�R-I andT�R-II in the whole epidermis in normal skin. Schmid et al. used formalin-fixed paraffin-embedded tissues, the same antibodies as were used in the present study and an additionalantibody to detect T�R-II, and an alkaline phosphatase-linked streptavidin detectionsystem. (Schmid et al. 1998.) In the present study, acetone-fixed frozen sections and aperoxidase-linked streptavidin detection system were used. It has been claimed that thealkaline phosphatase-linked streptavidin detection system provides greater sensitivitythan the peroxidase-linked streptavidin detection system (Elenitsas et al. 1997). This maysuggest that the sensitivity of the detection system used in the present study was too lowto detect T�R-I and T�R-II in upper epidermis. The fact that T�R-I mRNA and T�R-IImRNA have been shown to be present in the whole epidermis and the earlier studieswhere TGF-�1 has been shown to influence the differentiation of keratinocytes in culturesupport this conclusion to some extent (Reiss & Sartorelli 1987, Matsumoto et al. 1990,Matsuura et al. 1994, Schmid et al. 1998). On the other hand, frozen sections aregenerally considered to give more reliable immunohistochemical results on membrane-bound and cytoplasmic antigens than formalin-fixed paraffin-embedded tissue sections.Furthermore, formalin-fixed paraffin-embedded skin sections were also investigated inthe present study, but no reasonable results were obtained. However, the above issue wasnot of major importance in the present study, because the main finding regarding theexpression of T�R-I and T�R-II was their downregulation in lesional, but not in non-lesional psoriatic epidermis.

No or negligible epidermal immunoreactivity was detected for T�R-I and T�R-II in allthe lesional psoriatic skin samples studied. A previous in situ hybridization study hasshown that T�R-II mRNA is present uniformly in the whole epidermis and equally in

61

normal and psoriatic epidermis (Matsuura et al. 1994). This may suggest that the lack ofepidermal positivity for T�R-II protein in our lesional psoriasis specimens is due to post-transcriptional events leading to decreased protein synthesis or rapid degradation. Theepidermal changes seen in transgenic mice that overexpressed dominant negative-T�R-IIin the epidermis resembled those in lesional psoriatic epidermis (Wang et al. 1997). Thefindings suggest that the downregulation of T�R-II and T�R-I in lesional psoriaticepidermis may lead to a loss of TGF-�-mediated growth inhibition. The increased serumTGF-�1 detected in psoriatic patients and the somewhat decreased binding of TGF-�1 bylesional dermal microvascular endothelial cells suggest that increased diffusion of TGF-�1 from circulation may partially compensate for the downregulation of T�R-I and T�R-II (Bonifati et al. 1996, Cai et al. 1996).

6.2. Effect of oral isotretinoin and topical betamethasone on TGF-� in suction blister fluid and serum (II)

In the present study, serum TGF-�1 was preferred to plasma TGF-�1 in detectingpossible changes in systemic TGF-�1 after isotretinoin treatment, because plasma TGF-�1 determinations would have been more prone to laboratory failure due to difficulties inpreparing completely platelet-free plasma and platelet degranulation. When interpretingthe following data, it is important to take into account that serum TGF-�1 does notdirectly represent circulating TGF-�1. TGF-�1 is known to be transferredtransplacentally to fetuses, and this transferred protein appears to play a critical role inembryogenesis (Letterio et al. 1994). In addition, downregulation of TGF-�2 has beenlinked to tretinoin-induced malformations in mouse embryos, and TGF-�s are known tostimulate new bone formation (Taylor et al. 1995, Bostrom & Asnis 1998). In view ofisotretinoin-induced side-effects, e.g. teratogenicity and hyperostosis (Orfanos et al.1997), and the above data, it is noteworthy that isotretinoin did not affect the serumconcentration of TGF-�1 or TGF-�2 in the present study. The fact that serum TGF-�1was not affected by isotretinoin treatment may also imply that the increase in circulatingTGF-�1 detected in prostate cancer patients following combination therapy withisotretinoin and �-interferon is due to a synergistic effect of the two drugs or,alternatively, to �-interferon solely (DiPaola et al. 1997).

As no change was detected in the serum TGF-�1 concentration after the isotretinointreatment, the 19% increase in the suction blister fluid TGF-�1 concentration seems to becompletely of local origin, and may hence reflect a several-fold increase in local TGF-�1secretion and synthesis and/or considerably decreased tissue binding. Suction blister fluidsamples were collected after only six weeks of treatment with moderate dosage (40 mg/day) (Orfanos et al. 1997). A longer treatment period and higher dosage might haveresulted in a greater increase in suction blister fluid TGF-�1. Also, if the suction blisterfluid samples had been collected from facial skin, where the potential of isotretinoin forhealing acne is greater than on abdominal skin, the change in the level of suction blisterfluid TGF-�1 might have been different (Orfanos et al. 1997). In view of the presentresults, the upregulating effect of isotretinoin on TGF-�1 may be one mechanism bywhich isotretinoin mediates its effects in skin. As more TGF-�2 determinations exceeded

62

the sensitivity level of the assay after than before the isotretinoin treatment, it can bepostulated that isotretinoin may have some upregulating effect on suction blister fluidTGF-�2, but this requires further clarification. Our results on TGF-�1 in skin are parallelto an earlier immunohistochemical study, where both topical tretinoin and topical irritantresulted in upregulation of TGF-�1 expression in human skin. As both treatments(tretinoin and irritant) caused similar erythematous reactions, the upregulation of TGF-�1was thought to be related to topical irritation. (Fisher et al. 1992.) It is noteworthy,however, that although no skin irritation was observed after the oral isotretinoin treatmentat the suction blister test sites in the present study, parallel upregulation of TGF-�1 wasdetected. It is therefore likely that retinoid dermatitis and vasodilatation do not explain thepresent finding.

The present results suggest that most, if not all, suction blister fluid TGF-�1 in normalskin is derived from circulation by diffusion. It has been shown previously that topicalglucocorticoid treatment decreases the diffusion of proteins from circulation to suctionblister fluid by 10-35% (Kiistala et al. 1986). The above data suggest that the 17%decrease in the suction blister fluid TGF-�1 concentration detected here afterbetamethasone pretreatment may be exclusively due to glucocorticoid-inducedvasoconstriction. As TGF-�s are known to stimulate extracellular matrix formation, thisdecrease may be one mechanism leading to glucocorticoid-induced dermal atrophy.

6.3. Integrity of BMZ in uninvolved dermatitis herpetiformis skin (III, IV)

The expression of BP180, �6 integrin, �4 integrin, nidogen, collagen �1�2 (IV), collagenVII, laminin 5, laminin �3 chain and laminin �1�1 chains in uninvolved skin ofdermatitis herpetiformis patients were comparable to the findings in normal skin controls.The results hence suggest that these antigens have no major structural defectspredisposing to blistering in uninvolved skin of dermatitis herpetiformis patients.The main finding in uninvolved dermatitis herpetiformis skin specimens in the presentstudy is the significantly decreased expression of BP230 and plectin/HD1 in BMZ in partof the patients studied (Table 4). Although immunofluorescence is semiquantitative bynature, the present results on BP230 and plectin/HD1 showed dramatic reductions inreactivity for the two proteins, indicative of true changes compared to normal skin. Theseimmunofluorescence results were obtained with monoclonal antibodies and consequentlyreflect the availability of single epitopes on these proteins. However, parallel results wereobtained with three different antibodies, while a number of other BMZ antibodies,including hemidesmosomal transmembrane protein BP180, gave normal staining resultsin these specimens. Both BP230 and plectin/HD1 are located in the hemidesmosomalinner plaque region (Borradori & Sonnenberg 1999). In the present EM studies, however,the hemidesmosomal inner plaques of uninvolved dermatitis herpetiformis skin (cases9U, 10U) and normal skin were apparently similar to each other, although the dermatitisherpetiformis skin samples were from patients showing dramatic reductions in

63

immunoreactivity for BP230 and plectin/HD1 in BMZ. Thus, the present results suggestdistinct molecular changes in BP230 and plectin/HD1 in uninvolved dermatitisherpetiformis skin, such that they are altered or diminished rather than completely absent.

The reduction in immunoreactivity for BP230 and plectin/HD1 varied from patient topatient, and the reductions in plectin/HD1 immunoreactivity were not entirely identical,as shown by the two antibodies (HD-121 and 7A8) used. The uninvolved dermatitisherpetiformis skin samples studied were drawn from hospital biopsy archives and hadoriginally been taken for diagnostic purposes. All the ten samples were from �healthy�skin, but the further specifications, e.g. the distance to the lesional area, the exact locationon the body surface, prior treatment, and diet, remained unknown. In future studies, it willbe of interest to find out if any of the above matters is related to the detected decrease inimmunoreactivity for BP230 and/or for plectin/HD1 in BMZ. In a previous study,regional variation has been described in the expression of bullous pemphigoid antigenwith patient sera (Goldberg et al. 1984). As BP230 has been shown to be the principalantigen detected by most bullous pemphigoid patient sera (Moll & Moll 1998), theexpression of BP230 and plectin/HD1 was studied here by body site in normal skincontrols using IE5 and HD-121 antibodies with the same dilutions that were used to stainthe uninvolved dermatitis herpetiformis skin specimens. No variation was detected intheir expression in BMZ in these studies. Thus, differences in the anatomical location ofthe uninvolved dermatitis herpetiformis skin specimens in the present study do notexplain the observed reductions in immunoreactivity for BP230 and plectin/HD1.Possible prior blistering and subsequent blister healing at the biopsy sites are not likely toexplain the observed reductions, either, as BP230 and plectin/HD1 already appearedduring the reepithelialization phase in healing suction blisters in the present study. Thehealing of suction blisters simulates the healing of dermatitis herpetiformis blisters tosome extent, as blister formation takes place within lamina lucida in both cases (Kiistala& Mustakallio 1967, Smith et al. 1992). The pathogenetic mechanisms behind theobserved changes in BP230 and plectin/HD1 immunoreactivity remain open. Thepossibilities include, among others, genetic changes. It is also possible that normal BP230and plectin/HD1 proteins could be influenced by primary changes in molecules notstudied here. For example, proteolytic events or accumulation of pathological proteinscould alter the immunoreactivity for BP230 and plectin/HD1. In view of the presentresults on decreased BP230 and plectin/HD1 in uninvolved dermatitis herpetiformis skin,it is interesting that the IgA deposits typical of the disease are found in a location clearlydifferent from those of the intracellular BP230 and plectin/HD1 proteins (Smith & Zone1993).

BP230 knockout experiments with subsequent skin blistering upon mechanical stressresulting from basal cell rupturing parallel to and just above the cell base as well asplectin/HD1 mutations in epidermolysis bullosa patients with tissue separation within thebasal keratinocytes at the level of the inner hemidesmosomal plaque suggest that thechanges detected here in the expression of BP230 and plectin/HD1 may be a factorcontributing or predisposing to blister formation in dermatitis herpetiformis skin (Guo etal. 1995, Uitto & Pulkkinen 1996). Changes in BP230 and plectin/HD1 may decrease theability of hemidesmosomes to function normally in uninvolved dermatitis herpetiformisskin, and the inflammatory infiltrate may hence find a structural weakness in BMZ withsubsequent cleavage and blister formation.

64

In a few specimens, endothelial immunoreactivity for plectin/HD1 was also evaluatedwith double staining experiments, using UEA-I lectin as an endothelial cell marker (Table4). Controversial results were obtained with the two plectin/HD1 antibodies, and anyfurther conclusions are therefore difficult. The HD-121 antibody showed decreasedimmunoreaction in endothelia in uninvolved dermatitis herpetiformis skin specimenscompared to normal skin, while the 7A8 antibody did not show any significantdifferences in endothelial immunoreactivity between the above-mentioned tissues. In allcontrols of normal skin, the HD-121 antibody showed clearly stronger immunoreactionsthan the 7A8 antibody in BMZ, suggesting that the HD-121 antibody presumably hashigher affinity for the antigenic epitopes. The overall endothelial immunoreactivity in thecontrol specimens was clearly weaker than the immunoreactivity in BMZ, and it is thusdifficult to demonstrate any decrease in immunoreactivity in the endothelium with theseantibodies, particularly the 7A8 antibody. This could well explain why the 7A8 antibodyfailed to show a decrease in immunoreactivity in the endothelium in the same way as theHD-121 antibody.

6.4. Reepithelialization rate in intact blisters and open woundsand modulation by calcipotriol (IV)

Reepithelialization was considerably slower in suction-induced intact blisters than inroofless open wounds. This was unexpected, as an opposite result has been reported in asimilar study setting in mice, and as it has been demonstrated in humans that occludedwounds heal faster than nonoccluded wounds (Krawczyk 1971, Nemeth et al. 1991). Thepresent result suggests that removal of blister roofs may accelerate the clinical healingprocess in bullous disease, e.g. bullous pemphigoid and junctional epidermolysis bullosa,where blister formation takes place within lamina lucida, as in suction blisters (Uitto &Pulkkinen 1996, Moll & Moll 1998). The reasons for the slower reepithelialization rate inintact blisters are not known, but may include the intact laminin sheath in the blisterbases, contact inhibition by the pressure of the blister fluid, a difference in protease orcytokine expression or in interstitial fluid calcium concentration, accumulation ofinhibitory compounds into the blister cavity, and a lack of inflammation in intact blisters.In future studies, it would be interesting to compare the reepithelialization rate in openwounds to that in punctured blisters allowed to drain without actually removing theblister roofs. This would give more information on the role of the blister fluid contentsand the blister roof. Calcipotriol pretreatment did not have any morphologic effect on theregenerating epidermis in open wounds. This was unexpected due to its potentantiproliferative effect on hyperproliferating psoriatic keratinocytes and its proliferation-modulating and differentiation-modulating effects on keratinocytes in culture (Svendsenet al. 1997, Kragballe & Wildfang 1990). The present result suggests that a diagnosticbiopsy or a surgical procedure can be made during calcipotriol treatment without delayedwound epithelialization.

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6.5. BMZ components and epithelial integrins in reepithelializationin intact blisters and open wounds (IV, V)

Considerable interindividual variation in immunoreactivity for BP180 was detected at theleading edge in 2- and 4-day-old samples. This variation was independent of the woundtype and the reepithelialization rate. This may suggest that BP180 is not directly involvedin keratinocyte attachment to the wound bed at the leading edge. This would be in linewith a previous study, where the initial adhesion of BP180-deficient keratinocytes toextracellular matrix proteins, e.g. laminin 5 and fibronectin, was not substantiallyimpaired (Borradori et al. 1998). In mucosal wounds, however, linear BMZ positivity hasbeen detected beneath the entire length of the regenerating epithelial tongue in contrast towhat is reported here (Dabelsteen et al. 1998). BP180 was often found in 2-day-oldblister roofs, but only once in a blister base or an open wound base in the present study.This and an earlier study, where bullous pemphigoid sera gave positive immunostainingonly on the epidermal side in fresh suction blisters, suggest that the so far unidentifiedbasement membrane interactions of the extracellular domain of BP180 are of loweraffinity than the interactions of the cytoplasmic domain (Woodley et al. 1983).

In 2-day-old samples, BP230 and plectin/HD1 were better expressed at the leadingedge of the regenerating epidermis in intact blisters than in open wounds. This maysuggest that the keratin cytoskeleton is linked to the basal plasma membrane of basal cellsat the leading edge via BP230 and plectin/HD1 earlier in the more slowlyreepithelializing intact blisters than in open wounds, which may partly explain the slowerreepithelialization rate in intact blisters compared to open wounds. In view of the fasterreepithelialization rate in open wounds, it is interesting that a retardation ofreepithelialization in cutaneous wound healing was detected in BP230 knockout mice(Guo et al. 1995). On the 4th day, linear BMZ immunoreactivity both in blisters and inopen wounds was detected for BP230 and plectin/HD1 at the leading edge, suggestingformation of hemidesmosomal inner plaques in the basal plasma membrane of basal cellsat the leading edge. No regional variation was detected in BP180, BP230 and plectin/HD1expression. The results on these proteins hence apply to the whole body surface.

In the present study, the expression of �4 integrin in open wounds and intact blisterswas comparable to the �4 expression earlier described in full-thickness cutaneous wounds(Cavani et al. 1993). This suggests that the expression of �4 integrin at the leading edge isindependent of the composition of the wound bed. In the present study, two differentantibodies (P1D6 and BIE5) were used to detect the �5 subunit of the fibronectinreceptor, �5�1 integrin (Eble 1997). In normal skin, the present results are in line with aprevious study using the same P1D6 antibody. However, in contrast to the previous study,where full-thickness skin wounds were studied, increased expression of �5 integrin wasnot detected in migrating keratinocytes in the present study with P1D6 antibody. (Cavaniet al. 1993.) This suggests that, in contrast to �4 integrin, migrating keratinocytesupregulate �5 integrin only upon contact with dermal structures in vivo. In contrast to theearlier studies using mouse tissues (Stepp et al. 1995, Stepp & Zhu 1997), the �9 subunitof tenascin-C- binding �9�1 integrin was poorly expressed in normal epidermis and notupregulated in regenerating epidermis in the present study, although tenascin-C wasstrongly upregulated (Yokosaki et al. 1998).

66

In some earlier studies, an increase in the expression of the �v�5 complex has beendetected with the P1F6 antibody predominantly in basal keratinocytes in migratingepidermis during excisional but not incisional human wound healing (Clark et al. 1996,Haapasalmi et al. 1996). In the present study, no or negligible �v�5 expression wasdetected in normal or regenerating epidermis with the same antibody. The present resultsupports the earlier assumptions that only major dermal trauma induces upregulation ofthe �v�5 complex in regenerating epidermis in vivo. In the present study, increasedimmunoreactivity for the integrin �5 subunit was detected predominantly in thesuprabasal cell layers in regenerating epidermis. This differs considerably from what hasbeen detected here and previously for the �v�5 complex and the integrin �v subunit (see2.6.2.). Previously, the level of the �5 subunit has been found to be higher than that of the�v subunit on a few cell lines, and the possibility of an additional �5-associated subunitother than �v has been discussed (Pasqualini et al. 1993). The reason for the controversyin distribution detected here remains open.

The present results for laminin 5 are parallel to a previous suction blister study, wherelaminin �2 chain synthesis was described at the leading edge (Kainulainen et al. 1998).However, no distinction between intact blisters and open wounds was made. In contrast tolaminin 5, cytoplasmic positivity was not detected for the laminin �5 chain or the laminin�1 chain at the leading edge, which would have suggested laminin 10 (�5�1�1) orlaminin 6 (�3�1�1) synthesis. No immunostaining was detected for the laminin �5 chainor the laminin �1 chain in the blister roof. For laminin 10 (�5�1�1), this suggests a higheraffinity to the basement membrane than to its potential keratinocyte receptors, e.g. �3�1integrin (Eble et al. 1998, Kikkawa et al. 1998). In normal skin, laminin 6 is thought toform complexes with laminin 5. The laminin 5 subunit of the complex has been shown tobind the �3�1 and �6�4 integrins. (Burgeson & Christiano 1997.) For laminin 6(�3�1�1), the �1 chain results suggest a higher affinity to the basement membrane than tothe laminin 5 subunit of the laminin 5-6 complex or, if the complex is not split duringblister formation, a higher affinity of the laminin 5-6 complex to the basement membranethan to its keratinocyte receptors. The discontinuous linear laminin 5 immunostaining inthe blister roof may detect intact/broken laminin 5, a ligand for �6�4 integrin, and/or thelaminin 5 subunit from split laminin 5-6 complexes. The cytoplasmic laminin 5immunostaining observed in the basal keratinocytes of the blister roof may reflect activesynthesis. Whether endogenous or exogenous laminin 5 retards or promotes keratinocytemigration has remained unclear (Zhang & Kramer 1996, O�Toole et al. 1997, Goldfingeret al. 1998). In the present study, the linear immunoreactivity for laminin 5, laminin �5chain and laminin �1 chain was always intact in the blister bases, whereas in the openwound bases it was often fragmentary. The fragmentation could be due to degradingproteases released from the inflammatory cells present in the wound base. It is possiblethat the intact laminin sheath may have inhibitory effects on the reepithelialization rate.

The distribution and intensity of the increased tenascin-C immunoreactivity was thesame both in plain open wounds and plain blister bases and beneath the regeneratingepidermis. This indicates that the previously described tenascin-C synthesis byregenerating keratinocytes was limited in the present samples to an extent which cannotbe distinguished from the tenascin-C expression of dermal origin by semiquantitativeimmunohistochemistry (Latijnhouwers et al. 1997). The present results for tenascin-Csuggest that keratinocyte-derived cytokines in suction blister fluid do not affect tenascin-

67

C synthesis in the upper dermis to a noticeable degree. The present results are inaccordance with some earlier studies where increased tenascin-C expression has beendescribed in perilesional skin in epidermolysis bullosa patients, and where mechanicalforce has been shown to induce tenascin-C production in fibroblasts, and show thatdermo-epidermal cleavage leads to increased tenascin-C expression of dermal origin(Chiquet-Ehrismann et al. 1994, Schenk et al. 1995). The detection of cytoplasmicimmunoreactivity for tenascin-C in regenerating epidermis is in accordance with anearlier study (Latijnhouwers et al. 1997). Neither suction-induced dermo-epidermalseparation at the level of lamina lucida nor reepithelialization of either wound typeaffected type VII collagen expression. This suggests that, in contrast to blister healing indermatitis herpetiformis, where blister formation also takes place within lamina lucida,type VII collagen is not a target for degrading proteases during blister or open woundhealing (Airola et al. 1997). Calcipotriol had no effect on the expression of BP180,BP230, plectin/HD1, �4 integrin, laminin 5 or tenascin-C in either regenerating or normalepidermis.

7. Conclusions

The present study suggests that most, if not all, TGF-�1 and TGF-�2 detected in suctionblister fluid obtained from normal skin has been diffused from systemic circulation. Asthe suction blister fluid TGF-�2 concentration represented only a fraction of the suctionblister fluid TGF-�1 concentration, TGF-�1 seems to have a predominant role inmaintaining homeostasis in healthy skin compared to TGF-�2. Co-expression of T�R-Iand T�R-II in the basal epidermis in normal skin suggests that T�R-I and T�R-II arecapable of forming functional receptor complexes in the basal epidermis for TGF-� toexert its growth-regulatory effects. The present results suggest that circulation is likely tobe a major source of TGF-� for TGF-� receptors in the basal epidermis. Thedownregulation of T�R-I and T�R-II in lesional psoriatic epidermis seen in the presentstudy may lead to a loss of TGF-� mediated growth inhibition. In view of theisotretinoin-induced side-effects, it is interesting that isotretinoin treatment did not havean effect on the serum concentration of TGF-�1 or TGF-�2 in the present study. Theisotretinoin-induced increase in the suction blister fluid TGF-�1 concentration suggeststhat upregulation of local TGF-�1 may be one mechanism by which isotretinoin mediatesits effects in skin. Topical betamethasone had a slight downregulating effect on suctionblister fluid TGF-�1, presumably due to glucocorticoid-induced local vasoconstriction.This downregulation may be one mechanism leading to glucocorticoid-induced dermalatrophy.

The present results suggest that no major structural defects predisposing to blisterformation in uninvolved dermatitis herpetiformis skin exist in BP180, �6 integrin, �4integrin, nidogen, collagen �1�2 (IV), collagen VII, laminin 5, laminin �3 chain, orlaminin �1�1 chains. The immunoreactivity for BP230 and plectin/HD1 was decresed inBMZ in several uninvolved dermatitis herpetiformis skin specimens, while electronmicroscopy revealed no alterations in the ultrastructure of hemidesmosomal innerplaques. The findings suggest distinct molecular changes in BP230 and plectin/HD1. Thismay be a factor contributing or predisposing to blister formation in dermatitisherpetiformis skin.

The present study shows that removal of the blister roof accelerates thereepithelialization rate in suction blisters. This suggests that the same phenomenon couldtake place in several bullous diseases. This study also shows that calcipotriol pretreatment

69

has no effect on the reepithelialization rate in open wounds, suggesting that a surgicalprocedure can be made during calcipotriol treatment without delayed woundepithelialization. Reepithelialization took place on a continuous laminin sheath in intactblisters, while the laminin sheath in open wounds was partially discontinuous. BP230 andplectin/HD1 were better expressed at the leading edge in intact blisters than in openwounds. The data suggest that the intact laminin sheath may have inhibitory effects on thereepithelialization rate and that the keratin cytoskeleton may be linked to the basal plasmamembrane of basal cells at the leading edge earlier in the more slowly reepithelializingintact blisters than in open wounds. BP180, �4 integrin, �v�5 integrin, �5 integrin, �9integrin, tenascin-C, and type VII collagen showed similar distribution in intact blistersand open wounds, suggesting that they are not involved in the retarded reepithelializationdetected in intact blisters. Integrin �v�5 and integrin �5 antibodies showed divergentexpressions in regenerating epidermis. Calcipotriol pretreatment had no effect on BP180,BP230, plectin/HD1, �4 integrin, laminin 5 or tenascin-C expression in eitherregenerating or normal epidermis. BP180, BP230, and plectin/HD1 expression showed noregional variation, and the results on these proteins hence apply to the whole bodysurface.

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