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Please be advised that this information was generated on 2021-10-01 and may be subject to change. Epidermal barrier dysfunction New insights and opportunities for therapeutical intervention Kucháreková M. ■ Epidermal barrier dysfunction: New insights and opportunities for therapeutical intervention Thesis University Medical Centre Nijmegen, the Netherlands With summary in Dutch -224 p, © 2003

ISBN ■ 90-9017339-0 NUR ■ 876 Druk ■ Ipskamp Printpartners, Enschede Vormgeving ■ Milan Tjioe

No part of this book may be reproduced by any mechanical, photographic or electronic process, nor may it be stored in a retrieval system, transmitted, or otherwise copied for public or private use, without written permission of the author. Epidermal barrier dysfunction New insights and opportunities for therapeutical intervention

Een wetenschappelijke proeve op het gebied van de Medische Wetenschappen

Proefschrift

ter verkrijging van de graad van doctor aan de Katholieke Universiteit Nijmegen, op gezag van de Rector Magnificus Prof. dr. C.W.P.M. Blom volgens besluit van het College van Decanen in het openbaar te verdedigen op

donderdag 4 december 2003 des namiddags om 3.30 uur precies

door

Martina Kucháreková

Geboren op 5 juni 1971 te Zvolen, Slowakije Promotor: Prof. dr. dr. P.C.M. van de Kerkhof Copromotor: Dr. P.G.M. van der Valk Dr. J. Schalkwijk

Manuscriptcommissie: Prof. dr. D.J. Ruiter (voorzitter) Prof. dr. Y. Hekster Prof. dr. P.J. Coenraads (RUG) Twijfel is ‘t begin van wijsheid (René Descartes)

Voor mijn ouders Venované mojim rodičom CONTENTS

Chapter 1 General Introduction 9 1.1 Structure and function of the epidermis 11 1.2 Selected inflammatory skin disorders associated with an altered 21 epidermal barrier 1.3 Methodology 37 1.4 Aims and questions of this thesis 43

Chapter 2 Skin barrier and lipid metabolism 61 2.1 Lack of upregulation of epidermal fatty acid binding protein (E- 63 FABP) in dithranol induced irritation 2.2 Epidermal expression of epidermal fatty acid binding protein (E- 75 FABP) distinguishes atopic dermatitis from psoriasis vulgaris

Chapter 3 Epidermis as a barrier to microorganisms 85 3.1 Specific upregulation of epidermal host-defence proteins in psoriasis 87 compared to atopic dermatitis

Chapter 4 Novel therapies on irritant contact dermatitis: Experimental 99 and clinical studies 4.1 The effect of a PDE-4 inhibitor (cipamfylline) in two human models 101 of irritant contact dermatitis 4.2 Effect of a lipid-rich emollient containing ceramide 3 in experimentally 115 induced skin barrier dysfunction 4.3 A randomized comparison of an emollient containing skin-related 133 lipids with a petrolatum-based emollient as adjunct in the treatment of chronic hand dermatitis

7 Chapter 5 Clinical aspects and topical treatment of dithranol irritation 147 as a unique form of irritant contact dermatitis 5.1 The effects of topical corticosteroids and a coal tar preparation on 149 dithranol induced irritation in patients with psoriasis 5.2 Dithranol irritation in psoriasis treatment: a study in 68 inpatients 159 5.3 Skin irritation during treatment of chronic plaque psoriasis with short 169 contact dithranol versus calcipotriol ointment

Chapter 6 General Discussion 179 6.1. Barrier repair strategies in the treatment of irritant contact 183 dermatitis 6.2. Barrier repair strategies in the treatment of atopic dermatitis 190 6.3. Barrier repair strategies in the treatment of psoriasis 192

General conclusions 194 Summary 201 Samenvatting 207 List of publications 213 Curriculum vitae 216 Dankwoord 218 Colour Illustrations 221

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1.1 STRUCTURE AND FUNCTION OF THE EPIDERMIS

1.1.1. Anatomy and organisation of human epidermis 1

The normal epidermis is a non-vascularized, terminally differentiating, stratified squamous epithelium, which is supplied of nutrients by diffusion through the underlying dermis and through the basement membrane. The major cell, making up 95% of the total, is the keratinocyte and its differentiation product, the corneocyte. Originating from a pool of cells with a limited proliferative potential, called transient amplifying cells, the newly formed keratinocytes move from the epidermal basement membrane towards the skin surface in two weeks. Microscopically we can distinguish the epidermis in four layers: stratum basale or stratum germinativum, stratum spinosum, stratum granulosum, and stratum corneum (SC). The stratum basale consists of a continuous one cell thick layer, attached to the basement membrane by hemidesmosomes and integrin receptors. The basal cells are small and cuboidal and have large, dark-staining nuclei. Immediately above the basal cell layer, the differentiating keratinocytes enlarge and form a layer, which is called the stratum spinosum because of the many desmosomes. In this layer the protein involucrin is expressed. The stratum granulosum consists of cells with microscopically visible intracellular structures called keratohyalin granules, which contain many proteins needed for terminal differentiation. This is the last living layer which is very active. The cytoplasm of these cells also contains lamellar granules or bodies. The outermost layer of the epidermis is the stratum corneum where cells (corneocytes) have lost nuclei and cytoplasmic organelles. The cells become flattened and have a highly insoluble cornified envelope within the plasma membrane, formed by cross-linking of the soluble precursor, protein. In many areas of the skin, especially of the palms and soles a fifth layer can be observed called the stratum lucidum. It is the lower part of the stratum corneum, which stains as a homogeneous eosinophilic zone. During the migration from the inner layer to the outer layers of the epidermis, the keratinocytes undergo the following processes of proliferation and differentiation.

Epidermal proliferation Corneocytes are continuously shed from the skin surface and replaced by cells from deeper layers. The increase of keratinocytes by proliferation in the basal and first suprabasal layer is under normal circumstances perfectly in balance with the loss from the outermost layer. This process is not observed by the naked eye and therefore it is called ‘desquamatio invisibilis’. In normal skin only 5-10 % of the basal cells is actively cycling.2 Several skin condition including psoriasis3 and wound healing are characterized by a strong increase of cycling cells. This phenomenon, called hyperproliferation, may serve the aim to repair, structurally and functionally, a damaged stratum corneum as quickly as possible. It may, however, also be a consequence of pathological processes, harmful to the subject. In irritant

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contact dermatitis (ICD), environmental influences are responsible for damage to either the cellular or the intracellular compartment. It may result in an increase in cellular proliferation. This adaptive response may be caused by a direct interaction of irritants with the keratinocytes or by effects on the intercellular lipid domain and subsequent functional disturbances. Increased permeability for water may be a trigger for increased proliferation.4 Because corneocytes lack a nucleus, adaptive phenomena are the result of cellular responses in deeper layers. The stratum corneum is called a dead layer, but it should be realized that the stratum corneum contain many active enzymes in order to desquamate in the extremely orchestrated way as it does. In this thesis skin irritation by sodium lauryl sulfate (SDS) and dithranol is studied. SDS brings about strong effects on the skin barrier in contrast with dithranol. Both are, however, strong irritants inducing hyperproliferation.5;6

Epidermal differentiation As the epidermal keratinocytes move through the epidermis after losing their attachment to the basal lamina, they undergo the complex process of terminal differentiation, also known as keratinization, to produce SC. During this process plasma membrane is replaced by a tough, insoluble proteinaceous envelope. This layer, which is called the cornified envelope, consists of cross-linked proteins. First the proteins involucrin, envoplakin, and periplakin are sequentially cross-linked by epidermal transglutaminases (TGases), that promote the formation of disulphide and (-glutamyl)lysine isopeptide bonds. Proteins such as loricrin, elafin, S100, and small proline-rich region proteins (SPRRs) are incorporated later to form a mature envelope.7-9 This leads to a highly rigid insoluble structure, which is embedded in lipids from the plasma membrane and lipids secreted by the granular cells. During epidermal differentiation, epidermal lipids undergo a dramatic change both in composition and localisation resulting from activities of multiple enzymes.10 Basal and spinous cells show a complex lipid composition with phospholipids as the major constituents. The phospholipids contain a high proportion of linoleic acid. With progressive differentiation of the epidermis into the stratum, a marked decrease in phospholipids is observed while the amount of sphingolipids (ceramides and glucosylceramides) and cholesterol proved to be increased.11;12 At the end of the differentiation, the content of ceramides and neutral lipids is increased from the inner to the outermost layer. Free sterol and free fatty acid contents are also significantly increased. A mixture of polar and neutral lipids is replaced by a more nonpolar mixture, including ceramides, free sterols and free fatty acids. Epidermal differentiation is of great importance for the integrity of the permeability barrier.13

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1.1.2 Epidermal functions

For terrestrial animals, the skin’s most important role is to act as a barrier. Cornified layers protect the skin from transepidermal water loss (TEWL) and loss of nutrients from inside. The stratum corneum also protects against penetration of potentially harmful substances, against the invasion of micro-organisms from the outside, and against UV light. In addition, the skin is involved in regulation of body temperature and in vitamin D production. In this thesis we focus on water-holding properties of epidermis and on the skin as a barrier to microorganisms.

1.1.2.1 Water barrier function In 1944 it was established that water barrier function of skin can be attributed to the most outer skin layer, the stratum corneum (SC)14 and later this was shown to be associated with multilayered lipid lamellae localized in the extracellular spaces.15;16 A unique, two-compartment model of the SC as a barrier is accepted, in which protein- rich enucleate cells, the corneocytes, are embedded within a continuous lipid-rich matrix.17 (Figure 1)

Figure 1: The stratum corneum can be represented as a two-compartment system, called by Elias as the ‘brick and mortar’ model.17 The bricks are the cornified cells with thickened membranes and the mortar is the intercellular lipid domein.

The lipids only account for about 10 % of the SC dry weight, but they are crucial in providing the barrier to water loss.18;19 They have unique chemical properties, high melting point and polarity, which result in the formation of water resistant lipid bilayers. This means that they form a hydrophobic compartment which encloses the carbon chains that separates them from the water. The hydrophilic head groups face the water and form a border between a hydrophobic phase and the water.19;20 SC lipids are primarily generated from exocytosis of lipid-containing granules called lamellar bodies (LB) or Odland bodies. The lipids of lamellar bodies are phospholipids, sphingomyeline, cholesterol sulfate, glucosylceramide and acylglucosylceramides. During epidermal differentiation, these lipids are processed to three types of lipids in the SC.18;21 At the final stage of keratinocyte differentiation, the lamellar body lipids are secreted into the stratum granulosum and form the bilayer structure.

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An important role in the structure of the barrier plays the hydrophobic envelope mainly composed of involucrine, loricrin and filaggrin, formed on the surface of the corneocytes.22 The lamellar sheets are covalently bound to the cornified envelope by ω-hydroxyceramide and ω-hydroxy fatty acids, forming a lipid envelope, which stabilised the intercellular domain.23;24 The unique organisation of the hydrophilic cells within the lipid, hydrophobic environment, makes this 10-20 µm layer extremely efficient as a barrier. Its water permeability is comparable with a plastic membrane and 1000 times more efficient than other membranes of living organisms.25 The lamellar structure of the intercellular domain shows a well-balanced range of lipids. If the lipid composition is disturbed qualitatively and/or quantitatively by environmental influences or by disease, barrier function will be hampered. In particular, the long chained ceramides are essential for the barrier function. It should, however, be emphasized that barrier function is both the result of the structured lipid domain and the ordering and function of the corneocytes.18 The intercellular domain is most important for the water barrier. The role of sebum in barrier function is probably very limited.26 We want to emphasize that SC barrier formation is a dynamic process. The skin is continuously exposed to environmental influences, which may influence barrier integrity. Initially, harm to the skin may lead to decrease of barrier function, but subsequently, to either recovery and hardening, or to disease, dependent on the size and nature of the assault and relevant constitutional factors of the exposed individual.27 Increased lipid synthesis, lamellar body secretion and lipid processing are aimed at barrier repair. If these adaptive phenomena fail, chronic barrier dysfunction may be the result. Moreover, the SC is the end product of epidermal differentiation. This means that, e.g. nutritional deficiencies, neuroendocrine responses or skin diseases, influencing epidemal growth and differentiation, may change the structure and function of the SC and may, therefore, induce barrier abnormalities.

Epidermal lipids The skin is a very active lipid-synthesising tissue, with a production rate of 100 mg per day. The lipid composition of the SC differs from the other layers of the epidermis and other tissues.21 As mentioned above, the SC is composed of three classes of lipids: free sterols, ceramides and free fatty acids. A small amount of cholesterol sulfate has also been reported as an intercellular lipid.28 Ceramides are the major constituents of the SC - more than 40% of total lipid mass29 - and represent a unique, heterogenous group of at least eight subclasses that differ from each other by the head group architecture and by the mean fatty acid chain lenght. They are composed of sphingosine base and fatty acid. The fatty acid chain lenght varies between 16 C atoms in ceramide 5 to C30-C34 atoms in ceramide 1.30 Ceramides play an important role in the barrier function. There are a number of experimental observation that indicate the role of ceramides in preventing water loss throught the SC.(Table 1) The profile of the various ceramides is probably an

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important determinant.31 Especially, ceramide 1 is assumed to be of importance barrier function due to its exceptional molecular structure contains linoleic acid linked to the long chain (C>30) ω-hydroxy fatty acid.32 Ceramides, next to their structural role in the SC can also have a bioactive, signaling function. They play a key role as lipid second messenger and have effects in multiple cell signaling pathways. They have been described as intracellular lipid messengers of the sphingomyelin cycle. Increased levels of intracellular ceramides induced cell differentiation and/or apoptosis and reduce cell proliferation. There is evidence that not only intracellular ceramides but also extracellular barrier-forming ceramides may affect epidermal proliferation and differentiation.33 The different biological effects of the ceramides are shown in Table 1.

Table 1 Activity of Ceramides (modified from34 ) Modulation of lamellar gel crystal of the horny layer lipids Opponent of cholesterol concerning interlayer distance Solution aid for cholesterol Augmentation of crystalline parts

Influence on the SC properties 35-38 Decrease of TEWL Increase of water content Decrease of skin scalines

Cellular response 33 Downregulation of cell proliferation Induction of cell differentiation Initiation of apoptosis Second messenger function

Fatty acids (FA) are important substrates for energy production, synthesis of phospholipids and participation in signal transduction pathways in mammalian cells.39 Fatty acids are a major component of the SC lipids, in which linoleic acid is the most abundant unsaturated fatty acid.40 It is generally accepted their important role in the maintenance of water barrier function of epidermis. Previous studies have demonstrated that both acute and chronic disruptions of the barrier stimulate epidermal fatty acid synthesis by approximately twofold.41;42 This increase of the synthesis of fatty acids can be accounted for by increases in the enzymatic activities of fatty acid synthase (FAS) and acetyl CoA carboxylase (ACC).43 Moreover, it has been shown

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that FA synthesis leads to the recovery of the barrier and the inhibition of enzymes of fatty acid synthesis delays barrier recovery.42;44 Barrier disruption also stimulates epidermal DNA synthesis leading to epidermal hyperplasia which requires fatty acids as components of various membrane structures in the epidermal cells themselves.45

E-FABP as fatty acid carrier Since FA are poorly soluble in water, special binding and transport proteins are required to increase FA solubility. In the circulation, FA are bound to albumin.46 Within the cells, FA are bound to cytosolic, nonenzymatic fatty-acid-binding-proteins (FABP’s), which belong to a family of low molecular mass (14-15 kDa) proteins displaying tissue-specific expression.47 In 1993, epidermal type FABP (E-FABP) with a high affinity for C-18 FA has been characterized in human epidermis.48 Later it was shown that E-FABP is also expressed in the mammary gland, lens, brain, liver, kidney, adipose tissue and some endothelial cells. The function of E-FABP in skin has not been elucidated. Since there is a close relationship between FA metabolism and keratinocyte differentiation, E-FABP expression may be related to the commitment of keratinocyte differentiation. There is some evidence indicating the role of E-FABP in water barrier function, however, its function only with respect to water barrier has been questioned. Normal human skin contains small amounts of E-FABP (<0.5 pmol/mg protein) localized to the stratum granulosum. In this layer the activity of rate limiting enzymes required for barrier lipid synthesis is highest.49 Upregulation of E-FABP has been observed in sodium dodecyl sulphate induced skin barrier dysfunction.50 Further, delayed recovery of TEWL after acetone application to the normal skin of E-FABP-deficient mice has been reported.51 These studies demonstrate that the role of E-FABP is associated with maintaining of the skin barrier function although the exact mechanism remains to be elucidated. In lesional psoriatic epidermis, the strong overexpression of E-FABP correlates with only a slight increase of transepidermal water loss. Since the levels of linoleic acid are strongly increased in psoriatic scale52;53 and linoleic acid has a high affinity for E-FABP, it is possible that expression of E-FABP in psoriasis is a ligand-depended process54 and is linked specifically to epidermal hyperproliferation and abnormal differentiation of this disease. The presence of E-FABP in the non-keratinized oral mucosa epithelium and its expression in tissues other than skin suggests that the role of E-FABP is not restricted to the skin barrier alone. In this thesis we studied the expression of E-FABP in specific skin conditions: (1) dithranol irritation human model in which the skin barrier for transepidermal water loss is not compromised and (2) lesional skin of atopic dermatitis and psoriasis to contribute to the elucidation of its epidermal function.

1.1.2.2 Antimicrobial properties of the epidermis An intact SC prevents invasion of the skin by normal skin flora or pathogenic microorganisms. However, both minor injury to the skin as well as skin diseases

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can provide portals of entry to microorganisms, particularly to streptococci and staphylococci.55 The innate immune system can immediately respond to this intrusion by helping to prevent further invasion. The innate immune system provides broad- spectrum recognition and rapid elimination of invading microorganism. This is distinct from the adaptive immune system (acquired immunity) where a host response is initiated only after antigen stimulation of specific lymphocyte subpopulations. Innate immunity encompasses a variety of host defense modalities ranging in complexity from simple inorganic molecules to cellular elements.56 (Table 2)

Table 2 Elements of innate immunity. Adapted from 56. Type Examples Inorganic molecules HCl, nitric oxide, hydrogen peroxide Simple organic molecules Fatty acids Antimicrobial peptides (<100 a.a) Cecropins, magainins, defensins, cathelicidins, SLPI, SKALP, dermcidin, cystatin A, calprotectin Antimicrobial proteins BPI, PLA2, lysozyme, complement Binding proteins Mannose binding protein, collectins Cytokines IL-10, IL-12, TNF-α Carbohydrates PGC-Glucan Cells Macrophages, neutrophils, natural killer cells, epithelia Toll-like receptors TLR1-TLR10

As a component of this system, an essential role of Toll-like receptors (TLRs) in microbial recognition has been established a few years ago. So far, ten mammalian TLRs have been identified which recognize distinct structural components of pathogens and evoke inflammatory responses. The TLRs function through signaling molecules, including MyD88, IL-1 receptor-associated kinase, TNF receptor associated factor 6, mitogen-activated protein (MAP) kinases and nuclear factor (NF)-κB.57 Some of TLRs were recently demonstrated in human epidermis.58 Further, antimicrobial peptides and proteins are an integral component of the innate immune system.59 They have been proposed to provide a first line of defense against infection by acting as ‘natural antibiotics’.60;61 A variety of tissues synthesize antimicrobial peptides including bone marrow cells, specialized epithelial cells, and ciliated epithelium.62;63 In the 1990s there has been a rapid expansion in the field of antimicrobial peptides. Over 300 peptides are reported.56 More recently, mammalian skin has emerged as a source for these molecules and is now a major point of interest.64

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Keratinocytes express constitutively or inducibly a number of antimicrobial peptides with bactericidal or bacteriostatic properties.65 Studies have revealed unique mechanisms of action for this class of host defense agents. Firstly, these molecules are able to kill a variety of microbial organisms. In response to injury, keratinocytes rapidly increase synthesis of these peptides that participate with circulating cells in killing potential pathogens. Secondly, antimicrobial peptides signal specific responses in the dermis and can modulate the activity of circulating neutrophils. Initially two classes of antimicrobial peptides have been discovered in mammalian skin: cathelicidins (PR-39 and LL-37)66 and β-defensins (hBD1,2 and 3).64;67 LL-37, hBD-2 are not normally present in human epidermis but are highly induced upon barrier disruption or chronic inflammation such as in psoriasis. In this thesis we have studied three host-defense proteins not restricted to these two classes; skin-derivated antileukoproteinase (SKALP), secretory leucocytose proteinase inhibitor (SLPI) and MRP-8 have been described and are associated with a broad spectrum of biological activity.68;69 We briefly describe the properties of these molecules.

Skin-derivated antileukoproteinase (SKALP)/elafin SKALP also known as elafin70, is a very cationic molecule, which is present in an intact form with a calculated molecular weight of approximately 9.9 kDa. Immunohistochemical studies have demonstrated the localisation of SKALP in different normal human tissues. SKALP appeared, in general, to be present in stratified squamous epithelia like tongue, gingiva, epiglottis, pharynx, oesophagus, cervix, vagina and hair follicles.71 Normal epidermis and skin adnexae are negative, except for the keratinocytes lining the epidermal part of the sweat gland ducts and the infundibular part of the hair follicle. SKALP expression in pathological skin conditions has been extensively studied. SKALP is known to be expressed in vivo when keratinocytes are exposed to inflammatory cytokines (psoriasis, wound healing) and after skin barrier disruption by tape stripping or detergents.72-74 In psoriasis, a strong cytoplasmic staining in the upper layers of the suprabasal compartment of epidermis was found, whereas the basal cell layer was negative.75 The expression of SKALP in atopic dermatitis is unknown. SKALP inhibits human leukocyte elastase with high affinity, but also porcine pancreatic elastase and proteinase 3, an elastin- degrading protein also derived from neutrophils. TNF-α was shown to be the most potent inducer of SKALP gene expression, whereas interferon-γ and transforming growth factor α showed a milder induction of SKALP gene expression.76 Recently, antimicrobial activities of SKALP have been demonstrated.68

Secretory Leucocyte Protease Inhibitor (SLPI) SLPI, originally described as Human Seminal Inhibitor I (HUSI-I) or Antileucoprotease (ALP)77;78 is a small, cationic protein that is known to be constitutively expressed by several glandular epithelia. SLPI inhibits leukocyte- derived proteinases, has anti-HIV-179, antibacterial80 and antifungal properties81 and

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interferes with the induction of synthesis of proinflammatory mediators in monocytes and macrophages.82 In human epidermis, SLPI is an inducible antimicrobial protein, which effectively inhibits growth of P.aeruginosa and S.aureus. SLPI expression in skin is stimulated by inflammation (psoriasis, wound healing) and tape stripping. It is suggested that SLPI plays a role in the innate immune skin system providing antimicrobial activity and an antiproteinase shield against proteolysis of dermal elastic fibres and structural epidermal proteins.69 SLPI expression in atopic epidermis has not been studied so far.

Migration Inhibitory Factor Related Protein (MRP8) MRP8 also known as calgranulin A, is a S100 Ca++-binding protein that forms the heterodimeric calprotectin complex together with MRP14.83 Calprotectin is active against intracellular bacteria and C.albicans.84;85 Both proteins are highly abudant in human neutrophils and monocytes and they are also found in elevated levels in plasma of patients with various inflammatory disorders such as chronic bronchitis, cystic fibrosis and rheumatoid arthritis.86 MRP 8 is found in keratinocytes in a variety of inflammatory conditions.87;88 Normal epidermis and skin adnexae are negative.89

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1.2 SELECTED INFLAMMATORY SKIN DISORDERS ASSOCIATED WITH AN ALTERED EPIDERMAL BARRIER

Abnormalities in epidermal function are present in many inflammatory skin disorders. Irritant contact dermatitis (ICD), atopic dermatitis (AD) and psoriasis vulgaris (PV) share common characteristics of impaired cutaneous permeability barrier function which could eitheir initiate or sustain these cutaneous disorders.90-92 In this thesis we have performed studies with respect to epidermal barrier in these inflammatory skin disorders. We focused on ICD as a clinical condition where disturbance of the skin barrier is in most forms a prominent feature and is very important in the pathogenesis of ICD.

1.2.1 Irritant contact dermatitis (ICD)

Irritant contact reaction is inflammatory reaction of the skin to an external agent or agents where, although inflammatory and immunological mediators may be activated, no memory T-cel function or antigen-specific immunoglobulins are involved. Irritant contact reactions include several inflammatory responses that follow damage to the skin. This damage may result from acute toxic insults to the skin, as with accidental exposures to acids, alkalis, etc., or they may arise from repeated and cumulative damage from marginal irritants, both physically and chemically.93

1.2.1.1 Clinical aspects of ICD ICD has a spectrum of clinical features. Recently, ten different forms of irritant contact dermatitis have been described.94

Acute ICD Acute irritation results from skin exposure to a strong irritant or caustic chemical, such as acids or alkaline solutions. Signs of acute ICD include erythema, edema and necrosis in the skin area of the irritant exposure. Irritation is often morphologically similar to allergic contact dermatitis and is thus difficult to diagnose. One distinction exists in the course of the reaction after the exposure to the causative agent. The healing of acute ICD is described as a decrescendo phenomenon, which is opposed to the crescendo phenomenon described in acute allergic contact dermatitis.

Delayed acute ICD The irritation is acute, but the inflammatory response is delayed approximately 8-48 h following the initial exposure. Dithranol may induce a delayed acute ICD. In chapter 5 of this thesis we focus on dithranol induced irritation. Hence, we would like to highlight this unique form of ICD. Dithranol irritation is the most important limiting factor of dithranol treatment, which has been a main therapeutic agent for the topical treatment of psoriasis since

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1916. It is generally assumed that dithranol irritation is caused by the formation of super-oxide radicals due to the loss of a hydrogen atom from the methylene group at position C-10.95 Dithranol irritation presents as a unique form of ICD. The prominent differences between dithranol and a detergent induced irritation are summarized in Table 3. Moreover, in the course of treatment with dithranol, the skin adapts and becomes less susceptible to the irritant effects of dithranol. This adaptative effect is also responsible for the decrease of the antipsoriatic effect after repeated exposures. Therefore increasing the concentration of exposure times is nessesary.96 Such an adaptation is in contrast with the effect of detergents, where repeated challenges cause summation of the irritant response.97;98 The adaptation during repeated applications of dithranol discloses an entirely different mechanism, which is suggested to be specific for dithranol irritation.99 However, the underlying mechanism of this adaptive response of the skin is not elucidated. We will discuss the clinical aspects of this adaptive effect of dithranol in chapter 5. The sensitivity of the skin to dithranol shows large variations from time to time, but also between individuals. The question arises whether it is possible to predict susceptibility to dithranol. In analogy to the minimal erythema dose (MED) for ultraviolet light, the determination of the minimal irritation dose (MID) may optimize finding of the starting dose.100;101 A polymorphism of the TNF-α gene is the first demonstrated genetic marker for irritant susceptibility in normal individuals.102 Whether this marker may contribute to screening of individuals deemed at risk of increased susceptibility to dithranol is unknown. Nevertheless, there are other known factors relevant to dithranol irritation like the vehicle103;104 application frequency105, and skin type.100 Sensitivity to dithranol varies with location, the face, body flexures, axilla, scrotum, breasts and inner sides of the thighs being the most vulnerable parts of the body. The psoriasis lesions are less sensitive to the oxidative dithranol irritation than the surrounding skin probably due to an increased extracellular free radical scavenging system (thioredoxin reductase).106 Whether the frequency of irritation depends upon the severity of disease before treatment is unknown and will be focus of interest in chapter 5. Inflammatory conditions as well as damage to the horny layer make the skin more vulnerable. Pretreatment with corticosteroids, which make the horny layer thinner, may make the skin more vulnerable and phototherapy (UVB radiation or PUVA), which makes the horny layer thicker, renders the skin less vulnerable.41 Because of the redness and pain of uninvolved skin strong responses to dithranol force to stop the treatment for one or more days. An interesting question is whether skin irritation results in a more-or less favourable clinical response to dithranol. In chapter 5 this question is adressed. Dithranol irritation can be treated by topical corticosteroids, although some authors state that it would be ineffective.41;107 Corticosteroids suppress inflammation and symptoms like redness and pain. However, steroids may also make the epidermis thinner and may make the skin more vulnerable for subsequent dithranol irritation.

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Consequently the use of corticosteroids may increase dithranol irritation in the long run and may influence the effectiveness of dithranol therapy. Emollients are useful to cool and soften the skin in case of irritation. Anti-inflammatory effects with emollients and other modalities like tar and non-steroidal anti-inflammatory drugs (indometacin, scopolamin) in dithranol irritation are not to be expected.107-109 Effect of BN 52021 a platelet activating factor antagonist has been described.110 Chapter 5 will focus on the modulation of dithranol irritation in a human experimental model with topical corticosteroids, a coal tar solution and emollients.

Table 3 Prominent differences between dithranol irritation and SDS induced irritation Dithranol irritation SDS induced irritation Individual susceptibility Atopy unknown yes 111 Skin colour unknown yes112;113 Skin phototype type 1 more sensitive unknown 100;107 Age no 105 yes 114 Gender no 105 no 98 Ethnic unknown no 115 Application site yes 41 yes 114 Season no yes 116 Horny layer thickness yes 105 no 117 TEWL normal 118 increased 119 Dose response no 96 yes 120;121 relationship Tolerance after repeated yes122 no 98 application

Irritant reaction The irritant reaction is characterised by a monomorphic presentation that includes either scaling, redness, vesicles or erosions, often localised to the dorsum of the hands and fingers. Upon healing, hardening of the skin occurs. Hairdressers, metal workers, and others exposed to wet work are the most affected groups.

Subjective/sensory irritation 93 Sensory irritation refers to a chemical-induced burning, stinging or itch sensation without accompanying erythema or edema. Histologically is usually

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unchanged. These sensations most commonly affect the head and neck and may present as one form of cosmetic intolerance. Some chemicals will cause sensations within seconds after contact. Such immediate-type stinging follows after exposure to chloroform and methanol. The sensation abates quickly following removal of the irritant substance. A number of chemicals will cause discomfort within 1-2 min, reaching a maximum in 5-10 min., and disappears slowly over the next half hour. Among the substances able to induce such a reaction known as delayed-type stinging are the sunscreen agent amyldimethyl-p-aminobenzoate, the insect repellant diethyltoluamide and several other agents and vehicles used in both cosmetics and medicaments.

Suberythematous irritation Suberythematous irritation indicates a state in which the irritation is not visually apparent, but is histologically visible upon microscopic examination. Common symptoms of this irritation include burning, itching or stinging.

Cumulative ICD Cumulative ICD is thought to be caused by repeated exposures to various weak irritant factors. These minor stimuli in itself may impair the skin, but this impairment is not strong enought to cause a visible skin disorders. If the skin is exposed to irritants before it completely repairs from the previous challenge, ICD may develop. After a series of cumulative insults, which may be of varying nature, the threshold may be surpassed. Beyond this level irritant dermatitis will supervene and even a low level of exposure will cause dermatitis.

Traumiterative ICD This form is similar to cumulative ICD with one exception. Repetitive exposure from only one type of irritant is involved in this irritant response.

Traumatic ICD Traumatic ICD is caused by acute skin trauma. Common symptoms include burns or lacerations. These are most commonly localised on the hands.

Pustular and acneiform dermatitis Tars, mineral oils, croton oils and naphtalene exposure may result in pustular and acneiform dermatitis, especially in atopic and seborrhoic patients.

Exsiccation eczematid Patients, especially elderly persons, who frequently shower tend to get this form of dermatitis. Common symptoms are intense itching, dry skin and ichthyosiform scaling.

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1.2.1.2 Pathogenesis of ICD ICD is a heterogenous disorder with many different mechanisms contributing to its pathogenesis. Impairment of the skin barrier is thought to play a dominant role in the pathogenesis of ICD. The integrity of the epidermal barrier is important in protecting the skin against irritants. Without penetration no damage to the skin can be done. A well-functioning epidermal barrier is of utmost importance. Therefore, to protect the skin against environmental influences, we must aim at optimal barrier integrity. The skin, although under most environmental conditions capable of protecting us, is not always a perfect barrier. Firstly, in some genetic123 or acquired skin disorders the barrier may indirectly have a deficient barrier capacity, resulting in increased penetration of chemicals and increased risks of contact reactions. In atopic individuals, a greater vulnerability to irritants is associated with endogenous SC lipid abnormalities.124-126 Also in non-atopic individuals with a higher tendency for skin irritation, baseline abnormalities in SC lipid composition has been observed.127 Secondly, environmental factors may also have a direct effect on the barrier. For example detergents may wash-out SC lipids, giving rise to damage to cellular components, but especially to the intercellular space of the SC. Abnormalities in the lipid profile of the SC has been observed after application of organic solvents or detergents.119;128;129 Also strong hydration may cause histological and electronmicroscopical changes.130 These abnormalities are consistent with impairment of the skin barrier. Direct and indirect effects of physical, chemical and biological factors on the integrity of the SC, may result in chronic insufficiency of the epidermal barrier and may lead to chronic diseases like irritant contact dermatitis.27 Some chemicals like dithranol have strong inflammatory effects on the skin, without direct effects 131on theSC , exposure to other chemicals like the detergent sodium dodecyl sufate (SDS) give rise to strong erythematous reactions, correlating with skin barrier impairment.117;132 Cytokines carry the focus of interest within contact dermatitis over the last 10 years. Cytokines form a heterogeneous group of short-range, cell-derived peptides which can be released by a variety of nucleated cells. Cytokines may act directly, as inducers and regulators of cell growth, division and differentiation, as stimulators of cell movement and migration and as controllers of cellular function and indirectly interaction via induced changes in the expression of adhesion molecules and receptor for cytokines.133 Resting keratinocytes produce some cytokines constitutively. A variety of environmental stimuli, such as tumor promoters, ultraviolet light and chemical irritants, can induce epidermal keratinocytes to release inflammatory cytokines (IL-1, TNF-α), chemotactic cytokines (IL-8, IP-10) growth promoting cytokines (IL-6, IL-7, IL-15, GM-CSF, TGF-alpha) and cytokines regulating humoral versus cellular immunity (IL-10, IL-12, IL-18).97 Of all the cytokines, TNF-α and IL-1 are the only cytokines capable of activating sufficient effector mechanisms to independently trigger cutaneous inflammation.134 The main pool of TNF-α lies within dermal mast cells, although keratinocytes produce TNF-α constitutively and

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could be stimulated to increase expression.135 Also Langerhans cells and melanocytes are capable of constitutive or inducible expression.136 TNF-α have important pro- inflammatory properties, such as the induction of adhesion molecules, production of other cytokines and up-regulation of MHC class I and II expression.137 In a variety of experimental models of ICD, the release of TNF-α has been demonstrated by a number of investigators.6;138;139 Piquet at al described TNF-α as a critical mediator in ICD.101;140 An important aspect of TNF-α production is an interindividual variability probably due to TNF-α gene-308 polymorphism which is recently associated with susceptibility to ICD.102 Remarkably, not all irritants are able to induce TNF-α, indicating that exposure to irritants can lead to specific cutaneous reactions depending on the nature of irritants.141 Data on expression of TNF-α in dithranol induced irritation are not available, but it is known that dithranol in vitro inhibits human monocytes to secrete TNF-α.142 Oxidative stress is a situation in which an inbalance in pro-oxidant/anti- oxidant equilibrium occurs within cells and tissues, resulting in damage to lipids, proteins, carbohydrates and DNA. Such inbalance may result from excessive endogenous generation of free radicals during physiologic processes, but may also occur through exposure to exogenous sources of free radicals such as tobacco smoke and ultraviolet radiation.143 Irritants may also generate reactive oxygen species, both directly and indirectly. Dithranol-induced skin irritation is a well known example of ICD to be caused by the direct generation of free radicals and oxygen radicals.144 Dithranol slightly elevates lipid peroxidation in mouse skin after topical application, demonstrated by the exhalation of ethane, which is produced from gamma-3 polyunsaturated fatty acids and by malondialdehyde production.145 In addition, singlet oxygen can accelerate lipid peroxidation by directly reacting with unsaturated fatty acyl moieties to give hydroperoxides, which was also already demonstrated for dithranol.146 Thus the peroxidative damage to membrane lipids after dithranol application is generally believed to be related to its antipsoriatic action as wel as induction of skin irritation.147 Furthermore, changes in the levels of the antioxidant enzymes, glutathione S-transferase (GST) and superoxide dismutase (SOD) provide evidence that oxidative stress is also a component of irritation induced by some detergents and that oxidative stress plays a more important role in the pathogenesis of ICD than previously thought.148;149 For most irritants, except dithranol, the major source of free radicals is of an endogenous origin. A possible source is formed by infiltrating neutrophils and macrophages which generate and release of reactive oxygen species.118;143 Interesting is an association between cytokines and oxidative stress. Oxidative stress is known to induce the upregulation of TNF-α, IL-1 and IL-8, but paradoxically, increased TNF-α induced oxidative stress.150 Other mediators such as prostaglandins, leukotriens, neuropeptides etc. seem of less importance for the development of ICD.

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Althought the nature, concentration and duration of contact with the irritant chemical are of primary importance, there are many other factors which are important in modification and/or enhancing the irritant respons. Table 4 summarizes these factors.93

Table 4 Factors which influence susceptibility to irritation Exogenous Endogenous Cofactors Chemical characteristics Individual susceptibilty Mechanical Molecular structure Atopy Thermal pH Race/skin colour Climatic Hydrophobicity Skin phototype Hormonal Inherent toxicity Age Concentration/dose Barrier function Penetration Repair capacity characteristics Vehicle Eczema elsewhere Solubility Other skin diseases Duration of contact Application site Type of contact

1.2.1.3 Treatment of ICD To date, there is no general agreement about how to treat ICD. The aim of this section is to provide a review on topical treatment of ICD, focusing on non-steroidal agents, corticosteroids and topical lipids.

New non-steroidal, anti-inflammatory modulators Cytokines are important in the development of ICD and inhibition of their function can inhibit the site of the inflammatory response. In this thesis the question is addressed whether topical cytokine inhibitors can influence skin irritation and repair of barrier damage. As mentioned before, of all cytokines, TNF-α is a critical mediator in ICD.101 Modulating TNF-α activity in the skin may provide therapeutic benefits for a variety of skin conditions. Recently anti-TNF-α and TNF-α receptor constructs have been shown to have clinical efficacy in the treatment of psoriasis.151 Pretreatment with anti-TNF-α antibodies or with topical N-acetylcysteine, which is an selective inhibitor of gene expression for TNF-α 152, is able to inhibit ICD in mice.140;153 This effect was not observed in human studies where N-acetylcysteine failed to inhibit ICD induced by SDS or dimethylsulfoxide.154 Xantine derivates could be new promising therapeutical agents targeting

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TNF-α and other cytokines. They function as phosphodiesterase inhibitors and are able to suppress the production of TNF-α and other cytokines in murine and human leukocytes and keratinocytes.155;156 A great advantage of the xantine derivates is their well-known pharmacokinetics and safety. They have been used therapeutically in humans with vascular disorders for more than two decades without major side effects.157 Chapter 4 will discuss the effect of cipamfylline, a new phosphodiesterase- 4 inhibitor, on human models of ICD. Macrolide antibiotics may improve dermatitis by suppressing the immune response. Tacrolimus inhibits activation of calcineurin in T-cells. Several studies have documented a benefit of topical tacrolimus in inflammatory dermatoses, e.g. atopic dermatits, psoriasis, allergic contact dermatitis, lichen planus etc.158 However in SDS induced ICD, the widely reported anti-inflammatory effect of tacrolimus could not be confirmed. In one experimental human study on ICD, tacrolimus did not accelerate healing, even enhanced skin irritation.159 Pimecrolimus, as the other member of macrolide antibiotics seems to be effective in chronic irritant hand dermatitis.160 Other new nonsteroidal anti-inflammatory modulators as antiflammin 1 peptide161, phenoxyacetic acid methyl ester162, azaspiranes163, activators of liver X receptor164, strontium165;166 have been shown to be effective in a variety of animal and human experimental models of ICD. A curious rapport about anti-inflammatory effect of acetone has been published.167 Since there is strong evidence that free radicals and reactive oxygen species are important in ICD, potent antioxidants has been examined to influence ICD. Quercin, as a bioflavinoid with antioxidant and anti-inflammatory activity, does not appear to increase barrier recovery in SDS induced ICD.168

Topical lipids The benefits of lipids in treatment of the skin were already known by Cleopatra who took her bath in donkey’s milk. From this ancient on time on, waxes, oils, amphiphilic lipidic material have been used in various dermatological and cosmetic formulations. Galen’s formula for cold cream is thought to be the first example of an emulsion written down in the London Pharmacopoeia from 1618. The original formula did not contain borax, which was introduced in 1890 for the manufacture of cold cream. Borax was discovered in California in 1859.169 Despite the extensive clinical experience with lipids in dermatological formulations and more than 40 years of skin lipid research, understanding of the interaction of topically applied lipids with skin barrier lipids is lacking. As mentioned before, lipids of the stratum corneum (SC) play a significant role in maintaining the permeability barrier of the skin. Changes in lipid composition in the SC are associated with different skin conditions and disorders. There is evidence that topical application of skin related lipids (ceramides, cholesterol, fatty acids) may improve the skin barrier function. Effects of topical application of SC lipids on ICD were evaluated by the group of Elias. Firstly, they demonstrated that

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the application of a single SC lipid and mixture with two SC lipids delayed barrier repair in mice. Secondly, the topical application of an equimolar three-component system of ceramides, cholesterol and fatty acid allows normal barrier repair.170 Finally they found the optimal molar ratio of the SC lipids that accelerated barrier recovery in acetone induced barrier impairment in mice.171 Out of a number of preparations, those with a lipid ratio most comparable to that present in the human skin barrier, is considered to be effective.172 They also compared the mixture of skin related lipids with petrolatum on the barrier repair process. Petrolatum repaired the barrier more quickly than the related lipids, but 2 or 4 h later, the application of a skin related lipid resulted in a better recovery than petrolatum.173 Using fluorescence and electron microscopy has also been demonstrated by that the mixture of cholesterol, fatty acid and ceramides rapidly traversed the SC with subsequent uptake into the epidermal nucleated layers170;171;173, whereas the applied petrolatum stayed in the SC. A new generation of emollients with specific lipid profiles and lipid contents adjusted to specific skin condition is under development, but the evidence for superiority to the traditional products in human is still lacking. In this thesis (chapter 4) the question is addressed whether certain mixture of skin related lipids is effective in human models of experimentally induced ICD and in some clinical conditions associated with barrier damage.

Corticosteroids Dermatologists use topical corticosteroids to treat numerous disorders, however their use in ICD remains controversial. The anti-inflammtory properties of corticosteroids could improve the dermatitis, however, their antiproliferative effects might slow recovery of the skin barrier and may enhance penetration of irritants. Several authors reported a lack of efficacy of topical corticosteroids in skin irritation, whereas others clearly showed such an effect.174-179 However, there are considerable differences between the designs of these studies with regard to corticosteroid potency, the duration and site of the irritation, open or closed application, and the concentration of the irritant, which apparently may influence the outcome of the study. Acute and repetitive open application testing on the hands of volunteers with several irritants are recommended in order to establish the efficacy of corticosteriods in ICD.180 In case of radiation dermatitis, barrier enhancing properties of topical corticosteroids have been observed. It is suggested that the positive effect of topical corticosteroids on barrier damage in this skin condition is caused by a primary reduction in the inflammatory response.181 In nonanonic acid - induced irritation which associated with minimaly skin barrier dysfunction corticosteroids has been shown to be effective.182 In SDS induced ICD, barrier dysfunction precedes inflammation and therefore damage to the skin barrier may only partly be a reflection of an inflammatory response. This may explain the modest or absent efficacy of a topical corticosteroid in models of ICD, since barrier dysfunction is considered to play a prominent role in its pathogenesis.

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1.2.2 Atopic dermatitis (AD)

AD is a chronic, itching skin disease with a relapsing course. It forms part of the atopic syndrome, also known as atopy. Clinical and laboratory criteria have been developed for the definition of AD. The most widely used are those of Hanifin and Rajka (1980). AD is one of the most common skin diseases with a life-time prevalence of up to 30 %. Its incidence is increasing in Western countries. The highest incidence is among children.183 AD starts usually in early chilhood. The infant phase starts during the third month and is characterised by dry red scaling areas on the cheeks and chin, sparing the perioral and paranasal region. More severe cases show generalised papulation, redness, scaling, vesicles and crustae. The childhood phase is characterised by inflammation in flexural areas, sides of the neck, wrists and ankles with depigmentation and the first signs of lichenification. Marks of the adult phase are flexural inflammation, hand and/ or foot dermatitis, periorbital erythema and lichenification of the anogenital area.184 Histological changes in AD are a matter of controversy. Many authors tend to regard the histological findings as nonspecific. Histological findings in AD can be classified as acute, subacute and chronic, according to the various stages of clinical evolution. Epidermal changes in early lesions are spongiosis, intercellular oedema with vesiculation, irregular hyperplasia. Dermal changes include oedema and perivascular and intervascular inflammatory infiltrates consisting of lymphocytes, macrophages, occasional by neutrophils, eosinophils and basophils. Long-standing lichenified lesions of AD show acanthosis of the epidermis, para- and orthokeratosis with little or no intercellular oedema. Dermal changes include papillary fibrosis, moderate perivascular and intervascular lymphohistiocytic infiltrate in the upper dermis and increase of mast cells.185 AD is generelly accepted as an immunologic disease (inside-outside hypothesis). Recent investigations have greatly increased the understanding of immunological mechanism involved in the pathogenesis of AD. A major immunological abnormality is the skewing, within the secondary immune organs of ThO cells towards Th2 lymphocytes, upon allergenic challenge. As a result, Th2 cells dominate the central responses and through their specific cytokine secretion profile, induce B cells to produce allergen-specific IgE. These molecules are secreted into the circulation and will then sensitize different organs, by binding via Fc R receptors on mast cells and dendritic cells (including Langerhans cells). T cells are activated by aeroallergens, food antigens, autoantigens and bacterial superantigens.186 The existence of defects in skin barier function in AD is well accepted, but the epidermal abnormality generally is viewed as a downstream consequence of the inflammatory phenotype.186 Whithout denying the significance of immunological phenomena in the pathogenesis of AD, some authors support the outside-inside

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hypothesis in which barrier dysfunction plays a major role in the pathogenesis in AD.187;188 Evidence for this hypothesis is provided by the observation that increased TEWL, abnormal lipid composition and changed levels of enzymes involved in lipid metabolism, are already present in the clinically uninvolved skin in the absence of clinical and histological signs of inflammation.189-193 Furthermore, disease activity correlates with the extent of barrier abnormalities as wel as with factors influencing SC barrier function (decreased humidity194, season, psychological stress195). Also the threefold increase in prevalence of AD over the past 30 years can be explained by barrier dysfunction: alteration of life-styles, such as excesive use of soaps and shampoos, changes in indoor climate enhance skin barrier dysfunction.91;196;197 It is possible that barrier disruption in AD is an etiologic factor for eliciting dermatitis following the easy penetration of irritants and antigens.192 Nevertheless, it remains difficult to determine whether barrier dysfunction is a primary initiator or onlya secondary event that participates in the vicious circle. A possible relationship of epidermal barrier dysfunction to the pathogenesis of AD is shown in Figure 2.198

Environmental Genetic Low Humidity Ichthyosis vulgaris Allergens (Type I and IV allergy) Dry Skin Irritants (e.g. water and detergents) Barrier Disturbed epidermal lipid Psychological Stress perturbation content (ceramides) Trauma LC + Penetration of allergens

Immunological Cytokine cascade Infection abnormalities

Inflammation, Epidermal hyperproliferation Cytokine cascade

Atopic Pruritus Infectious complications eczema

Figure 2 Relationship of epidermal barrier dysfunction to the pathogenesis of atopic dermatitis. (Source198)

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Several studies on barrier function and lipid composition have been performed in patients with AD, and the important findings are: (1) increased TEWL both in clinical involved and uninvolved skin199;200 (2) decreased water content 201;202 (3) reduced water binding capacity 203 (4) decrease in the total amount of extractable SC lipids 189;204 (5) morphological abnormal lamellar bodies.205 The best investigated lipid fractions in AD epidermis are ceramides as they represent the main lipid component of permeability barrier.206 Although the results vary in the different studies, the total ceramide amount in involved and uninvolved skin of AD patients is significantly reduced.189;190;207 Among the ceramide fractions, ceramide 1 and 3 are most significantly reduced.126 The biologic basis of the ceramide deficiency may include abnormal expression of the enzymes involved in sphingomyelin metabolisme 191-193, the colonisation of AD skin by ceramidase-secreting bacteria208 or a disturbed extruding mechanism of LB’s. As ceramides are important determinants involved in the water-holding properties209;210 and barrier function of SC211;212, the insufficiency of ceramides provide an etiologic bassis for the barrier-disruption in AD patients. For some authors, the ceramide- deficiency in AD make a strong basis to explain some parts of pathogenesis in terms of biochemical mechanisms involved in lipid metabolism.192 The relevance of superantigens in the induction of dermatitis in AD patients has recently been underlined.213 AD is known to be associated with a high prevalence of skin infections, particularly with Staphylococcus aureus (S. aureus).214 Disease severity has been associated with the presence of S. aureus in the skin of children with AD215;216 and with the presence of IgE to superantigens.217 The important role of S. aureus is supported by observation that even in patients with AD whithout bacterial superinfection, the severity of the disease is more reduced by a combination of antistaphylococcal antibiotics and topical corticosteroids as compared to topical corticosteroids alone.218;219 The mechanisms underlying the increased colonisation of atopic skin with S. aureus is unknown. Impairment of the phagocyte system214, specific binding ofS. aureus to the inflammation mediated by type 2 helper T cells220, sphingosine-deficiency221, decreased secretion of IgA and alkaline pH in the skin of patients with AD222 are mentioned to explain the increased incidence of infections in these patients. Further, it is known that S. aureus grows in colonies in the upper layers of the epidermis between keratinocytes.223 This suggests that increase in S.aureus could be the result of failure of the innate immune defense system of atopic skin to restrict the growth of the organisms. Recently it was shown, that epidermis of AD patients expresses low levels of the antimicrobial proteins LL-37 and β-defensis-2, compared to psoriasis patients. It was speculated that a insufficiency in the expression of inflammation-induced antimicrobial peptides may explain the susceptibility of patients with AD to skin infection.224 In this thesis (chapter 3) we extended this observation to three other known host defence proteins (SKALP, SLPI and MRP8) to further investigate this phenomenon. Therapeutic agents for AD are limited. Topical corticosteroids are the mainstay of the treatment of AD, but their use in a chronic and relapsing condition is

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unsatisfactory due to potential toxicity, particularly in small children. A large variety of topical steroids is available. The potency of these corticosteroids ranges from mild to very potent.225 Recently new therapies has been introduced in the management of AD.226 Tacrolimus and pimecrolimus are new macrolide-type inflammatory cytokine inhibitors, with a mechanism of action similar to cyclosporin. In contrast to cyclosporin A, they are effective in the topical treatment of AD due to their relatively small size. Both compounds are effective against itch, one of the most prominent problems of patients with AD. In contrast to topical corticosteroids, these compounds seems to have a more favorable side-effect profile.129;158;160;227 Topical phosphodiesterase (PDE) inhibitors are also used experimentally in the therapy of AD.228;229 PDE inhibitors are cAMP-elevating agents as they block the PDE enzymes that convert cAMP to 5’AMP, leading to the intracellular acumulation of cAMP. It is known that intracellular elevation of cAMP has anti-inflammatory properties. The role of the cyclic nucleotide phosphodiesterases (PDE) in the pathogenese of AD was proposed more than 20 years ago as a possible explanation for the rapid enzymatic breakdown of cAMP found in leucocytes from AD patients. PDE 4 inhibitors act as anti-inflammatory agents by blocking the increased PDE activity found in AD. Besides the blocking, other pathways of the anti-inflammatory activity of PDE inhibitors should be taken into consideration. This includes IFN-γ which has been detected in chronic phase and bacterial infections, which through the production of superantigens may play a role in AD pathogenesis.230

1.2.3 Psoriasis

Psoriasis is a chronic, non-infectious, inflammatory disease of the skin. The prevalence ranges between 1 and 3% of the population.231;232 The course of the disease is unpredictable : it may last from a few weeks to a whole lifetime. Psoriasis is considered to be a polygenic and multifactorial disease. A hereditary component plays a role, but the mechanism of inheritance remains unclear.233 A family history for psoriasis is present in one third of the patients. There is a high association between psoriasis and certain HLA-types.234 In addition, a variety of external factors has been accepted as triggering factors in predisposed patients.235;236 It is known that local irritation or injury to the skin can result in the induction of a new psoriatic lesions (Köbner phenomenon).237 The cutaneous lesions in psoriasis are well-defined, elevated and erythematous plaques. There is a silvery, non-coherent scaling, which differs in intensity in the various patterns of psoriasis. The morphology of psoriasis shows a considerable variation. Clinical presentations are: guttate psoriasis, annular psoriasis, pustular psoriasis, erythrodermic psoriasis and chronic plaque psoriasis. The last one is the most common form of psoriasis, making up 90% of the cases.238 In addition to the skin lesions, extracutaneous manifestations can develop: in 30-50% of the patients

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nail abnormalities are present and a minority of the patients also suffers from psoriatic arthropathy.239 Increased proliferation, altered differentiation and cutaneous inflammation are the histopathologic changes that characterise psoriasis lesions. The histological aspects may vary considerably, depending on the stage of the lesion. The early plaque shows polymorphonuclear leucocytes penetrating into the stratum spinosum and corneum, forming Munro microabcesses and Kogoj pustuls, respectively. In a fully developed psoriatic lesion the histopathological features consist of ortho- and parakeratosis, disappearance of the granular layer, elongated rete ridges, oedematous papillary dermis and dilated and tortuous capillaries.240;241 Despite many investigations, the exact pathogenesis of psoriasis is still not known. It is beyond the scope of this thesis to discuss all different hypotheses on the pathogenesis of psoriasis. It is clear that both epidermis and dermis are needed for the appearance of psoriatic lesions. In psoriasis, dysfunction of water holding properties of epidermis has been observed. Water barrier function is impaired causing an increase in TEWL and these observations are consistent with disturbances in lipid metabolism. Since there is a close relationship between lipid metabolism and keratinocyte differentiation, these changes in lipid metabolism are probably a direct consequence of epidermal hyperproliferation and abnormal differentiation. In psoriatic scale, the same relative content of ceramides as in normal SC were found, however, chromatographic separation of ceramide species displayed a different pattern of ceramides distribution.35;242-244 In psoriatic scales, cer 1, cer 3 and cer 6 show a statistically significant decrease vs. normal SC, while some ceramides (cer 2 and cer 5) are increased. A comparable pattern of ceramide distribution has not been observed in atopic dermatitis. These biochemical changes correlate with ultrastructural studies on psoriasis where an extremely narrow intracellular space of SC and pathological lamellae has been found. Likewise, these morphological findings are psoriasis-specific and could not be observed in atopic dermatitis.245 Also covalently bound lipids, which have principal functions in the formation of a lipid envelope on the outer surface of keratinized cells, show different proportion from those found in normal SC. An elevated level of bound linoleic acid associated with alterations in the covalently-bound ceramides has been described. However, the psoriatic scale contains a similar total concentration of the covalently bound lipids.52 This different pattern of ceramide fraction and covalent bound lipids is possible linked specifically to the abnormal differentiation in psoriasis. Although the relative free fatty acid content is decreased remarkably (46%) in psoriatic scales, compared with normal human SC 242, psoriatic scale shows increased levels of bound linoleic acids and other esterified derivates of linoleic acid. One can divide the treatment of psoriasis in topical therapy and systemic treatment. Examples of topical therapy are vitamin D3-analogues (calcipotriol, tacalcitol and calcitriol), corticosteroids, coal tar and dithranol. In case of more

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widespread psoriasis, phototherapy and photochemotherapy are indicated. In severe cases systemic treatment with for example methotrexate, retinoids or cyclosporin is indicated.246;247 One purpose of this thesis is to investigate aspects and the management of dithranol induced irritation, which is a common side effect of dithranol therapy. Hence, a short description of dithranol therapy in psoriasis will be provided. Dithranol, a synthetic analogue of chrysarobin, has been used for over 80 years in the treatment of chronic plaque psoriasis. Dithranol is a yellow powder that can be incorporated in a cream, ointment, paste or stick. Dithranol application can be carried out according to many different regimes for instance the Ingram regime. The time honoured 24 hour dithranol appliactions are supposed to be most effective but it requires hospitalisation. Short contact applications and cream formulations have simplified dithranol treatment. Dithranol has two drawbacks: skin irritation and temporary discoloration of the skin and permanent discoloration of garments, furniture and sanitary. To avoid this permanent discoloration, precautions have to be taken to avoid contact between dithranol and these materials.95;248 Dithranol irritation is discussed as a part of irritant contact dermatitis.(see page 21).

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1.3 METHODOLOGY

1.3.1 Experimental human models of ICD In this thesis the effect of topical treatment on ICD was studied in four different established models of experimentally induced skin irritation.

1.3.1.1 Sodium dodecyl sulphate (SDS) In experimental dermatology, the anionic detergent, sodium dodecyl sulphate, is commonly used as irritant in models for ICD. SDS meets the requirements of an irritant for experimental use because it causes no systemic toxicity, no cosmetic inconvenience to the exposed subjects and it is not a carcinogen nor sensitiser.98 Guidelines regarding the SDS exposure test have been established and described by Tupker et al.249 Barrier dysfunction occurs after exposure to SDS as assessed by TEWL as a parameter for barrier function impairment.250;251 This barrier impairment is associated with cell biological changes. The cell biological response of the keratinocytes include (1) an increased proliferative rate as measured by Ki-67 positive cells (2) an induction of involucrin and (3) an upregulation of E-FABP.50 The effect of topical treatment in this thesis was studied in two different established models of SDS- induced skin irritation.50;252 Firstly we used a single exposure of 1% sodium dodecyl sulphate. We used a 1% dilution of SDS (w/v). An aliquot of 150 μl of SDS was pipetted on the patches, consisting of a 1,5 cm2 piece of absorbent, non-woven fabric on a 4.0 cm2 piece of impermeable plastic foil. 174 The subjects were patch tested on investigating sites on the lower back for 24 hours. After removal of the patches, the tested ointment was applied daily over the area of previously SDS exposed skin, dried at room temperature for 30 minutes. To mimic the development of ICD in daily life we used repetitive application of 0.2% SDS. 150 μl of SDS (0.2%) (w/v), was pipetted on the patches which of a 1.5 cm2 piece of absorbent, non-woven fabric on a 4 cm2 piece of impermeable plastic foil. The SDS patches were daily fixed at the investigation site to the lower back during 4 h for 4 consecutive days. After removal of the patches, the tested ointment was applied daily on the area of previous SDS exposed skin.

1.3.1.2 Tape stripping We used as another model for barrier disruption tape stripping. After tape stripping an increase in TEWL4 and induction of a hyperproliferative/inflammatory responses was observed, which include (1)epidermal proliferation as measured by Ki-67 positive cells, (2) upregulation of cytokeratin 16, (3) expression of SKALP and (4) an increase of density of epidermal Langerhans cells.253-255 Also lipid abnormalities after tape stripping have been described.256 As such it offers a suitable method to study influence of therapy on barrier dysfunction associated with epidermal cell biological changes. We used adhesive tape (Tesafilm) which was applied with gentle pressure to

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the exposed areas and then removed. This procedure was repeated until the skin was glistening.

1.3.1.3 Models of dithranol irritation Recently, two human experimental models (single and repetitive application) of dithranol irritation have been developed. The dynamics in epidermal changes after repeated challenge are comparable with those after single application, involving (1) an induction of the cornified envelope precursor protein involucrin (2) the cross-linking enzyme transglutaminase I and (3) an increase of epidermal proliferation. Uninvolved skin of patients with psoriasis is recommended to perform studies on the interference of dithranol irritation by various therapeutical agents.6 In our experiments we studied the response of topical treatment after repeated applications of dithranol cream on uninvolved skin of psoriasis patients. We applied the cream on 2 cm Ø of the lower back for 1 hour, after which the dithranol cream was removed with water. We repeated it once daily during 4 consecutive days.

1.3.2 Measurement of water barrier function: TEWL Transepidermal water loss (TEWL) is the constitutive steady state water vapour loss from the skin, theoretically excluding sweat gland activity. It is considered to be the result of passive diffusion through the skin, the diffusional flux obeying the physicochemical laws of passive transport through a membrane.257 TEWL measurement is a frequently used method to asses the function of skin barrier. An evaporimeter (Courage & Khaza, Germany) is used in this study. (see Figure 3)

Figure 3 Tewameter TM 210

Its operating principle is based on the method of gradient estimation. The measuring probe head of this instrument contains two sensor units, placed in in the enclosure of an open cylinder. Each sensor unit consist of a humidity transducer and

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a thermistor. The probe head is applied parallel to the skin, the sensor units being positioned af a different distance to the skin surface (3 and 9 mm). Thus, the signals of the humidity transducer and thermistors can be used to compute the partial pressure of the water vapour and the temperature at these positions above the skin surface. The partial pressure gradient and water vapour loss rate can thus be calculated.258 (see Figure 4)

Figure 4 The measuring principle. The law of diffusion is discovered by Adolf Fick : dm/dt = -D.A.dc/dx. The diffusion flow dm/dt indicates the mass 2per cm being transported in a period of time. It is a proportional to the area A and the change of concentration per distance dc/dx. D is the diffusion coefficient of water vapour in the air. This law is only valid within a homogenious diffusion zone, which is approximately formed by a hollow cylinder. The resulting density gradient is measured indirectly by two pairs of sensors (temperature and relative humidity) and is analysed by a microprocessor.

TEWL is by definition diffusional water loss through the epidermis. Therefore sweat gland activity should be eliminated as much as possible by measuring under neutral environmental conditions. In order to avoid thermal sweating and emotional sweating subjects should be put at ease for 15-30 minutes in a room with a constant temperature of 20-22 °C and a relative humidity of about 40%.259 In case there is still sweating despite these recommondations sweating can be blocked by a 1-hour occlusive application of scopolamine hydrobromide.260 In order to measure pure diffusional water loss one should avoid desorption (loss of excessive unbound water), for example by measuring not too early (< 1 hour) after removing patches in irritancy testing.261 Many environment-related variables influence water diffusion through the horny layer. The most important variables are environmental factors like (i) ambient air temperature which influences skin temperature and consequently the horny layer barrier function, (ii) air humidity which influences the water vapour gradient over the skin and the barrier function characteristics of the SC horny layer and (iii) seasonal variation. The latter is a very important variable, since the skin is often dehydrated by low indoor relative humidity in the winter in many parts of the world. Low relative humidity has a strong effect on skin hydration and consequently on barrier function. Also personal factors including age, sex, skin colour and anatomical site may be of influence.

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To avoid instrument-related disturbance of the microclimate amongst others direct light, heat, draft and pressure of the probe should be avoided. Pinnagoda et al defined guidelines for accurate and reproducible measurements.259

1.3.3 Visual grading Erythema can be assessed by means of colorimetry or visual grading. A previous study showed a good correlation between the visual scoring, with colorimetry and with other parameter like TEWL. In order to investigate the effect of therapies in ICD we used visual grading to determine the erythema. Semi-quantitative grading system of erythema, already described in other studies50, was used: 0-no response; 1- slight, patchy redness; 2-diffuse mild redness, 3-moderate redness, 4-intense redness; 5-intense redness with oedema.

1.3.4 Immunohistochemistry Tabel 1 summarizes epidermal markers we used to investigate the cell biological effects of therapeutical agents highlighted in this thesis in diverse models of ICD. (Chapter 4 and 5)

Tabel 1.Overwiew of used epidermal immunohistochemicals markers Immunohistochemical markers (target / antibody) eai 1 / 16 Keratin / Involucrin / minase Transgulta- / E-FABP Langerhans cells/Dako T6 Anti-E-FABP Ki76 / MIB-1 Anti-TGM

Models MON 150 LL025

SDS Acute (single dose + + + - + - 24 hrs (1%)) Chronic (repeated + + + - + - dose (0.2%)) Tape stripping + + + - - + Dithranol model + - + + - - (repeated dose 2%)

1.3.4.1 Epidermal proliferation Ki-67 antigen is essential for cell proliferation and has therefore been widely used as a proliferation marker. It is a protein doublet, consisting of 345 and 395-kDa proteins and it accumulates in the cell nuclei from G1 to mitosis, after which is rapidly dissociated.262 Ki67 expression, assessed by MIB-staining is a most valuable approach

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to assess epidermal proliferation263 and we used this marker to investigate the efficacy of selected treatments on ICD in the present thesis.

1.3.4.2 Epidermal differentiation A set of monoclonal antibodies directed against several cellular markers for keratinocyte differentiation was selected:

Cytokeratin 16 Keratinocytes in the basal layer of the epidermis express cytokeratin 5, 14 and a variable amount of cytokeratin 15. In contrast, in suprabasal compartment cytokeratins 1, 2, 10 and 11 are expressed. In hyperproliferative epidermis cytokeratin 6 and 16 are expressed.264 Cytokeratin 16 is found in hyperproliferative skin but also in damaged and recovering skin and is thought to be associated with disturbance of the epidermal integrity.265-267 In this thesis, the monoclonal antibody that was used is LL025, that is specific for the expression of cytokeratin 16.268

Involucrin The corneocytes possess an envelope of cross-linked proteins surroundings a network of keratins. Involucrin is a soluble cytoplasmic protein precursor which is synthesised in the stratum granulosum and is cross-linked in the SC under influence of the enzyme transglutaminase. It has been demonstrated that involucrin is a precursor in the early phase and loricrin in the late phase in the formation of the cornified envelope.269 Immunohistochemical studies show upregulation of involucrin expression in the suprabasal compartment of the epidermis in numerous skin conditions, characterised by impaired barrier function.50;270;271 In this thesis we used well-characterized monoclonal antibody MON-150 to study the expression of involucrin.272

1.3.4.3 Langerhans cells Langerhans cells (LCs) initiate T-cell-dependent immunity against foreign antigens. It has been postulated that epidermal exposure to any penetrating substance activates the local LC system, including antigen uptake and presentation. Interestingly, the epidermal LCs density also correlates with the integrity of the permeability barrier. Barrier disruption with tape stripping, SDS, acetone lead to an increase of epidermal LCs and the level of increase correlates with the extent of impairment of the permeability barrier. Immunohistochemical studies show that LCs are usually located in suprabasal cell layers. The dendrites of the LCs are orientated mainly towards the stratum granulosum. Using monoclonal antibodies DAKO-T6 (1:100, DAKO A/S, DENMARK), we made an analysis of the presence of LCs.

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1.3.4.4 Epidermal Fatty Acid Binding Protein (E-FABP) E-FABP belongs to a family of low molecular mass (14-15kDa) proteins. In 1993, epidermal type FABP (E-FABP) with a high affinity for C-18 fatty acid has been characterised in human epidermis.48 It is assumed that E-FABP is involved in the transport of fatty acids which play an important role in the epidermis at the structural and the functional level.273 For detailed description see page 16. A polyclonal rabbit antiserum against E-FABP used in this thesis was prepared as previously described.49

1.3.4.5 Host defense proteins To investigate the expression of host defense molecules we examined 3 antimicrobial proteins: SKALP, SLPI and MRP8. For detail description see pag 8. The dual function proteins SLPI and elafin were stained using polyclonal antibodies that we have previously generated. MRP8 was stained with a polyclonal rabbit antiserum, kindly provided by dr P. Madsen. Arhus, Denmark.

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1.4 AIMS AND QUESTIONS OF THIS THESIS

I. To expand the information on the water barrier of the epidermis with respect to lipid metabolism ▪ What is the expression of an epidermal fatty acid carrier in skin conditions with impairment of barrier function? Can we find evidence for the role of this carrier in skin barrier repair?

II. To study the antimicrobial properties of epidermis in inflammmatory skin disorders ▪ Are there differences in expression of epidermal host-defence proteins between psoriasis and atopic dermatitis?

III. To investigate novel therapies on irritant contact dermatitis (ICD) focusing on the improvement of water barrier impairment ▪ What is the effect of the phosphodiesterase-4 inhibitor (cipamfylline), introduced as a new anti-inflammatory modulator, on experimentally induced ICD?

▪ What is the effect of topical skin related lipids on experimentally induced ICD?

▪ Can we confirm superiority of topical skin related lipids in skin disorders with impaired barrier dysfunction?

IV. Clinical aspects and topical treatment of dithranol irritation as an unique form of ICD ▪ What is the optimal treatment of dithranol induced ICD? ▪ Adaptation as a clinical phenomenon in dithranol irritation ▪ Is dithranol-induced skin irritation correlated with clinical outcome?

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188. Elias,P.M. and Feingold,K.R., Does the tail wag the dog? Role of the barrier in the pathogenesis of inflammatory dermatoses and therapeutic implications.Arch.Dermatol 2001. 137: 1079-1081. 189. Imokawa,G., Abe,A., Jin,K., Higaki,Y., Kawashima,M., and Hidano,A., Decreased level of ceramides in stratum corneum of atopic dermatitis: an etiologic factor in atopic dry skin? J Invest Dermatol. 1991. 96: 523-526. 190. Yamamoto,A., Serizawa,S., Ito,M., and Sato,Y., stratum corneum lipid abnormali- ties in atopic dermatitis. Arch.Dermatol Res. 1991. 283: 219-223. 191. Murata,Y., Ogata,J., Higaki,Y., Kawashima,M., Yada,Y., Higuchi,K., Tsuchiya,T., Kawainami,S., and Imokawa,G., Abnormal expression of sphingomyelin acylase in atopic dermatitis: an etiologic factor for ceramide deficiency?J.Invest Dermatol. 1996. 106: 1242-1249. 192. Hara,J., Higuchi,K., Okamoto,R., Kawashima,M., and Imokawa,G., High-expres- sion of sphingomyelin deacylase is an important determinant of ceramide deficiency leading to barrier disruption in atopic dermatitis. J.Invest Dermatol. 2000. 115: 406- 413. 193. Okamoto,R., Arikawa,J., Ishibashi,M., Kawashima,M., Takagi,Y., and Imokawa,G., Sphingosylphosphorylcholine is upregulated in the stratum corneum of patients with atopic dermatitis. J.Lipid Res. 2003. 44: 93-102. 194. Sato,J., Denda,M., Chang,S., Elias,P.M., and Feingold,K.R., Abrupt decreases in environmental humidity induce abnormalities in permeability barrier homeostasis. J Invest Dermatol. 2002. 119: 900-904. 195. Altemus,M., Rao,B., Dhabhar,F.S., Ding,W., and Granstein,R.D., Stress-induced changes in skin barrier function in healthy women. J Invest Dermatol. 2001. 117: 309-317. 196. Williams,H.C., Forsdyke,H., Boodoo,G., Hay,R.J., and Burney,P.G., A protocol for recording the sign of flexural dermatitis in children. Br.J.Dermatol. 1995. 133: 941-949. 197. Rajka,G., Atopic dermatitis. Correlation of environmental factors with frequency. Int.J.Dermatol. 1986. 25: 301-304. 198. Proksch,E. and Elias,P.M., Epidermal Barrier in Atopic Dermatitis. In Bieber,T. and Leung,D.Y.M. (Eds.) Atopic Dermatitis. Marcel Dekker,Inc, New York 2002, pp 123-143. 199. Werner,Y. and Lindberg,M., Transepidermal water loss in dry and clinically normal skin in patients with atopic dermatitis. Acta Derm.Venereol. 1985. 65: 102-105. 200. Loden,M., Olsson,H., Axell,T., and Linde,Y.W., Friction, capacitance and transepi- dermal water loss (TEWL) in dry atopic and normal skin. Br.J Dermatol. 1992. 126: 137-141. 201. Werner,Y., The water content of the stratum corneum in patients with atopic derma- titis. Measurement with the Corneometer CM 420. Acta Derm.Venereol. 1986. 66: 281-284. 202. Berardesca,E., Fideli,D., Borroni,G., Rabbiosi,G., and Maibach,H., In vivo hydra- tion and water-retention capacity of stratum corneum in clinically uninvolved skin in atopic and psoriatic patients. Acta Derm.Venereol. 1990. 70: 400-404. 203. Werner,Y., Lindberg,M., and Forslind,B., The water-binding capacity of stratum corneum in dry non-eczematous skin of atopic eczema. Acta Derm.Venereol. 1982. 62: 334-337.

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204. Mustakallio,K.K., Kiistala,U., Piha,H.J., and Nieminen,E., Epidermal lipids in Besnier’s prurigo (atopic eczema). Ann.Med.Exp.Biol.Fenn. 1967. 45: 323-325. 205. Fartasch,M. and Diepgen,T.L., The barrier function in atopic dry skin. Disturbance of membrane-coating granule exocytosis and formation of epidermal lipids? Acta Derm.Venereol.Suppl (Stockh) 1992. 176: 26-31. 206. Macheleidt,O., Kaiser,H.W., and Sandhoff,K., Deficiency of epidermal protein- bound omega-hydroxyceramides in atopic dermatitis. J.Invest Dermatol. 2002. 119: 166-173. 207. Melnik,B., Hollmann,J., and Plewig,G., Decreased stratum corneum ceramides in atopic individuals--a pathobiochemical factor in xerosis? Br.J.Dermatol. 1988. 119: 547-549. 208. Ohnishi,Y., Okino,N., Ito,M., and Imayama,S., Ceramidase activity in bacterial skin flora as a possible cause of ceramide deficiency in atopic dermatitis. Clin.Diagn.Lab Immunol. 1999. 6: 101-104. 209. Imokawa,G. and Hattori,M., A possible function of structural lipids in the water- holding properties of the SC. J.Invest Dermatol. 1985. 84: 282-284. 210. Imokawa,G., Akasaki,S., Hattori,M., and Yoshizuka,N., Selective recovery of de- ranged water-holding properties by stratum corneum lipids. J Invest Dermatol. 1986. 87: 758-761. 211. Grubauer,G., Feingold,K.R., Harris,R.M., and Elias,P.M., Lipid content and lipid type as determinants of the epidermal permeability barrier. J Lipid Res. 1989. 30: 89- 96. 212. Holleran,W.M., Feingold,K.R., Mao,O.M., and et al, Regulation of epidermal sphingolipid synthesis by permeability barrier function. J Lipid Res. 1991. 32: 1151- 1158. 213. Skov,L., Olsen,J.V., Giorno,R., Schlievert,P.M., Baadsgaard,O., and Leung,D.Y., Application of Staphylococcal enterotoxin B on normal and atopic skin induces up- regulation of T cells by a superantigen-mediated mechanism. J.Allergy Clin.Immunol. 2000. 105: 820-826. 214. Christophers,E. and Henseler,T., Contrasting disease patterns in psoriasis and atopic dermatitis. Arch.Dermatol.Res. 1987. 279 Suppl: S48-S51. 215. Bunikowski,R., Mielke,M.E., Skarabis,H., Worm,M., Anagnostopoulos,I., Kolde,G., Wahn,U., and Renz,H., Evidence for a disease-promoting effect of Staph- ylococcus aureus-derived exotoxins in atopic dermatitis. J.Allergy Clin.Immunol. 2000. 105: 814-819. 216. Zollner,T.M., Wichelhaus,T.A., Hartung,A., Von Mallinckrodt,C., Wagner,T.O., Brade,V., and Kaufmann,R., Colonization with superantigen-producing Staphylococ- cus aureus is associated with increased severity of atopic dermatitis. Clin.Exp.Allergy 2000. 30: 994-1000. 217. Bunikowski,R., Mielke,M., Skarabis,H., Herz,U., Bergmann,R.L., Wahn,U., and Renz,H., Prevalence and role of serum IgE antibodies to the Staphylococcus au- reus-derived superantigens SEA and SEB in children with atopic dermatitis. J.Allergy Clin.Immunol. 1999. 103: 119-124. 218. Leyden,J.J. and Kligman,A.M., The case for steroid--antibiotic combinations. Br.J.Dermatol. 1977. 96: 179-187.

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219. Lever,R., Hadley,K., Downey,D., and MacKie,R., Staphylococcal colonization in atopic dermatitis and the effect of topical mupirocin therapy. Br.J.Dermatol. 1988. 119: 189-198. 220. Cho,S.H., Strickland,I., Boguniewicz,M., and Leung,D.Y., Fibronectin and fi- brinogen contribute to the enhanced binding of Staphylococcus aureus to atopic skin. J.Allergy Clin.Immunol. 2001. 108: 269-274. 221. Arikawa,J., Ishibashi,M., Kawashima,M., Takagi,Y., Ichikawa,Y., and Imokawa,G., Decreased levels of sphingosine, a natural antimicrobial agent, may be associated with vulnerability of the stratum corneum from patients with atopic dermatitis to colonization by Staphylococcus aureus. J.Invest Dermatol. 2002. 119: 433-439. 222. Imayama,S., Shimozono,Y., Hoashi,M., Yasumoto,S., Ohta,S., Yoneyama,K., and Hori,Y., Reduced secretion of IgA to skin surface of patients with atopic dermatitis. J.Allergy Clin.Immunol. 1994. 94: 195-200. 223. Morishita,Y., Tada,J., Sato,A., Toi,Y., Kanzaki,H., Akiyama,H., and Arata,J., Possible influences of Staphylococcus aureus on atopic dermatitis-- the colonizing features and the effects of staphylococcal enterotoxins. Clin.Exp.Allergy 1999. 29: 1110-1117. 224. Ong,P.Y., Ohtake,T., Brandt,C., Strickland,I., Boguniewicz,M., Ganz,T., Gallo,R.L., and Leung,D.Y., Endogenous antimicrobial peptides and skin infections in atopic dermatitis. N.Engl.J.Med. 2002. 347: 1151-1160. 225. Hanifin,J.M. and Tofte,S.J., Update on therapy of atopic dermatitis. J Allergy Clin Immunol. 1999. 104: S123-S125. 226. Hanifin,J.M. and Chan,S., Biochemical and immunologic mechanisms in atopic der- matitis: new targets for emerging therapies. J.Am.Acad.Dermatol. 1999. 41: 72-77. 227. Ponec,M., Boelsma,E., Weerheim,A., Mulder,A., Bouwstra,J., and Mommaas,M., Lipid and ultrastructural characterization of reconstructed skin models. Int.J.Pharm. 2000. 203: 211-225. 228. Hanifin,J.M., Chan,S.C., Cheng,J.B., Tofte,S.J., Henderson,W.R., Jr., Kirby,D.S., and Weiner,E.S., Type 4 phosphodiesterase inhibitors have clinical and in vitro anti- inflammatory effects in atopic dermatitis.J Invest Dermatol 1996. 107: 51-56. 229. Griffiths,C.-E.M., Van Leent,E.-J.M., Gilbert,M., and Traulsen,J., Randomized comparison of the type 4 phosphodiesterase inhibitor cipamfylline cream, cream vehicle and hydrocortisone 17-butyrate cream for the treatment of atopic dermatitis. Br.J.Dermatol. 2002. 147: 299-307. 230. Santamaria,L.F., Role of Cyclic Nucleotide Phosphodiesterases in Atopic Dermatitis. In Bieber,T. and Leung,D.Y. (Eds.) Atopic Dermatitis. Marcel Dekker, Inc, New York 2003, pp 491-501. 231. Farber,E.M. and Nall,L., Epidemiology: Natural history and genetics. In Roenigk,H.H. and Maibach,H. (Eds.) Psoriasis. Marcel Dekker, New York 1985, pp 141-186. 232. Zachariae,H., Epidemiology and genetics. In Mier,P.D. and van de Kerkhof,P.C. (Eds.) Textbook of psoriasis. Churchill Livingston, Edinburgh 1986, pp 4-12. 233. van Steensel,M.A. and Steijlen,P.M., Genetics of psoriasis. Clin.Dermatol 1997. 15: 669-675.

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234. Olerup,O., Fredrikson,S., Olsson,T., and Kam-Hansen,S., HLA class II genes in chronic progressive and in relapsing/remitting multiple sclerosis. Lancet 1987. 2: 327. 235. Tagami,H., Triggering factors. Clin.Dermatol. 1997. 15: 677-685. 236. Park,B.S. and Youn,J.I., Factors influencing psoriasis: an analysis based upon the extent of involvement and clinical type. J.Dermatol 1998. 25: 97-102. 237. Shelley,W.B. and Arthur,R.B., Biochemical and physiological clues to the nature os psoriasis. Arch.Dermatol 1958. 78: 14-29. 238. van de Kerkhof,P.C.M., Clinical Features. In Mier,P.D. and van de Kerkhof,P.C.M. (Eds.) Textbook of psoriasis. Churchill Livingstone, Edinburgh 1986, pp 13-39. 239. Farber,E.M. and Nall,L., Nail psoriasis. Cutis 1992. 50: 174-178. 240. Ragaz,A. and Ackerman,A.B., Evolution, maturation, and regression of lesions of psoriasis. New observations and correlation of clinical and histologic findings. Am.J.Dermatopathol. 1979. 1: 199-214. 241. Stadler,R., Schaumberg-Lever,G., and Orfanos,C.E., Histology. In Mier,P.D. and van de Kerkhof,P.C. (Eds.) Textbook of Psoriasis. Churchil Livingston, London 1986, pp 40-54. 242. Motta,S., Sesana,S., Monti,M., Giuliani,A., and Caputo,R., Interlamellar lipid dif- ferences between normal and psoriatic SC. Acta Derm.Venereol.Suppl (Stockh) 1994. 186: 131-132. 243. Motta,S., Monti,M., Sesana,S., Caputo,R., Carelli,S., and Ghidoni,R., Ceramide composition of the psoriatic scale. Biochim.Biophys.Acta 1993. 1182: 147-151. 244. Motta,S., Sesana,S., Ghidoni,R., and Monti,M., Content of the different lipid classes in psoriatic scale. Arch.Dermatol.Res. 1995. 287: 691-694. 245. Fartasch,M., Epidermal Barrier in disorders of the skin. Microsc.Res.Tech. 1997. 38: 361-372. 246. van de Kerkhof,P.C.M., The management of psoriasis. Neth.J.Med. 1998. 52: 40-45. 247. van de Kerkhof,P.C.M., Steegers-Theunissen,R.P., and Kuipers,M.V., Evaluation of topical drug treatment in psoriasis. Dermatology 1998. 197: 31-36. 248. Tsai,J.C., Feingold,K.R., Crumrine,D., Wood,L.C., Grunfeld,C., and Elias,P.M., Permeability barrier disruption alters the localization and expression of TNF alpha/ protein in the epidermis. Arch.Dermatol.Res. 1994. 286: 242-248. 249. Tupker,R.A., Willis,C., Berardesca,E., Lee,C.H., Fartasch,M., Agner,T., and Serup,J., Guidelines on sodium lauryl sulfate (SLS) exposure tests. A report from the Standardization Group of the European Society of Contact Dermatitis. Contact Dermatitis 1997. 37: 53-69. 250. Wilhelm,K.P., Freitag,G., and Wolff,H.H., Surfactant-induced skin irritation and skin repair: evaluation of a cumulative human irritation model by noninvasive tech- niques. J.Am.Acad.Dermatol. 1994. 31: 981-987. 251. Wilhelm,K.P., Freitag,G., and Wolff,H.H., Surfactant-induced skin irritation and skin repair. Evaluation of the acute human irritation model by noninvasive techniques. J.Am.Acad.Dermatol. 1994. 30: 944-949. 252. Le,M., Cals,S., Schalkwijk,J., and van,d., V, An immunohistochemical study on mild skin irritation induced by a single application of a low-molarity sodium dodecyl sulfate solution: keys to the prevention of irritant contact dermatitis. Curr.Probl.Dermatol. 1996. 25: 67-77.

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253. van der Valk,P.G.M. and Maibach,H.I., A functional study of the skin barrier to evaporative water loss by means of repeated cellophane-tape stripping. Clin.Exp Der- matol. 1990. 15: 180-182. 254. Alkemade,J.A., Molhuizen,H.O., Ponec,M., Kempenaar,J.A., Zeeuwen,P.L., de Jongh,G.J., Vlijmen-Willems,I.M., van Erp,P.E., van de Kerkhof,P.C., and Schalkwijk,J., SKALP/elafin is an inducible proteinase inhibitor in human epidermal keratinocytes. J.Cell Sci. 1994. 107 ( Pt 8): 2335-2342. 255. Proksch,E., Brasch,J., and Sterry,W., Integrity of the permeability barrier regulates epidermal Langerhans cell density. Br.J Dermatol. 1996. 134: 630-638. 256. Chatenay,F., Corcuff,P., Saint-Leger,D., and Leveque,J.L., Alterations in the com- position of human stratum corneum lipids induced by inflammation.Photodermatol.P hotoimmunol.Photomed. 1990. 7: 119-122. 257. Blank,I.H., Cutaneous barriers. J Invest Dermatol. 1965. 45: 249-256. 258. Nilsson,G.E., Measurement of water exchange through skin. Med.Biol.Eng Comput. 1977. 15: 209-218. 259. Pinnagoda,J., Tupker,R.A., Agner,T., and Serup,J., Guidelines for transepidermal water loss (TEWL) measurement. A report from the Standardization Group of the Eu- ropean Society of Contact Dermatitis. Contact Dermatitis 1990. 22: 164-178. 260. Pinnagoda,J., Tupker,R.A., Coenraads,P.J., and Nater,J.P., Transepidermal water loss with and without sweat gland inactivation. Contact Dermatitis 1989. 21: 16-22. 261. Friebe,K., Effendy,I., and Loffler,H., Effects of skin occlusion in patch testing with sodium lauryl sulphate. Br.J Dermatol. 2003. 148: 65-69. 262. Gerdes,J., Li,L., Schlueter,C., Duchrow,M., Wohlenberg,C., Gerlach,C., Stahmer,I., Kloth,S., Brandt,E., and Flad,H.D., Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67.Am.J.Pathol. 1991. 138: 867-873. 263. Rose,D.S., Maddox,P.H., and Brown,D.C., Which proliferation markers for routine immunohistology? A comparison of five antibodies. J.Clin.Pathol. 1994. 47: 1010- 1014. 264. Watanabe,S., Wagatsuma,K., Ichikawa,E., and Takahashi,H., Abnormal distribu- tion of epidermal protein antigens in psoriatic epidermis. J.Dermatol. 1991. 18: 143- 151. 265. Paladini,R.D., Takahashi,H., Bravo,N.S., and Coulombe,P.A., Onset of re-epitheli- alization after skin injury correlates with a reorganization of keratin filaments in wound edge keratinocytes; defining a potential role for keratin 16.J.Cell.Biol. 1996. 132. 266. Hodak,E., Gottlieb,A.B., Anzilotti,M., and Krueger,J.G., The insulin-like growth factor 1 receptor is expressed by epithelial cells with proliferative potential in human epidermis and skin appendages: correlation of increased expression with epidermal hyperplasia. J.Invest Dermatol. 1996. 106: 564-570. 267. Kallioinen M, Koivukangas V, Jarvinen M, and Oikarinen A, Expression of cytok- eratins in regenerating human epidermis. Br.J.Dermatol. 1995. 133. 268. Castelijns,F.A., Gerritsen,M.J., van Erp,P.E., and van de Kerkhof,P.C., Immuno- histochemical assessment of keratin 16 on paraffin- embedded sections of normal and hyperproliferative skin: monoclonal antibodies Ks8.12 and LL025 in a comparative study. Arch.Dermatol.Res. 1999. 291: 59-63. 269. Watt,F.M., Involucrin and other markers of keratinocyte terminal differentiation. J.Invest.Dermatol. 1983. 81: 100s-3s.

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270. Ghadially,R., Reed,J.T., and Elias,P.M., stratum corneum structure and function cor- relates with phenotype in psoriasis. J.Invest Dermatol. 1996. 107: 558-564. 271. Le,T.K., Schalkwijk,J., van de Kerkhof,P.C., van Haelst,U., and van der Valk,P.G., A histological and immunohistochemical study on chronic irritant contact dermatitis. Am.J Contact Dermat. 1998. 9: 23-28. 272. van Duijnhoven,J.L., Schalkwijk,J., Kranenborg,M.H., van Vlijmen Willems,I.M., Groeneveld,A., van Erp,P.E., Timmer,E.D., de Jongh,G.J., and van de Ven,W.J., MON-150, a versatile monoclonal antibody against involucrin: characterization and applications. Arch.Dermatol.Res. 1992. 284: 167-172. 273. Masouye,I., Saurat,J.H., and Siegenthaler,G., Epidermal fatty-acid-binding protein in psoriasis, basal and squamous cell carcinomas: an immunohistological study. Der- matology 1996. 192: 208-213.

60 Chapter 1 2 Skin barrier and lipid metabolism

This chapter was based on the following publications:

Lack of upregulation of epidermal fatty acid binding protein (E-FABP) in dithranol induced irritation M. Kucharekova, W.H.P.M. Vissers, J. Schalkwijk, P.C.M. van de Kerkhof, P.G.M. van der Valk Eur J Dermatol;2003;13(3):254-257

Epidermal expression of epidermal fatty acid binding protein (E-FABP) distinguishes atopic dermatitis from psoriasis vulgaris M. Kucharekova, J. Schalkwijk, W.A.M. Blokx, A. Dogan-Bayram, P.G.M. van der Valk Submitted

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62 63 Chapter 2 Skin barrier and lipid metabolism

2.1 LACK OF UPREGULATION OF EPIDERMAL FATTY ACID BINDING PROTEIN (E-FABP) IN DITHRANOL INDUCED IRRITATION

Abstract

The exact role of epidermal fatty acid binding protein (E-FABP) in skin is unknown. A restoration of barrier function may be associated with an upregulation of E-FABP. Moreover, E-FABP is upregulated in a variety of cells in response to oxidative stress. A recent observation that dithranol induced irritation is not associated with skin barrier impairment prompted us to investigated the expression of E-FABP in this skin condition to elucidate the unknown function of this protein in skin. This study shows lack of E-FABP upregulation after a single application of dithranol on uninvolved skin of patients with psoriasis. The lack of expression of E- FABP in dithranol irritation correlates with the unimpaired skin barrier function as assessed by measurements of TEWL.

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Introduction

E-FABP has been characterized as a molecule with a molecular weight of 15 kDA. It is a member of the mammalian intracellular lipid binding protein family (LBP), which plays a variety of roles in intracellular fatty acid, retinoid, bile acid and sterol trafficking.1 The function of E-FABP in skin has not been elucidated. It is assumed that this protein is involved in the transport of fatty acids (FA) which are the most important substrates for (1) energy production, (2) formation of phospholipids, i.e the maintenance of membrane lipid structures and (3) participation in signal transduction pathways.2 The upregulation of E-FABP expression is associated with a continuous high fatty acid transport, needed for the restoration of barrier function.3 A study on the cell biological alterations during irritant contact dermatitis strongly suggested that E-FABP expression is upregulated after barrier disruption.4 However, its function only with the respect to skin barrier has been questioned.5;6 The upregulation of the E-FABP gene expression in response to oxidative stress and the generation of reactive oxygen species has been shown in a variety of cell types.1 The expression of E-FABP in dithranol irritation has not been studied before. Dithranol-induced irritation is an unique type of skin irritation. It is caused by anthralin free radicals and oxygen radicals.7 Remarkably, a single application of dithranol which induces skin irritation, does not compromise the skin barrier as assessed by measurement of transepidermal water loss.8 The aim of the present study was to investigate the expression of E-FABP in a well-established model of free radical induced skin irritation.

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Material and methods

Subjects Nine patients with chronic stable psoriasis participated in the study (5 females, 4 males, age range 50-70 years, the mean of age 55,7 years). They had no history of other skin disease. None of the patients used any systemic medication. Permission of the Ethical Committee and written informed consent from all volunteers were obtained. The experiment was conducted from May to June 2002.

Application of dithranol The dithranol 2% cream (dithranol 69g, cetiol V 660g, cetomacrogol wax 450g, liquid petrolatum 450g, salicylic acid 30g, sorbic acid 4,5g, ascorbic acid 1,5g, demineralised water up to 3000g) was applied once on one selected site of 2 cm Ø of uninvolved skin of lower back of patients with psoriasis. The application time was 1 hour, after which the dithranol cream was removed with water. This model of dithranol irritation was already described and used before.9 Patients were not allowed to use any topical medication on the investigated site during the study.

Immunohistochemical assessment A total of 18 punch biopsies (3 mm diameter) were taken from the investigated- sites of nine patients after visual reading and TEWL measurements at 48 hours and at day 10. After 4 h fixation in formalin the samples were stored in alcohol 70% until embedding in paraffin was done. Then the samples were sectioned at 6 μm for immunohistochemical staining. The slides were deparaffinized by incubation in a histosafe for 2 times 15 minutes, followed by dipping in an ethanol series from 100% to 50%. After washing in phosfate-buffer saline (PBS) the sections were pre-incubated with normal goat serum (20%) for 15 minutes. Then all sections were incubated for 60 minutes with the anti-E-FABP (kindly provided by dr. Siegenthaler10) at the dilution of 1:100. After washing with PBS, sections were incubated for 30 minutes with biotinylated goat-anti-rabbit IgG (dilution 1:200). Another washing step with PBS was carried out, and then complexes were formed between biotin and avidin using the Vectastain Elite ABC peroxidase kit, according to the manufacturer’s instructions (Vector Laboratories, Burlingame, USA). Sections were developed with amino- ethylcarbazol. All sections were subsequently counterstained with hematoxylin 1%.

Evaluation Visual scoring. The clinical changes at the exposure sites were scored after 48 hours and after 10 days and rated as follows: 0 = no erythema, 1 = minimal erythema, 2 = moderate erythema, 3 = marked erythema and 4 = marked erythema with oedema.

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Transepidermal water loss measurements TEWL measurements were performed using an evaporimeter (Tewameter TM 210, Courage and Khazaka, Koln, Germany). Measurement were performed according to the guidelines described by the Standardisation Group of the European Society of Contact Dermatitis.11 Room temperature was kept between 20 and 22 °C and relative humidity between 40 and 56%. TEWL measurements were performed before exposure on day 0 and at 48 hours and at 10 days after exposure to dithranol.

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Results

Visual assessment and transepidermal water loss measurements Table 1 summarises the visual scores (mean ± SEM) and the TEWL (mean ± SEM) measurements following dithranol application (n=9). All patients clearly developed erythema after 48 hours of dithranol application which was also observed at day 10. In none of the patients oedema or vesicle formation was seen and no complains of pain or itching were reported. The baseline values of TEWL of the uninvolved skin of psoriatic patients were comparable to the values of the healthy skin as reported in the literature.12 There was no significant increase in TEWL at 48 hour and at 10 days compared to baseline.

The expression of E-FABP Following single application of dithranol on uninvolved psoriatic skin (n=9), strong cytoplasmic staining in the stratum granulosum and the upper spinous layer was observed at 48 hours and at 10 days.(Figure 1a) This pattern of E-FABP expression is the same as in normal skin (Figure 1b).5 The staining of the untreated lesional psoriatic skin that we used as a positive control, showed the expression of this protein in all suprabasal cells (Figure 1c) as already observed before.3 No staining was observed with the pre-imune serum in normal human skin.

Table 1 Transepidermal water loss (g/m²/h; mean ± SEM) en erythema score (mean ± SEM) following a single application of dithranol 2% after 48 hours and 10 days. (n=9) TEWL (mean ± SEM) Erythema (mean ± SEM) Baseline 6,7 ± 1,3 0 48 hours 6,1 ± 0,7 1,7 ± 0,2 10 days 6,6 ± 0,9 1,4 ± 0,2

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Figure 1 Immunohistochemical � expression of E-FABP. Following single application of dithranol, cytoplasmic staining in the stratum granulosum and the upper spinous layer was observed (a) This pattern of E-FABP expression is the same as in normal human skin (b), whereas untreated lesional psoriatic skin demonstrated E-FABP expression in all suprabasal cells (c).

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Discussion

The expression of epidermal fatty acid binding protein (E-FABP) was studied in dithranol induced irritation of uninvolved skin of psoriasis patients using immunohistochemical methods. In the present study, after a single application of dithranol, we observed a pronounced erythema after 48 hours in line with other studies.13-15 As observed also in a previous study8, dithranol did not increase TEWL indicating that single application of dithranol does not compromise the skin barrier. After dithranol application, we observed epidermal expression of E-FABP equal to normal human skin. These results show that the expression of E-FABP in dithranol irritation correlates with a normal function of the skin. The upregulation of E- FABP has been observed in skin conditions associated with the impairment of skin barrier like sodium dodecyl sulphate induced skin irritation4, psoriasis3 and atopic dermatitis (unpublished data). Furthermore, delayed recovery of TEWL after acetone application to the skin of E-FABP-deficient mice has been reported.16 All these studies demonstrate that the role of E-FABP is associated with maintaining of the skin barrier function although the exact mechanism for this function of E-FABP remains to be further elucidated. In contrast, the presence of E-FABP in the non-keratinized oral mucosa epithelium and its expression in tissues other than skin suggests that the role of E-FABP is not restricted to the skin barrier alone. Our finding that in dithranol irritation the epidermal barrier function and E-FABP expression is normal, suggests that increase E-FABP expression is linked to barrier function rather than to skin irritation. Recently, a new function of E-FABP as an antioxidant protein has been reported.1 E-FABP was identified as a molecular target for 4-Hydroxynonenal (4- HNE). 4-HNE is the most cytotoxic unsaturated aldehyde that is produced by lipid peroxidation that occurs in response to oxidative stress.17 The identification of E- FABP as a target molecule for a well known end-product of lipid peroxidation could indicate that the E-FABP functions as an antioxidant protein by scavenging reactive lipids.1 E-FABP is found in cells exposed to high oxidative stress, including the retina, lens, lung and tongue.18 The skin is also particularly vulnerable to the oxidative stress due to (i) its constant exposure to high oxygen tension, and (ii) the presence of considerable amounts of polyunsaturated fatty acid. 19 20 Indeed, E-FABP is observed to be upregulated in the skin conditions associated with oxidative stress.21-24Also dithranol-induced skin irritation is strongly suggested to be caused by the generation of dithranol free radicals and oxygen radicals.25 There is some evidence, that the release of active oxygen species from dithranol could initiate lipid peroxidation in cellular membranes of the skin. Firstly, dithranol slightly elevates lipid peroxidation in mouse skin after topical application, demonstrated by the exhalation of ethane, which is produced from gamma-3 polyunsaturated fatty acids26 and by malondialdehyde production.27 In addition, singlet oxygen can accelerate lipid peroxidation by directly reacting with unsaturated fatty acyl moieties to give hydroperoxides, which was also

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already demonstrated for dithranol.28 Thus the peroxidative damage to membrane lipids after dithranol application is generally believed to be related to its antipsoriatic action as well as induction of skin irritation.25;29 Interestingly, the presumed generation of free radical following dithranol application does not cause the induction of a protective endogenous mechanism such as enhanced expression of E-FABP. In the context of oxidative stress, the lack of upregulation of E-FABP observed in this study may be due to some reasons. Although 4-HNE was found to be responsible for the damaging effects associated with lipid peroxidation in various tissues17 including skin,30-33 there are no studies demonstrating that 4-HNE is generated by dithranol-induced lipid peroxidation and therefore the enhanced expression of E- FABP as a target for 4-HNE is not observed. The scavenging of the 4-HNE by other skin antioxidant systems like glutathione S-transferase22 or superoxide dismutase34 35, both observed to be modified after dithranol application, should be considered. In conclusion, in this study the lack of upregulation of the expression of E- FABP in dithranol irritation correlates with the lack of skin barrier dysfunction as assessed by measurements of TEWL, suggesting a role for E-FABP in the maintenance of the skin barrier.

Acknowledgement We thank Prof. Dr. Klaus Müller (Institute of Pharmaceutical Chemistry, University of Münster) for the critical reading of this manuscript.

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References

1. Bennaars-Eiden,A., Higgins,L., Hertzel,A.V., Kapphahn,R.J., Ferrington,D.A., and Bernlohr,D.A., Covalent modification of epithelial fatty acid binding protein by 4-hydroxynonenal in vitro and in vivo: Evidence for a role in antioxidant biology. J.Biol.Chem. 2002. 2. Schurer,N.Y., Implementation of fatty acid carriers to skin irritation and the epidermal barrier. Contact Dermatitis 2002. 47: 199-205. 3. Kuijpers,A.L., Bergers,M., Siegenthaler,G., Zeeuwen,P.L., van de Kerkhof,P.C.M., and Schalkwijk,J., Skin-derived antileukoproteinase (SKALP) and epidermal fatty acid-binding protein (E-FABP): two novel markers of the psoriatic phenotype that re- spond differentially to topical steroid. Acta Derm Venereol 1997. 77: 14-19. 4. Le,M., Schalkwijk,J., Siegenthaler,G., van de Kerkhof,P.C.M., Veerkamp,J.H., and van der Valk,P.G.M., Changes in keratinocyte differentiation following mild irritation by sodium dodecyl sulphate. Arch.Dermatol.Res. 1996. 288: 684-690. 5. Masouye,I., Saurat,J.H., and Siegenthaler,G., Epidermal fatty-acid-binding protein in psoriasis, basal and squamous cell carcinomas: an immunohistological study. Dermatol- ogy 1996. 192: 208-213. 6. Masouye,I., Hagens,G., Van Kuppevelt,T.H., Madsen,P., Saurat,J.H., Veerkamp,J.H., Pepper,M.S., and Siegenthaler,G., Endothelial cells of the human microvasculature express epidermal fatty acid-binding protein. Circ.Res. 1997. 81: 297- 303. 7. Kemeny,L., Ruzicka,T., and Braun Falco,O., Dithranol: a review of the mechanism of action in the treatment of psoriasis vulgaris. Skin.Pharmacol. 1990. 3: 1-20. 8. Snater,E., Janssen,E.A., van der Valk,P.G.M., and van de Kerkhof,P.C.M., Transepi- dermal water vapour loss is not increased during and following dithranol irritation. Br J Dermatol 1995. 132: 908-912. 9. Swinkels,O.Q., Prins,M., Vlijmen-Willems,I.M., Gerritsen,M.J.P., van der Valk,P.G.M., and van de Kerkhof,P.C.M., The response of uninvolved skin of patients with psoriasis to single and repeated applications of dithranol cream: an immunohisto- chemical assessment. Skin Pharmacol.Appl.Skin Physiol 2002. 15: 385-392. 10. Siegenthaler,G., Hotz,R., Chatellard-Gruaz,D., Jaconi,S., and Saurat,J.H., Charac- terization and expression of a novel human fatty acid-binding protein: the epidermal type (E-FABP). Biochem.Biophys.Res.Commun. 1993. 190: 482-487. 11. Pinnagoda,J., Tupker,R.A., Agner,T., and Serup,J., Guidelines for transepidermal wa- ter loss (TEWL) measurement. A report from the Standardization Group of the European Society of Contact Dermatitis. Contact Dermatitis 1990. 22: 164-178. 12. Oestmann,E., Lavrijsen,A.P., Hermans,J., and Ponec,M., Skin barrier function in healthy volunteers as assessed by transepidermal water loss and vascular response to hexyl nicotinate: intra- and inter-individual variability. Br J Dermatol 1993. 128: 130- 136. 13. Juhlin,L., Factors influencing anthralin erythema.Br.J.Dermatol. 1981. 105 Suppl 20. 14. Lawrence,C.M. and Shuster,S., Mechanism of anthralin inflammation. I. Dissociation of response to clobetasol and indomethacin. Br.J.Dermatol. 1985. 113: 107-115.

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15. Swinkels,O.Q., Prins,M., Birker,L.W., Gerritsen,M.J.P., van der Valk,P.G.M., and van de Kerkhof,P.C.M, The response of normal human skin to single and re- peated applications of dithranol cream: an immunohistochemical assessment. Skin Pharmacol.Appl.Skin Physiol 2002. 15: 262-269. 16. Owada,Y., Takano,H., Yamanaka,H., Kobayashi,H., Sugitani,Y., Tomioka,Y., Suzuki,I., Suzuki,R., Terui,T., Mizugaki,M., Tagami,H., Noda,T., and Kondo,H., Altered water barrier function in epidermal-type fatty acid binding protein-deficient mice. J Invest Dermatol 2002. 118: 430-435. 17. Esterbauer,H., Schaur,R.J., and Zollner,H., Chemistry and biochemistry of 4-hy- droxynonenal, malonaldehyde and related aldehydes. Free Radic.Biol.Med. 1991. 11: 81-128. 18. Kingma,P.B., Bok,D., and Ong,D.E., Bovine epidermal fatty acid-binding protein: determination of ligand specificity and cellular localization in retina and testis.Biochem - istry 1998. 37: 3250-3257. 19. Trenam,C.W., Blake,D.R., and Morris,C.J., Skin inflammation: reactive oxygen spe- cies and the role of iron. J.Invest Dermatol 1992. 99: 675-682. 20. Schallreuter,K.U. and Wood,J.M., Free radical reduction in the human epidermis. Free Radic.Biol.Med. 1989. 6: 519-532. 21. Trouba,K.J., Hamadeh,H.K., Amin,R.P., and Germolec,D.R., Oxidative stress and its role in skin disease. Antioxid.Redox.Signal. 2002. 4: 665-673. 22. Willis,C.M., Britton,L.E., Reiche,L., and Wilkinson,J.D., Reduced levels of glutath- ione S-transferases in patch test reactions to dithranol and sodium lauryl sulphate as demonstrated by quantitative immunocytochemistry: evidence for oxidative stress in acute irritant contact dermatitis. Eur.J.Dermatol. 2001. 11: 99-104. 23. Raab,W.P., Ingram methods: the precusor of photochemotherapy. Br J Dermatol 1981. 105 (suppl.20): 77-81. 24. Antille,C., Sorg,O., Lubbe,J., and Saurat,J.H., Decreased oxidative state in non-le- sional skin of atopic dermatitis. Dermatology 2002. 204: 69-71. 25. Muller,K., Antipsoriatic anthrones: Aspect of oxygen radical formation, challenges and prospects. Gen.Pharmac. 1996. 27: 1325-1335. 26. Muller,K., Wiegrebe,W., and Younes,M., Formation of active oxygen species by dithranol,III.Dithranol, active oxygen species and lipid peroxidation in vivo. Arch.Pharm.(Weinheim) 1987. 320: 59-66. 27. Finnen,M.J., Lawrence,C.M., and Shuster,S., Anthralin increases lipid peroxide for- mation in skin and free radical scavengers reduce anthralin irritancy. B.S.I.D.Annunal Meeting 1984. 683: 717. 28. Muller,K., Eibler,E., Mayer,K.K., Wiegrebe,W., and Klug,G., Dithranol, singlet oxy- gen and unsaturated fatty acids. Arch.Pharm.(Weinheim) 1986. 319: 2-9. 29. Muller, Antipsoriatic and proinflammatory action of anthralin (Implication for the role of oxygen radicals). Biochem.Pharmacol. 1997. 53: 1215-1221. 30. Valacchi,G., Van,D., V, Schock,B.C., Okamoto,T., Obermuller-Jevic,U., Cross,C.E., and Packer,L., Ozone exposure activates oxidative stress responses in murine skin. Toxicology 2002. 179: 163-170. 31. Tanaka,N., Tajima,S., Ishibashi,A., Uchida,K., and Shigematsu,T., Immunohisto- chemical detection of lipid peroxidation product protein-bound acrolein and 4-hydrox- ynonenal protein in actinic elastosis of photodamaged skin. Arch.Dermatol Res. 2001. 293: 363-367.

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32. Hiratsuka,A., Saito,H., Hirose,K., and Watabe,T., Marked expression of glutathione S-transferase A4-4 detoxifying 4-hydroxy-2(E)-nonenal in the skin of rats irradiated by ultraviolet B-band light (UVB). Biochem.Biophys.Res.Commun. 1999. 260: 740-746. 33. Um,S.C., Suzuki,S., Toyokuni,S., Uchida,K., Hiai,H., and Nishimura,Y., Formation of 4-hydroxy-2-nonenal-modified proteins and 3-nitro-L-tyrosine in rat island skin flaps during and after ischemia. Ann.Plast.Surg. 1999. 42: 293-298. 34. Hsieh,G.C. and Acosta,D., Dithranol-induced cytotoxicity in primary cultures of rat epidermal keratinocytes. I. The role of reactive oxygen species. Toxicol.Appl.Pharmacol. 1991. 107: 16-26. 35. Willis,C.M., Reiche,L., and Wilkinson,J.D., Immunocytochemical demonstration of reduced Cu,Zn-superoxide dismutase levels following topical application of dithranol and sodium lauryl sulphate: an indication of the role of oxidative stress in acute irritant contact dermatitis. Eur.J.Dermatol. 1998. 8: 8-12.

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74 75 Chapter 2 Skin barrier and lipid metabolism

2.2 EPIDERMAL EXPRESSION OF EPIDERMAL FATTY ACID BINDING PROTEIN (E-FABP) DISTINGUISHES ATOPIC DERMATITIS FROM PSORIASIS VULGARIS

Abstract

Psoriasis vulgaris (PV) and atopic dermatitis (AD) can usually be clearly differentiated. Chronic lesions in atopic dermatitis, however, may sometimes be very similar to psoriasis clinically and/or histologically. Various features of psoriatic lesions such as increased proliferation rates and altered keratinocyte differentiation can be found in chronic AD lesions as well. Here we studied the expression and localization of epidermal fatty acid binding protein (E-FABP) also known as psoriasis associated fatty acid binding protein (PA-FABP) in chronic AD lesions, and compared this with the known expression pattern in chronic PV lesions. E-FABP was highly upregulated in both diseases compared to normal skin, but the pattern was markedly different. In psoriatic epidermis the entire suprabasal compartment was strongly positive, whereas in AD the upper layers of the stratum spinosum were positive while the intensity of the staining gradually decreased towards the basal layer. No significant differences were found between PV and AD epidermis with respect to cellular proliferation (Ki-67 expression). Although the biological significance of this difference is not clear at present, our findings suggest that expression of this protein might be useful in the differentiation between AD and PV.

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Introduction

Psoriasis vulgaris (PV) and atopic dermatitis (AD) are two well-defined entities, which usually can be differentiated clinically.1-3 If the clinical expression is indefinite, histology may help. The spongiform pustules of Kogoj and Munro microabscesses, in typical cases confluent parakeratosis, suprapapillary thinning and hyperplasia of the rete ridges are diagnostic for psoriasis.1 The most characteristic histological features of atopic dermatitis are spongiosis, irregular epidermal thickening with accentuation of the rete pattern, and a partially parakeratotic and orthokeratotic epidermis.4 These histological features are most apparent in acute AD lesions. In chronic lesions like e.g. on the palmar side of the hands clinical distinction may be difficult. Histological differentiation may also be difficult and sometimes impossible, since in chronic AD and PV lesions typical diagnostic histological features may be missing. In chronic AD parakeratosis can be found while spongiosis may be lacking. Psoriasiform hyperplasia is invariably present with a sparse superficial perivascular lymphocytic infiltrate.5 Because the histopathology based on H&E staining is often not helpful in cases of uncertain clinical diagnosis, new immunohistochemical markers could be useful for differentiation. The epidermis of the psoriatic lesion shows a vast number of changes.6 These changes can be visualized by immunohistochemical staining with Ki- 67, a marker for proliferation, and with markers for differentiation-associated proteins such as involucrin7, filaggrin8 and transglutaminase.9 In normal skin the expression of these markers is restricted to the stratum granulosum. In contrast, involucrin and transglutaminase are strongly upregulated in psoriasis, whereas filaggrin is often diminished or absent. Epidermal fatty acid binding protein (E-FABP) also known as psoriasis associated fatty acid binding protein (PA-FABP) was originally described to be upregulated in psoriatic epidermis. It is assumed that E-FABP is involved in the transport of epidermal lipids with high affinity for C - 18 fatty acids (FA) which play an important role in the epidermis at the structural and the function level.10;11 Since marked differences in lipid composition of stratum corneum in both diseases has been decribed12-14, E-FABP is an interesting molecule to study because this protein is involved in distinct pathways of abnormal lipid metabolism. The aim of this study was to compare the expression of E-FABP in AD and PV. E-FABP was highly upregulated in both diseases compared to normal skin, but the pattern was markedly different.

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Material and methods

Subjects Nine patients (5 male and 4 females, mean age 48.1 years, age range 27-68) with clinical diagnosis of moderate to severe plaque psoriasis and ten patients (3 male and 7 females, mean age 33.8 years, age range 17- 54) with clinical diagnosis of moderate to severe atopic dermatitis participated. None of patient received systemic therapy, UVB therapy or topical corticosteroids at the time of biopsy taking. Punch biopsies were taken from chronic psoriasis plaques and from subacute to chronic atopic dermatitis lesions. Care was taken to exclude acute lesions with vesicles. Permission of the medical ethical committee and written informed consent of all patients were obtained.

Histological methods Biopsies were fixed in buffered 4% formalin for 4 hours and processed for routine histology. Tissues were embedded in paraffin and 5-µm sections were cut. Sections were stained with haematoxylin-eosin (H&E) or processed for immunohistochemical staining using an indirect immunoperoxidase technique with avidin biotin complex enhancement. We studied epidermal proliferation with an antibody directed against the Ki-67 antigen (MIB-1, Immunotech, SA, Marseilles, France). Anti-E-FABP serum was obtained as previously described.6 Paraffin section were deparaffinised, rehydrated and immunostaining was performed. An indirect peroxidase technique was used for staining with the antiserum against E-FABP. First, the section was pre-incubated with normal swine serum and then incubated with anti E-FABP staining at a dilution of 1:100 for 60 min. After incubation with peroxidase-conjugated swine-anti-rabbit Ig, the sections were developed with amino- ethylcarbazol as the chromogenic substrate. For staining of the Ki-67 antigen sections were pre-incubated with normal horse serum. Subsequently, slides were incubated with anti-Ki-67 antibodies in a dilution of 1:50 for 60 minutes. After incubation with biotinylated horse-anti-mouse IgG (dilution 1: 200) for 30 minutes, complexes were formed between biotin and avidin using the Vectastain Elite ABC peroxidase kit. The sections were developed with metal-enhanced diaminobenzidin as the chromogenic substrate.

Histological grading Epidermal proliferation was measured by counting the number of Ki-67 positive nuclei per mm length of the section. E-FABP staining in the epidermis was assessed using a five-point scale: 0=no staining, 1=sporadic staining, 2=weak staining, 3=moderate staining, 4=pronounced staining.

Statistical analysis The Mann-Whitney U-test was used for statistical analysis. Differences with P

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values <0.05 were considered statistically significant.

� � Figure 1 H&E staining of atopic dermatitis lesion (a) and psoriasis lesion (b). Immunohistochemical localisation od E-FABP in atopic dermatitis (c,e) and psoriasis (d,f). Note the differences in localisation and staining intensity between atopic dermatitis and psoriasis.

� � Epidermal proliferation as assessed by Ki-67 of atopic dermatitis lesions (g) and psoriatic lesion (h).

� �

� �

Table 1 Mean value of the marker for epidermal proliferation and EFABP ± SEM. Antigen Psoriasis Atopic dermatitis p-value E-FABP 3.8 ± 0.15 2.1 ± 0.18 < 0.001 Ki-67 89 ±16.0 85 ±10.3 Not significant E-FABP staining was assessed using a five-point scale: 0=no staining,1=sporadic staining, 2=weak staining, 3=moderate staining, 4=pronounced staining. Ki-67 staining was measured by counting the number of positive nuclei per mm length of the section.

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Results

Histopathology (Figure 1a,b) We examined the following histological features of the epidermis in both diseases: parakeratosis, orthokeratosis, spongiosis, elongated rete ridges, acanthosis and the presence of neutrophils in the SC. Histological examination confirmed that AD lesions were of the chronic/subacute type. Parakeratosis was found in all patients and in 7/10 AD lesions mostly with a patchwise distribution. Orthokeratosis was found in 9/10 AD lesions and 6/9 PV lesions. Spongiosis was clearly present in 10/10 AD lesions, but was also found in 7/9 PV lesions, although less pronounced. Microabcesses with neutrophils were found in 6/9 PV lesions, but in none of the AD lesions. Acanthosis was found in all lesions of both diseases. Uniform elongation of rete ridges was observed in 6/9 of PV lesions. In AD lesions irregular elongation of rete ridges was found.

Immunohistochemical staining Semiquantitative data on the staining pattern of Ki-67 (a measure of proliferating cells) and E-FABP are presented in Table 1. We observed similar patterns of Ki-67 in PV and AD (Figure 1g-h), indicating that in both diseases the basal layers of the epidermis were in a hyperproliferative state. We found marked differences in intensity and distribution in E-FABP expression between PV and AD lesions. In lesional psoriatic skin the well-known pattern of a strong, uniform, cytoplasmic E-FABP staining was found in all suprabasal layers of the epidermis, the basal layer being completely negative. (Figure 1d,f) In AD lesions the expression of E-FABP was increased compared to normal skin and restricted to the stratum granulosum. In all AD lesions we found strong staining of the stratum granulosum and upper spinous layers, which gradually decreased in intensity towards the basal layer. The first suprabasal layers of AD lesions were invariably negative for E-FABP in contrast to PV lesions (Figure 1c,e).

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Discussion

We studied the expression of E-FABP in AD and PV. In normal epidermis E- FABP expression is restricted to trace amounts in the granular layer, but is strongly upregulated in psoriatic epidermis15;16. We observed an increased E-FABP expression in AD compared to normal skin and we found significant differences in expression between AD and PV as well, although the pattern of expression differed. In contrast to psoriasis, we observed a diffuse pattern of expression decreasing in intensity to the deeper layers. The first suprabasal layers were negative. We did not find differences in the number of cycling cells between AD and PV indicating that the expression of E-FABP does not unconditionally correlate with epidermal proliferation. We conclude that E-FABP expression in lesional skin may discriminate AD from PV. The interpretation of these differences is limited by the unknown function of E-FABP. The animal model of E-FABP deficient mice could be helpful in understanding its specific function, however E-FABP deficient mice show normal development without metabolic disorders. Apparently other members of the FABP family like e.g. H-FABP compensate for the E-FABP deficiency.17 It is assumed that E-FABP is involved in the transport of fatty acids (FA) which play an important role in the epidermis at the structural and the function level.15 FA are important for energy production, phospoholipid formation and signal transduction.18 FA are poorly soluble in water and therefore binding to proteins is required to increase solubility. Within cells, E-FABP binds FA and has a particularly high affinity to C-18 fatty acids like linoleic acid. Therefore, E-FABP may be involved in intracellular FA transport. In psoriasis a decrease of free FA has been reported. It is attractive to speculate, that the strong over-expression of E-FABP in psoriatic epidermis represents a compensatory mechanism to repair this defect. This decrease in free FA appears to be a distinctive biochemical feature of psoriasis.19;20 Lesional atopic skin is also characterised by changes in the epidermal barrier lipids, but systematic data in FA (i.e. linoleic acid) are scarce and controversial. Mustakallio et al. found increased levels of free FA in affected atopic epidermis.21 In contrast, in dry non-eczematous atopic skin, the total amount of free FA is significantly decreased.22 Recent data show that the total amount of free FA is unchanged in lesional skin, but the amount of free very long chain fatty acids (>24-C) was remarkably reduced.23 There is a close relationship between lipid metabolism and keratinocyte differentiation.24 We speculate that the upregulation of E-FABP in lesional psoriasis skin is directly a consequence of prominent epidermal changes characterised by hyperproliferation and abnormal differentiation. In atopic dermatitis the upregulation of E-FABP may be the result of disease-specific changes in lipid metabolism since abnormal lipid composition and changed levels of enzymes involved in lipid metabolism are already present in the uninvolved skin.12;25-28 Nevertheless, further research on FA metabolism in both diseases is required to confirm this hypothesis. We conclude that over-expression of E-FABP is associated with both AD

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and PV lesional skin, however in two different ways. Differences in epidermal lipid metabolism in lesional skin may account for the difference. Since the metabolism of fatty acid in epidermis of both diseases is not exactly clarified, it is difficult to speculate about the metabolic pathways of this epidermal fatty acid carrier. Whether the findings of this study can be used to differentiate both diseases when clinical and histological findings fail to do so, requires further study.

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References

1. Lever,W. and Scaumburg-Lever,G., Non-infectious erythematous, papular, and squa- mous diseases. Histopathology of the skin. J B Lippincott Copmany, Philadelphia 2002, pp 136-163. 2. Braun-Falco,O. and Christophers,E., Structural aspects of the enitial psoriatic lesions. Arch Dermatolpathol Forsch 1974. 251: 95-110. 3. Ragaz,A. and Ackerman,A.B., Evolution, maturation, and regression of lesions of pso- riasis. Am J Dermatopathol 1979. 1: 199-214. 4. Marks,R., The pathology and pathogenesis of the eczematous reaction. In Marks,R. (Ed.) Eczema. Martin Dunitz Ltd, London 1992, pp 21-37. 5. Ackerman,A.B., Troy, J. L., Rosen, L. B., Jerasutus, S., White, R. C., and King, D. F., Differential Diagnosis in Dermatopathology ll. Lea & Febiger, Philadelphia 1988. 6. Kuijpers,A.L., Bergers,M., Siegenthaler,G., Zeeuwen,P.L., van de Kerkhof,P.C., and Schalkwijk,J., Skin-derived antileukoproteinase (SKALP) and epidermal fatty acid-binding protein (E-FABP): two novel markers of the psoriatic phenotype that re- spond differentially to topical steroid. Acta Derm Venereol 1997. 77: 14-19. 7. Murphy,G.F., Flynn,T.C., Rice,R.H., and Pinkus,G.S., Involucrin expression in normal and neoplastic human skin: a marker for keratinocyte differentiation. J.Invest.Dermatol. 1984. 82: 453-457. 8. Kanitakis,J., Ramirez,A., and Reano,A., Filaggrin expression in normal and patho- logical skin: a marker of keratinocyte differentiation. Virchows Arch (A) 1988. 412: 375-382. 9. Michel,S. and Demarchez,M., Localization and in vivo activity of epidermal trans- glutaminase. J.Invest.Dermatol. 1988. 90: 472-474. 10. Siegenthaler,G., Hotz,R., Chatellard-Gruaz,D., Didierjean,L., Hellman,U., and Saurat,J.H., Purification and characterization of the human epidermal fatty acid-binding protein: localization during epidermal cell differentiation in vivo and in vitro. Biochem.J 1994. 302: -71. 11. Masouye,I., Saurat,J.H., and Siegenthaler,G., Epidermal fatty-acid-binding protein in psoriasis, basal and squamous cell carcinomas: an immunohistological study. Dermatol- ogy 1996. 192: 208-213. 12. Yamamoto,A., Serizawa,S., Ito,M., and Sato,Y., stratum corneum lipid abnormalities in atopic dermatitis. Arch.Dermatol Res. 1991. 283: 219-223. 13. Motta,S., Monti,M., Sesana,S., Mellesi,L., Ghidoni,R., and Caputo,R., Abnormality of water barrier function in psoriasis. Role of ceramide fractions. Arch.Dermatol. 1994. 130: 452-456. 14. Motta,S., Monti,M., Sesana,S., Caputo,R., Carelli,S., and Ghidoni,R., Ceramide composition of the psoriatic scale. Biochim.Biophys.Acta 1993. 1182: 147-151. 15. Masouye,I., Saurat,J.H., and Siegenthaler,G., Epidermal fatty-acid-binding protein in psoriasis, basal and squamous cell carcinomas: an immunohistological study. Dermatol- ogy 1996. 192: 208-213. 16. Siegenthaler,G., Hotz,R., Chatellard-Gruaz,D., Jaconi,S., and Saurat,J.H., Charac- terization and expression of a novel human fatty acid-binding protein: the epidermal type (E-FABP). Biochem.Biophys.Res.Commun. 1993. 190: 482-487.

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17. Owada,Y., Takano,H., Yamanaka,H., Kobayashi,H., Sugitani,Y., Tomioka,Y., Suzuki,I., Suzuki,R., Terui,T., Mizugaki,M., Tagami,H., Noda,T., and Kondo,H., Al- tered water barrier function in epidermal-type fatty acid binding protein-deficient mice.J Invest Dermatol 2002. 118: 430-435. 18. Schurer,N.Y., Implementation of fatty acid carriers to skin irritation and the epidermal barrier. Contact Dermatitis 2002. 47: 199-205. 19. Motta,S., Sesana,S., Monti,M., Giuliani,A., and Caputo,R., Interlamellar lipid differ- ences between normal and psoriatic SC. Acta Derm.Venereol.Suppl (Stockh) 1994. 186: 131-132. 20. Wertz,P.W., Madison,K.C., and Downing,D.T., Covalently bound lipids of human SC. J.Invest Dermatol. 1989. 92: 109-111. 21. Mustakallio,K.K., Kiistala,U., Piha,H.J., and Nieminen,E., Epidermal lipids in Bes- nier’s prurigo (atopic eczema). Ann.Med.Exp.Biol.Fenn. 1967. 45: 323-325. 22. Melnik,B., Hollmann,J., Hofmann,U., Yuh,M.S., and Plewig,G., Lipid composition of outer stratum corneum and nails in atopic and control subjects. Arch.Dermatol Res. 1990. 282: 549-551. 23. Macheleidt,O., Kaiser,H.W., and Sandhoff,K., Deficiency of epidermal protein-bound omega-hydroxyceramides in atopic dermatitis. J.Invest Dermatol. 2002. 119: 166-173. 24. Rawlings,A.V., Scott,I.R., Harding,C.R., and Bowser,P.A., stratum corneum moisturi- zation at the molecular level. J Invest Dermatol 1994. 103: 731-741. 25. Imokawa,G., Abe,A., Jin,K., Higaki,Y., Kawashima,M., and Hidano,A., Decreased level of ceramides in stratum corneum of atopic dermatitis: an etiologic factor in atopic dry skin? J Invest Dermatol. 1991. 96: 523-526. 26. Murata,Y., Ogata,J., Higaki,Y., Kawashima,M., Yada,Y., Higuchi,K., Tsuchiya,T., Kawainami,S., and Imokawa,G., Abnormal expression of sphingomyelin acylase in atopic dermatitis: an etiologic factor for ceramide deficiency? J.Invest Dermatol. 1996. 106: 1242-1249. 27. Hara,J., Higuchi,K., Okamoto,R., Kawashima,M., and Imokawa,G., High-expres- sion of sphingomyelin deacylase is an important determinant of ceramide deficiency leading to barrier disruption in atopic dermatitis. J.Invest Dermatol. 2000. 115: 406- 413. 28. Okamoto,R., Arikawa,J., Ishibashi,M., Kawashima,M., Takagi,Y., and Imokawa,G., Sphingosylphosphorylcholine is upregulated in the stratum corneum of patients with at- opic dermatitis. J.Lipid Res. 2003. 44: 93-102.

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84 Chapter 2 3 Epidermis as a barrier to microorganisms

This chapter was based on the following publication:

Specific upregulation of epidermal host-defence proteins in psoriasis compared to atopic dermatitis M. Kucharekova, P.G.M. van der Valk, W.A.M. Blokx, A. Dogan-Bayram, P. Hiemstra, P.C.M. van de Kerkhof, J. Schalkwijk Submitted

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86 87 Chapter 3 Epidermis as a barrier to microorganisms

3.1 SPECIFIC UPREGULATION OF EPIDERMAL HOST- DEFENCE PROTEINS IN PSORIASIS COMPARED TO ATOPIC DERMATITIS

Abstract

Recently it was shown that lesional epidermis of atopic dermatitis patients expresses low levels of the antimicrobial peptides LL-37 and β-defensin-2, compared to psoriasis patients. Here we have investigated whether this is a general phenomenon for host defence proteins, and how specific it is for this class of molecules. Using immunohistochemistry, we examined chronic lesions of atopic dermatitis and psoriasis patients with respect to epidermal expression of SKALP/elafin, SLPI and MRP-8, three proteins associated with a broad spectrum of biological activity in cutaneous host defence. In addition, we examined markers of normal differentiation (involucrin) and other markers of the activated/hyperproliferative epidermal phenotype such as cytokeratin 16 and Ki-67. We found that chronic lesions of psoriasis and atopic dermatitis patients are remarkably similar with respect to cellular proliferation (Ki-67 staining) and expression of structural proteins (cytokeratin 16, involucrin). In contrast, the dual-function host defence proteins SLPI and SKALP/elafin, which have antiproteinase and antimicrobial properties, were found to be strongly expressed throughout the stratum spinosum of psoriasis lesions. In atopic dermatitis lesions we found only weak, mostly patchy expression of SLPI and SKALP/elafin. MRP8 was continuously expressed in the epidermis of both diseases, but staining intensity was weaker in atopic dermatitis than in psoriasis. We conclude that psoriatic epidermis expresses high levels of host defence proteins compared to atopic dermatitis epidermis, and this phenomenon appears to be specific for these proteins as several other markers for epidermal activation showed no differences. It remains to be investigated whether the difference in expression of these epidermal proteins is caused by genetic polymorphisms in cutaneous stress response pathways, or induced by differences in the cellular infiltrate in psoriasis compared to atopic dermatitis.

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Introduction

The stratum corneum of normal human epidermis is a crosslinked structure of insoluble proteins and lipids and as such represents a barrier against invasion by microbial agents. In addition to this physical barrier, human keratinocytes express a number of molecules that are part of the innate immune system and participate in recognition of “microbial non-self” and subsequent killing of invading microorganisms.1 Some of the toll-like receptors (TLR), which are involved in recognition of conserved microbial patterns, were recently demonstrated in human epidermis.2 The skin appears to be a particularly rich source of effector molecules as many proteins with bactericidal or bacteriostatic properties are constitutively or inducibly expressed in human epidermis. These include human β-defensin (hBD)-1, 2, 3 and 4, secretory leukocyte proteinase inhibitor (SLPI), LL-37, SKALP/elafin, dermcidin, calprotectin and cystatin A.3-6 Serial analysis of gene expression (SAGE) on cultured keratinocytes or human skin biopsies has revealed that some of these molecules are found at high levels of expression.7 There is evidence from genetic studies in mice that these molecules are vital for maintaining epidermal integrity.8;9 Elafin, LL-37, hBD-2 and calprotectin are not normally present in human epidermis but are highly induced upon barrier disruption or chronic inflammation such as in the skin disease psoriasis. In psoriasis the on-switch of these genes appears to be part of a stress response also referred to as regenerative maturation, which includes hyperproliferation and induction of marker genes such as cytokeratin (CK) 6, 16 and 17 and psoriasin. Psoriasis vulgaris and atopic dermatitis are two distinct clinical entities characterised by various different histological features depending on the stage of the lesion. These histological characteristics are most apparent in fully developed acute lesions. In the chronic phase of the disease the histopathological differences are less pronounced and both diseases are characterized by acanthosis and a mononuclear infiltrate. A striking difference is the high frequency of bacterial colonization of non-lesional atopic dermatitis skin and recurrent skin infections by bacterial, fungal and viral pathogens in lesional atopic dermatitis skin.10 In contrast, a large epidemiological study on disease concomitance in psoriasis revealed that psoriasis patients have an increased resistance to bacterial infections compared to controls and atopic dermatitis patients.11 Recently, a study was published showing that expression levels of two antimicrobial proteins hBD-2 and LL-37 are significantly decreased in lesional atopic dermatitis epidermis compared to lesional psoriatic epidermis.12 It was speculated that a relative deficiency in expression of innate immunity genes in atopic dermatitis patients could account for the susceptibility to skin infection with Staphylococcus aureus. Here we addressed the following two questions: (1) Is the difference in expression of host defence genes between psoriasis and atopic dermatitis patients a general phenomenon?, In other words: are all known host defence genes overexpressed in psoriasis? (2) How specific is the observed difference? In other

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words: is expression of genes not involved in host defence also altered? We therefore compared the presence of elafin, SLPI and MIF-related protein 8 (MRP8) in biopsies from psoriasis and atopic dermatitis patients. Although the presence of these molecules has been demonstrated in psoriasis, no systematic comparison has been made sofar.5;13 Elafin and SLPI, originally discovered as inhibitors of leukocyte serine proteinases14;15, have antimicrobial activity against a variety of microorganisms.4;16 MRP8 is a S100 Ca++-binding protein that forms the heterodimeric calprotectin complex together with MRP14. Calprotectin is active against C.albicans and intracellular bacteria presumably via binding of Zn++ ions.17 Our data indicate that in psoriasis and atopic dermatitis, expression patterns of these proteins are different as shown by immunohistochemistry; other established markers of the regenerative phenotype (Ki-67, involucrin, CK16) show equal distribution and expression levels.

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Materials and Methods

Subjects The study participants included 9 patients with a clinical diagnosis of moderate to severe chronic plaque psoriasis (5 male and 4 females, mean age 48,1, range 27- 68) and 10 patients with clinically moderate to severe atopic dermatitis (3 male and 7 females, mean age 33,8, range 17- 54). All patients were eligible for systemic treatment, UVB-treatment or hospitalisation. None of the patients received systemic therapy or UVB-therapy at the time of biopsy taking. Punch biopsies were taken from chronic psoriasis plaques and from subacute to chronic atopic dermatitis lesions. Care was taken to exclude acute lesions or lesions with crusts or vesicles. Biopsies from normal skin were obtained from healthy volunteers. The study was approved by the medical ethical committee and all patients gave written informed consent.

Histological methods Biopsies were fixed in buffered 4% formalin for 4 h and processed for routine histology. Tissues were embedded in paraffin and 5- m sections were cut. Sections were stained with haematoxylin-eosin (H&E) or processed for immunohistochemical staining using an indirect immunoperoxidase technique with avidin biotin complex enhancement. For the immunohistochemical stainings a panel of monoclonal and polyclonal antibodies was used: an antibody directed against the Ki-67 antigen was used to investigate epidermal proliferation, (MIB-1, Immunotech, SA, Marseilles, France). To assess epidermal differentiation we used monoclonal antibodies against involucrin (MON-150), cytokeratin 16 (LL025, Novocastra Laboratories Ltd, Newcastle upon Tyne, UK). The dual function proteins SLPI and elafin were stained using polyclonal antibodies that we have previously described.5 MRP8 was stained with a polyclonal rabbit antiserum, kindly provided by dr P.Madsen, Aarhus, Denmark.

Grading of immunohistochemical staining Epidermal proliferation was measured by counting the number of Ki-67 positive nuclei per mm, by an observer who was unaware of the patient history (average of 3 measurements per section). Expression of elafin, SLPI, involucrin, CK16 and MRP8 was assessed in two ways. First the distribution was scored on a 3 point scale: 0: absent, 1: patchy, 2: continuous. All markers with the exception of SLPI and elafin showed a continuous distribution in all samples studied. An additional parameter was the positively stained area of the epidermis, which was assessed by calculating the average ratio of positive cell layers/total cell layers of the epidermis between two dermal papillae (mean of 3 measurements).

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Results

Histopathology Biopsies taken from chronic lesions of both patient groups were analysed for parakeratosis, orthokeratosis, spongiosis, elongated rete ridges, acanthosis and the presence of neutrophils. Histology confirmed that atopic dermatitis lesions were of the chronic/subacute type. Acanthosis and elongation of rete ridges was observed in nearly all biopsies. Regular elongation of rete ridges, however, was found in 6/9 psoriasis patients and in none of the atopic dermatitis patients. Parakeratosis was found in all psoriasis patients, and in 7/10 atopic dermatitis patients mostly with a patchwise distribution. Spongiosis was clearly present in 10/10 atopic dermatitis patients, but was also found in 7/9 psoriasis patients, although less pronounced. Microabcesses with neutrophils were found in 6/9 psoriatic patients, but in none of the atopic dermatitis patients.

Host defence proteins To investigate if overexpression of host defence molecules as reported for hBD-2 and LL-37 is a general phenomenon, we examined 3 other antimicrobial proteins: elafin, SLPI and MRP8. SLPI staining in normal skin revealed aweak presence in the stratum granulosum (Figure 1a), whereas in psoriasis a strong staining throughout the upper layers of the stratum spinosum was found (Figure 1c). In epidermis of atopic dermatitis patients SLPI staining was variable (Figure 1b). In some biopsies it showed a discontinuous distribution and in others it resembled normal skin. In some cases a few layers of the stratum spinosum were stained. Semi- quantitative scoring for distribution and intensity indicated a significant difference between the two diseases (see Table 1). Elafin was absent in normal human epidermis (Figure 1d) but is highly expressed in psoriatic epidermis as shown in Figure 1f. In the epidermis of atopic dermatitis patients we observed a similar pattern as found for SLPI; the distribution was often discontinuous, and the intensity as measured by the ratio of positive cell layers over the total number of cell layers was significantly different between the two diseases (Figure 1e and Table 1). Staining for MRP8 was absent in normal skin (Figure 1g) but showed a continuous staining of the suprabasal compartment in both diseases (Figures 1h and 1i). Staining intensity, however, was more intense in psoriasis, and often extended to the first suprabasal layer Figure( 1i). The difference was of borderline significance Table( 1).

Differentiation related proteins We now asked whether other epidermal proteins that are not known to be associated with host defence showed similar patterns. The structural protein involucrin is present in the stratum granulosum of normal epidermis (not shown), and is highly expressed throughout the stratum spinosum of psoriatic epidermis (Figure 2b). Figure 2a and 2b show that the staining pattern is similar between psoriasis and

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SLPI

Elafin

MRP8

Normal skin Atopic dermatitis Psoriasis

Figure 1 Host defense proteins in human skin Immunohistochemical staining of normal skin (a-d-g), atopic dermatitis skin (b-e-h) and psoriatic skin (c-f-i) for SLPI (a-c), elafin (d-f) and MRP8 (g-i). Note the difference in elafin andSLPI localization and staining intensity, between psoriasis and atopic dermatitis. Bar: 50 μm

Figure 1 Kucharekova et al atopic dermatitis. No significant difference was found (Table 1). The cytoskeletal protein CK16 is absent from normal epidermis (not shown) but is highly induced in psoriasis as shown previously by others. As such it has been one of the earliest recognized markers for the regenerative differentiation pathway in epidermis. Figure 2 and Table 1 show that there is no significant difference in CK16 staining between the two diseases.

Epidermal proliferation It could be argued that the observed difference in expression level of host defence proteins is a consequence of gross differences in epidermal structure, the number of cell layers or proliferative status of the tissues. Although histopathological differences exist, features such as parakeratosis, acanthosis and spongiosis were observed in both conditions (see above). To investigate cellular proliferation we performed Ki-67 staining which is a marker for cycling cells. Figures 2e and

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Table 1 Semi-quantitative scoring of immunohistochemical staining Protein Score Psoriasis Atopic dermatitis Elafin distribution 2.0 + 0.0* 0.8 + 0.4 ratio 0.48 + 0.14* 0.23 + 0.11 SLPI distribution 2.0 + 0.0$ 1.1 + 0.6 ratio 0.49 + 0.10# 0.28 + 0.16 MRP8 distribution 2.0 + 0.0 2.0 + 0.0 ratio 0.78 + 0.12¶ 0.50 + 0.30 Involucrin distribution 2.0 + 0.0 2.0 + 0.0 ratio 0.59 + 0.11 0.64 + 0.05 CK16 distribution 2.0 + 0.0 2.0 + 0.0 ratio 0.85 + 0.14 0.84 + 0.10 Ki-67 positive cells per mm 89 + 45 85 + 45 Immunohistochemical staining was quantified as indicated in the Method section Figure s represent mean +/- standard deviation of 10 atopic dermatitis patients and 9 psoriasis patients. Values for elafin, SLPI and MRP8 staining were significantly different between psoriasis and atopic dermatitis: *p<0.001, $p<0.01, #p<0.02, ¶p<0.05. No significant differences were found for involucrin, CK16 and Ki67. Mann-Whitney U-test

Figure 2 Differentiation and proliferation in psoriasis and atopic dermatitis InvolucrinInvolucrin Immunohistochemical staining of atopic dermatitis skin (a-c-e) and psoriatic skin (b-d-f) for involucrin (a-b), CK16 (c-d) and Ki-67 (e-f). Note the similar localization and staining intensities of these markers CK16CK16 in the two diseases. Bar: 100 μm

Ki-67Ki-57

AtopicAtopic dermatitisdermatitis PsoriasisPsoriasis

92 Figure 2 Kucharekova et al 93 Chapter 3 Epidermis as a barrier to microorganisms

2f, and Table 1 show that the epidermis of atopic dermatitis patients is equally hyperproliferative as is the case in psoriasis.

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Discussion

A recent study by Ong et al has raised the intriguing possibility that the increased susceptibility of atopic dermatitis patients for bacterial infections may be related to low expression levels of antimicrobial peptides (when compared to psoriasis patients).12 In our present study we have extended their observations to three other host defence proteins, and we show that this phenomenon is not found for two other differentiation related proteins without a known function in host defence. It should be noted, however, that there is at least one other epidermal protein (E-FABP) not associated with host defence, which also shows a differential expression pattern between psoriasis and atopic dermatitis (see chapter 2 of this thesis). How could we interpret the remarkable finding that there appears to be a difference in cutaneous innate immunity between these two diseases? First, one could ask whether expression of epidermal host defence genes is low in atopic dermatitis or high in psoriasis. A comparison with healthy individuals has not been performed in either study, for the obvious reason that it is not easy to obtain a suitable chronic inflammatory T-cell mediated control lesion in otherwise healthy volunteers. It is conceivable to address this experimentally, using the induction of antimicrobial proteins in response to standard injury of normal skin, or uninvolved skin of psoriasis and atopic dermatitis patients. The tape-stripping model would be an interesting option in this respect. This would, however, not address the question whether it is a cell-autonomous process or not, in other words: is the difference in epidermal innate immunity governed by the inflammatory infiltrate, or is it an intrinsic property of the keratinocyte? This could be investigated in keratinocyte cultures following stimulation with the appropriate factor to induce innate immunity genes in vitro. Previous studies have shown that serum, TNF-alpha and detergents can induce elafin gene expression in cultured keratinocytes.18;19 These studies and reports on elafin induction in other cellular systems have indicated that its expression is dependent on p38 MAPkinase and NFκB signalling.20 hBD2 and LL37 were shown to be induced by various agents including IL-1, TNF-α, IL-6 and bacterial products. 21;22 Taken together these data suggest that several of these antimicrobial proteins may be expressed following Toll-like receptor (TLR) signaling or in the context of a Th1 immune response, where TNF- α is a dominant cytokine. Interestingly, an active TLR-4 receptor has recently been described in keratinocytes.23 It remains to be investigated whether the actual tissue levels of TNF-α or activity of TLR signalling pathways are higher in psoriasis than in atopic dermatitis lesions. Alternatively, keratinocytes from psoriatic patients could be genetically programmed to respond earlier (at lower thresholds) or more vigourously to stimuli such as TNF-α or microbial products. In this case, one would expect that the psoriasis loci that have been identified in extensive linkage studies performed over the last years, would yield candidate genes associated with innate immunity. The NOD2 gene would be a good candidate but this has been recently excluded for psoriasis.24 It is interesting to note that, in culture, differences in chemokine production have been

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found between psoriasis and atopic dermatitis keratinocytes.25 This study suggests that psoriatic keratinocytes intrinsically produce more IL-8, MCP-1 and IP-10 than cells from atopic dermatitis patients. It could be argued that a comparison between any two diseases would reveal differences in immunolocalization of host defence genes, because of the differences in disease stage and severity, and the specific architecture of the affected epidermis in a particular disease. When, for instance, an acute lesion of atopic dermatitis is compared with a psoriatic lesion, the comparison would be less meaningful because of the reasons mentioned above. We took care, however, to study chronic lesions of both patient groups. Although the clinical diagnosis of the patients was clear-cut, the lesions showed a number of histopathological similarities, as indicated above. Some of the biopsies (5/19) could not be unambiguously classified on the basis of histopathology alone. Cellular proliferation and acanthosis measured as the number of epidermal cell layers were not different between the two diseases. A remarkable finding was that CK16, an established marker of keratinocyte activation in the context of hyperproliferaton, was not differentially expressed. It is conceivable that psoriasis and atopic dermatitis patients represent the extremes of a population. Psoriasis patients may have a genetic background that predisposes towards a Th1 dominated T-cell population in response to microbial stimuli and they might carry polymorphisms in TNF-α and NFκB signaling that favour expression of epidermal host defence genes; on the other hand atopic dermatitis patients favour a Th2 response26 and could possibly carry genetic polymorphisms conferring a poor epidermal innate immune response towards environmental microbial stimuli. The fact that psoriasis and atopic dermatitis are very common diseases, strongly suggests that these predisposing genes confer a certain evolutionary advantage. A large population based study by Henseler et al has shown that psoriasis patients have an increased resistance to cutaneous bacterial infections.11 In this light, psoriasis could be the evolutionary cost of having too many, otherwise favourable genetic polymorphisms. To summarize, we think that the observations made by Ong et al12 and the data presented here together with the recently proposed interaction between innate and adaptive immunity, can provide new insights in the immunobiology of common inflammatory skin diseases. The notion that polymorphisms in genes dealing with environmental (microbial) stimuli play a role in these conditions may enable us to gain greater insight in the pathogenesis of these diseases and even lead to more specific therapeutic avenues.

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References

1. Gallo,R.L. and Huttner,K.M., Antimicrobial peptides: an emerging concept in cutane- ous biology. J.Invest Dermatol. 1998. 111: 739-743. 2. Curry,J.L., Qin,J.Z., Bonish,B., Carrick,R., Bacon,P., Panella,J., Robinson,J., and Nickoloff,B.J., Innate immune-related receptors in normal and psoriatic skin. Arch.Pathol.Lab Med. 2003. 127: 178-186. 3. Harder,J., Bartels,J., Christophers,E., and Schroder,J.M., A peptide antibiotic from human skin. Nature 1997. 387: 861. 4. Simpson,A.J., Maxwell,A.I., Govan,J.R., Haslett,C., and Sallenave,J.M., Elafin (elastase-specific inhibitor) has anti-microbial activity against gram-positive and gram- negative respiratory pathogens. FEBS Lett. 1999. 452: 309-313. 5. Wingens,M., van Bergen,B.H., Hiemstra,P.S., Meis,J.F., Vlijmen-Willems,I.M., Zeeuwen,P.L., Mulder,J., Kramps,H.A., van Ruissen,F., and Schalkwijk,J., Induc- tion of SLPI (ALP/HUSI-I) in epidermal keratinocytes. J.Invest Dermatol. 1998. 111: 996-1002. 6. Zaiou,M. and Gallo,R.L., Cathelicidins, essential gene-encoded mammalian antibiot- ics. J.Mol.Med. 2002. 80: 549-561. 7. Jansen,B.J., van Ruissen,F., de Jongh,G., Zeeuwen,P.L., and Schalkwijk,J., Serial analysis of gene expression in differentiated cultures of human epidermal keratinocytes. J.Invest Dermatol. 2001. 116: 12-22. 8. Ashcroft,G.S., Lei,K., Jin,W., Longenecker,G., Kulkarni,A.B., Greenwell-Wild,T., Hale-Donze,H., McGrady,G., Song,X.Y., and Wahl,S.M., Secretory leukocyte pro- tease inhibitor mediates non-redundant functions necessary for normal wound healing. Nat.Med. 2000. 6: 1147-1153. 9. Nizet,V., Ohtake,T., Lauth,X., Trowbridge,J., Rudisill,J., Dorschner,R.A., Pestonjamasp,V., Piraino,J., Huttner,K., and Gallo,R.L., Innate antimicrobial peptide protects the skin from invasive bacterial infection. Nature 2001. 414: 454-457. 10. Leung,D.Y. and Bieber,T., Atopic dermatitis. Lancet 2003. 361: 151-160. 11. Henseler,T. and Christophers,E., Disease concomitance in psoriasis. J.Am.Acad.Dermatol. 1995. 32: 982-986. 12. Ong,P.Y., Ohtake,T., Brandt,C., Strickland,I., Boguniewicz,M., Ganz,T., Gallo,R.L., and Leung,D.Y., Endogenous antimicrobial peptides and skin infections in atopic der- matitis. N.Engl.J.Med. 2002. 347: 1151-1160. 13. Gabrielsen,T.O., Dale,I., Brandtzaeg,P., Hoel,P.S., Fagerhol,M.K., Larsen,T.E., and Thune,P.O., Epidermal and dermal distribution of a myelomonocytic antigen (L1) shared by epithelial cells in various inflammatory skin diseases. J.Am.Acad.Dermatol. 1986. 15: 173-179. 14. Thompson,R.C. and Ohlsson,K., Isolation, properties, and complete amino acid se- quence of human secretory leukocyte protease inhibitor, a potent inhibitor of leukocyte elastase. Proc.Natl.Acad.Sci.U.S.A 1986. 83: 6692-6696. 15. Wiedow,O., Schroder,J.M., Gregory,H., Young,J.A., and Christophers,E., Elafin: an elastase-specific inhibitor of human skin. Purification, characterization, and complete amino acid sequence. J.Biol.Chem. 1990. 265: 14791-14795. 16. Hiemstra,P.S., Maassen,R.J., Stolk,J., Heinzel-Wieland,R., Steffens,G.J., and Dijkman,J.H., Antibacterial activity of antileukoprotease. Infect.Immun. 1996. 64: 4520-4524.

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17. Ganz,T. and Lehrer,R.I., Antimicrobial peptides of leukocytes. Curr.Opin.Hematol. 1997. 4: 53-58. 18. Pfundt,R., van Ruissen,F., Vlijmen-Willems,I.M., Alkemade,H.A., Zeeuwen,P.L., Jap,P.H., Dijkman,H., Fransen,J., Croes,H., van Erp,P.E., and Schalkwijk,J., Con- stitutive and inducible expression of SKALP/elafin provides anti-elastase defense in hu- man epithelia. J.Clin.Invest 1996. 98: 1389-1399. 19. Pfundt,R., Wingens,M., Bergers,M., Zweers,M., Frenken,M., and Schalkwijk,J., TNF-alpha and serum induce SKALP/elafin gene expression in human keratinocytes by a p38 MAP kinase-dependent pathway. Arch.Dermatol.Res. 2000. 292: 180-187. 20. Bingle,L., Tetley,T.D., and Bingle,C.D., Cytokine-mediated induction of the hu- man elafin gene in pulmonary epithelial cells is regulated by nuclear factor-kappaB. Am.J.Respir.Cell Mol.Biol. 2001. 25: 84-91. 21. Liu,A.Y., Destoumieux,D., Wong,A.V., Park,C.H., Valore,E.V., Liu,L., and Ganz,T., Human beta-defensin-2 production in keratinocytes is regulated by interleukin-1, bacte- ria, and the state of differentiation. J.Invest Dermatol. 2002. 118: 275-281. 22. Tsutsumi-Ishii,Y. and Nagaoka,I., NF-kappa B-mediated transcriptional regulation of human beta-defensin-2 gene following lipopolysaccharide stimulation. J.Leukoc.Biol. 2002. 71: 154-162. 23. Song,P.I., Armstrong,C.A., and Ansel,J.C., Human Normal Keratinocytes Express Biologically Functional CD14 and Toll-Like Receptors. J.Invest Dermatol. 2003. 121: 217-218. 24. Nair,R.P., Stuart,P., Ogura,Y., Inohara,N., Chia,N.V., Young,L., Henseler,T., Jenisch,S., Christophers,E., Voorhees,J.J., Nunez,G., and Elder,J.T., Lack of asso- ciation between NOD2 3020InsC frameshift mutation and psoriasis. J.Invest Dermatol. 2001. 117: 1671-1672. 25. Giustizieri,M.L., Mascia,F., Frezzolini,A., De Pita,O., Chinni,L.M., Giannetti,A., Girolomoni,G., and Pastore,S., Keratinocytes from patients with atopic dermatitis and psoriasis show a distinct chemokine production profile in response to T cell-derived cy- tokines. J.Allergy Clin.Immunol. 2001. 107: 871-877. 26. Hamid,Q., Boguniewicz,M., and Leung,D.Y., Differential in situ cytokine gene expres- sion in acute versus chronic atopic dermatitis. J.Clin.Invest 1994. 94: 870-876.

98 Chapter 3 4 Novel therapies on irritant contact dermatitis: Experimental and clinical studies

This chapter was based on the following publications:

The effect of a PDE-4 inhibitor (Cipamfylline) in two human models of irritant contact dermatitis M. Kucharekova, M. Hornix, T. Ashikaga, S. T’kint, G.J. de Jongh, J. Schalkwijk, P.C.M. van de Kerkhof, P.G.M. van der Valk Arch Derm Res;2003;295(1):29-32

Effect of a lipid-rich emollient containing ceramide 3 in an experimentally induced skin barrier dysfunction M. Kucharekova, J. Schalkwijk, P.C.M. van de Kerkhof, P.G.M. van der Valk Contact Dermatitis;2002;46(6):331-338

A randomized comparison of an emollient containing skin-related lipids with a petrolatum-based emollient as adjunct in the treatment of chronic hand dermatitis M. Kucharekova, P.C.M. van de Kerkhof, P.G.M. van der Valk Contact dermatitis; In Press

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4.1 THE EFFECT OF A PDE-4 INHIBITOR (CIPAMFYLLINE) IN TWO HUMAN MODELS OF IRRITANT CONTACT DERMATITIS

Abstract

The treatment of irritant contact dermatitis (ICD) is a major problem in clinical dermatology. New therapeutic approaches have to be considered. Recently, phosphodiesterase 4 (PDE-4) inhibitors have been introduced as non-steroidal, anti- inflammatory agents. These agents inhibit the secretion of the cytokines, which are thoughts to be involved in the pathogenesis of ICD. We investigated the effect of a new selective PDE-4 inhibitor (cipamfylline), in human models using single and repeated exposures to an irritant in a blind, randomized pilot study with healthy volunteers. We compared the effect of cipamfylline-ointment with a strong corticosteroid (betamethasone -17-valerate) and with a placebo-ointment. Ten volunteers were patch tested at four investigation sites with sodium dodecyl sulfate (1%) for 24 hours. In a model that simulates chronic damage, eleven volunteers were patch tested with sodium dodecyl sulphate (0.2%) for 4 hours a day for 4 consecutive days. The investigation sites were treated once a day with the above mentioned agents. One site was left untreated. We used erythema scoring, measurements of transepidermal water loss (TEWL) and several immunohistochemical markers for epidermal proliferation and differentiation. Repetitive application revealed that betamethasone-17-valerate caused a statistically significant reduction of erythema and TEWL compared to cipamfylline and placebo. Furthermore, we observed a significant suppression of proliferating cells and cytokeratin 16 expression at the site treated with betamethason compared to the other sites. In the model for acute ICD no significant differences were seen between the investigated sites. Our data shows that betamethasone-17-valerate may be modulating the response in ICD. In these human models of ICD we could not confirm the efficacy of cipamfylline. Clinical studies are needed before refuting of PDE-4 inhibitors in ICD with certainty.

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Introduction

Irritant contact dermatitis (ICD) has been defined as a non-immunological inflammatory response of the skin due to one or more external factors.1 ICD may be classified into two main types. 1. Acute irritant contact dermatitis may develop from a single exposure of the skin to a strong irritant in a relatively short time. 2. Chronic irritant contact dermatitis is thought to be caused by repeated exposures to various irritant factors by a repetition of one or more impairing external factors. ICD is a major cause of occupational disability, it results in loss of productivity, medical expenses and disability payment.2 Various therapies have been tried but treatment remains unsatisfactory. New therapeutic approaches, therefore, still need to be considered. Recently, phosphodiesterase-4 (PDE-4) inhibitors have been introduced as anti-inflammatory agents. Phosphodiesterases (PDEs) are a family of enzymes responsible for the hydrolysis of the intracellular messengers, cyclic AMP (cAMP) and cyclic GMP (cGMP) causing their inactivation. The PDE-family consists of at least 9 isoenzymes with different selectivity for tissue of localisation. PDE-4 is localised in all inflammatory cells.3 The inhibition of PDE-4 and the subsequent increase in cAMP levels may be of significant value in controlling inflammatory disorders. In this way, PDE-4 inhibitors are able to inhibit inflammatory cytokine production and modulate immune and inflammatory responses.4 Clinical and preclinical studies with PDE-4 inhibitors have shown that these agents reduce inflammation in atopic dermatitis 4;5 and in models of immunological inflammation.6-8 These promising results in atopic inflammation prompted us to study the effect of PDE-4 inhibitors in ICD. PDE-4 inhibitors may be potential therapeutic agents in ICD because of several reasons. Upon skin irritation enhanced production of cytokines has been reported.9-11These cytokines may act as mediators to elicit an inflammatory response and/or coordinate metabolic responses for barrier repair. Thus, cytokines may be important candidates controlling irritant contact reaction and inhibition of their function may diminish an ensuing inflammatory response.12 In particular, the proinflammatory cytokine, tumor necrosis factor - alpha (TNF-α), is thought to be critical in ICD responses.2;10;13-15The expected effects of PDE-4 inhibitors on ICD could be due to their ability to inhibit TNF-α6 and the production of other cytokines as IL-123, IL-4.6 Such a non-steroidal, anti-inflammatory approach is a promising therapy of ICD and may be even superior to dermatocorticosteroid therapy because it may not only modulate the inflammatory response, but is also not likely to induce skin atrophy and barrier impairment. In the present study we investigated the effect of cipamfylline, a new selective inhibitor of phosphodiesterase-4 enzyme in human models using single and repeated exposures to a detergent (SDS) in a blind, randomized pilot study with healthy volunteers. We compared the effect of cipamfylline ointment with a strong dermatocorticosteroid (betamethasone-17-valerate) and with a placebo ointment.

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Barrier function disruption occurs after exposure to detergents as SDS and this impairment may be relevant to ICD. Therefore, we investigated repair of skin barrier function by measuring transepidermal water loss. We also performed clinical erythema scoring. The development of erythema and increasing of TEWL in SDS induced irritation, is associated with increased number of cycling cells16 and upregulation of differentiation-associated proteins (e.g. involucrin, epidermal fatty acid binding protein).17 These aberrations can be visualised by immunohistochemical staining of different marker proteins. We used these immunohistochemical markers to investigate potential cell-biological effects of the above mentioned therapeutical agents. To assess the changes on epidermal proliferation, we used an immunohistochemical marker of epidermal growth, known as Ki-67 antigen, present in cycling cells. Furthermore, we observed the expression of two terminal differentiation proteins (involucrin, epidermal-fatty acid binding protein) and the expression of cytokeratin 16 which is present in hyperproliferative conditions.18;19

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Material and Methods

Subjects 21 healthy volunteers with no history of skin diseases (6 male, 15 female, mean age 48.1, ranging from 24 to 69 years) participated in the study. After approval by the Medical Ethics Committee, informed consent was obtained from all subjects. The study was conducted in January -March 2002.

Skin exposure to sodium dodecyl sulphate and topical formulations Experimentally induced skin irritation was studied in two different models. Model A consisted of a single exposure of 1% sodium dodecyl sulphate. We used a 1% dilution of SDS (w/v). An aliquot of 150 μl of SDS was pipetted on the patches, consisting of a 1,5 cm2 piece of absorbent, non-woven fabric on a 4.0 cm2 piece of impermeable plastic foil.20 10 subjects were patch tested on 4 investigating sites on the lower back for 24 hours. After removal of the patches, the ointment was applied daily over the area of previously SDS exposed skin, dried at room temperature for 30 minutes. The ointment was applied for 3 consecutive days from day 1 to day 3, once a day. Model B consisted of repetitive application of 0.2% SDS for 4 consecutive days.150 μl of SDS (0.2%) (w/v), pipetted on the patches, consisting of a 1.5 cm2 piece of absorbent, non-woven fabric on a 4 cm2 piece of impermeable plastic foil, which was daily fixed at the same site to the back of 11 subjects during 4 h for 4 consecutive days from day 0 to day 3. After removal of the patches, the ointment was applied daily over the area of previous SDS exposed skin, dried at room temperature for 30 minutes. The ointments were applied for 4 consecutive days from day 0 to day 3, once a day. The following formulations were used for both models: cipamfylline ointment (cipamfylline 2.5 mg/g, macrogol-2 stearyl ether, white soft paraffin, liquid paraffin, and propylene glycol), placebo ointment (macrogol-2 stearyl ether, white soft paraffin, liquid paraffin, propylene glycol) and betamethason-17-valerate ointment (1mg/g). One site was untreated. Applications were performed according to the randomisation code.

Clinical scoring and transepidermal water loss (TEWL) measurements For both models visual grading and TEWL were assessed at day 0 to day 4. TEWL was measured with a Tewameter TM 210, according to standard guidelines.21 Room temperature varied from 18º C until 22º C and relative humidity from 38% to 56%. Semi-quantitative grading system of erythema, already described in other study17, was used: 0-no response; 1-slight, patchy redness; 2-diffuse mild redness, 3- moderate redness, 4-intense redness; 5-intense redness with oedema.

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Biopsy procedures A total of 80 punch biopsies (3 mm diameter) were taken from each of the patch-test sites of 20 volunteers after visual reading and TEWL measurements at day 4. After 4 h fixation in formalin the samples were stored in alcohol 70% until embedding in paraffin was done. Then the samples were sectioned at 6 μmfor immunohistochemical staining.

Immunohistochemical methods Biopsy sections were evaluated light microscopically using haematoxylin- eosin staining according to Mayer and immunohistochemical staining. For the immunohistochemical staining a panel of monoclonal and polyclonal antibodies was used. To assess epidermal proliferation, an antibody directed against the Ki-67 antigen was used (MIB-1, DAKO A/S Denmark). To assess epidermal differentiation three markers were used: a monoclonal antibody against involucrin (MON-150)22 a monoclonal antibody against cytokeratin 16 (LL025, Novocastra, Newcastle, United Kingdom), and a polyclonal antibody against the epidermal-fatty-acid-binding- protein (anti-E-FABP).23 The slides were deparaffinized by incubation in histosafe for 2 times 15 minutes, followed by dipping in an ethanol series from 100% to 50%. After washing in phosfate-buffer saline (PBS) the sections were pre-incubated with normal horse serum (20%) for MIB-1, LL025, MON-150; and with normal goat serum (20%) for anti-E-FABP for 15 minutes. Before this pre-incubation the sections were pre-treated with citrate buffer in case of MIB-1, LL025 and MON-150. Furthermore, an antigen retrieval method in a microwave oven was necessary for MIB-1, LL025 and MON- 150. Then all sections were incubated for 60 minutes with the following primary antibodies: MIB-1, anti-E-FABP, LL025 and MON-150 at the dilution of 1:50, 1: 400, 1:10 and 1:40, respectively. After washing with PBS, sections were incubated for 30 minutes with biotinylated horse-anti-mouse IgG or goat-anti-rabbit IgG (dilution 1:200). Another washing step with PBS, and then complexes were formed between biotin and avidin using the Vectastain Elite ABC peroxidase kit, according to the manufaturer’s instructions (Vector Laboratories, Burlingame, USA). Sections were developed with amino-ethylcarbazol as the chromogenic substrate or metal-enhanced diaminobenzidin. All sections were subsequently counterstained with hematoxylin 1%.

Histological grading Semiquantative assessment of the histology was carried out by two observers who were unaware of which sections accounted for which treatment. Epidermal proliferation was measured by counting the number of Ki-67 positive nuclei per mm length of the section. Of every section 3 fields were counted, and we calculated mean ± SEM. Involucrin staining was assessed by calculating the ratio of positively stained cell layers, compared to the total number of cell layers, at a representative

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interpapillary and papillary area. The expression of E-FABP was scored as already mentioned before on page 77. CK-16 was graded: 0- no staining, 1- sporadic staining, 2- minimal staining, 3- moderate staining, 4- moderate/pronounced staining, 5- pronounced staining.

Statistical analysis Data are reported as means ± SEM. The differences were analysed with statistica 5.0 using ANOVA/MANOVA. In case of a significant difference further analysis was performed by use of the Duncan-test. A p-value < 0.05 was supposed to be statistically significant.

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Results

Clinical response and TEWL measurements Single exposure of human skin to SDS (1%) resulted in an erythematous reaction and increase of TEWL on all investigated sites, gradually decreasing to day 4. No significant differences in erythema or TEWL was found between the treated and untreated sites. Repetitive exposure to SDS (0.2%) induced an increase of erythematous reaction and TEWL values, enhancing in intensity up to day 4. There was a significant smaller increase of erythema and TEWL values in the betamethason-17-valerate treated sites compared to the cipamfylline and placebo, but not compared to the untreated site. (Figure 1a,b)

Histology and immunohistochemistry

H&E staining At day 4 we observed in both models mild to moderate parakeratosis, spongiosis and acanthosis and a mild to moderate perivascular infiltrate. No apparent differences were seen between the various treated sites.

Epidermal proliferation The MIB-1 antibody was used to stain the Ki-67 antigen in cycling cells. Keratinocytes with positively stained nuclei were located in the basal and the first suprabasal layers of the epidermis. Skin exposed to SDS in the acute and chronic model is characterised by an increase in the number of MIB-1 positive nuclei. In the acute model no significant differences were found in cycling cells/mm between the various investigated sites (data not shown). Repetitive exposure of SDS (0,2%) resulted in untreated skin in an increase in the number of cycling cells/mm to a maximum of 48,9 ± 8,3 (mean ± SEM). A marked, significant decrease in the cycling cells/mm was observed in the betamethasone-17-valerate treated sections compared to the sites treated with cipamfylline, placebo and not treated site . (Figure 2a)

Epidermal differentiation In both models, exposure to SDS, caused an increased expression of involucrin, E-FABP and cytokeratin 16. None of the treatment affected cytokeratin 16 expression in the acute model. (data not shown) In the chronic model, the expression of cytokeratin 16 after treatment with betamethasone differed significantly from cipamfylline treated site, placebo and not treated site, respectively (Figure 2b). In both our models we did not find any significant differences between the investigated sites with respect to expression of involucrin and epidermal fatty acid binding protein. (data not shown)

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Figure 1 (a) Erythema score in Repetitive model model of repetitive application of

2.5 SDS (0.2%). Erythema was assessed * using a five-point scale: 0-no 2 Cipamfylline response; 1-slight, patchy redness; 1.5 Placebo 2-diffuse mild redness, 3-moderate 1 Betamethasone redness, 4-intense redness; 5-intense Not treated Erythema score 0.5 redness with oedema. * Significant

0 difference between the site treated Day 0 Day 1 Day 2 Day 3 Day 4 with betamethasone compared to the sites treated with cipamfylline (p=0.013) and placebo (p=0.015), but not compared to the not treated Repetitive model site. 2 60 (b)TEWL values (g/m /h) in model 50 * of repetitive application of SDS Cipamfylline 40 (0.2%). * Significant difference Placebo 30 between the site treated with

TEWL Betamethasone 20 betamethasone compared to the sites Not treated 10 treated with cipamfylline (p=0.017) 0 and placebo (p=0.01), but not Day 0 Day 1 Day 2 Day 3 Day 4 compared to the not treated site.

Figure 2 Immunuhistochemical Cycling cells staining. (a) The number of proliferative cell (Ki-67) per mm 70 60 length of the section after repetitive 50 application of 0.2% SDS. The values * 40 are mean ± SEM. * Significant 30 difference between the sites treated 20

cell number/mm with betamethasone compared to 10 the sites treated with cipamfylline 0 Cipamfylline Placebo Betamethasone Not treated (p=0.02), placebo (p=0.006) and not treated site (p=0.031). (b) Cytokeratin 16 after repetitive application of 0.2% SDS. The Cytokeratin 16 expression of CK 16 in epidermis was assessed using a five-point 4 scale: 0- no staining, 1- sporadic 3 staining, 2- minimal staining, 3- moderate staining, 4- moderate/ 2 * pronounced staining, 5- pronounced score (1-5) 1 staining. * Significant difference between the site treated with 0 Cipamfylline Placebo Betamethasone Not treated betamethasone compared to the sites treated with cipamfylline (p=0.006), placebo (p=0.018) and not treated site (p=0.023).

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Discussion

In the present study, the induction of irritant reactions by single and repetitive exposure to SDS and the repair with or without treatment was studied using visual scoring, TEWL and markers of epidermal proliferation and differentiation. Single exposure to SDS resulted in an erythematous reaction and an increase in TEWL values on all investigated sites after 24 hours, gradually decreasing till day 4. The repetitive model showed an increase in erythematous reaction and TEWL, increasing in intensity up to day 4. The acute and repetitive exposure to SDS not only induced histological changes in the epidermal cell compartment, but also upregulated the expression of markers for epidermal differentiation and proliferation in line with a previous study.20 Treatment of skin irritation with cipamfylline ointment, placebo and betametasone-17- valerate after single, 24 hour exposure of SDS, did not significantly decrease erythema, TEWL and also did not significantly modulate epidermal proliferation assessed by Ki-67 and epidermal differentiation assessed by the expression of involucrin, E-FABP and cytokeratin 16. Therefore, it can be concluded that these treatments did not modulate SDS damage in this model. The present study confirms a study by Levin who also failed to show an effect of corticosteroids in the acute model of ICD.24 Repetitive exposure to SDS, however, provides a model which is more similar to irritant contact dermatitis. The validity of single and repeated application of SDS as a model for ICD has been discussed before.20 In contrast to one single application, repetitive application revealed that betamethasone-17-valerate caused a statistically significant reduction of erythema and TEWL values compared to cipamfylline and placebo and a tendency of a reduction as compared to the untreated site. In order to elucidate the potential of a corticosteroid and cipamfylline in ICD we studied epidermal proliferation and differentiation parameters using immunohistochemical markers. An inhibition of Ki-67 and cytokeratin 16 was shown following betamethasone-17-valerate treatment compared to cipamfylline treated, placebo treated and untreated sites. However, again cipamfylline did not show any effect on the expression of these markers. Previously, several authors reported a lack of efficacy of topical corticosteroids on skin irritation, whereas others clearly showed such an effect.20;24-27 However, there are considerable differences between the designs of these studies with regard to corticosteroid potency, the duration and site of the irritation, open or closed application, the concentration of the irritant, which apparently may influence the outcome of the study. Acute and repetitive open application testing on the hands of volunteers with several irritant and corticosteroid formulations are recommended in order to establish the efficacy of corticosteroids in ICD.28 Consistent with the study of Le 20, who studied the effect of mild corticosteroid (triamcinolon acetonide cream) in the repetitive model of ICD, we found betamethasone-17-valerate to reduce the number of cycling cells significantly. But in contrast to this study we also

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find a significant decrease in erythema, TEWL and cytokeratin 16 expression. Such might be due to the fact that betamethasone-17-valerate is more potent compared to triamcinolon acetonide. In various entities of dermatitis (e.g. radiation dermatitis29, dithranol irritation30, nickel contact dermatitis31) a beneficial clinical effect of topical corticosteroids has been observed. Schmutz et al suggested that the positive effect of topical corticosteroids on barrier function in radiation dermatitis is caused by a reduction in the inflammatory response.29 In ICD barrier dysfunction precedes inflammation and therefore damage to the skin barrier may only be partly a reflection of an inflammatory response. This may explain the modest or absent efficacy of a topical corticosteroid or other anti-inflammatory treatment in some models of ICD, since barrier dysfunction is considered to play a role in its pathogenesis.32 In contrast to a mild effect of betamethasone-17-valerate on the clinical severity and the pronounced antiproliferation effect of betamethasone-17-valerate in the repetitive model of ICD, cipamfylline proved to have no effect on erythema, TEWL and epidermal proliferation and differentiation. On the other hand in atopic dermatitis, PDE 4 inhibitors have been shown to be effective.4;8 In this respect there are pronounced differences between ICD and atopic dermatitis. Indeed, costicosteroids and more recently tacrolimus are effective treatments of atopic dermatitis 33-35 but have a limited or no efficacy in ICD at all. In contrast, tacrolimus has been shown to enhance experimentally induced contact dermatitis.36 The rationale for the potential use of PDE-4 inhibitors in ICD was based on the ability of PDE-4 to inhibit TNF-α and the production of other cytokines which are enhanced in ICD. 9;10;15;37 However, some animal studies demonstrated that neither TNF-α nor IL-1 alone are essential for cutaneous pathology induced by barrier perturbation. In these studies it is suggested that cytokines do not mediate any of the responses induced by barrier disruption and their modulation would not be likely to result in significant alteration in skin barrier response.38 Therefore, it is most likely, that the effect of cipamfylline in irritant skin reaction is limited to the anti-inflammatory action. In summary, this present study supports our view that effective topical treatment of skin irritation is arduous. Our data shows that betamethasone-17-valerate may be modulating the response in ICD. However, we could not confirm any action of cipamfylline on SDS induced irritation.

Acknowledgment We would like to thank Leo Pharma BV for providing the cipamfylline ointment and cipamfylline ointment vehicle (placebo).

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References

1. Malten,K.E., Thoughts on irritant contact dermatitis. Contact Dermatitis 1981. 7: 238- 247. 2. Allen,M.H., Wakelin,S.H., Holloway,D., Lisby,S., Baadsgaard,O., Barker,J.N., and McFadden,J.P., Association of TNFA gene polymorphism at position -308 with suscep- tibility to irritant contact dermatitis. Immunogenetics 2000. 51: 201-205. 3. Doherty,A.M., Phosphodiesterase 4 inhibitors as novel anti-inflammatory agents. Curr.Opin.Chem.Biol. 1999. 3: 466-473. 4. Hanifin,J.M., Chan,S.C., Cheng,J.B., Tofte,S.J., Henderson,W.R., Jr., Kirby,D.S., and Weiner,E.S., Type 4 phosphodiesterase inhibitors have clinical and in vitro anti- in- flammatory effects in atopic dermatitis.J Invest Dermatol 1996. 107: 51-56. 5. Ferrer,L., Alberola,J., Queralt,M., Brazis,P., Rabanal,R., Llenas,J., and Puigdemont,A., Clinical anti-inflammatory efficacy of arofylline, a new selective phos- phodiesterase-4 inhibitor, in dogs with atopic dermatitis. Vet.Rec. 1999. 145: 191-194. 6. Griswold,D.E., Webb,E.F., Badger,A.M., Gorycki,P.D., Levandoski,P.A., Barnette,M.A., Grous,M., Christensen,S., and Torphy,T.J., SB 207499 (Ariflo), a second generation phosphodiesterase 4 inhibitor, reduces tumor necrosis factor alpha and interleukin-4 production in vivo. J Pharmacol Exp Ther 1998. 287: 705-711. 7. Cohan,V.L., Showell,H.J., Fisher,D.A., Pazoles,C.J., Watson,J.W., Turner,C.R., and Cheng,J.B., In vitro pharmacology of the novel phosphodiesterase type 4 inhibitor, CP- 80633. J Pharmacol Exp Ther 1996. 278: 1356-1361. 8. Ehinger,A.M., Gorr,G., Hoppmann,J., Telser,E., Ehinger,B., and Kietzmann,M., Effects of the phosphodiesterase 4 inhibitor RPR 73401 in a model of immunological inflammation.Eur J Pharmacol 2000. 392: 93-99. 9. Nickoloff,B.J. and Naidu,Y., Perturbation of epidermal barrier function correlates with initiation of cytokine cascade in human skin. J Am Acad Dermatol 1994. 30: 535-546. 10. Wood,L.C., Jackson,S.M., Elias,P.M., Grunfeld,C., and Feingold,K.R., Cutane- ous barrier perturbation stimulates cytokine production in the epidermis of mice. J.Clin.Invest 1992. 90: 482-487. 11. Hoefakker,S., Caubo,M., ‘t Erve,E.H., Roggeveen,M.J., Boersma,W.J., van Joost,T., Notten,W.R., and Claassen,E., In vivo cytokine profiles in allergic and irritant contact dermatitis. Contact Dermatitis 1995. 33: 258-266. 12. Thestrup,P.K. and Halkier,S.L., Mechanism of irritant contact dermatitis. In van der Valk,P.G. and Maibach,H.I. (Eds.) The irritant contact dermatitis syndrome. CRC press, Inc, 1996, pp 305-309. 13. Lisby,S., Muller,K.M., Jongeneel,C.V., Saurat,J.H., and Hauser,C., Nickel and skin irritants up-regulate tumor necrosis factor-alpha mRNA in keratinocytes by different but potentially synergistic mechanisms. Int.Immunol. 1995. 7: 343-352. 14. Wood,L.C., Stalder,A.K., Liou,A., Campbell,I.L., Grunfeld,C., Elias,P.M., and Feingold,K.R., Barrier disruption increases gene expression of cytokines and the 55 kD TNF receptor in murine skin. Exp Dermatol 1997. 6: 98-104. 15. Piguet,P.F., Grau,G.E., Hauser,C., and Vassalli,P., Tumor necrosis factor is a critical mediator in hapten induced irritant and contact hypersensitivity reactions. J Exp Med. 1991. 173: 673-679.

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16. Proksch,E., Holleran,W.M., Menon,G.K., Elias,P.M., and Feingold,K.R., Barrier function regulates epidermal lipid and DNA synthesis. Br.J Dermatol. 1993. 128: 473- 482. 17. Le,M., Schalkwijk,J., Siegenthaler,G., van de Kerkhof,P.C., Veerkamp,J.H., and van der Valk,P.G., Changes in keratinocyte differentiation following mild irritation by sodium dodecyl sulphate. Arch.Dermatol.Res. 1996. 288: 684-690. 18. Castelijns,F.A., Gerritsen,M.J., van Erp,P.E., and van de Kerkhof,P.C., Immunohis- tochemical assessment of keratin 16 on paraffin- embedded sections of normal and hy- perproliferative skin: monoclonal antibodies Ks8.12 and LL025 in a comparative study. Arch.Dermatol.Res. 1999. 291: 59-63. 19. de Mare,S., van Erp,P.E., and van de Kerkhof,P.C., Epidermal hyperproliferation assessed by the monoclonal Ks8.12 on frozen sections. J.Invest.Dermatol. 1989. 92: 130-131. 20. Le,T.K., De Mon,P., Schalkwijk,J., and van der Valk,P.G., Effect of a topical corticos- teroid, a retinoid and a vitamin D3 derivative on sodium dodecyl sulphate induced skin irritation. Contact Dermatitis 1997. 37: 19-26. 21. Pinnagoda,J., Tupker,R.A., Agner,T., and Serup,J., Guidelines for transepidermal wa- ter loss (TEWL) measurement. A report from the Standardization Group of the European Society of Contact Dermatitis. Contact Dermatitis 1990. 22: 164-178. 22. van Duijnhoven,J.L., Schalkwijk,J., Kranenborg,M.H., van Vlijmen Willems,I.M., Groeneveld,A., van Erp,P.E., Timmer,E.D., de Jongh,G.J., and van de Ven,W.J., MON-150, a versatile monoclonal antibody against involucrin: characterization and ap- plications. Arch.Dermatol.Res. 1992. 284: 167-172. 23. Siegenthaler,G., Hotz,R., Chatellard-Gruaz,D., Didierjean,L., Hellman,U., and Saurat,J.H., Purification and characterization of the human epidermal fatty acid-binding protein: localization during epidermal cell differentiation in vivo and in vitro. Biochem.J 1994. 302: -71. 24. Levin,C., Zhai,H., Bashir,S., Chew,A.L., Anigbogu,A., Stern,R., and Maibach,H., Efficacy of corticosteroids in acute experimental irritant contact dermatitis? Skin Res.Technol. 2001. 7: 214-218. 25. van der Valk,P.G.M. and Maibach,H.I., Do topical corticosteroids modulate skin irrita- tion in human beings? Assessment by transepidermal water loss and visual scoring. J Am Acad Dermatol 1989. 21: 519-522. 26. Berardesca,E., Acute irritant dermatitis: effect on short-term topical corticoid treatment. In Surber C, Elsner P, and Bircher A (Eds.) Exogenous dermatology. Karger, Basel 1995, pp 86-90. 27. Ramsing,D.W. and Agner,T., Efficacy of topical corticosteroids on irritant skin reac- tions. Contact Dermatitis 1995. 32: 293-297. 28. Levin,C. and Maibach,H.I., Corticosteroids of clinical value in acute and cumulative experimental irritant dermatitis in humans? In Maibach,H.I., Bashir,S., and McKibbon (Eds.) Evidence - based dermatology. 2002, pp 143-148. 29. Schmuth,M., Wimmer,M.A., Hofer,S., Sztankay,A., Weinlich,G., Linder,D.M., Elias,P.M., Fritsch,P.O., and Fritsch,E., Topical corticosteroid therapy for acute radia- tion dermatitis: a prospective, randomized, double-blind study. Br J Dermatol 2002. 146: 983-991.

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30. Swinkels,O.Q., Kucharekova,M., Prins,M., Gerritsen,M.J., van der Valk,P.G.M., and van de Kerkhof,P.C.M., The effects of topical corticosteroids and a coal tar prepa- ration on dithranol induced irritation in patients with psoriasis. Skin Pharmacol Appl.Skin Physiol 2003. 16: 12-17. 31. Levin,C. and Maibach,H.I., An overview of the efficacy of topical corticosteroids in experimental human nickel contact dermatitis. Contact Dermatitis 2000. 43: 317-321. 32. van der Valk,P.G.M., Kruis-de Vries,M.H., Nater,J.P., Bleumink,E., and de Jong,M.C., Eczematous (irritant and allergic) reactions of the skin and barrier function as determined by water vapour loss. Clin Exp Dermatol 1985. 10: 185-193. 33. Hanifin,J.M., Atopic dermatitis in infants and children. Pediatr.Clin North Am 1991. 38: 763-789. 34. Ruzicka,T., Assmann,T., and Homey,B., Tacrolimus: the drug for the turn of the mil- lennium? Arch.Dermatol. 1999. 135: 574-580. 35. Hanifin,J.M., Ling,M.R., Langley,R., Breneman,D., and Rafal,E., Tacrolimus ointment for the treatment of atopic dermatitis in adult patients: part I, efficacy. J.Am.Acad.Dermatol 2001. 44: S28-S38. 36. Fuchs,M., Schliemann-Willers,S., Heinemann,C., and Elsner,P., Tacrolimus en- hances irritation in a 5-day human irritancy in vivo model. Contact Dermatitis 2002. 46: 290-294. 37. Newby,C.S., Barr,R.M., Greaves,M.W., and Mallet,A.I., Cytokine release and cyto- toxicity in human keratinocytes and fibroblasts induced by phenols and sodium dodecyl sulfate. J Invest Dermatol 2000. 115: 292-298. 38. Man,M.Q., Wood,L., Elias,P.M., and Feingold,K.R., Cutaneous barrier repair and pathophysiology following barrier disruption in IL-1 and TNF type I receptor deficient mice. Exp Dermatol 1999. 8: 261-266.

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4.2 EFFECT OF A LIPID-RICH EMOLLIENT CONTAINING CERAMIDE 3 IN EXPERIMENTALLY INDUCED SKIN BARRIER DYSFUNCTION

Abstract

In the present study we compared the effect of a ceramide 3-containing emollient (Locobase® Repair) with a control emollient (vaselinum album/cremor lanette ana) and untreated damaged skin using clinical, bioengineering and immunohistochemical methods in two different models of experimentally induced skin barrier dysfunction. In model A (n=13) skin barrier dysfunction was inflicted at three investigation sites by tape stripping. In model B (n=13), the volunteers were patch tested at three investigation sites with sodium dodecyl sulfate (0.2%) for 4 h a day for 4 consecutive days. The investigation sites were treated once a day with the above-mentioned agents. Daily, irritant reaction was assessed by erythema scoring and measurements of transepidermal water loss (TEWL). After 5 days punch biopsies were taken from all sites. Immunohistochemical assessment was carried out with respect to epidermal proliferation, epidermal differentiation and Langerhans cells. Tape stripping resulted in an erythematous reaction and an increase of TEWL associated with upregulation of cycling cells, involucrin and expression of cytokeratin 16. Ceramide 3 - containing emollient significantly decreased at day 4 the erythema score compared to the untreated site. TEWL and cycling cells are significantly decreased in comparison with the untreated site and the site treated with control emollient. Repetitive exposure to SDS induced a variable degree of erythema, gradual increase of TEWL, an increase of cycling cells, and upregulation of involucrin, cytokeratin 16, E-FABP and SKALP/elafin. The treatment with the control emollient significantly prevented erythema, increase of TEWL and cycling cells at day 4 compared to the untreated site. In summary, the present study demonstrated that, both tested emollients improve skin barrier in different conditions compared to the untreated skin and that there is some indication that formulations containing skin- related lipids might be of benefit in barrier disruption following tape stripping. Different models and clinical trials are needed to establish in specific conditions the usefulness of emollients containing skin-related lipids.

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Introduction

Emollients have been used in the treatment of dry, scaly skin conditions for over 2000 years. Although we are starting to understand how emollients work, only a few trial data have been published demonstrating their efficacy.1-4 Theoretically, emollients may act on three different levels of the epidermis: 1. an immediate effect by adding a lipid mixture to the skin surface (occlusion) 2. an intermediate effect by adding a lipid mixture to the intercellular spaces 3. a delayed effect by providing lipids to the epidermal cells, enabling these to enhance the natural lipid production and lipid release. 5

Most emollients act on the first two levels listed. An ideal emollient would be one that possesses all three of these qualities. A promising approach in this respect may be the incorporation of lipids chemically related to the physiological content of the intercellular domain of the horny layer in a topical formulation. Recently, findings on the composition of intercellular lipids and the effects of well-defined preparation of these lipids on the skin barrier have made this approach feasible. Firstly, it was shown that the topical application of an equimolar three- component system of ceramides, cholesterol and fatty acid allows normal barrier repair in acetone-treated murine skin as compared to delayed barrier repair by lipids alone or by other combinations. In these animal studies it has also been demonstrated by fluorescence and electron microscopy that the mixture of cholesterol, fatty acid and ceramides rapidly traversed the SC with subsequent uptake into the epidermal nucleated layers.6;7 Secondly, it was shown that acceleration of barrier repair can be achieved if the relative contribution of any of these ingredients is increased.8 Finally, it has been suggested that cholesterol, ceramides, palmitate and linoleate in the composition of 4.3 : 2.3 : 1 : 1.08 is the most optimal ratio for barrier recovery. This lipid formulation causes a markedly accelerated skin barrier recovery following barrier disruption in mice.9 To correlate animal data with human skin, different lipid formulations containing varying molar ratios of these lipids were tested in limited numbers of patients. In some combinations a noticeable improvement in skin suppleness was observed.10 These experimental studies showed evidence that emollients containing certain combinations of key physiologic lipids may have therapeutic benefits in conditions involving skin barrier dysfunction.11 However, studies that document the efficacy of such lipid compositions in barrier disturbance in humans either clinically or in models are scarce. The limited number of available human studies, using bioengineering methods to assess barrier function, is contradicting.10;12-17 In the present study we investigated the efficacy of a ceramide 3-containing emollient (Locobase® Repair). It is claimed to be a formulation which contains an optimal composition of skin- related lipids in barrier recovery. We studied this claim by comparing Locobase®

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Repair with vaselinum album/cremor lanette I ana (control emollient) and untreated damaged skin in barrier recovery capacity. We investigated skin barrier function by measuring transepidermal water loss. We also performed clinical erythema scoring and (immuno)histological studies of experimentally induced skin barrier dysfunction. The skin barrier dysfunction was inflicted by tape stripping or exposure of the skin to a low concentration of sodium dodecyl sulfate (SDS) in a cumulative exposure model. Beneficial effects of both emollients using clinical, bioengineering and immunohistochemical methods were observed depending on the type of injury.

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Materials and methods

Subjects A total of 26 healthy volunteers without past or present history of skin diseases (11 male, 15 female, mean age 38, ranging from 20 to 61) were included in the study. Written informed consent was obtained from all participants and the study was approved by the local Ethics committee. The study was conducted in January 2000.

Methods Experimentally induced barrier dysfunction was studied in two different models: A. single tape stripping B. repetitive exposure to low concentration of sodium dodecyl sulfate (SDS)

Model A: Mechanical irritation by single tape stripping: On baseline (day 0) a plastic template with three windows (2,5 cm diameter) was applied to three sites of the lower back of 13 participants. The exposed areas were at least 2.5 cm apart. Adhesive tape (Tesafilm ) was applied with gentle pressure to the exposed areas (site 1, 2, 3) and then removed. This procedure was repeated until the skin was glistening. Model B: 13 participants were patch tested with 0.2% SDS once daily for 4 hours on each of 4 consecutive days from Monday to Thursday (days 0-3). 150 µl of SDS was applied to our self-made patches consisted of a 1.5 cm2 piece of absorbent, non-woven fabric fitted into a flat round silver chamber with an internal diameter of 30 mm. The chambers were applied on the three sites of the lower back (min. distance 2,5 cm) and hold in place with tape (Fixomull stretch, Beiersdorf AG, Hamburg). After the removal of all SDS chambers, the skin was washed with cold running water and gently patted dry with a paper towel.

Topical application of the emollients The exposed areas were treated once daily for 7 days in model A and for 5 days in model B according to the following scheme: Site 1 control emollient (vaselinum album/cremor lanette I ana, Dutch pharmacopoeia) Site 2 untreated Site 3 ceramide 3-containing emollient (Locobase® Repair, Yamanouchi Pharma B.V.)

Evaluation Clinical assessment and Transepidermal Water Loss (TEWL) measurements Erythema was evaluated by the investigator daily according to the scoring system: 0-no response; 1-slight, patchy redness; 2-diffuse mild redness, 3-moderate redness, 4-intense redness; 5-intense redness with oedema. TEWL were measured by TEWAMETER TM 210 (Robinson electronic Ltd.)

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according to the guidelines from the Standardisation Group of the European Society of Contact Dermatitis.18 Measurements were performed following a waiting period of 30 min. Measurements were taken more than 12 h after application of the emollients. Room temperature varied from 19 °C to 22 °C and relative humidity from 40% to 50%. TEWL measurements were performed: Model A: at day 0 - before (baseline TEWL) and at various moments after tape stripping: post-exposure (dag 0), and 24 hours (day 1), 48 hours (day 2), 96 hours (day 4), 168 hours (day 7) after barrier disruption. Model B: at day 0 - before SDS exposure (baseline TEWL), after 24 hours (day 1), 48 hours (day 2), 96 hours (day 4). The measurements at 24 h, 48 h and 96 hours were performed before the following exposure of SDS.

Biopsy procedures Model A: 24 punch biopsies (3mm) were taken under local anaesthesia at 48 (day 2) and 96 hours (day 4) after tape stripping from all investigated sites of eight participants. The biopsy specimens were embedded in Tissue TEK II OTC compound, immediately snap-frozen in liquid nitrogen and stored at -80 C until further use. Sections of 6 μm were cut and fixed during 10 minutes with acetone-ether (60/ 40vol.%) for the MIB-1 staining and for the other staining with cold acetone only. Model B: 24 punch biopsies (3mm) were taken under local anaesthesia at 96 hours (day 4) after repetitive application of SDS from all investigated sites of eight participants. After 24 h fixation in formalin, the samples were embedded in paraffin, sectioned at 6 μm and prepared for staining.

Histological methods Biopsy sections were evaluated light microscopically using haematoxylin- eosin staining and immunohistochemical stainings. For the immunohistochemical stainings a panel of monoclonal and polyclonal antibodies dependent on model was used. Model A: To assess epidermal proliferation, an antibody directed against the Ki-67 antigen was used (MIB-1, 1:50, Immunotech, SA, Marseilles, France). To assess epidermal differentiation, monoclonal antibodies against involucrin (MON- 150, 1:15, Dept. Clin. Chem., St. Elisabeth Hospital, Tilburg, The Netherlands) and monoclonal antibody against cytokeratin 16 (LL025, 1:10, Novocastra Laboratories Ltd, Newcastle upon Tyne, UK) were used. Using monoclonal antibodies DAKO-T6 (1:100, DAKO A/S, DENMARK), made analysis of the presence of Langerhans cells. For all monoclonal antibodies, an indirect immunoperoxidase technique was used. Model B: All markers except the marker for Langerhans cells have been used. Besides that, we used two other differentiation markers. A polyclonal antibody against skin-derived-antileukoproteinase (anti-SKALP, 1:500) and a polyclonal antibody against epidermal-fatty-acid-binding-protein (anti-E-FABP, 1:100, ) have been used.

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For all antibodies, except for MIB-1 and anti-E-FABP, an indirect immunoperoxidae technique was used. An avidin-biotin immunoperoxidase technique was carried out for MIB-1 and anti-E-FABP.

Statistical analysis The differences were analysed by the Wilcoxon test for erythema score and immunohistochemical scoring. P<0.05 was chosen as level of significance.

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Results

Clinical response and TEWL measurements Tape stripping of human skin resulted in an erythematous reaction. After tape stripping TEWL strongly increased and then gradually decreased to day 7. The time course of the visual grading of erythema and TEWL measurements is shown in Figure 1a and Figure 1b, respectively. The erythema score was significantly decreased at day 4 on the site treated with ceramide 3 - containing emollient compared to the untreated site, but not significantly compared to the side treated with control emollient. TEWL value was significantly decreased at day 4 on the site treated with ceramide 3- containing emollient compared to the control emollient and untreated site. Repetitive exposure of human skin to SDS induced a variable degree of erythema. The erythematous response increases in intensity up to day 4 in 9 participants (Figure 2a). No strong erythematous reactions were observed. Four participants did not have any erythematous reaction at all. However, a gradual increase of TEWL values with maximum at day 4 was observed in all participants (Figure 2b). Treatment with both the ceramide 3-containing emollient and the control emollient prevented erythema and the increase of TEWL values. The treatment with the control emollient significantly prevented erythema and increase of TEWL value at day 4 compared to untreated site.

Immunohistochemistry

Epidermal proliferation We observed an increase of the number of cycling cells at 48 and 96 hours after tape stripping at all sites (Figure 3a). At the site treated with ceramide 3 - containing emollient the number of cycling cells is significantly reduced in comparison to the untreated site and to the site treated with control emollient. Repetitive exposure to SDS induced an increased number of cycling cells after 4 days at all investigated sites (Figure 3b). The site treated with control emollient showed a significant lower number of cycling cells at day 4 in comparison with the untreated site.

Epidermal differentiation In the present study, tape stripping and SDS application induced clear expression of cytokeratin 16. However, no significant effect of any of the treatment was noticed. Expression of involucrin was slightly upregulated in both models. However, treatment with neither of the emollients significantly altered involucrin expression (data not shown). With respect to E-FABP and SKALP, we observed upregulation of E-FABP and expression of SKALP exclusively in patients with slight erythema after SDS application. We did not see any effects of the treatment on these markers.

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4 Figure 1a Erythema score in tape 3.5 stripping model. 3

2.5 control emollient 2 untreated * Significant difference between the 1.5 * Locobase Repair site treated with Locobase® Repair

Erythema score 1 and the untreated site. (p<0.03) 0.5 0 day1 day2 day4 day7

90 80 control emollient Figure 1b TEWL measurements 2 70 untreated (g/m /h) in tape stripping model 60 Locobase Repair 50 40 * Significant difference between the TEWL 30 site treated with Locobase® Repair 20 * 10 vs. the untreated site 0 (p<0.03) and Locobase® Repair vs. . p. xp ay1 ay2 ay4 ay7 control emollient (p<0.03) ex d d d d ree ost p p

1,8 Figure 2a Erythema score in model of repetitive application of SDS 1,6 (0.2%) 1,4 1,2 * * Significant difference between the 1 control emollient site treated with control emollient untreated 0,8 Locobase Repair and the untreated site (p<0.03)

erythema score erythema 0,6

0,4

0,2

0 preexpos. day1 day2 day3 day4

45 Figure 2b TEWL measurements in

40 model of repetitive application of

35 * SDS (0.2%)

30

25 control emollient * Significant difference between the untreated site treated with control emollient 20 Locobase Repair

TEWL (g/m/h) TEWL and the untreated site (p<0.05) 15

10

5

0 preexpos. day1 day2 day3 day4

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Langerhans cells (LCs) We investigated the expression of Langerhans cells 48 and 96 hours after tape stripping. We observed at all sites an increase of epidermal LCs after epidermal injury. No significant differences were found in the exposed treated and untreated skin. Table s 1, 2 and 3 summarise the responses using various clinical, physiological and immunohistochemical parameters.

Figure 3a Cycling cells (Ki-67) 100 48 hours and 96 hours after tape 80 stripping 60 control emollient untreated 40 Locobase Repair We found a significant effect of Locobase® Repair compared to Cycling cells/mm 20 control emollient (p<0.03) 0 48 hours 96 hours and the untreated site (p<0.01). The time in hours sampling time was not significant.

Figure 3b Cycling cells (Ki-67) 96 50 45 hours after repetitive application of 40 SDS (0.2%). 35 30 * 25 * Significant difference between the 20 site treated with control emollient 15 Cycling cells/mm 10 and the untreated site 5 (p< 0.03). 0 control emollient untreated Locobase Repair

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Table 1 Interference of Locobase® Repair and control emollient with tape stripping induced changes Parameters Locobase® Control P1- value P2-value Repair emollient (Vaselinum/ cr. Lanette) Erythema ↓↓ ↓ 0.018 TEWL ↓↓ ↓ 0.012 0.020 Ki 67 ↓↓ ↓ 0.018 0.003 Involucrin ↔ ↔ Cytokeratin 16 ↔ ↔ Langerhans cells ↔ ↔ ↓↑ = tendency of an inhibition or stimulation, ↓↓↑↑ = statistically significant inhibition or stimulation, ↔ = no effect P1 is the p value for difference between the site treated with Locobase® Repair and the untreated site P2 is the p value for difference between the site treated with Locobase® Repair and control emollient P values only mentioned if significant

Table 2 Interference of Locobase® Repair and control emollient with SDS induced changes

Parameters Locobase®Repair Control emollient P- (Vaselinum/cr. Lanette) value Erythema ↓ ↓↓ 0.027 TEWL ↓ ↓↓ 0.032 Ki 67 ↓ ↓↓ 0.013 Involucrin ↔ ↔ Cytokeratin 16 ↔ ↔ SKALP/elafin ↔ ↔ E-FABP ↔ ↔ ↓↑ = tendency of an inhibition or stimulation, ↓↓↑↑ = statistically significant inhibition or stimulation, ↔ = no effect P is the p-value for difference between the site treated with control emollient and the untreated site. P-values only mentioned if significant

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Table 3 Characteristic clinical, TEWL and immunohistochemical changes following repetitive application of SDS and tape stripping in our study Parameters Repetitive application of Tape stripping SDS (0,2%) Erythema gradual increase ↑↑ TEWL gradual increase ↑↑ Ki 67 ↑ ↑↑ Cytokeratin 16 ↑ ↑↑ Involucrin ↑ ↑ SKALP/elafin ↑ ND E-FABP ↑ ND Langerhans cells ND ↑ in epidermis ND= not done

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Discussion

In both models, following a single course of tape stripping and repeated applications of SDS, skin barrier repair was accelerated by the two lipid formulations (Locobase® Repair and vaselinum album/cremor lanette I ana). In the acute model of barrier disruption (tape stripping) Locobase® Repair accelerated repair of barrier damage significantly. In the cumulative SDS-model, vaselinum album/cremor lanette I ana recovered significantly the repair of damaged skin barrier. In the tape stripping model we observed a significant suppression of proliferating cells at the site treated with Locobase® Repair, whereas in the SDS models we observed a significant suppression of proliferating cells on the site treated with the emollient-control. The decrease of proliferating cells correlated with suppression of erythema score and TEWL. We did not observe any modulation of cytokeratin 16, involucrin, E-FABP, SKALP and Langerhans cells after the application of both emollients. The results are in line with the results of an animal study using a comparable lipid mixture. In the animal experiment, the tested lipid mixture accelerated barrier recovery after tape stripping but not after SDS related damage.9 Several different mechanisms can explain the discrepancy of results between the models. Firstly, horny layer damage after detergent application differs from tape stripping induced injury. Tape stripping disrupts barrier function by removing the SC mechanically, detergents disrupt barrier function by removing and rearranging SC lipids. It is suggested that the quantity and spectrum of lipids removed from the SC, is detergent - specific. It is possible that detergents such SDS, may cause damage to the nucleated layers of the epidermis, which cannot be corrected by providing lipids alone. The difference in efficacy of tested emollients between the tested models supports the hypothesis that the ability of the lipid mixture to accelerate barrier repair depends on the specific damage, requiring damage-specific correction.9 Secondly, the limited efficacy of Locobase® Repair in SDS model compared to control emollient could be matter of ratio of the lipid components. It is known that not all types of topical formulations, which contain lipids chemically related to the physiological content of the intercellular domain of the SC, are effective. Out of a number of preparations, those with a lipid ratio most comparable to that present in the human skin barrier, is considered to be effective.16 It is unknown whether lipid ratio of Locobase® Repair, which consists of cholesterol, semisynthetic ceramide 3, oleic acid and palmitic acid, mimics the lipid ratio in the human skin. Furthemore, ceramide 3 is the exclusive ceramide that is incorporated in Locobase® Repair. Ceramides represent a unique, heterogeneus group of at least six ceramides that differ from each other by the head group architecture and by the mean fatty acid chain length. Other ceramides in the SC are probably needed to provide the SC with a water-resistant lipid multilayer. It is assumed that especially ceramide 1 is of major importance for proper SC barrier function.19 Because of its unusual structure, which contains linoleic acid linked to the long chain (C>30) omega hydroxy fatty acid, ceramide 1 is of great

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importance for the formation of the lateral lipid packing as well as the long range lamellar ordering in SC.19;20 Studies of lipid composition of atopic skin showed a lowered level a ceramides, especially ceramide 1, suggesting its important role in barrier function.21-23 Concerning SDS-induced irritation, only a few references can be found with respect to the correlation between SC abnormalities and SDS irritation.24;25 Nevertheless, the data imply that ceramide 1 is an important ceramide, and it is unknown whether emollients with ceramide 3 could sufficiently replace the role of ceramide 1 and other natural occuring ceramides. In human studies the beneficial effect of emollients on the barrier function in SDS-induced skin irritation and tape stripping using clinical and bioengineering methods has been established.15-17;26 In one of these studies the effect of a ceramide 3 -containing emulsion was investigated in which no additional positive effect was observed on barrier repair in SDS induced skin irritation. However, the authors did found positive effects on TEWL when damaged skin is treated with o/w emulsions enriched with mixtures of ceramides, cholesterol and fatty acids.16 In two other studies, a lipid mixture containing ceramide 3, cholesterol and fatty acids (Locobase® Repair) was compared with different emollients which contain non-physiological lipids on SDS induced skin irritation. In these studies, Locobase® Repair was of no advantage compared to non-physiological lipids containing emollient with respect to acceleration of barrier recovery after SDS application, which is in line of our results.15;17 To the best of our knowledge there is only one clinical study on the beneficial effect of emollients containing skin-related lipids on tape stripped human skin.17 In this study Locobase® Repair was not superior to petrolatum in contrast with our results. Probably the methods used to inflict barrier dysfunction were not similar. In our study the barrier dysfunction after tape stripping was more severe which may explain the lack of the superior effect of Locobase® Repair in their study. In addition to the clinical assessment and TEWL measurements, we investigated cell biological changes using markers for epidermal proliferation (MIB-l) epidermal differentiation (LL025, MON 150, anti-E-FABP, anti-SKALP) and Langerhans cells (T6). The disturbances in epidermal proliferation, epidermal differentiation and Langerhans cells can be associated with impaired skin barrier function in some skin diseases and experimentally induced skin barrier irritation. 27-33 Using these immunohistochemical markers which are supposed to be associated with skin barrier dysfunction, we wanted to compare the cell biological effects of emollient treatment on experimentally induced skin barrier dysfunction. In our models of skin barrier dysfunctions we observed differences in the dynamics of proliferating cells after treatment with emollients. With regards to the other markers, we observed downregulation after skin barrier dysfunction, but we did not see any significant differences in the expression of these markers after application of the investigated emollients. According to some authors the accelerated recovery of barrier disruption after emollients could be due to a semiocclusive effect of the emollient, which restricts

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water evaporation from the skin thereby allowing the skin to recover.(15) However, the role of occlusion has been questioned.34;35 We hypothesise that the limited efficacy of Locobase® Repair as compared to other more occlusive emollient may not be related to limited barrier recovery but rather to limited protection against repeated exposure to a detergent. The fatty components of the emollient - control may form a better physical barrier than the components of Locobase® Repair. These results support the hypothesis that the optimal barrier repair therapy should be based on understandings of the specific damage and recovery.17;36 We suggest that an ideal emollient does not exist and that a specific emollient will not be appropriate for all clinical purposes. In summary, the present study demonstrates that emollients improve skin barrier in different conditions compared to untreated skin and that there is some indication that formulations containing skin-related lipids might be of benefit in barrier disruption by tape stripping. To demonstrate a beneficial effect of emollients containing skin-related lipids, clinical studies are mandatory. An emollient protecting against all kinds of physical and chemical injuries is probably not realistic.

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References

1. Ramsing,D.W. and Agner,T., Preventive and therapeutic effects of a moisturizer. An experimental study of human skin. Acta Derm.Venereol. 1997. 77: 335-337. 2. Halkier,S.L. and Thestrup,P.K., The efficacy of a moisturizer (Locobase) among clean- ers and kitchen assistants during everyday exposure to water and detergents. Contact Dermatitis 1993. 29: 266-271. 3. Ghadially,R., Halkier,S.L., and Elias,P.M., Effects of petrolatum on stratum corneum structure and function. J.Am.Acad.Dermatol. 1992. 26: 387-396. 4. Loden,M., Barrier recovery and influence of irritant stimuli in skin treated with a mois- turizing cream. Contact Dermatitis 1997. 36: 256-260. 5. Halkier,S.L., Occupational skin diseases. Contact dermatitis, supplement No 1 1996. 35. 6. Man,M.Q., Feingold,K.R., and Elias,P.M., Exogenous lipids influence permeability barrier recovery in acetone-treated murine skin. Arch.Dermatol. 1993. 129: 728-738. 7. Mao,Q.M., Brown,B.E., Wu,P.S., Feingold,K.R., and Elias,P.M., Exogenous non- physiologic vs physiologic lipids. Divergent mechanisms for correction of permeability barrier dysfunction. Arch.Dermatol. 1995. 131: 809-816. 8. Man,M.M., Feingold,K.R., Thornfeldt,C.R., and Elias,P.M., Optimization of physi- ological lipid mixtures for barrier repair. J.Invest Dermatol. 1996. 106: 1096-1101. 9. Yang,L., Mao,Q.M., Taljebini,M., Elias,P.M., and Feingold,K.R., Topical stratum cor- neum lipids accelerate barrier repair after tape stripping, solvent treatment and some but not all types of detergent treatment. Br.J Dermatol. 1995. 133: 679-685. 10. Thornfeldt, C. R., Elias, P. M., and Feingold, K. R. Lipids for Epidermal Moisturiza- tion and Repair of Barrier Function. (5,693,899). 7-1-1997. US. 11. Imokawa G, Skin Moisturizers:Development and Clinical Use of Ceramides. In Loden,M. and Maibach,H.I. (Eds.) Dry skin and Moisturizes: Chemistry and Function. CDC Press LLC, 2000, pp 269-298. 12. Imokawa, Moisturizes used in dermatology fields.J.Clin.Dermatology 1998. 56: 87-96. 13. Imokawa,G. and Akasaki,S., Water-retaining function in the stratum corneum and its recovery properties by sythetic pseudo-ceramides. J.Soc.Cosmet.Chem. 1989. 40: 273- 285. 14. Imokawa G, Function of epidermal sphingolipids and their application. Fragrance J. 1990. 4: 26-34. 15. Held,E., Lund H, and Agner,T., Effect of different moisturizers on SLS-irritated human skin. Contact Dermatitis 2001. 44: 229-334. 16. De Paepe,K., Derde,M.P., Roseeuw,D., and Rogiers,V., Incorporation of ceramide 3B in dermatocosmetic emulsions: effect on the transepidermal water loss of sodium lauryl sulphate-damaged skin. J Eur.Acad.Dermatol.Venereol. 2000. 14: 272-279. 17. Loden,M. and Barany,E., Skin-identical lipids versus petrolatum in the treatment of tape-stripped and detergent-perturbed human skin. Acta Derm Venereol 2000. 80: 412- 415. 18. Pinnagoda,J., Tupker,R.A., Agner,T., and Serup,J., Guidelines for transepidermal wa- ter loss (TEWL) measurement. A report from the Standardization Group of the European Society of Contact Dermatitis. Contact Dermatitis 1990. 22: 164-178.

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19. Bouwstra,J.A., Gooris,G.S., Dubbelaar,F.E., Weerheim,A.M., Ijzerman,A.P., and Ponec,M., Role of ceramide 1 in the molecular organization of the stratum corneum lipids. J.Lipid Res. 1998. 39: 186-196. 20. Wertz,P.W. and Downing,D.T., Acylglucosylceramides of pig epidermis: structure de- termination. J.Lipid Res. 1983. 24: 753-758. 21. Yamamoto,A., Serizawa,S., Ito,M., and Sato,Y., stratum corneum lipid abnormalities in atopic dermatitis. Arch.Dermatol Res. 1991. 283: 219-223. 22. Melnik,B., Hollmann,J., Hofmann,U., Yuh,M.S., and Plewig,G., Lipid composition of outer stratum corneum and nails in atopic and control subjects. Arch.Dermatol Res. 1990. 282: 549-551. 23. di Nardo,A., Wertz,P., Giannetti,A., and Seidenari,S., Ceramide and cholesterol com- position of the skin of patients with atopic dermatitis. Acta Derm.Venereol. 1998. 78: 27-30. 24. Fulmer A.W and Kramer G.J, stratum corneum lipid abnormalities in surfactant-in- duced dry scaly skin. J.Invest Dermatol. 1986. 86: 598-602. 25. di Nardo,A., Sugino,K., Wertz,P., Ademola,J., and Maibach,H.I., Sodium lauryl sul- fate (SLS) induced irritant contact dermatitis: a correlation study between ceramides and in vivo parameters of irritation. Contact Dermatitis 1996. 35: 86-91. 26. De Paepe,K., Hachem, and Vanpee,E.e.al., Beneficial effects of a skin tolerance-tested moisturizing cream on the barrier function in experimentally-elicited irritant and allergic contact dermatitis. Contact Dermatitis 2001. 44: 337-343. 27. Le,T.K., Schalkwijk,J., van de Kerkhof,P.C., van Haelst,U., and van der Valk,P.G., A histological and immunohistochemical study on chronic irritant contact dermatitis. Am.J Contact Dermat. 1998. 9: 23-28. 28. Le,T.K., De Mon,P., Schalkwijk,J., and van der Valk,P.G., Effect of a topical corticos- teroid, a retinoid and a vitamin D3 derivative on sodium dodecyl sulphate induced skin irritation. Contact Dermatitis 1997. 37: 19-26. 29. Le,M., Schalkwijk,J., Siegenthaler,G., van de Kerkhof,P.C., Veerkamp,J.H., and van der Valk,P.G., Changes in keratinocyte differentiation following mild irritation by sodium dodecyl sulphate. Arch.Dermatol.Res. 1996. 288: 684-690. 30. Proksch,E., Brasch,J., and Sterry,W., Integrity of the permeability barrier regulates epidermal Langerhans cell density. Br.J Dermatol. 1996. 134: 630-638. 31. Castelijns,F.A., Gerritsen,M.J., van Erp,P.E., and van de Kerkhof,P.C., Immunohis- tochemical assessment of keratin 16 on paraffin- embedded sections of normal and hy- perproliferative skin: monoclonal antibodies Ks8.12 and LL025 in a comparative study. Arch.Dermatol.Res. 1999. 291: 59-63. 32. de Mare,S., van Erp,P.E., and van de Kerkhof,P.C., Epidermal hyperproliferation assessed by the monoclonal Ks8.12 on frozen sections. J.Invest.Dermatol. 1989. 92: 130-131. 33. Gerritsen,M.J., Rulo,H.F., Arnold W.P, and van de Kerkhof,P.C., Response of the clinically uninvolved skin of psoriatic patients to tape stripping during cyclosporin A treatment. Br.J Dermatol. 1994. 130: 181-188. 34. van de Kerkhof,P.C.M., de Mare,S., and Arnold W.P, Epidermal regeneration and oc- clusion. Acta Derm Venereol Stockh 1995. 75: 6-8. 35. Denda,M., Wood,L.C., Elias,P.M., and Feingold,K.R., The epidermal hyperplasia as- sociated with barrier disruption by acetone treatment or tape stripping cannot be attrib- uted to increased water loss. Arch.Dermatol Res. 1996. 288: 230-238.

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36. Elias,P.M. and Feingold,K.R., Does the tail wag the dog? Role of the barrier in the pathogenesis of inflammatory dermatoses and therapeutic implications. Arch.Dermatol 2001. 137: 1079-1081.

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4.3 A RANDOMIZED COMPARISON OF AN EMOLLIENT CONTAINING SKIN-RELATED LIPIDS WITH A PETROLATUM-BASED EMOLLIENT AS ADJUNCT IN THE TREATMENT OF CHRONIC HAND DERMATITIS

Abstract

Hand dermatitis is a multifactorial skin disorder in which skin barrier impairment is involved in the pathogenesis. The development of topical agents that improve skin barrier function is therefore a promising approach for the management of hand dermatitis. Topically applied lipids may interfere with skin barrier function, and emollients containing skin-related lipids have been suggested to facilitate repair of the skin barrier. However, evidence for the superiority of emollients containing skin-related lipids over the more traditional emollients is still lacking. The aim of this study was to compare an emollient containing skin-related lipids (Locobase® Repair) with a traditional petrolatum-based emollient for the management of hand dermatitis. Adult males and females (n=30) with mild to moderate chronic hand dermatitis were treated twice daily for 2 months either with an emollient containing skin-related lipids or with a petrolatum-based emollient. In the case of exacerbation, the patients of both treatment groups were allowed to use a mild corticosteroid according to instructions. Both treatment regimes significantly improved clinical symptoms of hand dermatitis as assessed by the investigator global assessment and hand eczema area and severity score. We did not observe significant differences in the improvement of clinical signs, itching, the patients’ assessment of efficacy, cosmetic acceptability or the usage of topical corticosteroids between both treatment groups. In conclusion, this study confirms that the frequent use of emollients may be useful in the therapy of hand dermatitis. However, we could not demonstrate the superiority of this particular emollient containing skin- related lipids in patients with chronic hand dermatitis.

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Introduction

Emollients may have various effects on the skin. They may form a covering film, which acts as a barrier for chemicals from the exterior and which restricts the loss of water and other essential substances from the interior.1 The product can be designated as a barrier cream if the primary goal is prevention of penetration.2;3 If the emollient restricts water loss, the stratum corneum is hydrated and the emollient can be designated as a moisturizer.4 If ingredients of the emollient penetrate the skin, these ingredients may interact with the lipids in the intercellular domain. Finally, ingredients of an emollient may enter corneocytes, eventually be metabolized and modify lipid secretory machinery of the SC.5;6 Recently, emollients containing skin-related lipids have been introduced. This new generation of emollients is believed to improve the skin barrier dysfunction by being incorporated into the multi-layered structure of the intercellular space or in cells of the SC. Chronic hand eczema is a common skin problem affecting approximately 2 to 10 % of all patients seen by dermatologists.7 It is a multifaceted disease in which both endogenous and environmental influences are often relevant.8 The impact of the function of the skin barrier in this disorder should be emphasized. A good skin barrier helps to avoid the penetration of both allergens and irritants. One of the putative goals in the treatment of this disease, therefore, may be improvement of skin barrier function by well-selected emollients. Animal studies have shown that topical mixtures of three key stratum corneum (SC) lipids, including ceramide, cholesterol and free fatty acid, when applied in optimized proportions accelerate barrier repair following a variety of external, acute or sustained perturbations.6;9;10 Human studies report the efficacy of different mixtures of these physiological lipids in experimentally induced skin barrier dysfunction 11-15 and in clinical conditions involving skin barrier disturbances.16-18 However, evidence for the superiority of emollients with a specific lipid profile over the more traditional emollients is still lacking. In a previous report we reported on the effects of an emollient containing skin- related lipids (Locobase® Repair) in experimentally induced skin barrier dysfunction. In that study, Locobase® Repair significantly accelerated repair of barrier damage in the acute model of barrier disruption (tape stripping) as assessed by clinical, bioengineering and immunohistochemical methods.14 This paper reports on a pilot study with Locobase® Repair in chronic hand dermatitis. We compared the effects of this emollient containing skin-related lipids with a petrolatum - based emollient (cremor vaselini lanette FNA). Here we studied clinical effects, cosmetic acceptability and the usage of topical corticosteroids.

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Materials and Methods

Patient selection 32 patients with bilateral chronic hand dermatitis entered the study after informed consent was obtained. The patients were recruited from a university hospital outpatient clinic between January 2002 and May 2002. Chronic hand dermatitis was defined as an eczematous eruption present for longer than 6 months. Patients meeting the following criteria were included: • mild to moderate disease (according to the investigator global assessment) • good response to class I or class II topical corticosteroids Exclusion criteria were: • clinically relevant allergic or irritant contact dermatitis and the inability to avoid exposure • severe and very severe hand dermatitis (according to the investigator global assessment) • severe vesiculation or bullae • history of contact urticaria and pustular diseases • recent therapy with potent topical corticosteroids (class III or IV) • recent systemic therapy or phototherapy

Methods This pilot study compared the efficacy of an emollient containing skin-related lipids (Locobase® Repair; Yamanouchi Pharma BV; contains ceramide 3, oleic and palmitic acid in a fatty cream base with petrolatum, aqua, paraffin, paraffinum liquidum, glycerin, sorbitan oleate, carnauba, cholesterol, carbomer, tromethamine in unknown ratio; lipid content 63%) and a traditional petrolatum-based emollient (Vas/Lan; Cremor vaselini lanette FNA, Bipharma, BV, The Netherlands; contains petrolatum, lanettewax, paraffinum liquidum, propylenglycol). On entry to the study all patients were interviewed regarding previous course and duration of the hand dermatitis, contact factors in their work and/or hobbies, atopy and current use of emollients and topical corticosteroids. We provided information on proper skin care related to personal circumstances. Patients were asked to discontinue the use of topical prescriptions and emollients for a wash-out period of 1 week. Patients were randomized into 2 groups: 17 participants were asked to use Locobase® Repair twice daily, 15 participants were asked to use petrolatum-based emollient twice daily. In the case of active dermatitis all patients were allowed to use mild corticosteroid (unguentum triamcinoloni 0.1 % FNA, Dutch pharmacopoeia) according to the following instructions. • In case of exacerbation of the dermatitis the patients were allowed to use the topical corticosteroid twice daily for a maximum of 1 week. After this week the use was tapered to once daily for a maximum of 4 days a week.

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• In the case of clearance the patient stopped the use of the topical corticosteroid and continued only with the emollient. • Patients were asked to record the use of the topical corticosteroid and emollients in a diary. Besides the study medication, no other topical treatment was allowed.

Clinical evaluation The following variables were used to monitor the efficacy of the treatment. The same study investigator blindly assessed the dermatitis at various time-points: at baseline, after 1 month and after 2 months. To this end we used the hand eczema area and severity score (HEAS) as described and used before 19: the hand was divided into 7 areas: the palm and the back of the hand and the 5 digits, each area having its own correction factor based on the relative size (palm and back of the hands 0.25 and the digits 0.1) Each area was evaluated for area on a scale of 0-4 (0=0%, 1=1-25%, 2= 26-50%, 3=51-75%, 4=76-100%). We scored each area for intensity of erythema, vesicles, papules, scaling, fissures, excoriations and hyperkeratosis on a 0-3 scale (0=none, 1=slight, 2=moderate, 3=severe). We calculated a score for each area by multiplying the % of the affected area with the correction factor and with the sum of the intensities of the symptoms. Finally, we calculated the sum score by adding up the scores of each area. Investigator global assessment (IGA): This assessment was made on a scale of 0-520: • Clear (0): no residual visible dermatitis • Almost clear (1): minimal erythema and/or scaling • Mild disease (2): clearly visible signs of dermatitis (erythema, scaling), with no hyperkeratosis, oedema, fissures or functional impact • Moderate disease (3): moderately severe signs of dermatitis, with a few fissures and moderate functional impairment. • Severe disease (4): marked signs of dermatitis, with oedema, fissures or functional impairment. • Very severe disease (5): marked signs of dermatitis with exdutation/crusting and very severe functional impairment.

Patients’ evaluation A questionnaire assessed the patient’s opinion on the course of the disease after treatment (1=worse, 2=no change, 3=minimal improvement, 4=moderate improvement, 5=marked improvement, 6=clearing or almost clearing). Patients assessed the cosmetic acceptability of the study-emollients and rated the products as ‘very poor’, ‘poor’, ‘acceptable ’, ‘good’, ‘excellent’ and compared this with past and current emollients.21 In this questionnaire, we also enquired about habits before and during the study in order to investigate the effect of the study protocol.

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The use of topical corticosteroids We asked the patient to specify when topical corticosteroids were used in a diary. The tubes of corticosteroids were collected for weighing to determinate the amount of medication used. At the end of the study patients were questioned regarding the use of corticosteroids.

Statistics We used the Mann-Whitney U test to test the differences in the HEAS and IGA between both treatment groups at baseline, at 1 month and at 2 months. For the differences in results between baseline and the end-of-the-study results for each treatment group, we used the Wilcoxon matched pairs test. For the patients’ assessment of efficacy, itching, cosmetic acceptability and the usage of corticosteroids we used the chi-square test of a contingency table.

Table 1 Demographics of randomized patients. Subjects baseline demographics and characteristics Patients (n) 32 Sex (n) 10 males, 22 females Age (years) 19-65, mean 39.15 Duration of dermatitis (years) 0.5 to 31, mean 10.10 Subject baseline disease characteristics Hand eczema area and severity score 3.9 Baseline IGA (0-5) 2.3 Atopy (n) 23 Atopic dermatitis 18 Bronchial asthma 7 Hay Fever 8

Hand surface involvement (n) Dorsum 1 Palms 2 Digits 4 Palms and digits 13 Dorsum and digits 9 Palms, dorsum and digits 3

Use of emollients 4 weeks before the study Twice or more per day 3 Daily 9 Sporadically 11 No use 9

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Results

Efficacy The profile of randomized patients (n=32) is shown in Table 1. 2 patients were lost to follow-up after the baseline visit due to unknown reasons. We used only their baseline characteristics. 2 patients from each group discontinued therapy during the study. One Locobase® Repair patient dropped out of the study at visit 1 due to deviation from the protocol. This patient was not able to use the emollient twice daily. The second dropout patient in the Locobase® Repair group and both dropout patients of the Vas/Lan group had to stop because of exacerbation of the hand dermatitis, which made it necessary to treat with a strong potency topical corticosteroid. The analysis at the end-of-treatment as assessed by the IGA and the HEAS involved 30 patients including the 4 dropout patients. We observed a statistically significant reduction in the IGA from baseline to end-of-treatment for both treatments (p=0.005 Locobase® Repair, p=0.007 Vas/Lan). We also observed a significant improvement in the HEAS in both groups (p=0.002 Locobase® Repair, p=0.02 Vas/Lan). We did not see any statistically significant difference between the group treated with Locobase® Repair and the group treated with Vas/Lan (Figures 1 and 2)

Patients’ assessment of efficacy Assessment by patient of the treatment response at the end of treatment is shown in Figure 3a. Approximately 2/3 of patients of both groups (67.7% Locobase® Repair, 69.2% Vas/Lan) indicated improvement or clearance of the hand eczema. There were no significant differences between the groups. The patients’ assessment of itching is shown in Figure 3b. At the end of treatment, itching improved or even disappeared in 75% of Locobase® Repair users and 69% of Vas/Lan users. There were no significant differences between the groups.

Cosmetic assessment All patients tolerated the emollients well. Evaluation of the patients opinion on cosmetic acceptability of the study-emollients showed that both groups rated the study-emollients as acceptable , good or excellent (1-5). Most patients (43.7%) using Locobase® Repair considered this product as good (score 4), while most patients (38.4%) using Vas/Lan considered it as acceptable (score 3). There was no significant difference in the patients’ assessment on the cosmetic acceptability between the groups (Figure 4). 12 % of Locobase® Repair users and 7.6% of Vas/Lan users rated the emollient as poor. 81 % considered Locobase® Repair as helpful in treating their skin condition, in comparison to 69% of patients using Vas/Lan. 53 % percent of both groups stated that the study-emollient was superior to their current emollient and most of them were likely to continue the use of the emollient (94% Locobase® Repair, 85% Vas/Lan). We found no significant differences between the groups.

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3 Figure 1 IGA - investigator global assessment. 0= clear 1= almost 2,5 clear 2= mild disease 3= moderate

2 disease 4= severe disease 5= very ** severe disease. * Significant decrease Locobase 1,5 * Vas/Lan of IGA in the group treated with IGA (0-5)

1 corticosteroid and Locobase Repair at visit 2 (end-of-treatment) 0,5 in comparison to baseline.(p=0.005)

0 ** Significant decrease of IGA in Baseline Visit 1 Visit 2 the group treated with corticosteroid and Vas/Lan at the visit 2 (end-of- treatment) in comparison to baseline. (p=0.007) No significant differences between the treated groups were found.

Figure 2 Hand eczema area and

5 severity score. For more details see

4 Methods. * Significant decrease Locobase Repair 3 ** Vas/Lan of IGA in the group treated with corticosteroid and Locobase 2 * Repair at visit 2 (end-of-treatment) 1 in comparison to baseline. (p=0.002) 0 Baseline Visit 1 Visit 2 ** Significant decrease of IGA in

Hand eczema area and severity score the group treated with corticosteroid and Vas/Lan at the visit 2 (end-of- treatment) in comparison to baseline (p=0.02). No significant differences between the treated groups.

100% Figure 3 Patients’ assessment of 80% (a) the response of hand eczema

60% (b) itching after 2 months of

40% treatment with Locobase Repair efficacy + corticosteroid or Vas/Lan + 20% corticosteroid. No significant Patient's assessment of 0% Locobase+steroid Vas/Lan+steroid differences were found. (a) worse no change minimal improvement moderate improvement marked improvement clearing or almost clearing

100%

80%

60%

40%

20%

0% Patients assessment of itching (b) Locobase+steroid Vas/Lan+steroid no itching before study worse no change improvement no itching

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The use of emollients and corticosteroids It appeared that 9 of the patients (28%) did not previously use emollients, whereas the remainder (72%) had used emollients with varying frequency. During the study most patients (56.2 % of Locobase® Repair group, 61.5 % of Vas/Lan group) used more of the emollient than in the period of 4 weeks before the study. As to the use of topical corticosteroids, the questionnaires showed that 62.2% of the Locobase® Repair group used less corticosteroids during the study than before the study, in comparison with 38.4% of Vas/Lan group. Almost half of the patients of the Vas/Lan group (46.1%) stated that they had had to use more corticosteroids during the study (Figure 5). An analysis of the diaries, in which patients recorded the use of corticosteroids, showed that the Locobase® Repair group used corticosteroids 1.78 days and the Vas/Lan group 1.72 days per week. On average, the amount of corticosteroids used during the study was 10.7 g for the Locobase® Repair group and 9.7 g for patients treated with Vas/Lan. These differences are not significant.

The effect of the protocol To elucidate the effect of the protocol of the study, we asked whether certain changes in daily life occurred during the study. The time the patients took over the care of their hands before the beginning of the study was for most patients (93%) 15 min per day. During the study 56.2% of the Locobase® Repair users spent more time on the care of their hands than before the study. The care-time of 37.5% of the patients remained unchanged. In the Vas/Lan group, 2/3 of the patients (69.2%) stated that the time did not change, while 37.5% of them spent more time than before the study. At the beginning of the study 88.9 % performed daily wet work activities, of whom 14 % spent even more then 2 h daily. The questionnaires showed that 22.3 % of all patients decreased wet work activities and also the frequency of washing of their hands during the study. 78 % of all patients did not change such habits at all.

Figure 4 Patients’ assessment 100% of cosmetic acceptability of 80% Locobase Repair and Vas/Lan. 60% No significant differences were

40% found.

acceptability 20%

0% Locobase Repair Vaseline/Lanette Patient's assessment of cosmetic

very poor poor acceptable good excellent

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Figure 5 Patients’ assessment of 100% the use of corticosteroids during 80% study compared to the period before

60% study. No significant differences were found. 40%

20%

The use of corticosteroids 0% Locobase Repair Vaselinum/Lanette

no change less more

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Discussion

In the present study, an emollient containing skin-related lipids (ceramide 3, cholesterol and free fatty acids) was compared with a traditional petrolatum- based emollient. Active lesions were treated with a mild topical corticosteroid. Both treatment regimens significantly improved clinical signs as assessed by the IGA score and the HEAS. However, there were no statistically significant differences in improvement between both study arms. Furthermore, we did not observe significant differences in the improvement of itching, the patient’s assessment of efficacy and cosmetic acceptability and the usage of topical corticosteroids between both treatment groups. Thus, the study shows that both emollients are effective in the therapy of hand dermatitis and both are cosmetically acceptable . The efficacy of traditional emollients containing petrolatum has already been confirmed in various skin conditions.22-26 Because of its hydrating properties, petrolatum has been considered as a standard emollient against which other moisturisers should be compared.27 In this study, the efficacy of Locobase® Repair was comparable with a petrolatum-containing emollient. Because of a lack of significant differences and a lack of statistical power due to small sample size, we cannot conclude whether Locobase® Repair is a more appropriate choice than the other emollient. The superiority of emollients containing skin-related lipids remains a point of discussion. Previous studies have shown that the ratio and the concentration of the skin-related lipids in the emollient are crucial to the rate of barrier recovery.28 It is known that not all types of topical formulations that contain skin related lipids are effective. Out of a number of preparations, those with a lipid ratio most comparable to that present in the human skin barrier are considered to be effective. 29 It is unknown whether the ratio and the concentration of lipid component of Locobase® Repair, which consists of cholesterol, semisynthetic ceramide 3, oleic acid and palmitic acid, is optimal for barrier repair in chronic hand dermatitis. Further, ceramide 3 is the only ceramide that is incorporated into Locobase® Repair. Research data indicate that ceramide 1 is also an important ceramide in skin and it is unknown whether ceramide 3 can sufficiently fill the need for ceramide 1 and other natural occurring ceramides.30 Furthermore, the effect of the application of skin-related lipids has varied depending on the method of barrier disruption. A promising effect of skin-related lipids was seen in experimental models of barrier dysfunction inflicted by tape stripping or an organic solvent. When the barrier was disrupted by treatment with detergents such as sodium dodecyl sulfate or ammonium lauryl sulfosuccinate, skin- related lipids did not accelerate barrier repair.9 This may indicate that the ability of the lipid mixture to accelerate barrier repair depends on the specific external damage, suggesting the need for damage-specific correction.31 In this study, we applied emollients in patients with chronic hand dermatitis, a skin condition characterized by a multifactor origin in

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which the skin barrier may be variably relevant. There is evidence that a barrier repair strategy should not only meet specific demands depending on the type of external damage, but should also be adjusted to endogenous deficiencies.17;32-34 In conclusion, this study confirms that, related to the methodological strategies applied, the frequent use of emollients may be useful in the therapy of hand dermatitis and that patients should be encouraged to use them. However the results of this pilot study do not support the concept of superiority of an emollient containing skin-related lipids in a heterogeneous group of patients with chronic hand dermatitis. Since the composition of emollients is important, it should be further investigated whether emollients with another composition of skin-related lipids have more efficacy in different clinical conditions.

Acknowledgements We thank M.E. Kooijmans-Otero for skilful technical assistance and all the patients who participated.

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References

1. Halkier,S.L., Occupational skin diseases. Contact dermatitis, supplement No 1 1996. 35. 2. Frosch,P.J. and Kurte,A., Efficacy of skin barrier creams (IV). The repetitive irritation test (RIT) with a set of 4 standard irritants. Contact Dermatitis 1994. 31: 161-168. 3. Draelos,Z.D., Hydrogel barrier/repair creams and contact dermatitis. Am J Contact Der- mat. 2000. 11: 222-225. 4. Loden,M. and Maibach, H. I., Dry skin and moisturizers. CRC Press, 2000. 5. Wertz,P.W. and Downing,D.T., Metabolism of topically applied fatty acid methyl esters in BALB/C mouse epidermis. J Dermatol.Sci 1990. 1: 33-37. 6. Mao,Q.M., Brown,B.E., Wu,P.S., Feingold,K.R., and Elias,P.M., Exogenous non- physiologic vs physiologic lipids. Divergent mechanisms for correction of permeability barrier dysfunction. Arch.Dermatol. 1995. 131: 809-816. 7. Menne,T. and Maibach, H. I., Hand eczema. CRC Press,Inc, Boca Raton 1994. 8. Diepgen,T.L. and Fartasch,M., General aspects of risk factors in hand eczema. In Menne,T. and Maibach,H.I. (Eds.) Hand eczema. CRC Press, Boca Raton 1994, pp 142-154. 9. Yang,L., Mao,Q.M., Taljebini,M., Elias,P.M., and Feingold,K.R., Topical stratum corneum lipids accelerate barrier repair after tape stripping, solvent treatment and some but not all types of detergent treatment. Br.J Dermatol. 1995. 133: 679-685. 10. Man,M.M., Feingold,K.R., Thornfeldt,C.R., and Elias,P.M., Optimization of physi- ological lipid mixtures for barrier repair. J.Invest Dermatol. 1996. 106: 1096-1101. 11. Imokawa,G. and Akasaki,S., Water-retaining function in the stratum corneum and its recovery properties by sythetic pseudo-ceramides. J.Soc.Cosmet.Chem. 1989. 40: 273- 285. 12. Loden,M. and Barany,E., Skin-identical lipids versus petrolatum in the treatment of tape-stripped and detergent-perturbed human skin. Acta Derm Venereol 2000. 80: 412- 415. 13. Lintner,K., Mondon,P., Girard,F., and Gibaud,C., The effect of a synthetic ceramide- 2 on transepidermal water loss after stripping or sodium lauryl sulfate treatment: an in vivo study. International Journal of Cosmetic Science 1997. 19: 15-25. 14. Kucharekova M, Schalkwijk,J., and van der Valk,P.G., Effects of a lipid-rich emol- lient containing ceramide 3 in an experimentally induced skin barrier dysfunction. Con- tact Dermatitis 2002. 46: 331-338. 15. De Paepe,K., Roseeuw,D., and Rogiers,V., Repair of acetone- and sodium lauryl sul- phate-damaged human skin barrier function using topically applied emulsions containing barrier lipids. J.Eur.Acad.Dermatol.Venereol. 2002. 16: 587-594. 16. Berardesca,E., Barbareschi,M., Veraldi,S., and Pimpinelli,N., Evaluation of efficacy of a skin lipid mixture in patients with irritant contact dermatitis, allergic contact derma- titis or atopic dermatitis: a multicenter study. Contact Dermatitis 2001. 45: 280-285. 17. Chamlin,S.L., Frieden,I.J., Fowler,A., Williams,M., Kao,J., Sheu,M., and Elias,P.M., Ceramide-dominant, barrier-repair lipids improve childhood atopic dermati- tis. Arch.Dermatol 2001. 137: 1110-1112. 18. Mortensen,J.T., Bjerring,P., and Cramers,M., Locobase Repair cream following CO laser skin resurfacing reduces interstitial fluid oozing. J Cosmetic @ Laser Ther 2001. 3: 155-158.

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19. Simons,J.R., Bohnen,I.J., and van der Valk,P.G., A left-right comparison of UVB phototherapy and topical photochemotherapy in bilateral chronic hand dermatitis after 6 weeks’ treatment. Clin.Exp.Dermatol 1997. 22: 7-10. 20. Hebert,A.A., Warken,K.A., and Cherill,R., Pimecrolimus cream 1%: a new devel- opment in nonsteroid topical treatment of inflammatory skin diseases. Semin.Cutan Med.Surg. 2001. 20: 260-267. 21. Griffiths,C.-E.M., Van Leent,E.-J.M., Gilbert,M., and Traulsen,J., Randomized com- parison of the type 4 phosphodiesterase inhibitor cipamfylline cream, cream vehicle and hydrocortisone 17-butyrate cream for the treatment of atopic dermatitis. Br.J.Dermatol. 2002. 147: 299-307. 22. Ghadially,R., Halkier,S.L., and Elias,P.M., Effects of petrolatum on stratum corneum structure and function. J.Am.Acad.Dermatol. 1992. 26: 387-396. 23. Halkier,S.L. and Thestrup,P.K., The efficacy of a moisturizer (Locobase) among clean- ers and kitchen assistants during everyday exposure to water and detergents. Contact Dermatitis 1993. 29: 266-271. 24. Wigger-Alberti,W. and Elsner,P., Petrolatum prevents irritation in a human cumulative exposure model in vivo. Dermatology 1997. 194: 247-250. 25. Ramsing,D.W. and Agner,T., Preventive and therapeutic effects of a moisturizer. An experimental study of human skin. Acta Derm.Venereol. 1997. 77: 335-337. 26. Odio,M.R., O’Connor,R.J., Sarbaugh,F., and Baldwin,S., Continuous topical ad- ministration of a petrolatum formulation by a novel disposable diaper. 2. Effect on skin condition. Dermatology 2000. 200: 238-243. 27. Flesch,P., Chemical basis of emollient function in horny layers. Proc Sci Sect Toilet Goods Assoc 1963. 40: 1-7. 28. Man,M.Q., Feingold,K.R., and Elias,P.M., Exogenous lipids influence permeability barrier recovery in acetone-treated murine skin. Arch.Dermatol. 1993. 129: 728-738. 29. Thornfeldt,C.R., Critical and Optimal Molar Ratios of Key Lipids. In Loden,M. and Maibach,H.I. (Eds.) Dry Skin and Moisturizes. CRC Press, Boca Raton 2000, pp 337- 346. 30. Bouwstra,J.A., Gooris,G.S., Dubbelaar,F.E., Weerheim,A.M., Ijzerman,A.P., and Ponec,M., Role of ceramide 1 in the molecular organization of the stratum corneum lipids. J.Lipid Res. 1998. 39: 186-196. 31. Elias,P.M. and Feingold,K.R., Does the tail wag the dog? Role of the barrier in the pathogenesis of inflammatory dermatoses and therapeutic implications. Arch.Dermatol 2001. 137: 1079-1081. 32. Press,M., Hartop,P.J., and Prottey,C., Correction of essential fatty-acid deficiency in man by the cutaneous application of sunflower-seed oil.Lancet 1974. 1: 597-598. 33. Zettersten,E.M., Ghadially,R., Feingold,K.R., Crumrine,D., and Elias,P.M., Opti- mal ratios of topical stratum corneum lipids improve barrier recovery in chronologically aged skin. J.Am.Acad.Dermatol. 1997. 37: 403-408. 34. Zettersten,E., Man,M.Q., Sato,J., Denda,M., Farrell,A., Ghadially,R., Williams,M.L., Feingold,K.R., and Elias,P.M., Recessive x-linked ichthyosis: role of cholesterol-sulfate accumulation in the barrier abnormality. J Invest Dermatol. 1998. 111: 784-790.

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146 Chapter 4 5 Clinical aspects and topical treatment of dithranol irritation as a unique form of irritant contact dermatitis

This chapter was based on the following publications:

The effects of topical corticosteroids and coal tar preparation on dithranol induced irritation in patients with psoriasis O.Q.J. Swinkels, M. Kucharekova, M. Prins, M.J.P. Gerritsen, P.G.M. van der Valk, P.C.M. van de Kerkhof Skin Pharmacol.Appl.Skin Physiol;2003;16(1):12-17

Dithranol irritation in psoriasis treatment: a study in 68 inpatients M. Kucharekova, L. Lieffers, P.C.M. van de Kerkhof, P.G.M. van der Valk Submitted

Skin irritation during treatment of chronic plaque psoriasis with short contact dithranol versus calcipotriol ointment M. Kucharekova, P.G.M. van der Valk, P.C.M. van de Kerkhof Manuscript in preparation

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148 149 Chapter 5 Clinical aspects and topical treatment of dithranol irritation

5.1 THE EFFECTS OF TOPICAL CORTICOSTEROIDS AND A COAL TAR PREPARATION ON DITHRANOL INDUCED IRRITATION IN PATIENTS WITH PSORIASIS

Abstract

Since more than 80 years dithranol is a mainstay in the treatment of psoriasis. Although a safe approach, the irritation of the clinically uninvolved perilesional skin remains a major limitation of this treatment. Corticosteroids and coal tar solution have an anti-inflammatory potential. The aim of the present study was to investigate the clinical and cell-biological effects of two topical corticosteroids and a coal tar preparation on dithranol irritation. During four consecutive days, dithranol 2% cream was equally applied to six uninvolved skin sites on the lower back of 9 patients with psoriasis. Dithranol was left on the skin for one hour, after which it was removed with water and soap and the skin was dried with a towel. Subsequently site one was treated with 0.05% clobetasol- 17-propionate ointment (CP), site two with unguentum cetomacrogolis (vehicle 1), site three with 0.005% fluticasonpropionate ointment (FP), site four with 10% coal tar solution in lanettewax cream (CTS), site five was left untreated (control) and site six was treated with lanettewax cream (vehicle 2). Every day erythema, oedema and vesicle formation was scored. On day 5, punch biopsies were taken from the six sites. The expression of epidermal proliferation, differentiation and inflammation markers and the clinical irritation scores indicate that the application of a strong potency corticosteroid (CP) is the best approach to minimalize dithranol irritation whereas CTS had virtually no effect on dithranol irritation using a 4 day experimental model.

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Introduction

Dithranol has been a main therapeutic agent for the topical treatment of psoriasis since 1916. Although it is a well-know irritant of the symptomless skin, it is a frequently applied treatment for almost a century. Although corticosteroids and coal tar solution (CTS) have been used to treat dithranol irritation, controlled studies on their effect are contradictory. Previously, it has been shown that pre-treatment with CP1 and betamethasone-valerate2 results in a reduction of the inflammatory reaction. On the other hand, Misch et al. suggested that dithranol erythema represents a betamethasone-17-valerate resistant form of inflammation.3 Puschman concluded that dithranol irritation is a hydrocortisone 1% and clobetasol-17-propionat resistant form of irritation.4 Juhlin observed no effect of betamethasone, while fluocinolone augmented the irritative response.5 Van de Kerkhof and Timmerman studied the effect of CP and crude coal tar on dithranol irritation in uninvolved skin: both agents did not influence dithranol-induced erythema, but in contrast had a synergistic effect on the induction of alkaline phosphatase (ALP) as a direct reflection of the metabolic activity of endothelial cells. The authors concluded that CP and crude coal tar are not indicated for the treatment of dithranol irritation but rather suggested that both agents aggravate dithranol irritancy.6 The aim of the present study was to investigate the clinical and cell biological changes in uninvolved skin of psoriatic patients after modulating dithranol irritation in an experimental model with topical corticosteroids, coal tar solution and their vehicles.

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Materials and methods

Nine patients with psoriasis affecting more than 5% of the body surface participated in the present study. The patients had no signs or a history of other diseases of the skin or keloid-forming scars. Unstable psoriasis, oral anti-psoriatic medication, antihistaminic’s, NSAID’s and immunosuppressiva were exclusion criteria. The group consisted of two females and seven males. Their ages were between 42 and 82 years. Approval of the medical ethical committee was obtained and informed consent forms were signed. Preliminary investigations in five subjects revealed a marked clinical response to 2% dithranol after one hour of application, while being well tolerated after repeated applications. In the present study the following model was applied to study dithranol irritation. For four consecutive days, dithranol 2% cream was equally applied to six uninvolved skin sites (Ø 3 cm) on the lower back of the nine patients. Dithranol was left in situ for one hour, after which it was removed by water and soap, and the skin was dried with a towel. Subsequently, site one was treated with 0.05% clobetasol-17- propionate ointment (CP), site two with unguentum cetomacrogolis approaching the ointment base of CP and 0.005% fluticasonpropionate ointment (FP) (vehicle 1), site three with FP, site four with 10% coal tar solution in vaselinum album cremor lanette I ana partes (CTS), site five was left untreated (control) and site six was treated with vaselinum album cremor lanette I ana partes (vehicle 2). During day 0, 1, 2, 3, before dithranol application and at day 4, 24 hours after the fourth dithranol application, just before taking the biopsies, clinical scoring was performed. On day 4, 3 mm punch biopsies were taken from the six sites. For detailed information on the biopsy procedure see previous publications.7

Clinical assessment Erythema, oedema and vesicle formation were scored on a five point scale: 0 = absent, 1 = minimal, 2 = moderate, 3 = marked and 4 = pronounced.

Immunohistochemical assessment The monoclonal antibody MIB-1 for nuclear expression of Ki-67 was chosen to assess epidermal proliferation. We used two differentiation markers: a monoclonal antibody against involucrin (MON-150) and a mouse monoclonal antibody anti-human keratinocyte transglutaminase. Three inflammation markers were used. Anti-elastase, as a monoclonal mouse antibody to human neutrophil elastase, was used to visualise polymorphonuclear leukocytes (PMN). T11 was used as a monoclonal antibody against T-lymphocytes. WT14 was used as a monoclonal antibody against CD14-positive cells, which represent monocytes and macrophages. An indirect peroxidase technique was used for MIB-1, MON 150, anti-

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transglutaminase and anti-elastase. For the T11 and WT14 staining the avidin-biotin method (ABC method) was used. For detailed information concerning immunohistochemistry, histological examination and statistical analysis we would like to refer to previous publication.7

Table 1 Mean clinical erythema scores ±(SEM) P-value (between different treatments on day four) Treatment Day 1 Day 2 Day 3 Day 4 P-value CP 0 ± (0) 0.4 ± (0.29) 1.1 ± (0.45) 1.6 ± (0.41) * Vehicle 1 0 ± (0) 1.3 ± (0.33) 2.1 ± (0.26) 2.7 ± (0.33) 0.001 FP 0 ± (0) 1.2 ± (0.27) 1.8 ± (0.43) 2.2 ± (0.32) 0.01 CTS 0.3 ± (0.23) 1.8 ± (0.32) 2.4 ± (0.29) 2.9 ± (0.26) < 0.001 0.01 Control 0.1 ± (0.11) 1.4 ± (0.29) 2.3 ± (0.23) 2.9 ± (0.26) < 0.001 Vehicle 2 0 ± (0) 1.3 ± (0.28) 2.0 ± (0.37) 2.4 ± (0.29) < 0.01 *At day 4 erythema scores at CP test sites were significantly decreased compared to the other 5 test sites.

Table 2 mean value ±(sem) of proliferation and differentiation markers 24 hours after the fourth dithranol application. TGM Papillary Treatment Interpapillary Interpapillary Involucrin Involucrin Papillary P-value P-value Ki-67 TGM

CP 5 ± (1.8) 0.28 ± 0.42 ± 0.30 ± 0.47 ± (0.06) (0.06) (0.05) (0.07) Vehicle 1 19.2 ± 0.26 ± 0.46 ± 0.05 0.27 ± 0.47 ± (6.8) (0.04) (0.05) (CTS) (0.03) (0.04) FP 13.7 ± 0.28 ± 0.49 ± 0.30 ± 0.55 ± (4.0) (0.04) (0.05) (0.04) (0.05) CTS 26.8 ± < 0.01 0.30 ± 0.55 ± 0.01 0.32 ± 0.55 ± (6.5) (CP) (0.04) (0.05) (CP) (0.03) (0.04) Control 23.3 ± 0.02 0.30 ± 0.51 ± 0.04 0.31 ± 0.46 ± (5.1) (CP) (0.03) (0.04) (CP) (0.02) (0.04) Vehicle 2 12.9 ± 0.26 ± 0.46 ± 0.05 0.26 ± 0.51 ± (4.2) (0.05) (0.05) (CTS) (0.04) (0.05) p-value (between different treatments) only mentioned if significant.

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Results

Clinical results Table 1 summarises erythema scores. In none of the volunteers oedema or vesicle formation were noticed. 48 hours after the first dithranol application all volunteers showed an erythematous reaction on the control site which increased up to day 4. 96 hours after the first dithranol application we noticed a maximum of irritation. P-values of the statistical analysis of the responses at day 4 of the six different test sites are given in Table 1. The erythematous reaction on the vehiculum 1 and the vehiculum 2 site were comparable to that on the control site on all days. CP suppressed the erythematous response in the majority of the volunteers on day 2. Two patients showed pronounced irritation. Even on day 3, there were still 5 volunteers who did not show any irritation, and on day 4 eight out of nine volunteers showed mild irritation while the ninth did not show irritation at all. On day four CP significantly reduced of erythema scores as compared to all other treatments. At the FP treated site irritation started on day 2 increasing further on day 3 in eight volunteers. However, on day 4 in none of the volunteers a significant reduction of the erythematous response to FP was observed as compared to the control site. At the CTS treated site we already observed pronounced irritation in 2 volunteers at day 1. On day 2, eight volunteers showed irritation and on day 3 all volunteers showed pronounced irritation. On day 4, we noted a significant increase of the erythematous response compared to the CP and the FP treated site. Remarkably, in one patient we observed sharply demarcated annular islands of normal appearing skin, even on day 4, within the erythematous test sites. . Cell biological results The cell biological results 24 hours after the fourth dithranol application (96 hours after the first) have been summarised inTable 2 and 3. The staining for epidermal proliferation (Ki67 staining) revealed less cycling epidermal cells on the CP treated site in comparison with the control site (p = 0.02) while the CTS treated site showed a significant higher number of cycling cells, in comparison with the site treated with CP (p < 0.01). Evaluating the epidermal differentiation markers we found a significant difference of the papillary involucrin ratio between the CP treated site and the control site (p = 0.04). The CTS treated site showed a significant higher ratio in comparison with the site treated with CP (p = 0.01), but also compared to the site treated with vehicle 1 (p = 0.05) and the site treated with vehicle 2 (p = 0.05). Transglutaminase expression revealed no statistically significant differences. With respect to inflammation, epidermal T cell accumulation at the CP treated site was found to be significantly reduced compared to the sites treated with vehicle 1, vehicle 2 and the control site (p = 0.02). The occurrence of monocytes and macrophages at the CP treated site was significantly reduced in the epidermis (p

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= 0.02) and diffuse in the dermis (p < 0.01) in comparison to the sites treated with vehicle 1, vehicle 2 and the control site. Anti-elastase (PMN) staining did not reveal significant differences between the test sites.

Table 3 Mean value ±(sem) of inflammation markers in the six treated sites 24 hours after the fourth dithranol application. Treatment T-lymphocytes Monocytes And Polymorphonuclear Macrophages Cells Epidermal Epidermal Epidermal Vascular Vascular Vascular Dermal Dermal Dermal Diffuse Diffuse Diffuse Peri- Peri- Peri-

CP 0.7 ± 1.1 ± 1.9 ± 0.7 ± 1.2 ± 2.0 ± 0.0 ± 0.9 ± 0.3 ± (0.2) (0.1) (0.2) (0.17) (0.15) (0) (0) (0.11) (0.17) Vehicle 1 1.0 ± 1.3 ± 2.4 ± 1.0 ± 1.8 ± 2.1 ± 0.0 ± 0.8 ± 0.4 ± (0) (0.2) (0.2) (0) (0.15) (0.11) (0) (0.15) (0.17) FP 0.9 ± 1.1 ± 2.0 ± 0.8 ± 1.7 ± 2.2 ± 0.1 ± 0.7 ± 0.0 ± (0.1) (0.1) (0) (0.17) (0.17) (0.15) (0.11) (0.17) (0) CTS 1.0 ± 1.3 ± 2.8 ± 1.0 ± 2.0 ± 2.6 ± 0.2 ± 0.9 ± 0.4 ± (0) (0.2) (0.3) (0) (0.17) (0.24) (0.15) (0.11) (0.17) Control 1.0 ± 1.4 v 2.7 ± 1.0 ± 1.9 ± 2.7 ± 0.1 ± 0.8 ± 0.2 ± (0) (0.2) 0.2) (0) (0.11) (0.23) (0.11) (0.15) (0.15) Vehicle 2 1.0 ± 1.2 ± 2.4 ± 1.0 ± 1.9 ± 2.2 ± 0.0 ± 0.9 ± 0.4 ± (0) (0.2) (0.3) (0) (0.11) (0.15) (0) (0.11) (0.17) Only significant p-values are mentioned. Semiquantitative T-lymphocytes scores: CP scores were significantly different compared to the sites treated with vehicle.1,2 And the control site: epidermal (p = 0.02 ) Semiquantitative monocytes and macrophages scores: cp scores were significantly different compared to the sites treated with vehicle.1,2 And the control site: epidermal (p = 0.02 ) And diffuse dermal (p < 0.01).

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Discussion

We investigated the clinical and cell biological changes after influencing dithranol irritation by topical corticosteroids, a coal tar solution and their vehicles in uninvolved skin of patients with psoriasis. Clinical observation showed that, although the majority of patients (eight out of nine) demonstrated an inflammatory reaction 48 hours after the first dithranol application, dithranol irritation was already manifest after 24 hours (1 patient). The maximal irritation was observed at day 4 (24 hours after the fourth dithranol application). The appearance of irritation 24-48 hours after dithranol application is in correspondence with literature.1;8 The response to dithranol is dependent on the concentration, application time and frequency of application. The sharply demarcated annular islands of normal appearing skin within a pronounced erythema of test sites even on day 4 in one patient is intriguing. It is attractive to speculate that previous psoriatic lesions or initial psoriatic involvement below the clinical threshold was present causing resistance to dithranol. CP treatment significantly suppressed the erythematous response to dithranol, whereas CTS seemed to have no effect. In a pilot study (n = 10) with the same design we found no effect of CTS, confirming the present findings. In an earlier study we investigated the influence of CP 0,05% ointment on dithranol short contact therapy showing a reduction of perilesional irritation at the cellular level although this effect was not expressed by reducing dithranol erythema.9 In the present study, coal tar solution had no inhibitory effect on dithranol irritation, confirming findings in several previous studies.6;10;11 Other authors found that combined therapy of dithranol and coal tar augmented the rate of clearance of psoriasis and diminished dithranol irritancy12;13 and explaining its widespread and long-established clinical use.14-18 It has been suggested that this effect is caused by the inactivation of dithranol by coal tar.19 The addition of coal tar results in a rapid oxidation of dithranol into its ineffective oxidation products, danthron and dithranol dimmer.20 The contradictory results may be explained by the fact that crude coal tar preparations and coal tar solution preparations are not identical concerning the described effects. Ki-67 is a suitable marker of dithranol irritation. De Zwart et al. observed a significant increase of Ki-67 positive cells after 48 hours of dithranol application.21 Epidermal proliferation, Ki-67staining, revealed less cycling epidermal cells on the site treated with CP in comparison with the control site (p = 0.02), while the site treated with CTS showed a significant higher number of cycling cells, in comparison with site 1 (p = 0.005). Evaluating the epidermal differentiation markers we found a significant difference of the papillary involucrin ratio between the site treated with CP and the control site (p = 0.04). The site treated with CTS showed a significant higher ratio in comparison with the site treated with CP (p = 0.01), the site treated with vehicle 1(p = 0.05) and the site treated with vehicle 2 (p = 0.05). Concerning inflammation, epidermal T cells, monocytes and macrophages and diffuse dermal monocytes and macrophages were found to be significantly reduced on the site treated with CP. Those

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effects underline the clinical results and confirm strong potency corticosteroids (CP) as the best treatment for dithranol irritation. The present study, using a model of 4 days short contact dithranol challenge of the uninvolved skin of patients with psoriasis, suggests that CP is effective in reducing dithranol erythema, whilst CTS is not. The role of CP and CTS in reducing dithranol irritation during treatment of psoriatic plaques remains controversial.

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References

1. Lawrence,C.M. and Shuster,S., Mechanism of anthralin inflammation. I. Dissociation of response to clobetasol and indomethacin. Br.J.Dermatol. 1985. 113: 107-115. 2. Lawrence,C.M. and Shuster,S., Mechanism of anthralin inflammation. 2. Effect of pretreatment with glucocorticoids, anthralin and removal of SC. Br.J.Dermatol. 1985. 113: 117-122. 3. Misch,K., Davies,M., Greaves,M., and Coutts,A., Pharmacological studies of anthra- lin erythema. Br.J.Dermatol. 1981. 105 Suppl 20. 4. Puschmann,M., [Anthralin erythema: effect of concentration, contact time, oxidation products and corticosteroids. Clinical, reflection photometry and electron microscopy studies]. Z.Hautkr. 1983. 58: 1646-1647. 5. Juhlin,L., Factors influencing anthralin erythema.Br.J.Dermatol. 1981. 105 Suppl 20. 6. van de Kerkhof,P.C.M. and Timmerman,M.G., The effect of clobetasol-17-propion- ate and crude coal tar on dithranol-induced inflammation. A clinical and biochemical study. Acta.Derm.Venereol. 1990. 70: 434-437. 7. Swinkels,O.Q., Prins,M., Birker,L., Gerritsen,M.J.P., van der Valk,P.G.M., and van de Kerkhof,P.C.M., The response of normal human skin single and repeated applica- tion of dithranol cream: an immunohistochemical assessment. Skin Pharmacol Appl.Skin Physiol 2002. 15: 262-269. 8. Kingston,T. and Marks,R., Irritant reactions to dithranol in normal subjects and psori- atic patients. Br J Dermatol 1983. 108: 307-313. 9. Swinkels,O.Q., Prins,M., Kucharekova,M., De Boo,T., Gerritsen,M.J.P., van der Valk,P.G.M., and van de Kerkhof,P.C.M., Combining lesional short-contact dithranol therapy of psoriasis with a potent topical corticosteroid. Br J Dermatol 2002. 146: 621- 626. 10. Duhra,P. and Ryatt,K.S., Lack of additive effect of coal tar combined with dithranol for psoriasis. Clin.Exp.Dermatol. 1988. 13: 72-73. 11. Downey,D.J. and Finlay,A.Y., Combined short contact crude coal tar and dithranol therapy for psoriasis. Clin.Exp.Dermatol. 1986. 11: 498-501. 12. Bratzke,B., Albrecht,G., and Orfanos,C.E., [Skin reaction to dithranol and its modifi- cation by the addition of tar (LCD)] Hautreaktion auf Dithranol und ihre Beeinflussung durch Teerzusatz (LCD). Hautarzt. 1987. 38: 356-360. 13. Steigleder,G.K. and Schulze,H.J., [A new Cologne therapy scheme--addition of tar to cignoline-salicylic acid-white vaseline therapy in psoriasis vulgaris]. Z.Hautkr. 1984. 59: 188, 191-188, 192. 14. Young,E. and van Weelden,H., Treatment of psoriasis with a combination of dithranol and coal tar [letter]. Clin.Exp.Dermatol. 1988. 13: 419-420. 15. Schulze,H.J. and Steigleder,G.K., [Experiences in half-side treatment with tar additive to cignolin-salicylic-acid-white-vaseline therapy of psoriasis vulgaris]. Z.Hautkr. 1984. 59: 654-656. 16. Schauder,S. and Mahrle,G., [Effect of coal tar on cignolin erythema--1 hour treatment of psoriasis with high-dose cignolin with and without tar]. Z.Hautkr. 1985. 60: 875-883. 17. Schulze,H.J., [Suppression of cignolin-induced erythema by tar]. Z.Hautkr. 1984. 59: 659-662.

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18. Lawrence,C.M., Finnen,M.J., and Shuster,S., Effect of coal tar on cutaneous aryl hydrocarbon hydroxylase induction and anthralin irritancy. Br.J.Dermatol. 1984. 110: 671-675. 19. Whitefield,M., Degradation of anthralin by coal tar [letter]. J Am Acad Dermatol 1987. 16: 629. 20. Muller,R., Naumann,E., Detmar,M., and Orfanos,C.E., [Stability of cignolin (dithra- nol) in ointments containing tar with and without the addition of salicylic acid. Oxidation to danthron and dithranol dimer]. Hautarzt 1987. 38: 107-111. 21. de Zwart,A.J., de Jong,E.M., and van de Kerkhof,P.C.M., Topical application of dithranol on normal skin induces epidermal hyperproliferation and increased Ks8.12 binding. Skin.Pharmacol. 1992. 5: 34-40.

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5.2 DITHRANOL IRRITATION IN PSORIASIS TREATMENT: A STUDY IN 68 INPATIENTS

Abstract

The irritative response of perilesional skin is a serious limitation of dithranol therapy in psoriasis. No data are available on the actual prevalence and severity of irritation during 24 hours dithranol treatment in an inpatient setting. Using a retrospective analysis of 68 patients with psoriasis visiting our inpatient department for dithranol treatment, the occurrence of dithranol irritation was studied. We found a relatively high frequency of dithranol irritation. Most irritation occurs at the start of the therapy with relatively low concentrations.

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Introduction

Dithranol (1,8-dihydroxy-9-anthrone), also known as cignolin or anthralin, has been a mainstay in the treatment of psoriasis for more than eight decades.1 It may be used in different formulations (ointment, paste, cream or stick) and application schemes. Dithranol can be applied for 24 hours, in paste Lassar or stiff paraffin on the psoriatic lesions, or in white petrolatum on the involved and the uninvolved skin (diffuse application). Application for 24 hours is practically impossible in an outpatient setting because of irreversible staining of garments and furniture. Although dithranol treatment is safe, the irritative response of perilesional and uninvolved skin is a serious limitation.2;3In case of irritation, application has to be withdrawn for several days and may result in a prolongation of the treatment. Alternative application schedules with a cream formulation in a short contact exposure seems to reduce the disadvanrage of staining of the classical 24 hours dithranol application.1;4 Because of short contact times, staining of clothes can be avoided and patients can be treated at home. Comparing different vehicles and treatment schedules of dithranol, 24 hours application of dithranol in a paste or ointment appears to be most effective. Inpatient treatment results in a significant faster clearance as compared to outpatient treatment.1 At our inpatient department of dermatology we routinely use dithranol ointment which is diffusely applied for 24 hours. So far, the information on the duration of dithranol irritation and the relationship of dithranol irritation with the duration of treatment has not been documented. Therefore, we set out to study the occurrence of irritation in our inpatient department. In particular we investigated the frequency and threshold concentration for irritation during treatment with 24 h application of dithranol ointment. In the present study we analyzed retrospectively the characteristics of dithranol irritation and interpreted the irritation in the light of available information on the characteristics of responses to other irritants.

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Material and methods

The present study is based on a retrospective analysis of data from records of patients visiting our in-patient department for psoriasis treatment during the period January 1999 until March 2001. In total 68 patients with generalised psoriasis were included and evaluated. These patients were treated with a dithranol ointment (dithranol in white petrolatum) as monotherapy for 24 hours. Dithranol ointment was applied diffusely over the skin (both involved and uninvolved skin) below the head and neck, except flexures. Tube-bandages were used to cover the dithranol application sites and to protect the patient’s clothes and environment. Dithranol was applied daily by trained nurses. The treatment was started with 0.1 % dithranol and if no irritation occurred the concentration was increased stepwise every three-four days (maximum concentration 5.0%) according to a written protocol used at our centre. Dithranol ointment was prescribed in the following concentration: 0.1%, 0.25%, 0.3%, 0.4%, 0.6%, 0.8%, 1.0%, 2.0%, 3.0%, and 5.0%. In case of irritation, physician decided to remain at the same concentration or to interrupt the dithranol treatment for a few days. The use of emollients was recommended during the cessation of treatment. Physicians were also the persons who determinate the re-introduction of dithranol therapy which wasusually started with a lower concentration. The treatment was continued until total clearance was reached or a considerable improvement had occurred which was acceptable for the patient. Patients who have used systemic therapy, phototherapy and topical treatments of corticosteroids and vitamin D3 analogues were excluded from the analysis. Information was collected concerning frequency of hospitalisation during the study period, the frequency and duration of “irritation-episodes” and the concentration which caused irritation. In this study an irritation-episode was defined as an episode of skin irritation, which required temporary cessation of dithranol therapy as assessed by a physician.

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Figure 1 Duration of dithranol 50 treatment interruption (days). 45 Mostly the irritation-episode was 40 35 short lasting (2 days or less). 30 25 20

Patients (%) Patients 15 10 5 0 1 2 3 4 >4 Days

Figure 2 Frequency of irritation- episodes. In this study an irritation- episode was defined as an episode 40 of skin irritation, which required temporary cessation of dithranol 30 therapy. 20 10 Patients (%) Patients 0 0x 1x 2x 3x 4x 5x Episodes of irritation

40 Figure 3 Frequency of irritation- episodes in relation to the different 35 concentration of dithranol. Note 30 that the most of irritation-episodes 25 occurred at the start of the therapy

20 with a relatively low concentration.

15

10

Frequency of irritation (%) 5

0 0,01 0,03 0,05 0,1 0,2 0,3 0,4 0,6 >0,8 Dithranol concentration (%)

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Results

We evaluated 68 patients with psoriasis who received at least one course of monotherapy with dithranol ointment during the period January 1999 until March 2001 at our inpatient department. 34 were male and 34 were female, ranging in age from 17 to 92 years. 64 were of Caucasian origin and 4 of Asian origin. The average number of hospitalisations per patient was 1.4 during the 26 months observation period. In total 172 irritation episodes were recorded during 96 hospitalisations. The average number of irritation episodes per hospitalisation was 1.9 and the mean duration requiring discontinuation of dithranol was 2.36 days. Table 1 summarises the hospitalisation data. Figure 1 shows the number of days patients were not treated because of irritation per episode. Mostly the irritation was short lasting. In 73% of the patients the irritation was 2 days or less. The percentage of patients who had to discontinue dithranol for 4 days or longer was 13%. Most patients (85.3%) experienced skin irritation at least once during the treatment, while 14.7% patients did not have skin irritation and completed the dithranol treatment without interruption (Figure 2). The frequency of irritation-episodes in relation to the concentration of dithranol in the vehicle is shown in Figure 3. Most episodes of irritation occurred at the start of the therapy with relatively low concentrations (0.1%- 0.2%).

Table 1 Data on hospitalisation and irritation Number of patients 68 Total hospitalisations (from January 1999 until March 96 2001) Hospitalisations per patient (mean ± SEM) 1.41 ± 0.08 Days of treatment period (mean ± SEM) 37.26 ± 1.84 Total irritation-episodes 172 Irritation-episodes per hospitalisation (mean ± SEM) 1.92 ± 0.18 Stop days per irritation-episode (mean ± SEM) 2.36 ± 0.71

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Discussion

In the analysis of 68 inpatients we observed in 85,3 % of the patients experienced skin irritation. Most of the patients had only one episode of dithranol irritation and the irritation was mild and caused only a short cessation of the therapy (up to 2 days). This is in line with our impression, that extreme irritation during this regime is rare. Only 13 % of the patients had to discontinue dithranol treatment for 4 days or longer for reasons of irritation. As may have been predicted, the number of patients experiencing irritations in this study is higher compared to available data on skin irritation taken from clinical trials where dithranol crème in short contact regime in outpatients was applied (48% - 72%).5-7 Short application time of dithranol relatively increases the bioavailability in the psoriasis plaque compared to the healthy skin. This phenomenon makes it possible to treat the patients with high concentration of dithranol, while side effects remain modest.8 The other possibility for the high prevalence of dithranol irritation in our study might be due to the high number of our patients with severe and instable disease. The sensitivity to dithranol in these patients is likely to be increased. Furthermore, most episodes of dithranol irritation occur at the start of the therapy with relatively low concentrations (0.1 - 0.2 %). Subsequently with further dosage increments episodes of irritation were less frequent. These results have confirmed the well known clinical experience that in the course of treatment with dithranol, the skin adapts and will be less susceptible to the irritant effects of dithranol. This adaptive effect is also responsible for the decrease of antipsoriatic potency of dithranol during prolonged treatment which requires increasing concentrations of dithranol.9 Such an adaptation is in striking contrast to e.g. sodium dodecyl sulphate irritation, where repeated challenges cause summation of the irritant response.10- 12 In case of dithranol irritation the possibility of a delayed type hypersensitivity should be reconciled.13-16 Differentiation between an irritant response or a delayed type hypersensitivity may be a problem as dithranol irritation occurs at low concentrations. Adaptation following repeated application is normally not seen with subsequent applications of a contact allergen. For example, repeated applications of diphenylcyclopropenone is a well known treatment of alopecia areata.17 These applications elicit a mild eczema and no tendency of habituation is observed in the vast majority of the patients. In fact, one study showed tolerance to the contact allergen only in 5.8% of the patients.18 Apparently, the adaptation during repeated applications of dithranol discloses an entirely different mechanism which is suggested to be specific for dithranol irritation.19 However, the underlying mechanism of this adaptive response of the skin has not been elucidated. Brandt and Mustakallio suggested that this adaptation may be due to exhaustion of anthrone-induced inflammatory mediators.20 Lawrence and Shuster hypothesised that this phenomenon may be caused by an unknown active adaptive mechanism. They found that tape stripping and a very low dose of dithranol induced tolerance of the skin to dithranol.21 Farkas et al. investigated repeated treatment with dithranol in a hyperproliferative human keratinocyte line.

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Their results indicated that the keratinocytes experienced tolerance (tachyphylaxis) to the antiproliferative effects of dithranol due to a selective elimination of the most dithranol-sensitive cells.9 Kemeny et al. recently demonstrated that a low dose of dithranol significantly suppressed dithranol induced mouse ear oedema, whereas it had no effect on croton oil- or dinitrofluorobenzene (DNFB)-induced dermatitis. Superoxide dismutase treatment cancel this tolerance-inducing effect of low dose dithranol, indicating that superoxide anion radicals are involved in the triggering the adaptive mechanism of dithranol.22 Further studies are needed to elucidate the exact mechanism of adaptation of the skin to dithranol which may be helpful in finding optimal therapeutic schedules with dithranol for the treatment of psoriasis. In conclusion, dithranol irritation is a phenomenon which occurs preferentially in an early phase of dithranol treatment. This habituation to dithranol irritation is in contrast with the response to other irritants.

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References

1. van de Kerkhof,P.C.M., Dithranol treatment in psoriasis:after 75 years, still going strong. European Journal of Dermatology 1991. 1: 79-88. 2. Kingston,T. and Marks,R., Irritant reactions to dithranol in normal subjects and psori- atic patients. Br J Dermatol 1983. 108: 307-313. 3. Misch,K., Davies,M., Greaves,M., and Coutts,A., Pharmacological studies of anthralin erythema. Br.J.Dermatol. 1981. 105 Suppl 20. 4. Wilson,P.D. and Ive,F.A., Dithrocream in psoriasis. Br.J.Dermatol. 1980. 103: 105- 106. 5. de Mare,S., Calis,N., den Hartog,G., and van de Kerkhof,P.C.M., Outpatient treat- ment with short-contact dithranol. The impact of frequent concentration adjustments. Acta Derm.Venereol 1989. 69: 449-451. 6. Hutchinson,P.E., Marks,R., and White,J., The efficacy, safety and tolerance of calci- triol 3 microg/g ointment in the treatment of plaque psoriasis: a comparison with short- contact dithranol. Dermatology 2000. 201: 139-145. 7. Berth-Jones,J., Chu,A.C., Dodd,W.A., Ganpule,M., Griffiths,W.A., Haydey,R.P., Klaber,M.R., Murray,S.J., Rogers,S., and Jurgensen,H.J., A multicentre, parallel- group comparison of calcipotriol ointment and short-contact dithranol therapy in chronic plaque psoriasis. Br.J.Dermatol. 1992. 127: 266-271. 8. Schaefer,H., Farber,E.M., Goldberg,L., and Schalla,W., Limited application pe- riod for dithranol in psoriasis. Preliminary report on penetration and clinical efficacy. Br.J.Dermatol. 1980. 102: 571-573. 9. Farkas,B., Bonnekoh,B., and Mahrle,G., Repeated treatment with dithranol induces a tolerance reaction in keratinocytes in vitro. Arch.Dermatol.Res. 1991. 283: 337-341. 10. Wilhelm,K.P., Freitag,G., and Wolff,H.H., Surfactant-induced skin irritation and skin repair: evaluation of a cumulative human irritation model by noninvasive techniques. J Am Acad Dermatol 1994. 31: 981-987. 11. Kligman, A. M and Wooding W.M. A methods for the measurement and evaluation of irritants on human skin. J Invest Dermatol 1967. 49: 78-94. 12. Lee, C. H and Maibach, H. I. The sodium lauryl sulfate model: an overview. Contact Dermatitis 1995. 33: 1-7. 13. de Groot,A.C. and Nater,J.P., Contact allergy to dithranol. Contact Dermatitis 1981. 7: 5-8. 14. Burden,A.D., Stapleton,M., and Beck,M.H., Dithranol allergy: fact or fiction?Contact Dermatitis 1992. 27: 291-293. 15. Di Landro,A., Valsecchi,R., and Cainelli,T., Contact allergy to dithranol. Contact Der- matitis 1992. 26: 49-50. 16. Prins,M., Swinkels,O.Q., Mommers,J.M., Gerritsen,M.J.P., and van der Valk,P.G.M., Dithranol treatment of psoriasis in dithranol-sensitive patients. Contact Dermatitis 1999. 41: 116-117. 17. Happle,R., Hausen,B.M., and Wiesner-Menzel,L., Diphencyprone in the treatment of alopecia areata. Acta Derm.Venereol 1983. 63: 49-52. 18. van der Steen,P.H., van Baar,H.M., Perret,C.M., and Happle,R., Treatment of alo- pecia areata with diphenylcyclopropenone. J Am Acad Dermatol 1991. 24: 253-257. 19. Parslew,R. and Friedmann,P.S., The irritancy of anthralin is inhibited by repeat appli- cations of a subirritant concentration. Br.J.Dermatol. 1999. 141: 469-474.

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20. Brandt,H. and Mustakallio,K.K., Irritation and staining by dithranol (anthralin) and related compounds. III. Cumulative irritancy and staining during repeated chamber test- ing. Acta Derm.Venereol. 1983. 63: 237-240. 21. Lawrence,C.M. and Shuster,S., Mechanism of anthralin inflammation. 2. Effect of pretreatment with glucocorticoids, anthralin and removal of SC. Br.J.Dermatol. 1985. 113: 117-122. 22. Kemeny,L., Farkas,A., and Dobozy,A., Low-dose dithranol treatment and tape strip- ping induce tolerance to dithranol in a mouse ear oedema model. Br J Dermatol 2002. 146: 764-769.

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5.3 SKIN IRRITATION DURING TREATMENT OF CHRONIC PLAQUE PSORIASIS WITH SHORT CONTACT DITHRANOL VERSUS CALCIPOTRIOL OINTMENT

Abstract

The efficacy of topical treatment with dithranol and calcipotriol in psoriasis is well-established. In an open randomized trial we studied aspects of skin irritancy of both treatments. 105 patients with chronic stable plaque psoriasis were included. 52 patients were treated with short contact dithranol and 53 patients were treated twice daily with calcipotriol ointment once daily. All patients were treated at the day-care centre. Skin irritation was observed in 48.1% of patients treated with dithranol and in 18.9% of patients treated with calcipotriol. We did not observe any significant correlation between Psoriasis Area and severity Index (PASI) on the entry of the study and frequency of skin irritation in both treatments. No significant correlation was observed between skin irritation and PASI-scores at the end of treatment. In conclusion, skin irritation during dithranol and calcipotriol treatment is probably not very relevant to the efficacy of these treatments in the studied population.

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Introduction

Treatment with dithranol has been a time-honored principle in the topical treatment of psoriasis, characterized by high efficacy and safety. However, staining and irritation are serious limitations of this treatment.1 In the majority of patients, skin irritation occurs during dithranol treatment when dose increasments are carried out. Irritation during dithranol has been suggested to be a prerequisite for efficacy. Topical treatment with calcipotriol has become a mainstay in the treatment of mild to moderate psoriasis. Calcipotriol is generally well tolerated although skin irritation is a side effect, which occurs in 10-25% of the patients if treated twice daily with calcipotriol ointment.2-6 So far, no studies have been reported suggesting that irritation due to calcipotriol is followed by enhanced or decreased efficacy. The aim of the present study was to characterize irritation during dithranol and calcipotriol treatment in order to find out (I) whether the frequency of irritation depends upon the severity as assessed by PASI before treatment and (II) whether patients with irritation have a more-or less favorable clinical response to calcipotriol and dithranol compared to patients without irritation.

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Materials and methods

Study design The trial was a multi-center, randomized; open, prospective, parallel-group comparison of two treatments and was performed in the Netherlands. Patients were instructed and treated at day-care centers in order to reach optimal compliance.

Subjects Out-patients, of both sex and aged 18 or over, with a minimum PASI score for extent of 2 in at least one body region, were recruited. Medical Ethics Committee approval was obtained prior to recruitment and all patients gave informed consent. Patients with guttate, pustular, erythrodermic or exfoliative psoriasis, patients who have received systemic antipsoriatic treatment or phototherapy within the last 6 weeks, patients with changes in their concurrent medication that could affect their psoriasis during the study period, patients with known or suspected hypercalcaemia, patients who are hypersensitive to components of either calcipotriol ointment or dithranol cream and females who were pregnant or not using adequate contraception were excluded.

Randomization Randomization was by computer-generated random numbers.

Study visit schedule Subjects attended a screening visit at which all treatments for psoriasis were discontinued except for emollients and/or descaling agents (5% salicylic acid in white petrolatum). After a 2-week washout period subjects attended for a baseline visit. Those who met all entry criteria were randomly allocated to one of two treatment groups. Subjects were seen at study visits by the same clinical investigator at 2, 4, 8 and 12 weeks of active treatment.

Treatment protocol A labor- and time intensive treatment concept for both treatments has been chosen that combines treatment of the patient in a day-care center with home treatment, aiming to improve self-treatment and treatment compliance. During the first week, the patients were treated and instructed at the day- care center daily. After this week patients were treated at the day-care center twice a week and treated themselves at home the other five days of the week. Calcipotriol ointment (50 g/g, Daivonex ointment, Leo Pharma BV) was applied twice daily to all lesions below the head and neck, not exceeding a maximum of 100 g per week. Dithranol cream (dithranol, cetiol V, cetomacrogol wax, liquid petrolatum, salicylic acid, sorbic acid, ascorbic acid, demineralized water) in appropriate

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concentrations (0.05%-0.1%-0.2%-0.3%-0.4%-0.6%-0.8%-1%-2%-3%) was applied to lesional and perilesional skin. Dithranol preparations were manufactured, certified and supplied by Tiofarma, the Netherlands. Dithranol was applied according to a short-contact treatment regime. We used a three days short contact application scheme starting with 15 minutes of application, followed by 30 to 45 minutes application time. After each application dithranol was washed off. After three days of 45 minutes application time the therapy was continued with a 15 minutes application of a one step higher concentration. Individual adjustments were allowed, according to the individual patient’s susceptibility. Patients who were intolerant to 0.1% dithranol cream, started their treatment with 0.05% dithranol cream. Face and scalp were excluded from this treatment. In case of irritation the treatment time was shortened or dithranol concentration was lowered. The duration of treatment in this study was 12 weeks. Because of the differences in the treatment schedules, it was not possible to blind the study.

Compliance Checks During the first week daily and subsequently twice a week the patients were treated at the day-care centre, which guaranties 100% compliance at these visits. The patient’s study compliance at home was monitored by asking the patient at all study visit if he/she had used the meditation as prescribed. If not, the degree of non- compliance was recorded.

Clinical Assessments At baseline, 2, 4, 8 and 12 weeks of treatment the severity was assessed using the modified PASI. The extent of psoriatic involment was recorded for each of the three areas: upper extremities, trunk and lower extremities (which correspond to 20, 30 and 40% of the body area, respectively) using the following 7-category scale: 0=no involment, 1=<10%, 2=10-29%, 3=30-49%, 4=50-69%, 5=70-89%, 6=90-100%. The head and neck were not assessed. The severity of the psoriasis lesions was recorded with respect to each of the following three clinical signs: redness, thickness, scaliness using the 5-category scale below: 0=no cutaneus involment,1=slight involment, 2=moderate involment,3=severe involment, 4=severest possible involment.

Skin Irritation In case of skin irritation the duration of irritation, the intensity (mild, moderate or severe) was recorded.

Statistics Patients with irritation and without irritation were compared with respect to the decrease in PASI-score during treatment using a student t-test.

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Tabel 1: Treatment duration (in days) before the first period of skin irritation Calcipotriol (n=53) Dithranol (n=52) Mean 30.7 26.3 SD 18.7 17.3 Minimum 8 0 Maximum 57 56

Tabel 2. The number and percentage of patients in each category of number of skin irritation episodes Calcipotriol (n=10) Dithranol (n=25) Number of Number of % Number of % skin irritation patients patients episodes 1 10 100 13 52 2 0 0 5 20 3 0 0 5 20 4 0 0 1 4 5 0 0 1 4

Tabel 3: Baseline PASI. No significant correlation was found between severity of psoriasis at the entry of the study and skin irritation in both treatments groups Calcipotriol (n=53) Dithranol (n=52) Skin irritation No skin Skin irritation No skin (n=10) irritation (n=25) irritation (n=43) (n=27) Mean 11.2 9.4 10.5 9.7 Median 9.8 8.6 10.7 9.0 SD 5.3 5.2 2.6 4.9

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Results

In total 52 patients were randomized in the dithranol treatment group and 53 in the calcipotriol group. Twenty-five (48.1%) of the 52 patients in dithranol group and ten (18.9%) of the 53 patients in the calcipotriol group experienced at least one episode of irritation. The treatment duration before the first episode of skin irritation for both treatments is shown in Table 1. Treatment duration before the occurrence of skin irritation was comparable in both groups. The Table 2 shows the number of episodes with skin irritations in calcipotriol and dithranol treated patients. During calcipotriol treatment only one single episode of irritation occurred and in the dithranol group 12 out of 25 patients had multiple episodes of irritation. In the calcipotriol group the average number of days with skin irritation was 2.3 days, in dithranol group 7.2 days. In four patients of the calcipotriol group skin irritation was the reason of withdrawal. In the dithranol group, three patients withdrew due to skin irritation. Regarding severity of irritation, two patients of the calcipotriol group experienced irritation as mild, seven patients as moderate and one patient as severe. In the dithranol group, twelve patients experienced the skin irritation as mild, eleven patients as moderate and one patient as severe. The severity had not been assessed in one patient. Table 3 and Table 4 show the baseline PASI-scores and the end of treatment PASI-scores of these patients with and without skin irritation, respectively. No significant differences were found with respect to the baseline PASI-scores and end of treatment PASI-scores between the treatment groups and between patients with or without skin irritation.

Table 4 End of treatment PASI. No significant correlation was found between severity of psoriasis at the end of study and skin irritation in both treatments groups Calcipotriol (n=53) Dithranol (n=52) Skin irritation No skin Skin irritation No skin (n=10) irritation (n=25) irritation (n=43) (n=27) Mean 5.3 3.2 3.8 3.2 Median 4.8 2.2 2.7 2.6 SD 3.4 3.5 3.1 3.1

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Discussion

In the analysis of 53 out-patients treated with short contact dithranol was observed that 48.1% of these patients experienced irritation. Half of these patients (52%) had only one episode of dithranol irritation and the irritation was mild. Occurrence of skin irritation during short contact dithranol considerably varies in literature (48% to 72 %).7-9 The frequency of skin irritation by calcipotriol ointment in this study was 18.9% and is in line with literature (10-25%).10-12 Although skin irritation was observed more often in the dithranol group, such was not reflected in a higher number of patients (n=3) who stopped compared to the calcipotriol group (n=4). Skin irritation during dithranol treatment is a major limitation of this time- honored treatment. Many unsuccesful attempts have been made to reduce the side effect of dithranol by changing the duration of the application or the vehicle, or by a combination of dithranol with other drugs. A marked irritation to dithranol may require stopping the treatment for one or more days. Previously, a significant negative correlation between the duration of cessation of treatment for reason of irritation and treatment days needed to achieve a clearance of skin in a population of patients with moderate to severe plaque psoriasis has been observed.13 However, in the present study we did not observe a correlation between PASI-scores at the end of treatment period and skin irritation. Therefore, although the duration of treatment required for clearing may be longer in patients with severe irritation, the efficacy assessed at the end of treatment was similar. Skin irritation during treatment with calcipotriol is the most frequent reported adverse event. Although the skin irritation during calcipotriol treatment is often mild, it may impair compliance and eventually result in the decreased efficacy. In literature some patients experienced deterioration of psoriasis accompanied by skin irritation of the uninvolved skin.7 In the present study, we did not observe a significant correlation between skin irritation and PASI-scores at the end of the treatment, although a tendency was observed that patients without skin irritation reached better results. The exact underlying pathogenic mechanism of the calcipotriol- and dithranol - induced skin irritation has not been elucidated. Both of them do not affect the skin barrier (non-corrosive type of skin irritation).14-16 It is generally assumed that dithranol irritation is caused by the formation of super-oxide radicals due to the loss of a hydrogen atom from the methylene group at position C-10.17 Regarding calcipotriol, recent studies have shown that the irritant profile of calcipotriol is dominated by vascular reactions. The special ointment, which is needed to keep calcipotriol stable , may be in part responsible for the skin irritation. Furthermore, in individual patients the possibility of allergic contact dermatitis should be reconciled.18-24 It has been suggested that patients with unstable relapsing psoriasis are more sensitive to dithranol irritation. PASI-scores, reflect severity of the disease and not necessarily indicate instability of the disease. However, in this study, the severity of

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the disease before treatment as assessed by PASI-scores did not correlate significantly with the frequency and the severity of irritation. In the calcipotriol group we did not observe a significant correlation between baseline PASI-scores and the frequency of irritation either. These results indicate that PASI-scores has not a predictive value for dithranol and calcipotriol induced skin irritation. In conclusion, skin irritation following dithranol and calcipotriol are common limitations to these treatments. In this study, irritation during these treatments showed a limited impact on the clinical efficacy. A baseline PASI-score does not have a predictive value for the development of skin irritation during these treatments in the population studied.

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References

1. van de Kerkhof,P.C.M., Dithranol treatment in psoriasis:after 75 years, still going strong. European Journal of Dermatology 1991. 1: 79-88. 2. Kragballe,K., Treatment of psoriasis by the topical application of the novel cholecalcif- erol analogue calcipotriol (MC 903). Arch.Dermatol. 1989. 125: 1647-1652. 3. Tham,S.N., Lun,K.C., and Cheong,W.K., A comparative study of calcipotriol oint- ment and tar in chronic plaque psoriasis. Br.J Dermatol. 1994. 131: 673-677. 4. Kragballe,K., Barnes,L., Hamberg,K.J., Hutchinson,P., Murphy,F., Moller,S., Ruzicka,T., and van de Kerkhof,P.C.M., Calcipotriol cream with or without concur- rent topical corticosteroid in psoriasis: tolerability and efficacy. Br.J Dermatol. 1998. 139: 649-654. 5. Molin,L., Cutler,T.P., Helander,I., Nyfors,B., and Downes,N., Comparative efficacy of calcipotriol (MC903) cream and betamethasone 17-valerate cream in the treatment of chronic plaque psoriasis. A randomized, double-blind, parallel group multicentre study. Calcipotriol Study Group. Br.J Dermatol. 1997. 136: 89-93. 6. Kragballe,K., Gjertsen,B.T., De Hoop,D., Karlsmark,T., van de Kerkhof,P.C.M., Larko,O., Nieboer,C., Roed-Petersen,J., Strand,A., and Tikjob,G., Double-blind, right/left comparison of calcipotriol and betamethasone valerate in treatment of psoriasis vulgaris. Lancet 1991. 337: 193-196. 7. Hutchinson,P.E., Marks,R., and White,J., The efficacy, safety and tolerance of calci- triol 3 microg/g ointment in the treatment of plaque psoriasis: a comparison with short- contact dithranol. Dermatology 2000. 201: 139-145. 8. de Mare,S., Calis,N., den Hartog,G., and van de Kerkhof,P.C.M., Outpatient treat- ment with short-contact dithranol. The impact of frequent concentration adjustments. Acta Derm.Venereol 1989. 69: 449-451. 9. Berth-Jones,J., Chu,A.C., Dodd,W.A., Ganpule,M., Griffiths,W.A., Haydey,R.P., Klaber,M.R., Murray,S.J., Rogers,S., and Jurgensen,H.J., A multicentre, parallel- group comparison of calcipotriol ointment and short-contact dithranol therapy in chronic plaque psoriasis. Br.J.Dermatol. 1992. 127: 266-271. 10. Tham,S.N., Lun,K.C., and Cheong,W.K., A comparative study of calcipotriol oint- ment and tar in chronic plaque psoriasis. Br.J Dermatol. 1994. 131: 673-677. 11. Kragballe,K., Barnes,L., Hamberg,K.J., Hutchinson,P., Murphy,F., Moller,S., Ruzicka,T., and van de Kerkhof,P.C.M., Calcipotriol cream with or without concur- rent topical corticosteroid in psoriasis: tolerability and efficacy. Br.J Dermatol. 1998. 139: 649-654. 12. Molin,L., Cutler,T.P., Helander,I., Nyfors,B., and Downes,N., Comparative efficacy of calcipotriol (MC903) cream and betamethasone 17-valerate cream in the treatment of chronic plaque psoriasis. A randomized, double-blind, parallel group multicentre study. Calcipotriol Study Group. Br.J Dermatol. 1997. 136: 89-93. 13. Swinkels,O.Q., Prins,M., Veenhuis,R.T., De Boo,T., Gerritsen,M.J.P., Van der Wilt,G.J., van de Kerkhof,P.C.M., and van der Valk,P.G.M., Effectiveness and side effects of UVB-phototherapy, dithranol inpatients therapy and a care instruction pro- gramme of short contact dithranol in moderate to severe psoriasis. An open randomised trial. submitted 2002.

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14. Fullerton,A., Avnstorp,C., Agner,T., Dahl,J.C., Olsen,L.O., and Serup,J., Patch test study with calcipotriol ointment in different patient groups, including psoriatic patients with and without adverse dermatitis. Acta Derm.Venereol. 1996. 76: 194-202. 15. Fullerton,A. and Serup,J., Characterization of irritant patch test reactions to topical D vitamins and all-trans retinoic acid in comparison with sodium lauryl sulphate. Evalu- ation by clinical scoring and multiparametric non-invasive measuring techniques. Br.J Dermatol. 1997. 137: 234-240. 16. Effendy,I., Kwangsukstith,C., Chiappe,M., and Maibach,H.I., Effects of calcipotriol on stratum corneum barrier function, hydration and cell renewal in humans. Br.J Der- matol. 1996. 135: 545-549. 17. Kemeny,L., Ruzicka,T., and Braun Falco,O., Dithranol: a review of the mechanism of action in the treatment of psoriasis vulgaris. Skin.Pharmacol. 1990. 3: 1-20. 18. Loffler,H., Effendy,I., and Happle,R., Skin susceptibility to dithranol: contact allergy or irritation? Eur.J Dermatol 1999. 9: 32-34. 19. Prins,M., Swinkels,O.Q., Mommers,J.M., Gerritsen,M.J.P., and van der Valk,P.G.M., Dithranol treatment of psoriasis in dithranol-sensitive patients. Contact Dermatitis 1999. 41: 116-117. 20. Yip,J. and Goodfield,M., Contact dermatitis from MC 903, a topical vitamin D3 ana- logue. Contact Dermatitis 1991. 25: 139-140. 21. Bruynzeel,D.P., Hol,C.W., and Nieboer,C., Allergic contact dermatitis to calcipotriol. Br.J Dermatol. 1992. 127: 66. 22. Steinkjer,B., Contact dermatitis from calcipotriol. Contact Dermatitis 1994. 31: 122. 23. de Groot,A.C., Contact allergy to calcipotriol. Contact Dermatitis 1994. 30: 242-243. 24. Molin,L., Contact dermatitis after calcipotriol and patch test evaluation. Acta Derm.Venereol. 1996. 76: 163-164.

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This thesis addresses several aspects of the epidermal barrier. The primary role of the epidermis is to form an efficient barrier for chemical, physical and microbiological threats. Epidermal growth takes care of the continuous adaptive formation of the stratum corneum (SC). The SC is a very efficient defence line against the penetration of chemicals, ultraviolet light and microorganisms. The barrier is not perfect and penetration of external agents may harm the viable layers of the epidermis. Also, external agents and environmental circumstances may cause direct harm to the SC. The injuries to the skin, both to the SC and the deeper layers, normally lead to regeneration and adaptation of the epidermal compartment. However, if the injury is severe or the epidermis is diseased, repair may be delayed or incomplete. If this is the case, disturbed epidermal differentiation will lead to sub-optimal barrier formation. Increased susceptibility to external agents may be the result and a vicious circle may ensue. Many skin diseases are associated with the impairment of epidermal barrier function. In this thesis we focused on three chronic skin disorders, irritant contact dermatitis (ICD), atopic dermatitis (AD) and psoriasis vulgaris (PV), more or less associated with skin barrier impairment. Since curative therapy of these diseases is still needed, the disturbed skin barrier is an attractive target for the development of new therapies. Therefore, new insights in the involvement of the impaired skin barrier in the pathogenesis of these diseases are necessary. In this final chapter we will discuss epidermal barrier function in these three disorders, in relation to our findings described earlier.

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6.1. BARRIER REPAIR STRATEGIES IN THE TREATMENT OF IRRITANT CONTACT DERMATITIS

Irritant contact dermatitis (ICD) may be caused by external factors directly influencing the barrier function of the SC. Occlusion and high ambient humidity may over-hydrate the skin, low ambient humidity may dry the skin and chemicals may damage the cellular and intercellular domain e.g. by washing out lipids. All these factors have an impact on the function of the SC, which may, per se, lead to skin irritation. External toxins may also penetrate the skin and give rise to damage to the viable layers of the epidermis with or without interaction with the SC. Damage to these deeper layers will lead to disturbed differentiation and SC formation. Skin irritation is under most circumstances quickly reversible. However, ICD may be very chronic and the question rises why healing takes often so much time despite strong reduction in skin exposure to irritants. Sustaining factors may involve persistent inflammation and/or persistent barrier disturbance. Therefore, anti- inflammatory therapy may be useful in accelerating the cure of the irritant dermatitis. Especially anti-inflammatory treatments which may have a beneficial effect on the function of the SC, or at least do not cause atrophy of the epidermal compartment such as dermatocorticosteroids, are good candidates. The development of such anti- inflammatory agents is hampered by the fact that more target sites could be relevant in the skin barrier repair. Furthermore, in most cases the interaction between target sites is unknown or debatable. Therefore, the elucidation of the relationship between anti- proliferative effects of such drugs and the skin barrier repair requires further studies. In most hyperproliferative conditions (e.g. psoriasis) the beneficial effect of the anti-proliferative and anti-inflammatory agents like dermatocorticosteroids has been shown.1 Contrary, in ICD associated with barrier impairment, the anti-proliferative effects of dermatocorticosteroids has been suggested to cause recovery delay of the skin barrier. 2;3

6.1.1. Cytokines as a therapeutical target

An attractive target for new anti-inflammatory agents is the modulation of cytokines, since up-regulation may both play a role in the pathogenesis of ICD and on repair processes after barrier disruption. The inhibition of their production may diminish the inflammatory response accompanying ICD.4 However, the interaction between cytokines, (especially TNF-α) and barrier repair remains to be clarified. Some animal studies show a marked increase in epidermal cytokines in response to barrier disruption suggesting their major role in skin barrier repair.5;6 In contrast, the development of TNF receptor type I and IL-1 receptor deficient mice, as a decisive model, raises doubts about the role of TNF-α and IL-1 in barrier repair. Since a normal phenotype and normal recovery after barrier disruption in these knock-out mice has

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been observed, some authors suggested that TNF-α is probably not essential in the process of skin repair.7 Therefore, the modulation of TNF-α and other cytokines has probably no effect on repair. This implies that the development of new anti- inflammatory agents targeting the improvement of the skin barrier, should be based on more precise insights in the pathogenesis of ICD. In this thesis, we investigated a new anti-inflammatory agent (cipamfylline), which by inhibition of phosphodiesterase-4 (PDE-4) is able to suppress cytokines.8 We know that PDE-4 inhibitors are able to suppress the production of cytokines (e.g. TNF-α) in keratinocytes. 9;10 PDE-4 inhibitors have been used successfully in the treatment of atopic dermatitis as well.11 Therefore, we considered a PDE-4 inhibitor as a good candidate for the treatment of ICD, also because this principle is unlikely to cause skin atrophy, the well-established side effect of topical corticosteroids. In our study described in paragraph 4.1, we could not confirm a clinical effect of cipamfylline in an acute and a cumulative exposure model using applications of a detergent (SDS). In contrast, a positive control (betamethasone-17-valerate) showed a beneficial effect on erythema scores and on barrier repair as assessed by transepidermal water loss (TEWL) in the cumulative exposure model. We observed an inhibition of the number of proliferating cells and an inhibition of the expression of cytokeratin 16 in betamethasone-17-valerate treated sites compared to the control sites. Again cipamfylline did not show any effect on these immunohistochemical parameters. In addition to the potential dual relation between cytokines and skin barrier repair, some methodological reasons for the lack of effect in our study can be taken into consideration: (1) The bioavailability of cipamfylline from the selected vehicle may be sub- optimal. In our study it was not possible to assess tissue-levels of the drug. (2) Therapeutic agents may suppress an inflammatory response, but do not repair cellular or architectural damage. The inflammatory response, although causing short-term clinical effects (symptoms) may be essential for long-term recovery. Anti-inflammatory drugs may suppress short-term effects, but aggravate the condition in the long run. In neither of these models we prolonged our observation until erythema cleared and baseline values of TEWL were reached. It may be very interesting to find out how many days are needed until complete recovery of the skin occurs upon different therapeutical agents and whether long term therapy with PDE-4 inhibitor can cause increased skin susceptibility to irritants as observed by some authors after long term use of dermato corticosteroids. (3) Since exposure to irritants can lead to specific cutaneous reactions depending on the nature of irritants we cannot exclude any efficacy of PDE-4 inhibitors in skin irritation caused by another irritant stimulus.

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(4) The difference in the course of SDS-induced skin irritation (complete healing in 1-2 weeks) and chronic ICD (healing in weeks to months) points out the limitations of these models and indicate that results of intervention in these models should be interpreted with caution until verified in clinical conditions.

In conclusion, PDE-4 inhibitors are not likely to induce skin atrophy, tachyphylaxis and rebound phenomena. Therefore, despite the negative results of our pilot study, it is still the possible that cipamfylline might be effective in the treatment of ICD and further clinical studies are recommended. Whether the therapeutic effects both in vivo models and in clinical trials will be beneficial or not, will be dependent on its intrinsic pharmacodynamic effect and its bioavailability.

6.1.2. Lipid metabolism as a therapeutical target

SC lipids play a crucial role in skin barrier and its water-holding capacity.12 The positive effect of topical lipids on the reinforcement of skin barrier in ICD has been documented in experimental studies.13 For long we considered lipids as part of the vehiculum with only physical effects on the SC. Lipids are occlusive and therefore may form a barrier for water, resulting in hydration of the skin and alteration of barrier characteristics.14 In recent years is became clear that lipids may penetrate the intercellular domain of the SC and may have specific effects on the composition of the lipid domain and on epidermal differentiation.15 A new generation of emollients with a specific lipid profile and a specific lipid content approaching the physiological situation is under development, but evidence for superiority in human to the traditional emollients remains to be provided. Based on the studies described in chapter 4, we can conclude that the application of topical lipids on ICD associated with skin barrier impairment is advisable. However, in our pilot clinical study where we investigated the efficacy of the emollients in heterogeneous group of hand dermatitis, we could not confirm the superiority of treatment with skin related lipids as we observed earlier in the in vivo models for ICD. We cannot rule out that long term treatment of ICD with skin related lipids may have some superior effects especially when investigated in a large number of patients with more clearly defined presentations of ICD. In the context of the results described in this thesis (chapter 4) some recommendations could be made concerning the use of topical lipids in ICD. It is very likely that an ideal emollient, effective in all clinical situations, does not exist. The pathogenesis-based barrier repair therapy should be based on understanding of the specific damage and recovery.16 Therefore, initially the purpose of application of emollients should be defined in an individual patient. If protection of healthy skin against a detergent is intended, an emollient with a high content of lipids before

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the exposure to irritants seems to be appropriated. Such emollients may provide a relative sufficient protection against cumulative exposure to a weak irritant. However, emollients with a high content of lipids are not convenient to use in all occupational or environmental settings. An emollient with less fat may suffice, although strong drying effects should be avoided. Of course, during the short exposure to a strong irritant, such an emollient is failing and some other kind of protection or a combination of protective measures should be considered. If emollients accelerate the skin barrier repair in already damaged skin, the correction with a three-component formulation of skin related lipids (cholesterol, ceramide, free fatty acid) in an optimized ratio could be of benefit. The results of our experimental study (paragraph 4.2) support the concept of supplementation with skin related lipids after mechanical damage. One should realize that such a recommendation is not applicable for all formulations consisting of skin related lipids, because of considerable variations of the ratio, concentration, and structuring between such products. This thesis does not provide enough evidence for the superiority of the formulation with skin related lipids, so their use cannot generally be recommended. It is still unknown whether formulation with skin related lipids can prevent irritant and allergic reactions of the skin and also whether they can enhance the therapeutical activity of other pharmacologically active agents. Some key questions regarding topical lipids still remain to be answered. Does an exchange between exogenous and endogenous lipids exist in human skin? Does incorporation of skin related lipids in epidermal cells exist? Is incorporation of skin related lipids in epidermal cells possible? Does the extent of occlusive effect influence the efficacy of the product? Further research should focus on these questions and on the development of standardized test models for topical lipids.

6.1.3. Development of new agents targeting skin barrier repair in ICD

New treatment modalities focusing on the improvement of barrier function should be based on new insights in homeostatic repair response in ICD. As described in chapter 1, lipids play a key role in the formation of skin barrier. In this thesis, topical application of suitable lipids as one method for skin barrier repair was investigated. Skin barrier repair can be accelerated not only by the topical application of lipids but also by regulation of nonlipid factors such as enzymes and ions. Lipid metabolism is regulated by a series of enzymes in the epidermis and each of them has its optimal pH and optimal ion balance.17 Calcium, magnesium and potassium in human epidermis play an important role in the lipid metabolism of the epidermis. It has been shown that the topical application of calcium or potassium impairs barrier repair, whereas magnesium or a mixture of calcium and magnesium salts accelerates the repair process.18 From the observation described in chapter 4 it is evident, that the treatment

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of ICD does not have one single mode of action. An important role of the nuclear hormone receptor PPAR-α (peroxysome-proliferator-activated receptor-α) on the formation and maintenance of epidermal lipid barrier has been demonstrated.19-22 It has also been shown that topical application of PPAR-α activators (clofibrate, linoleic acid) accelerate barrier recovery after tape stripping or acetone treatment.23 Regulation of the nuclear hormone receptor could open a new possibility for improvement of the skin barrier function. In this respect, the elucidation of the function of epidermal fatty acid carriers can possibly provide an intervention target, since the interaction between epidermal fatty acid carriers and PPAR-α has been demonstrated.24 Furthermore, the relationship between the skin barrier function and the histamine H1 and H2 receptor has been reported. The topical application of histamine H1 and H2 receptor antagonist accelerated the barrier repair. Histamine itself delayed the barrier repair.25 Although the mechanism of the relationship between the histamine receptors and the barrier repair process has not been clarified, these findings provide another perspective target for treatment of ICD. In general, topical treatment is preferable above systemic treatment in ICD, because systemic (immunosuppressive or immunoregulatory) treatment may cause severe side effects. On the other hand, the potential irritancy of a topical formulation and/or its ingredients is possibly a considerable disadvantage of topical treatment. In highly irritable skin and/or in vesicobullous diseases systemic treatment should be considered for breaking a vicious circle. To date, no sufficient systemic treatment of ICD is available. In this context, modulation of retinoid X receptors (RXR’s) could be an alternative since an important role of retinoid X receptors (RXR’s) for the SC has been suggested.26 From the therapeutical agents, 9-cis-Retinoid Acid (9-cis-RA) has a particular pattern of binding and activating retinoid X receptors (RXR’s). Oral 9- cis-RA has already been investigated in the treatment of chronic hand dermatitis with promising results.27;28 Whether oral 9-cis-RA can really accelerate the barrier repair is unknown and could be the focus of further experimental and clinical studies. In conclusion, different signaling pathways could be targets in the development of effective treatment. Replenishing of suitable lipids is one of the possibilities. But also the pharmacological regulation of the endogenous homeostatic process of the epidermal inflammation, proliferation and differentiation may be effective in the improvement of the skin barrier. Further studies on the pathogenesis of ICD and further insights in the mode of action of potential pharmacological agents are important investments for patients of tomorrow.

6.1.4. Prevention

Topical therapy of ICD is unsatisfactory and arduous. Not only the disappointing results of the pharmacological agents, but also a risk of skin irritation from the topical formulations prompt us to look for other approaches. ICD is one

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example of skin diseases where sufficient primary and secondary prevention could significantly decrease the prevalence of this disease with a major social impact. Therefore, preventive measurements in ICD are required and should be a main interest of further research. Preventive measurements include (1) identification of individuals with a higher risk of ICD and (2) identification and avoidance of potential irritants.

(1)Identification of individuals with a higher risk of ICD

Individuals with an atopic dermatitis or a history of atopic dermatitis have an increased susceptibility to irritants.29 However, increased responsiveness to irritants is not only restricted to such susceptible phenotypes but can be extended to the healthy, nonatopic population. In the non-atopic population it is difficult to identify individuals with a predisposition to over-response to irritants, because reliable prospective data for such risk persons are scarce. Many investigators have attempted to identify factors, which affect reactivity to irritants, as summarized in the Table 4 of chapter 1. Possible underlying biological mechanisms should be considered. Among them, a genetic predisposition to inflammation is under current attention since gene polymorphisms seem to play an important role.30-32 Since cytokines have been implicated in the pathogenesis of ICD, it is not unreasonable to speculate that interindividual variations in the level of their production give rise to variations in the intensity of an inflammatory response. Recently the first description of a nonatopic genetic marker for irritant susceptibility in normal individuals has been published. A polymorphisms at position-308 of the TNF-α gene has been associated with susceptibility to experimentally induced ICD in response to SDS.33 Such suggests that cytokine genotyping may be an important screening method for use in occupations associated with high risk of ICD in the future. In chapter 1 we already introduced oxidative stress as a component involved in ICD, especially in dithranol induced skin irritation. There is evidence that polymorphisms in genes encoding several of the antioxidant enzymes, have an influence on the extent of cellular damage associated with oxidative stress.30 The genetic variability of the capacity of an individual’s skin to scavenge free radicals may also be responsible for variation in dithranol induced skin irritation.

(2) Identification and avoidance of potential irritants

Although complete avoidance of contact with irritants is usually not needed, strict reduction may be necessary and could lead to improvement of chronic ICD. This reduction can be achieved by appropriate use of gloves and protective clothing. For household tasks, rubber or PVC gloves should suffice. When protective gloves are used for more than 10 minutes, cotton gloves should be worn underneath. It is important to take off the gloves on a regular basis, because sweating may aggravate

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existing dermatitis.34 The gloves can create an occlusive environment, which is an irritant situation in itself and may impair the barrier function.35;36 If an irritant gets into the glove, the occlusive milieu leads to an increased penetration and stronger irritant reactions.37 In an occupational setting, the most appropriate glove for prevention will depend upon the nature of the irritant stimuli, because penetration of chemicals differs strongly between different materials.38;39 Substitution of non-irritating agents could be another protective measure. The most common example is a soap substitute.40 Theoretically, a barrier cream often called ‘invisible gloves’ may protect against well-defined chemical substances. Since ICD is only rarely caused by one substance and stimulus41, barrier cream must protect against a wide spectrum of irritant stimuli during the entire period of exposure. Until now, barrier creams are usually not tested sufficiently in vivo against a combination of different irritants.42;43 Therefore, their use is limited and cannot be generally promoted, because of a false sense of security is offered.44 Furthermore, it has to be kept in mind that barrier creams may have an irritant potential on their own, therefore their use cannot be generally recommended. Summarizing we can state that the prevention by avoidance of skin contact to irritants is a rewarding approach. Because patients or employees in high-risk jobs are often not aware of the exposure and/or the nature of irritant, education in preventive action and instruction in proper skin care should be offered.

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6.2. BARRIER REPAIR STRATEGIES IN THE TREATMENT OF ATOPIC DERMATITIS

Most treatment strategies for atopic dermatitis (AD) such as corticosteroids, calcineurin-inhibitors (tacrolimus) are targeted primarily at the immunopathogenesis of AD. These approaches result in secondary improvement of the barrier function that parallels the clinical response. The involvement of skin barrier impairment in the pathogenesis of AD is unambiguous. The skin barrier for chemicals, physical influences and micro-organisms is perturbed. (For more details see chapter 1) Involved and uninvolved skin of AD patients exhibit a barrier deficit which correlates with a reduction in the ceramide fraction of the intercellular lipid domain.45;46 This abnormality is so prominent that some authors consider AD as a ceramide-deficient disease.47 Although emollients are mainstays in the management of AD, currently available emollients do not correct this prominent SC lipid abnormality. Ceramide- dominant formulation of skin related lipids may represent a rational intervention.16 Preliminary trials with a ceramide-dominant formulation significantly reduced clinical symptoms in children with recalcitrant AD.48;49 Further controlled studies are needed to validate this approach in the therapy of AD. In addition, from a number of studies it can be concluded that bacterial superantigens contribute to the pathogenesis and exacerbation of AD.50-52 The mechanisms underlying the increased bacterial colonization of atopic skin are poorly understood. As mentioned in chapter 1, the skin is the first line of defence against invasion of microbial agents. The failure of the innate immune defence system of atopic skin against bacterial invasion should be considered. Recently, it was speculated that insufficient expression of antimicrobial peptides, as elements of the innate immune system, may be involved in the pathogenesis of the increased susceptibility to bacterial infection in atopic patients.53 In this thesis (chapter 3) we extended this observation to other antimicrobial peptides (SKALP, SLPI and MRP8). Consistent with the study by Ong et al we found a decreased level of several of these peptides in AD versus psoriasis, although expression of differentiation markers involucrin and keratin 16 was comparable in both conditions. This finding provides a strong evidence for a significant role of host defence proteins in superinfections in AD. Identification of the molecules involved in the process of enhanced bacterial colonization may have important implications in the approach to manage and develop new therapies for AD. Whether keratinocytes in AD have an intrinsic defect in the control of bacterial colonization by polymorphisms in genes encoding antimicrobial peptides or driving their expression, may become a fascinating new field of research. The possibility that bacterial overgrowth can exacerbate acute AD provides a rationale for the use of antibacterial therapy in patients with AD. Topical anti-septic may be of value, but may irritate the skin.54 Topical antibiotics such as 2% mupirocine ointment or 2% sodium fusidate ointment may be considered.55 Due to increased

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risk of bacterial resistance, it is important to combine antimicrobial therapy with effective skin care since it is well established that the excoriated inflamed skin of AD predisposes to S. aureus colonization. Therefore the use of antibiotic therapy must be carried out with good skin hydration to restore skin barrier function and effective anti-inflammatory therapy to reduce skin inflammation.56 From this point of view the combination of topical corticosteroids with antibiotic in an optimal vehiculum can be recommended.57 Regarding the vehiculum, a non-irritative basis should be guaranteed. Recently, it has been demonstrated that levels of sphingosine (as a natural antimicrobial agent) are significantly reduced in epidermis of AD patients.58 Focusing on the improvement of the antibacterial defence system, adding sphingosine to the lipid formulations may be an important therapeutic approach for reduction of bacterial growth in atopic skin.

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6.3. BARRIER REPAIR STRATEGIES IN THE TREATMENT OF PSORIASIS

It is evident that psoriasis has a complex pathogenesis. A role for keratinocytes, endothelium as well as T-lymphocytes has been shown, which provide multiple targets for anti-psoriatic treatment. As mentioned in chapter 1, psoriasis is associated with skin barrier impairment. The improvement of skin barrier and implementation of barrier repair strategies could play an important role in the antipsoriatic therapy. The first anti-psoriatic therapy that interferes with barrier function, are emollients that are widely used in the treatment of psoriasis.14 Generally, emollients may interact with the epidermis in several ways. These complex interactions comprise physical effects like occlusions, direct effects on the SC and indirect effects on epidermal differentiation and hyperproliferation. In psoriasis, especially emollients with occlusive properties are widely used to soften the scaly hyperkeratotic surface and enhance the penetration of other topical therapies.59 Barrier abnormalities in psoriasis are consistent with disturbances in the lipid metabolism.60-63 Theoretically, topical lipids may correct these disturbances. It is not unreasonable to speculate that an emollient with a specific lipid profile could be advantageous in the correction of these psoriasis-specific lipid abnormalities. Unfortunately, since little is known about the exact underlying lipid disorder in psoriasis, it is impossible to apply the principle of a disease specific barrier strategy, as in atopic skin. Therefore, further research on the lipid metabolism in this skin disease is needed. In de context of disturbed lipid metabolism another interesting target could be the pharmacological modulation of PPAR-α (peroxysome-proliferator-activated receptor α). PPAR-α is a key-transcription factor involved in the control of the epidermal lipid barrier24 and is downregulated in involved psoriatic epidermis.22 Further studies should demonstrate if the modulation of PPAR-α with selective agonists (e.g. free fatty acid) may have a beneficial action on the impaired skin barrier of psoriasis. We already mentioned that emollients could enhance the penetration of topical treatment. Besides occlusive emollients, this could be achieved using an occlusive dressing in particular a hydrocolloid dressing.64 In a psoriatic plaque, a combination of a hydrocolloid dressing with topical corticosteroids is very effective.65;66 This provides evidence for a new pharmacological principle with an intensified topical anti-psoriatic action, increased bioavailability, decreased dose and providing optimal patient compliance. In addition, occlusion has an anti-psoriatic potential on its own.67 This therapy stimulates regeneration of the disturbed granular layer and reduces epidermal hyperproliferation, a hallmark of psoriasis.68;69 The exact mechanisms have not been elucidated. It has been suggested that occlusion restores the skin barrier, which leads to recovery of the keratinization process and a reduction in psoriatic hyperproliferation. Contrary, some animal and human experimental studies demonstrated that occlusion, which prevents water loss, does not prevent the development of these epidermal

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abnormalities70;71, denying the need of skin barrier repair in the therapy of psoriasis. Despite of these conflicting results, many clinical studies demonstrated the beneficial effect of occlusion in psoriasis.

6.3.1. The relevance of skin barrier function in psoriasis treatment

Topical treatment of psoriasis is an obvious choice. Because of frequent skin irritation by anti-psoriatic treatment, topical formulations should be used, which do not compromise the skin barrier and give raise to skin irritation. This is impossible in the case of dithranol where the skin irritation parallels its therapeutic effect. Attempts have been made to reduce this side effect by changing the treatment time or the vehicle (e.g. without salicylic acid). Penetration of the epidermis is enhanced in a lipophilic vehicle. The other possibility is a pharmacological modulation of dithranol irritation. As dithranol induces inflammatory changes in normal skin and topical corticosteroids have anti-inflammatory effects, it is likely that this treatment represents a complementary approach. The experimental study described in chapter 5 suggests that a short intervention with a superpotent corticosteroid is an alternative to suppress dithranol irritation. The role of topical costicosteroids in the modulation of dithranol irritation is controversial. On the one side, corticosteroids may cause in short time the clinical and subjective improvement of skin irritation. On the other side, it is suggested that sensitivity to dithranol depends on the status of the skin barrier.72 The frequent use of costicosteroids may influence the skin barrier and make skin more vulnerable to skin irritation. From this aspect, new anti-inflammatory agents without these negative effects on the skin barrier could be promising. Furthermore, in this thesis (chapter 5) we demonstrated a relatively high frequency of skin irritation during dithranol treatment. In paragraph 5.3 it was shown that dithranol irritation and the treatment results assessed by PASI-scores were not associated. Therefore, modulation of dithranol irritation is unlikely to improve the therapeutic efficacy, however reduction of dithranol irritation by dermatocorticosteroids and other anti-inflammatory treatments can be useful to reduce the subject’s discomfort. Controlled, long-term studies with different therapeutical approaches in dithranol irritation are needed.

Conclusion

In conclusion, the field of barrier repair therapies is still in its infancy. Very few studies have evaluated the clinical efficacy of barrier repair therapy. A lot of work remains to be done before barrier repair therapeutics will have an important position in the treatment of skin diseases. There is no doubt that the current efforts in understanding the skin barrier repair will lead to the design of new agents, which specifically target various abnormalities of the skin barrier repair contributing to these skin conditions.

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GENERAL CONCLUSIONS

These general conclusions are based on the answers to the questions as we defined in the introduction.

1.1 In the present thesis it has been shown that expression of E-FABP in dithranol induced irritation is comparable to the normal skin. This lack of upregulation of E-FABP in such an non-corrosive type of irritation (not associated with skin barrier impairment), in contrast to the upregulation following the skin barrier impairment in SDS induced irritation, indirectly suggests a possible role of E-FABP in the maintenance of epidermal skin repair.

1.2 The expression of E-FABP in atopic skin shows clear differences between normal human skin and psoriasis. E-FABP is a good candidate for immunohistochemical differentiation of both inflammatory disorders.

2.1 The pattern of expression of some antimicrobial proteins in the epidermis is different in atopic dermatitis and psoriasis. These differences may explain the susceptibility of patients with atopic dermatitis to skin infections. Psoriatic epidermis expresses high levels of antimicrobial proteins compared to atopic dermis.

3.1 Cipamfylline, a strong phosphodiesterase-4 inhibitor does not show an effect on skin barrier repair in SDS induced irritation.

3.2 Topical lipids improve skin barrier in different conditions compared to untreated skin. An ideal lipid formulation effective in all clinical situations does not exist. The optimal barrier repair therapy should be based on understandings of the specific damage and recovery. There is some indication that formulations containing skin related lipids might be of benefit in mechanical barrier disruption.

3.3 Frequent use of topical lipids may be useful in the therapy of hand dermatitis and patients should be encouraged to use them. However, we cannot confirm the concept of superiority of skin related lipids in a heterogeneous group of patients with chronic hand dermatitis.

4.1 Using a repetitive model of dithranol irritation we observed that the potent corticosteroids are effective in reducing dithranol erythema, whereas coal tar solution does not.

4.2 Dithranol induced skin irritation occurs preferentially in an early phase of dithranol treatment.

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4.3 Skin irritation during short contact therapy with dithranol has not a relevant impact on the treatment results. The PASI does not have any predictive value for dithranol induced skin irritation.

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References

1. van de Kerkhof,P.C.M., Steegers-Theunissen,R.P., and Kuipers,M.V., Evaluation of topical drug treatment in psoriasis. Dermatology 1998. 197: 31-36. 2. Parillo,J. and Faucu,A., Mechanisms of glucocorticoid action on immune processes. Ann Rev Pharmacol Toxicol 1979. 19: 179-201. 3. Epstein,E., Hand dermatitis: practical management and current concepts. J.Am.Acad.Dermatol 1984. 10: 395-424. 4. Thestrup,P.K. and Halkier,S.L., Mechanism of irritant contact dermatitis. In van der Valk,P.G. and Maibach,H.I. (Eds.) The irritant contact dermatitis syndrome. CRC press, Inc, 1996, pp 305-309. 5. Wood,L.C., Jackson,S.M., Elias,P.M., Grunfeld,C., and Feingold,K.R., Cutane- ous barrier perturbation stimulates cytokine production in the epidermis of mice. J.Clin.Invest 1992. 90: 482-487. 6. Nickoloff,B.J. and Naidu,Y., Perturbation of epidermal barrier function correlates with initiation of cytokine cascade in human skin. J Am Acad Dermatol 1994. 30: 535-546. 7. Man,M.Q., Wood,L., Elias,P.M., and Feingold,K.R., Cutaneous barrier repair and pathophysiology following barrier disruption in IL-1 and TNF type I receptor deficient mice. Exp Dermatol 1999. 8: 261-266. 8. Griswold,D.E., Webb,E.F., Badger,A.M., Gorycki,P.D., Levandoski,P.A., Barnette,M.A., Grous,M., Christensen,S., and Torphy,T.J., SB 207499 (Ariflo), a second generation phosphodiesterase 4 inhibitor, reduces tumor necrosis factor alpha and interleukin-4 production in vivo. J Pharmacol Exp Ther 1998. 287: 705-711. 9. Strieter,R.M., Remick,D.G., Ward,P.A., Spengler,R.N., Lynch,J.P., III, Larrick,J., and Kunkel,S.L., Cellular and molecular regulation of tumor necrosis factor-alpha pro- duction by pentoxifylline. Biochem.Biophys.Res.Commun. 1988. 155: 1230-1236. 10. Schwarz,A., Krone,C., Trautinger,F., Aragane,Y., Neuner,P., Luger,T.A., and Schwarz,T., Pentoxifylline suppresses irritant and contact hypersensitivity reactions. J Invest Dermatol 1993. 101: 549-552. 11. Hanifin,J.M., Chan,S.C., Cheng,J.B., Tofte,S.J., Henderson,W.R., Jr., Kirby,D.S., and Weiner,E.S., Type 4 phosphodiesterase inhibitors have clinical and in vitro anti- inflammatory effects in atopic dermatitis.J Invest Dermatol 1996. 107: 51-56. 12. Elias,P.M., Lipids and the Epidermal Permeability Barrier. Arch.Dermatol Res. 1981. 270: 95-117. 13. Agner,T. and Held,E., Moisturizes: Evidence for Efficacy? In Maibach,H.I., Bashir,S., and McKibbon,A. (Eds.) Evidence-Based Dermatology. BC Decker Inc, London 2002, pp 233-243. 14. Ghadially,R., Halkier,S.L., and Elias,P.M., Effects of petrolatum on Stratum corneum structure and function. J.Am.Acad.Dermatol. 1992. 26: 387-396. 15. Yang,L., Mao,Q.M., Taljebini,M., Elias,P.M., and Feingold,K.R., Topical Stratum corneum lipids accelerate barrier repair after tape stripping, solvent treatment and some but not all types of detergent treatment. Br.J Dermatol. 1995. 133: 679-685. 16. Elias,P.M. and Feingold,K.R., Does the tail wag the dog? Role of the barrier in the pathogenesis of inflammatory dermatoses and therapeutic implications. Arch.Dermatol 2001. 137: 1079-1081. 17. Denda,M., Role of lipids in skin barrier function. In Forster,T. (Ed.) Cosmetic Lipids and the Skin Barrier. Marcel Dekker, Inc, New York 2002, pp 97-120.

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18. Denda,M., Katagiri,C., Hirao,T., Maruyama,N., and Takahashi,M., Some magnesi- um salts and a mixture of magnesium and calcium salts accelerate skin barrier recovery. Arch.Dermatol Res. 1999. 291: 560-563. 19. Hanley,K., Komuves,L.G., Bass,N.M., He,S.S., Jiang,Y., Crumrine,D., Appel,R., Friedman,M., Bettencourt,J., Min,K., Elias,P.M., Williams,M.L., and Feingold,K.R., Fetal epidermal differentiation and barrier development In vivo is accelerated by nuclear hormone receptor activators. J.Invest Dermatol 1999. 113: 788- 795. 20. Komuves,L.G., Hanley,K., Man,M.Q., Elias,P.M., Williams,M.L., and Feingold,K.R., Keratinocyte differentiation in hyperproliferative epidermis: topical application of PPARalpha activators restores tissue homeostasis. J.Invest Dermatol 2000. 115: 361-367. 21. Komuves,L.G., Hanley,K., Lefebvre,A.M., Man,M.Q., Ng,D.C., Bikle,D.D., Williams,M.L., Elias,P.M., Auwerx,J., and Feingold,K.R., Stimulation of PPARal- pha promotes epidermal keratinocyte differentiation in vivo. J.Invest Dermatol 2000. 115: 353-360. 22. Rivier,M., Safonova,I., Lebrun,P., Griffiths,C.E., Ailhaud,G., and Michel,S., Dif- ferential expression of peroxisome proliferator-activated receptor subtypes during the differentiation of human keratinocytes. J.Invest Dermatol 1998. 111: 1116-1121. 23. Feingold,K.R., Role of nuclear hormone receptors in regulating epidermal differentia- tion. Society of Cosmetic Chemists 1999. 50: 30-31. 24. Rivier,M., Castiel,I., Safonova,I., Ailhaud,G., and Michel,S., Peroxisome prolif- erator-activated receptor-alpha enhances lipid metabolism in a skin equivalent model. J.Invest Dermatol 2000. 114: 681-687. 25. Ashida,Y., Denda,M., and Hirao,T., Histamine H1 and H2 receptor antagonists ac- celerate skin barrier repair and prevent epidermal hyperplasia induced by barrier disrup- tion in a dry environment. J.Invest Dermatol 2001. 116: 261-265. 26. Imakado,S., Bickenbach,J.R., Bundman,D.S., Rothnagel,J.A., Attar,P.S., Wang,X.J., Walczak,V.R., Wisniewski,S., Pote,J., Gordon,J.S., and ., Targeting expression of a dominant-negative retinoic acid receptor mutant in the epidermis of transgenic mice results in loss of barrier function. Genes Dev. 1995. 9: 317-329. 27. Bollag,W. and Ott,F., Successful treatment of chronic hand eczema with oral 9-cis- retinoic acid. Dermatology 1999. 199: 308-312. 28. Larsen, F, Galewicz, D, Ruzicka, T., Horvath, A., Coenraads, P. J., Harsch, M., Brown, T., and Zultak, M. Alitretinoin (9-cis retinoic acid) is effective and well-toler- ated in the treatment of refractory chronic hand dermatitis. J Eur.Acad.Dermatol.Vene reol. 2003. Abstract 29. Agner,T., Susceptibility of atopic dermatitis patients to irritant dermatitis caused by sodium lauryl sulphate. Acta Derm.Venereol. 1991. 71: 296-300. 30. Willis,C.M., Variability in responsiveness to irritants: thoughts on possible underlying mechanisms. Contact Dermatitis 2002. 47: 267-271. 31. Hajeer,A.H. and Hutchinson,I.V., TNF-alpha gene polymorphism: clinical and bio- logical implications. Microsc.Res.Tech. 2000. 50: 216-228. 32. Hajeer,A.H. and Hutchinson,I.V., Influence of TNF-αlpha gene polymorphisms on TNF-αlpha production and disease. Hum.Immunol. 2001. 62: 1191-1199.

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33. Allen,M.H., Wakelin,S.H., Holloway,D., Lisby,S., Baadsgaard,O., Barker,J.N., and McFadden,J.P., Association of TNFA gene polymorphism at position -308 with sus- ceptibility to irritant contact dermatitis. Immunogenetics 2000. 51: 201-205. 34. Bourke,J., Coulson,I., and English,J., Guidelines for care of contact dermatitis. Br J Dermatol 2001. 145: 877-885. 35. Ramsing,D.W. and Agner,T., Effect of glove occlusion on human skin. (I). short-term experimental exposure. Contact Dermatitis 1996. 34: 1-5. 36. Ramsing,D.W. and Agner,T., Effect of glove occlusion on human skin (II). Long-term experimental exposure. Contact Dermatitis 1996. 34: 258-262. 37. van der Valk,P.G.M. and Maibach,H.I., Post-application occlusion substantially increases the irritant response of the skin to repeated short-term sodium lauryl sulfate (SLS) exposure. Contact Dermatitis 1989. 21: 335-338. 38. Mathias,C.G., Prevention of occupational contact dermatitis. J Am.Acad.Dermatol. 1990. 23: 742-748. 39. Mellstrom,G.A. and Bowman,A., Protective gloves. In Kanerva,L., Elsner,P., Wahlberg,J.E., and Maibach,H. (Eds.) Handbook of Occupational Dermatology. Springer, Berlin 2000, pp 416-425. 40. Lauharanta,J., Ojajarvi,J., Sarna,S., and Makela,P., Prevention of dryness and ec- zema of the hands of hospital staff by emulsion cleansing instead of washing with soap. J Hosp.Infect. 1991. 17: 207-215. 41. Wigger-Alberti,W., Krebs,A., and Elsner,P., Experimental irritant contact dermatitis due to cumulative epicutaneous exposure to sodium lauryl sulphate and toluene: single and concurrent application. Br.J.Dermatol 2000. 143: 551-556. 42. Frosch,P.J. and Kurte,A., Efficacy of skin barrier creams (IV). The repetitive irritation test (RIT) with a set of 4 standard irritants. Contact Dermatitis 1994. 31: 161-168. 43. Frosch,P.J., Schulze-Dirks,A., Hoffmann,M., and Axthelm,I., Efficacy of skin bar- rier creams (II). Ineffectiveness of a popular “skin protector” against various irritants in the repetitive irritation test in the guinea pig. Contact Dermatitis 1993. 29: 74-77. 44. Loffler,H. and Effendy,I., Prevention of irritant contact dermatitis. Eur J Dermatol 2002. 12: 4-9. 45. Yamamoto,A., Serizawa,S., Ito,M., and Sato,Y., Stratum corneum lipid abnormalities in atopic dermatitis. Arch.Dermatol Res. 1991. 283: 219-223. 46. di Nardo,A., Wertz,P., Giannetti,A., and Seidenari,S., Ceramide and cholesterol composition of the skin of patients with atopic dermatitis. Acta Derm.Venereol. 1998. 78: 27-30. 47. Hara,J., Higuchi,K., Okamoto,R., Kawashima,M., and Imokawa,G., High-expres- sion of sphingomyelin deacylase is an important determinant of ceramide deficiency leading to barrier disruption in atopic dermatitis. J.Invest Dermatol. 2000. 115: 406- 413. 48. Chamlin,S.L., Frieden,I.J., Fowler,A., Williams,M., Kao,J., Sheu,M., and Elias,P.M., Ceramide-dominant, barrier-repair lipids improve childhood atopic derma- titis. Arch.Dermatol 2001. 137: 1110-1112. 49. Chamlin,S.L., Kao,J., Frieden,I.J., Sheu,M.Y., Fowler,A.J., Fluhr,J.W., Williams,M.L., and Elias,P.M., Ceramide-dominant barrier repair lipids alleviate childhood atopic dermatitis: changes in barrier function provide a sensitive indicator of disease activity. J Am.Acad.Dermatol. 2002. 47: 198-208.

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50. Leyden,J.J., Marples,R.R., and Kligman,A.M., Staphylococcus aureus in the lesions of atopic dermatitis. Br.J Dermatol. 1974. 90: 525-530. 51. Leung,D.Y., Atopic dermatitis: new insights and opportunities for therapeutic interven- tion. J.Allergy Clin.Immunol. 2000. 105: 860-876. 52. Leung,D.Y., Atopic dermatitis and the immune system: the role of superantigens and bacteria. J.Am.Acad.Dermatol. 2001. 45: S13-S16. 53. Ong,P.Y., Ohtake,T., Brandt,C., Strickland,I., Boguniewicz,M., Ganz,T., Gallo,R.L., and Leung,D.Y., Endogenous antimicrobial peptides and skin infections in atopic dermatitis. N.Engl.J.Med. 2002. 347: 1151-1160. 54. Stalder,J.F., Fleury,M., Sourisse,M., Allavoine,T., Chalamet,C., Brosset,P., and Litoux,P., Comparative effects of two topical antiseptics (chlorhexidine vs KMn04) on bacterial skin flora in atopic dermatitis. Acta Derm.Venereol.Suppl (Stockh) 1992. 176: 132-134. 55. Lever,R., Hadley,K., Downey,D., and MacKie,R., Staphylococcal colonization in atopic dermatitis and the effect of topical mupirocin therapy. Br.J.Dermatol. 1988. 119: 189-198. 56. Stalder,J.F., Fleury,M., Sourisse,M., Rostin,M., Pheline,F., and Litoux,P., Local steroid therapy and bacterial skin flora in atopic dermatitis. Br.J Dermatol. 1994. 131: 536-540. 57. Wachs,G.N. and Maibach,H.I., Co-operative double-blind trial of an antibiotic/ corticoid combination in impetiginized atopic dermatitis. Br.J Dermatol. 1976. 95: 323-328. 58. Arikawa,J., Ishibashi,M., Kawashima,M., Takagi,Y., Ichikawa,Y., and Imokawa,G., Decreased levels of sphingosine, a natural antimicrobial agent, may be associated with vulnerability of the Stratum corneum from patients with atopic dermati- tis to colonization by Staphylococcus aureus. J.Invest Dermatol. 2002. 119: 433-439. 59. Greaves,M.W. and Weinstein,G.D., Treatment of psoriasis. N.Engl.J Med. 1995. 332: 581-588. 60. Motta,S., Monti,M., Sesana,S., Mellesi,L., Ghidoni,R., and Caputo,R., Abnormality of water barrier function in psoriasis. Role of ceramide fractions. Arch.Dermatol. 1994. 130: 452-456. 61. Motta,S., Sesana,S., Monti,M., Giuliani,A., and Caputo,R., Interlamellar lipid dif- ferences between normal and psoriatic SC. Acta Derm.Venereol.Suppl (Stockh) 1994. 186: 131-132. 62. Motta,S., Monti,M., Sesana,S., Caputo,R., Carelli,S., and Ghidoni,R., Ceramide composition of the psoriatic scale. Biochim.Biophys.Acta 1993. 1182: 147-151. 63. Motta,S., Sesana,S., Ghidoni,R., and Monti,M., Content of the different lipid classes in psoriatic scale. Arch.Dermatol.Res. 1995. 287: 691-694. 64. Zhai,H. and Maibach,H.I., Effects of skin occlusion on percutaneous absorption: an overview. Skin Pharmacol.Appl.Skin Physiol 2001. 14: 1-10. 65. Friedman,S., Management of psoriasis vulgaris with a hydrocolloid occlusive dress- ing. Arch Derm 1987. 123: 1046-1052. 66. van de Kerkhof,P.C.M., Chang,A., van der Walle,H.B., Vlijmen-Willems,I., Boezeman,J.B., and Huigen-Tijdink,R., Weekly treatment of psoriasis with a hydro- colloid dressing in combination with triamcinolone acetonide. A controlled compara- tive study. Acta Derm Venereol. 1994. 74: 143-146.

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67. Fry,L., Almeyda,J., and McMinn,R.M., Effect of plastic occlusive dressings on pso- riatic epidermis. Br.J Dermatol. 1970. 82: 458-462. 68. Baxter,D.L. and Stoughton,R.B., Mitotic index of psoriatic lesions treated with an- thralin, glucocorticosteriod and occlusion only. J Invest Dermatol. 1970. 54: 410-412. 69. van Vlijmen Willems,I.M., Chang,A., Boezeman,J.B., and van de Kerkhof,P.C.M., The immunohistochemical effect of a hydrocolloid occlusive dressing (DuoDERM E) in psoriasis vulgaris. Dermatology. 1993. 187: 257-262. 70. Denda,M., Wood,L.C., Elias,P.M., and Feingold,K.R., The epidermal hyperplasia associated with barrier disruption by acetone treatment or tape stripping cannot be at- tributed to increased water loss. Arch.Dermatol Res. 1996. 288: 230-238. 71. van de Kerkhof,P.C.M., de Mare,S., and Arnold W.P, Epidermal regeneration and occlusion. Acta Derm Venereol Stockh 1995. 75: 6-8. 72. Kemeny,L., Ruzicka,T., and Braun Falco,O., Dithranol: a review of the mechanism of action in the treatment of psoriasis vulgaris. Skin.Pharmacol. 1990. 3: 1-20.

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Summary

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SUMMARY

The subject of the present thesis is the epidermal barrier. An intact epidermal barrier prevents the penetration of chemicals, protects against physical factors and the invasion of microorganisms. We focused on three inflammatory skin diseases, irritant contact dermatitis (ICD), atopic dermatitis (AD) and psoriasis vulgaris (PV), with a special emphasis on the skin barrier as a target for therapeutic strategies. In the general introduction (chapter 1), several aspects of the structure and the function of the stratum corneum (SC) are described. Information on the development of the skin barrier, with an emphasis on lipid metabolism and the intercellular lipid domain, are given. In paragraph 1.2 a brief review is given on clinical features, histopathology, pathogenesis and available therapies of the three selected inflammatory skin diseases. We outline the impaired skin barrier and its putative involvement in the pathogenesis of these skin disorders. In ICD the disturbance of the skin barrier is a prominent feature and likely to be important in its pathogenesis. ICD is, therefore, reviewed in more detail. In paragraph 1.3 of this chapter we describe four different models of ICD as well as the relationship between these models and skin barrier impairment. We give a short review of the clinical and cell-biological changes in the epidermis, associated with these models and the effects of different topical treatments. We used transepidermal water loss (TEWL) measurements to assess the barrier function of the skin. To evaluate the clinical response we used visual scoring. Cell-biological changes, associated with impaired epidermal proliferation and differentiation, were studied by means of relevant immunohistochemical staining of samples of treated and untreated skin. In order to extend our knowledge of the repair of the epidermal barrier, we investigated the expression of epidermal fatty acid binding protein (E-FABP), a protein which is supposed to be involved in the epidermal barrier formation and lipid trafficking, in different skin conditions. In chapter 2 the results of these studies are described. In paragraph 2.1 we describe the expression of E-FABP in dithranol induced irritation. The expression proved to be comparable to normal skin. The lack of up-regulation of E-FABP in such a non-corrosive type of irritation, not associated with skin barrier impairment, is in contrast to the up-regulation of E-FABP in SDS induced skin irritation, suggesting a possible role of E-FABP in epidermal barrier repair. In the study, described in paragraph 2.2, we compared the expression of E- FABP in AD and PV. E-FABP proved to be highly up-regulated in both diseases compared to normal skin, but the pattern was markedly different. The human epidermis harbors a wide range of proteins, which are potentially involved in innate immunity and host defense against microbes. The innate immune system provides broad-spectrum recognition and rapid elimination of invading microorganism. In AD, a skin disorder associated with bacterial super-infections, a failure of the innate immune defense system of skin may be involved. The hypothesis is

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that an insufficiency in the expression of inflammation-induced antimicrobial proteins in the epidermis explains the susceptibility of patients with AD to skin infection. Skin- derived-antileukoproteinase (SKALP/elafin) and secretory leukoprotease inhibitor (SLPI), proteins that are likely to play crucial roles in regulating inflammatory responses, are also part of the innate immune system. In chapter 3 we describe a study in which we found a lower expression of these peptides in AD as compared to PV. This finding provides evidence for the role of a lack of up-regulation of host defense proteins in AD, explaining at least in part the increased susceptibility for bacterial superinfections of lesional skin of patients with AD. Identification of molecules involved in the prevention of bacterial colonization may be important in the development of new therapeutic approaches for AD. In chapter 4 we describe studies on the evaluation of new therapies for ICD. Anti-inflammatory agents, without negatively affecting the SC barrier function, are promising candidates in the therapy of ICD. An attractive target for new anti-inflammatory agents is modulation of the expression of cytokines, since up-regulation of cytokines may both play a role in the pathogenesis of ICD and on repair processes after barrier disruption. The inhibition of their production may diminish the inflammatory response accompanying ICD. In this thesis, we describe study on a topical anti-inflammatory agent (cipamfylline), which by inhibition of phosphodiesterase-4 (PDE-4) is able to suppress the production of skin inflammation. Paragraph 4.1 describes a study, which evaluates the efficacy of cipamfylline in two human experimental models of ICD using clinical evaluation, transepidermal water loss and immunohistochemical methods. Despite the promising results of PDE-4 inhibitors in AD, we did not show any effects of this agent in the two models of ICD. Possible reasons for the lack of efficacy of this agent are discussed. SC lipids play a crucial role in skin barrier function. Normal synthesis and processing of lipids are important for optimal epidermal barrier formation. Topical application of suitable lipids may putatively improve the barrier function. The efficacy of topical lipids can be evaluated either in experimental or in clinical studies. Paragraph 4.2 describes an experimental study with healthy volunteers investigating the efficacy on clinical parameters and barrier repair of a traditional, petrolatum- based emollient and an emollient containing skin related lipids. With two different models of experimentally induced skin irritation and barrier impairment we attempted to simulate different types of occupational and environmental irritant effects. Tape stripping was chosen as a model for acute barrier perturbation. To mimic the development of chronic ICD, we used well-established model of repeated exposure of low concentration of a detergent (SDS). In both models repair was accelerated by the treatment with emollients. In the acute model of barrier disruption (tape stripping) the emollient containing skin related lipids accelerated the repair of barrier damage significantly in contrast with the petrolatum-base emollient. In the cumulative SDS- model, the petrolatum-based emollient, not the emollient with skin related lipids, seemed to protect the skin against SDS-irritation.

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Supplementary to the experimental studies, we performed a clinical, pilot- study in chronic hand dermatitis. In hand dermatitis, because of frequent exposure to environmental factors, barrier repair is likely to be of importance in the healing process. Paragraph 4.3 describes a randomized, open study in 32 patients with mild to moderate chronic hand dermatitis. The aim of the study was to compare the emollients in terms of clinical efficacy and dermatocorticosteroid-sparing effects. Patients were treated twice daily during two months with the emollient containing skin related lipids or with a traditional petrolatum-based emollient. In case of exacerbation, the patients of both treatment groups were allowed to use a mild dermatocorticosteroid. In line with the results of the previous experimental studies we observed that both treatment regimens significantly improved clinical signs of the dermatitis. However, wedid not observe significant differences in the improvement of clinical signs, itching, the patient’s assessment of efficacy and the usage of topical corticosteroids between both treatment groups. Most patients considered both emollients as cosmetically acceptable . Based on the results of these studies we conclude that the use of topical lipids in chronic hand dermatitis and in diseases associated with barrier dysfunction is advisable. However, the information provided in this thesis does not support the superiority of emollients with skin related lipids. It is still unknown whether emollients with skin related lipids are superior in the prevention of irritant and allergic reactions and whether they are superior in enhancing barrier repair. In chapter 5 we focus on dithranol irritation as a unique form of ICD. In paragraph 5.1 the effects of a topical corticosteroid, a coal tar solution and their vehicles on dithranol irritation of uninvolved skin of patients with psoriasis are described. The results indicate that a short intervention with a potent dermatocorticosteroid is effective in reducing dithranol irritation. However, a possible negative effect on SC barrier function, cannot be excluded. In paragraph 5.2 we describe the frequency of dithranol irritation in traditional 24-hour applications. Most irritation reactions occur at the start of the therapy with relatively low concentrations. In paragraph 5.3 we describe the frequency of skin irritation in short contact dithranol treatment in comparison to skin irritation in calcipotriol treatment. Although many patients experience skin irritation in both treatments, the reactions are mild and do not seem to have a relevant impact on treatment duration and results. In the general discussion (chapter 6) future perspectives on barrier repair strategies are discussed.

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Samenvatting

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SAMENVATTING

Het onderwerp van dit proefschrift is de huidbarrière, die voornamelijk wordt gevormd door de hoornlaag, het differentiatieproduct van de opperhuid (epidermis). Een intacte barrière voorkomt het binnendringen van chemische stoffen, beschermt tegen fysische factoren en behoedt voor de invasie van micro-organismen. Wij hebben ons, in het voorliggende proefschrift, gericht op drie inflammatoire huidziekten, namelijk orthoërgisch eczeem (irritant contact dermatitis, ICD), constitutioneel eczeem (atopic dermatitis, AD) en psoriasis vulgaris (PV), waarbij speciaal aandacht wordt gegeven aan het functioneren van de epidermale barrière en de wijze waarop therapeutica de barrière kan verbeteren. In de algemene inleiding (hoofdstuk 1), worden meerdere aspecten van de structuur en de functie van het stratum corneum (SC) beschreven. Informatie over de vorming van de huidbarrière wordt verschaft, waarbij de nadruk is komen te liggen op het metabolisme van de epidermale vetten en de opbouw van de intercellulaire ruimte in de hoornlaag. In paragraaf 1.2 wordt een kort overzicht gegeven van de klinische kenmerken, de histopathologie, de pathogenese en de beschikbare therapieën van de drie geselecteerde inflammatoire huidziekten. In hoofdlijnen wordt de vermoedelijke betrokkenheid van de huidbarrière in de pathogenese van deze huidaandoeningen beschreven. Bij ICD is de verstoring van de huidbarrière het grootst en vervult het waarschijnlijk de belangrijkste rol. ICD wordt daarom uitvoeriger belicht. In paragraaf 1.3 van dit hoofdstuk beschrijven wij vier verschillende modellen voor het nabootsen van ICD, alsmede de relatie tussen het model en de verstoring van de huidbarrière. Een kort overzicht van de klinische en celbiologische veranderingen in de epidermis, die geassocieerd zijn met deze modellen, wordt gegeven en we beschrijven de effecten van de verschillende topische behandelingen. Met behulp van transepidermaal waterverlies (TEWL) metingen wordt een kwantitatieve mate verkregen voor de functie van de huidbarrière. Om de klinische respons te evalueren, scoorden wij het erytheem semi-kwantitatief. Celbiologische veranderingen, geassocieerd met verstoorde epidermale proliferatie en differentiatie, werden bestudeerd met behulp van immunohistochemische kleuringen op relevante epitopen in behandelde en onbehandelde huidmonsters. Om onze kennis van het herstel van de epidermale barrière te vergroten, onderzochten wij de expressie van het epidermale vetzuurbindend-eiwit (E-FABP), een eiwit dat geacht wordt betrokken te zijn bij de epidermale barrièrevorming en transport van vetten, in verschillende huidcondities. In hoofdstuk 2 worden de resultaten van deze studies beschreven. In paragraaf 2.1 behandelen wij de expressie van E-FABP in dithranol geïnduceerde huidirritatie. De expressie blijkt vergelijkbaar te zijn met die van normale huid. Het gebrek aan opregulatie van E-FABP in een dergelijk niet-corrosief type van irritatie, niet geassocieerd met verstoring van de huidbarrière, is tegengesteld aan de opregulatie van E-FABP in huidirritatie geïnduceerd door blootstelling aan een wasactieve stof met een krachtig huidbarrière

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verstorend effect. Deze bevinding is suggestief voor een mogelijke rol van E-FABP in het herstel van de epidermale barrière. In paragraaf 2.2 beschrijven wij de expressie van E-FABP bij AD en PV. E-FABP bleek sterk opgereguleerd in beide ziekten in vergelijking tot de normale huid, maar het patroon van expressie was duidelijk verschillend. Het aangeboren afweersysteem heeft een breed spectrum aan mogelijkheden om micro-organismen te herkennen en te verwijderen. De menselijke epidermis bevat een wijd scala aan eiwitten, die mogelijk betrokken zijn bij dit aangeboren systeem. In AD, een huidaandoening geassocieerd met bacteriële superinfecties, zou het falen van het aangeboren afweersysteem betrokken kunnen zijn. De hypothese is dat door een insufficiënte expressie van antimicrobiële eiwitten in de epidermis patiënten met AD meer vatbaar zijn voor huidinfecties. De in de huid tot expressie komende eiwitten, antileukoproteïnase (SKALP/elafin) en secretorisch leukoprotease inhibitor (SLPI), vervullen waarschijnlijk ook een rol in het aangeboren imuunsysteem. In hoofdstuk 3 beschrijven wij een studie waarbij een lagere expressie van deze peptiden gevonden werd in AD in vergelijking met PV. De toegenomen vatbaarheid voor bacteriële superinfecties van de huid van patiënten met AD zou (voor een deel) hierdoor kunnen worden verklaard. Identificatie van moleculen, die betrokken zijn bij de preventie van bacteriële kolonisatie, kan van belang zijn voor de ontwikkeling van nieuwe therapeutische middelen in de behandeling van AD. In hoofdstuk 4 beschrijven wij studies ter evaluatie van nieuwe therapieën voor ICD. Ontstekingsremmende middelen, zonder een negatieve invloed op de barrièrefunctie, zijn veelbelovend in de therapie van ICD. Een aantrekkelijk doel voor nieuwe anti-inflammatoire middelen is de modulatie van de expressie van cytokines, aangezien de regulatie van cytokines mogelijk een rol speelt in de pathogenese van ICD, maar mogelijk ook op herstelprocessen na barrièreverstoring. Het afremmen van hun productie zou de inflammatoire respons bij huidirritatie kunnen verminderen. In dit proefschrift beschrijven wij een studie naar een topisch anti-inflammatoire middel (cipamfylline), die door remming van het enzym fosfodiesterase-4 (PDE-4) in staat is de productie van huidinflammatie te onderdrukken. Paragraaf 4.1 beschrijft een studie naar de effectiviteit van cipamfylline in twee humane experimentele modellen voor ICD. Ondanks de veelbelovende resultaten van PDE-4 remmers in AD, konden wij geen effecten van dit middel aantonen in de twee modellen voor ICD. Mogelijke oorzaken voor het gebrek aan effectiviteit van dit middel worden bediscussieerd. SC lipiden spelen een cruciale rol in de huidbarrière. Topicaal gebruik van geschikte lipiden zou mogelijk de barrièrefunctie kunnen verbeteren. De effectiviteit van topische lipiden kunnen worden geëvalueerd aan de hand van experimentele, dan wel klinische studies. Paragraaf 4.2 beschrijft een experimentele studie met gezonde vrijwilligers, waarin de effecten op klinische parameters en barrièreherstel onderzocht werden van een traditioneel, vaseline bevattend emolliens en een emolliens met lipiden, die gelijkenis vertonen met de van nature in het SC voorkomende lipiden. Met twee verschillende experimentele modellen voor huidirritatie en barrièrebeschadiging

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probeerden wij verschillende typen van beroepsmatige en omgevingsafhankelijke irritatieve effecten na te bootsen. Tape-stripping werd gekozen als model voor acute mechanische barrièrebeschadiging. Het nabootsen van de ontwikkeling van chronische ICD werd bewerkstelligd door middel van een bekend model van herhaalde blootstelling aan lage concentraties detergens (sodium-dodecyl-sulphate, SDS). In beide modellen werd het herstel versneld door de behandeling met emolliëntia. In het acute model voor barrièrebeschadiging (tape-stripping) werd het herstel van barrièreschade significant versneld door een emolliens met de SC gerelateerde lipiden, echter niet door het vaselinebevattende emolliens. In het cumulatieve SDS-model leek het vaseline-bevattende emolliens beter te beschermen tegen SDS geïnduceerde irritatie dan het emolliens met SC gerelateerde lipiden. Aanvullend bij de experimentele studies, voerden wij een klinische pilot- studie uit bij chronisch handeczeem. Bij handeczeem is het aannemelijk dat het barrièreherstel belangrijk is bij het genezingsproces. Paragraaf 4.3 beschrijft een gerandomiseerde, open studie met 32 patiënten met een matige tot milde vorm van chronische handeczeem. Het doel van de studie was het vergelijken van het effect van twee verschillende emolliëntia. Patiënten werden tweemaal daags gedurende twee maanden behandeld met het emolliens met de SC gerelateerde lipiden of met het vaseline-bevattende emolliens. In geval van verslechtering mochten patiënten in beide groepen een licht dermatocorticosteroïd gebruiken. In overeenstemming met de resultaten uit de voorgaande experimentele studies zagen wij dat beide behandelingswijzen een significante verbetering in het klinisch beeld van het eczeem tot gevolg hadden. Echter, wij constateerden geen significante verschillen tussen beide groepen met betrekking tot het klinisch beeld, jeuk, eigen inschatting van het effect en het gebruik van dermatocorticosteroïden. De meeste patiënten waren van mening dat beide emolliëntia cosmetisch acceptabel waren. Gestoeld op de resultaten van deze studies concluderen wij dat het gebruik van topische lipiden bij chronische handeczeem en bij aandoeningen geassocieerd met epidermale barrièredysfunctie geadviseerd kan worden. Echter, de informatie verkregen door de studies beschreven in dit proefschrift, onderschrijft niet de superioriteit van emolliëntia met SC gerelateerde lipiden. In hoofdstuk 5 vestigen wij de aandacht op dithranolirritatie als een unieke vorm van ICD. In paragraaf 5.1 worden de effecten van een corticosteroïdzalf, een koolteeroplossing in een zalfbasis en de vehicula van beide middelen op dithranolirritatie van de onbehandelde huid van patiënten met psoriasis bestudeerd. De resultaten tonen aan dat een kortdurende interventie met een krachtig dermatocorticosteroïd effectief is in het reduceren van dithranolirritatie. Echter, een mogelijk negatief effect op de barrièrefunctie kan niet worden uitgesloten. In paragraaf 5.2 beschrijven wij de frequentie van huidirritatie bij traditionele 24-uurs behandelingen. De meeste huidirritaties doen zich voor bij het begin van de therapie met relatief lage concentraties. In paragraaf 5.3 beschrijven wij de frequentie van huidirritatie bij kort contact dithranolbehandeling en vergelijken het voorkomen

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bij calcipotriolbehandeling. Hoewel vele patiënten klachten van huidirritatie hebben bij beide behandelingen, zijn de reacties overwegend mild en lijken zij geen relevante invloed te hebben op met name de behandelduur. In de algemene discussie (hoofdstuk 6) worden toekomstperspectieven van nieuwe therapeutische strategieën gericht op het huidbarrièreherstel bij inflammatoire huidziekten bediscussieerd.

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List of publications

Curriculum vitae

212 List of publications List of pubications

LIST OF PUBLICATIONS

Kucharekova M, van der Valk PGM Toxische epidermale necrolyse Nederlands Tijdschrift voor Dermatologie en Venereologie 2001;11(4)

Swinkels OQJ, Prins M, Kucharekova M, de Boo Th, Gerritsen MJP, van der Valk PGM, van de Kerkhof PCM Combining lesional short-contact dithranol therapy of psoriasis with a potent topical corticosteroid British Journal of Dermatology 2002;146(4):621-626

Kucharekova M, Schalkwijk J, van de Kerkhof PCM, van der Valk PGM The effect of a lipid-rich emollient containing ceramide 3 in an experimentally induced skin barrier dysfunction Contact Dermatitis 2002;46(6):331-338

Swinkels OQJ, Kucharekova M, Prins M, Gerritsen MJP, van der Valk PGM, van de Kerkhof PCM The effects of topical corticosteroids and coal tar preparation on dithranol induced irritation in patients with psoriasis Skin Pharmacology and Applied Skin Physiology 2003;16(1):12-17

Kucharekova M, Hornix M, Ashikaga T, T’kint S, de Jongh GJ, Schalkwijk J, van de Kerkhof PCM, van der Valk PGM The effect of a PDE-4 inhibitor (Cipamfylline) in two human models of irritant contact dermatitis Archives of Dermatological Research 2003;295(1):29-32

Kucharekova M, Vissers WHPM, Schalkwijk J, van de Kerkhof PCM, van der Valk PGM Lack of upregulation of epidermal fatty acid binding protein in dithranol induced irritation European Journal of Dermatology 2003;13(3):254-257

Kucharekova M, van de Kerkhof PCM, van der Valk PGM A randomised comparison of an emollient containing skin related lipids with a petrolatum-based emollient as adjunct in the treatment of chronic hand dermatitis Contact Dermatitis. In Press

214 215 List of publications List of pubications

Kucharekova M, Schalkwijk J, Blokx WAM, Dogan-Bayram A, van der Valk PGM Epidermal expression of epidermal fatty acid binding protein (E-FABP) distinguishes atopic dermatitis from psoriasis vulgaris Submitted

Kucharekova M, van der Valk PGM, Blokx WAM, Dogan-Bayram A, Hiemstra P, van de Kerkhof PCM, Schalkwijk J Specific upregulation of epidermal host-defence proteins in psoriasis compared to atopic dermatitis Submitted

Kucharekova M, Lieffers L, van de Kerkhof PCM, van der Valk PGM Dithranol irritation in psoriasis treatment: a study of 68 inpatients Submitted

Zweers MC, Bristow J, Steijlen PM, Dean WB, Hamel BC, Otero M, Kucharekova M, Boezeman JB, Schalkwijk J Haploinsufficiency of TNXB Is Associated With Hypermobility Type Ehlers-Danlos Syndrome American Journal of Human Genetics 2003;73:214-217

Peeters ACTM, Kucharekova M, Timmermans J, van den Berkmortel FWPJ, Boers GHJ, Novakova IRO, Egging D, den Heijer M, Schalkwijk J A clinical and cardiovascular survey of Ehlers-Danlos syndrome patients with complete deficiency of Tenascine-X Submitted

214 215 Curriculum vitae

CURRICULUM VITAE

Martina Kucháreková werd op 5 juni 1971 geboren in de Slowaakse stad Zvolen en groeide op in Spišská Nová Ves. In 1989 behaalde zij daar haar diploma aan het Gymnásium kpt.J.Nálepku. Aansluitend startte zij met de studie geneeskunde aan de Universiteit P.J.Šafárik te Košice in Slowakije. Deze studie geneeskunde heeft ze vervolgd aan de Karel Universiteit te Praag in Tsjechië waar ze in 1995 haar arts diploma heeft verworven. Vanaf 1995 woont ze in Nederland waar ze in november 1999 haar artsexamen aan de Katolieke Universiteit te Nijmegen heeft behaald. Al tijdens de studie in Praag werd haar interesse voor de dermatologie gewekt. Aan deze interesse werd gevolg gegeven met een wetenschappelijke stage op de afdeling Dermatologie in het Universitair Medisch Centrum St Radboud Nijmegen. Na haar artsexamen ging ze aldaar onder leiding van Prof.dr.dr. P.C.M. van de Kerkhof en Dr. P.G.M. van der Valk werken. Hier werden vervolgens de onderzoeken verricht die tot dit proefschrift hebben geleid. Zij zal vanaf januari 2004 als assistent in opleiding werkzaam zijn bij de afdeling Dermatologie onder leiding van Prof.dr. P.M. Steijlen in het Academisch Ziekenhuis Maastricht. Zij is sedert 13. 9. 2002 gelukkig getrouwd met Michel van Geel en is de trotse moeder van dochter Emilia (17-06-2003).

216 Curriculum vitae

Dankwoord

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DANKWOORD

Dit proefschrift is tot stand gekomen met de wetenschappelijke of persoonlijke hulp van velen, die elk op hun eigen, unieke wijze een bijdrage hebben geleverd. Het is onmogelijk om op deze plaats iedereen persoonlijk hiervoor te bedanken, maar een aantal mensen wil ik toch graag afzonderlijk noemen.

Allereerst dank aan de inspirators achter dit proefschrift. Professor Peter van de Kerkhof, mijn promotor, wil ik bedanken voor het in mij gestelde vertrouwen en de steun om dit promotietraject af te ronden. Wat ik altijd bewonderde was uw ontembare enthousiasme, creativiteit en effectiviteit als het om onderzoek ging. Op cruciale momenten bracht u mij op de goede weg wanneer ik de rode draad in mijn onderzoek even was verloren. Bedankt ook voor de praktische tips en razendsnelle beoordeling van de manuscripten. Uiterarad wil ik mijn eerste co-promotor Pieter van der Valk danken, die me de kans bood onderzoek te doen op de afdeling dermatologie en die daarmee de eerste steen legde voor deze promotie. Beste Pieter, gedurende mijn promotie tijd heb ik je leren kennen als een prettig persoon met sterke principes en een kritische blik. Ook daardoor was het voor mij zeer uitdagend om jouw wetenschappelijke ideeën in uitvoerbare projecten te kneden, voorzien van mijn eigen stempel. Ook al is met dit proefschrift voor mij de laatste steen gelegd, hopelijk bouw jij nog jaren door aan uitdagende wetenschappelijke projecten. Bedankt voor alles. Joost Schalkwijk, mijn tweede co-promotor, ben ik veel dank verschuldigd. Joost, ondanks het feit dat je in het begin niet betrokken was bij mijn onderzoek, was je er altijd om advies te geven. Ook was je degene die me met beide voeten op de grond terug wist te zetten, met name bij wetenschappelijke verklaringen van mijn immunohistochemische experimenten.

Marisol Kooijmans-Otero, researchverpleegkundige, zonder jouw hulp is het uitvoeren van patiëntgebonden onderzoek op de afdeling bijna onmogelijk. Niet alleen jouw kennis en praktische tips, maar vooral jouw “luisterende oor” hebben mijn promotietraject zeer aangenaam gemaakt. Ook al is een onderzoek meestal een collectieve gebeurtenis, jou lukte het om me dagen lang te isoleren op onderzoekskamer 3 waar ik met gevoelens van Robinson Crusoé de TEWL-meting volgens de richtlijnen kon uitvoeren. Deze tijdelijke, persoonlijke metamorfose was mede mogelijk door het op de deur geplakte, gezag uitstralende berichtje (Niet storen - TEWL meting), die de meeste collega’s niet gelijk konden ontcijferen en die daardoor twijfelend hun deurklopje uitstelden. Milan Tjioe, Jürgen Smit, Manon Franssen (destijds Agnio’s); bedankt voor alle gezellige, bemoedigende en inspirerende momenten op het lab, in de koffiekamer of tijdens de lunch. Onder het genot van een drankje werden de promotieperikelen meestal uitgebreid verder besproken in een van de Nijmeegse horeca-instellingen.

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Milan, jou wil ik apart bedanken omdat je de opmaak van mijn proefschrift uit handen hebt willen nemen en omdat je de rol van paranimf bij mijn promotie hebt aanvaard. Onvergetelijk is ook jouw hulp in mijn levenslange strijd tegen mijn aartsvijand Reference Manager. Quintus Swinkels. Ik wil je bedanken voor al die late avonden wanneer je structureel het gebruikelijke tijdstip van avondeten negeerde en er nog was om mij van advies te voorzien. In die late uurtjes kon ik voor een sociale raad, een opbeurend gesprek of een opfrissend woord (b.v. “laat maar zitten die promotie”) bij jou terecht. Gelukkig heb ik niet altijd naar je geluisterd... Sara T’ kint, als een tijdelijke bewoner van kamer 007 wil ik je bedanken voor de leuke tijd. In het jaar dat je bij ons vanuit de Vrije Universiteit Brussel een stage kwam lopen is het ons gelukt “de boze wereld” buiten de deur van kamer 007 te houden en kleurige gesprekken te voeren, niet uitsluitend maar over dermatologie. Directe collega’s (AGNIO’s en AGIO’s); Marieke, Carine, Astrid, Monique, Fransje, Rob, Roland, Mandy, Maurice, Michelle, Tim, Wynand, José, Angelina, Saskia, Mirella en Cerpil, bedankt voor de prettige samenwerking en de mogelijkheid om indien nodig (b.v. vanwege een deadline voor inclusie voor een nieuwe klinische trial) bij jullie hulp te vragen. Alle collega’s van het lab, bedankt voor alle gezelligheid. Yvonne van Vlijmen, bedankt voor je hulp bij de verschillende immunohistochemische kleuringen. Al tijdens mijn wetenschappelijke stage leerde je me de kleurtechnieken en het snijden van vriescoupes. Ik kon altijd bij je terecht met vragen over rare verdunningen, verkeerde antilichamen, etc. Gijs, gelukkig had je begrip voor mijn structurele weigering om statistische methoden te leren. Zonder jouw hulp met statistiek zou de totstandkoming van dit proefschrift nog langer duren. Patrick, bedankt voor de gezellige uurtjes binnen en buiten werktijd en je niet te evenaren humor. Alle stafleden, bedankt voor jullie steun en jullie interesse in mijn onderzoek. Mayke Hornix, Aynur Dogan, Lonneke Lieffers dank ik voor het vele werk dat zij gedaan hebben als wetenschappelijke stagiaires.

Hartelijk dank aan alle baliemedewerkers, fotografen, administratief en verplegend personeel van de verpleegafdeling en de polikliniek dermatologie voor de prettige samenwerking tijdens mijn AGNIO-periode. Speciaal wil ik hierbij de medewerkers van de Arbeids- en Tijdsintensieve Behandeling (ATB) noemen die zich hebben ingezet voor het goede verloop van de MCO-studie (ó, die ellende met het noteren van lege ditranoltubes van 40 patiënten gedurende 1,5 jaar). Verder wil ik Marja Geurts niet vergeten die samen met me aan de wieg van de Handeczeem poli (HAHAND) heeft gestaan.

Beste patiënten en gezonde vrijwilligers, bedankt voor het geduld dat jullie hadden met die buitenlandse dokter die met een raar kastje en een stokje (TEWL- apparaat) bepaalde cijfers heeft afgelezen van de onderzoeksplekken op jullie ruggen.

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Leuk was anders; liggen zonder te bewegen (om aan de kamertemperatuur te wennen), geen koffie (want dat stimuleert transpireren), geen praatjes (want dat stoort), maar toch zijn jullie trouw tijdens het hele experiment gekomen, ongelovig! Erg bedankt, zonder jullie zou dit proefschrift nooit tot stand zijn gekomen.

Peter Paul van Geel wil ik hartelijk danken voor de aanvaarding van de rol van paranimf bij mijn promotie.

Mijn ouders, milí rodičia, d’akujem Vám za lásku s ktorou ste ma pod vrcholmi neprekonatel’ných Vysokých Tatier vychovali.

Lieve Michel, bedankt voor al je steun en de stoïcijnse rust waarmee jij me altijd in de realiteit terugbracht. Met name de laatste maanden hebben best veel van je gevraagd omdat we naar Limburg zijn verhuisd en omdat ons duo met Emilia werd versterkt. Voor alles bedankt.

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Colour Illustrations

220 Colour Illustrations Colour Illustrations

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Chapter 2 - paragraph 2.1 - figure 1 Chapter 2- paragraph 2.2 - figure 1 - page 78 - page 68

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SSLPILPI

SKALP/Elafin Elafin

MRP8 MRP8

Normal skin Atopic dermatitis Psoriasis

Chapter 3 - paragraph 3.1- figure 1 - page 92

Figure 1 Kucharekova et al InvolucrinInvolucrin

CCK16K16

Ki-67 Ki-57

Atopic dermatitis Psoriasis Chapter 3 - paragraph 3.1 - figure 2 - page 93

Figure 2 Kucharekova et al 222 223 224