Skin Barrier Function

Current Problems in Dermatology Editors: P. Itin, G.B.E. Jemec Vol. 49 Skin Barrier Function

Editor T. Agner Skin Barrier Function Current Problems in Dermatology

Vol. 49

Series Editors

Peter Itin Basel Gregor B.E. Jemec Roskilde Skin Barrier Function

Volume Editor

Tove Agner Copenhagen

26 figures, 6 in color, and 14 tables, 2016

Basel · Freiburg · Paris · London · New York · Chennai · New Delhi · Bangkok · Beijing · Shanghai · Tokyo · Kuala Lumpur · Singapore · Sydney Current Problems in Dermatology

Prof. Tove Agner Bispebjerg University Hospital Department of Dermatology Copenhagen (Denmark)

Library of Congress Cataloging-in-Publication Data

Names: Agner, Tove, editor. Title: Skin barrier function / volume editor, Tove Agner. Other titles: Current problems in dermatology ; v. 49. 1421-5721 Description: Basel ; New York : Karger, 2016. | Series: Current problems in dermatology, ISSN 1421-5721 ; vol. 49 | Includes bibliographical references and indexes. Identifiers: LCCN 2015047342| ISBN 9783318055856 (hard cover : alk. paper) | ISBN 9783318055863 (electronic version) Subjects: | MESH: Skin Physiological Phenomena | Epidermis--physiology Classification: LCC QM481 | NLM WR 102 | DDC 612.7/9--dc23 LC record available at http://lccn.loc.gov/2015047342

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VII Preface Agner, T. (Copenhagen)

Section I: Basic Parameters

1 Filaggrin and Skin Barrier Function Kezic, S. (Amsterdam); Jakasa, I. (Zagreb) 8 Stratum Corneum Lipids: Their Role for the Skin Barrier Function in Healthy Subjects and Atopic Dermatitis Patients van Smeden, J.; Bouwstra, J.A. (Leiden) 27 Importance of Tight Junctions in Relation to Skin Barrier Function Brandner, J.M. (Hamburg) 38 Antimicrobial Peptides, Infections and the Skin Barrier Clausen, M.-L.; Agner, T. (Copenhagen) 47 Biological Variation in Skin Barrier Function: From A (Atopic Dermatitis) to X (Xerosis) Danby, S.G. (Sheffield)

Section II: External Factors Influencing Skin Barrier

61 Methods for the Assessment of Barrier Function Antonov, D.; Schliemann, S.; Elsner, P. (Jena) 71 In vivo Raman Confocal Spectroscopy in the Investigation of the Skin Barrier Darlenski, R. (Sofia); Fluhr, J.W. (Berlin) 80 Irritants and Skin Barrier Function Angelova-Fischer, I. (Lübeck) 90 Skin Barrier Function and Allergens Engebretsen, K.A.; Thyssen, J.P. (Hellerup) 103 Penetration through the Skin Barrier Nielsen, J.B. (Odense); Benfeldt, E. (Roskilde); Holmgaard, R. (Copenhagen)

Section III: Optimizing Skin Barrier Function

112 Treatments Improving Skin Barrier Function Lodén, M. (Solna)

V Section IV: Clinical Aspects of Skin Barrier Function

123 Standards for the Protection of Skin Barrier Function Giménez-Arnau, A. (Barcelona) 135 The Role of the Skin Barrier in Occupational Skin Diseases Kasemsarn, P. (Carlton, Vic.; Bangkok); Bosco, J.; Nixon, R.L. (Carlton, Vic.) 144 Wet Work and Barrier Function Fartasch, M. (Bochum) 152 Saving the Barrier by Prevention Weisshaar, E. (Heidelberg)

159 Author Index 160 Subject Index

VI Contents Preface

The skin barrier is important to human life. Phys- terplay between the immune system and barrier ically, it protects us from external threats such as function; it has been documented that impaired infectious agents, chemicals, systemic toxicity barrier function leads to up-regulation of certain and allergens; internally, the skin helps to main- interleukins, which again influence the amount tain homeostasis and to protect from enhanced of natural moisturizing factor. Other very im- loss of water from the body. Both of these are life- portant players in the maintenance of an intact saving qualities. Another aspect is the esthetic ap- barrier are lipids, in particular long-chained cer- pearance of the skin, which should not be under- amides. Tight junctions are important for main- estimated. Since ancient times, a well-hydrated taining the structure of the stratum corneum, and skin texture has been treasured, with tradition antimicrobial peptides help to fight infectious featuring bathing in milk as the ultimate luxury to agents. Clarification of up- and down-regulation, secure soft and moisturized skin. and interaction between all the factors involved, Since in the last decade filaggrin mutations will be an important future research subject. were discovered as a major risk factor for atopic The permeability of the skin barrier is of out- dermatitis, an inflammatory skin disease with a most importance for the penetration of toxic particularly impaired barrier function, research chemicals, but also for pharmacological treat- in skin barrier function has escalated. The skin ment. Precise and reproducible experimental barrier is located in the stratum corneum, made models exist, but are difficult to transfer to hu- up of flattened, anucleated cells and a highly or- man real-life exposures due to great heterogene- ganized lipid matrix. The filaggrin protein helps ity between individuals. to maintain an organized barrier, and loss-of- The skin barrier function is hampered in many function mutations in the filaggrin gene are a ma- (most?) skin diseases, with atopic dermatitis and jor determinant of filaggrin expression in the ichthyosis being the most obvious ones. The clin- skin. Natural moisturizing factor, an amorphous ical look of these diseases is characterized by dry, substance highly important for hydration of the scaly and fissured skin, and experimentally by in- stratum corneum, is a degradation product of fil- creased transepidermal water loss and a decreased aggrin. An extremely interesting finding is the in- amount of natural moisturizing factor.

VII Environmental as well as inherited factors may function, this has yet to be reflected in the treat- affect the barrier function negatively, and irritant ment of xerosis and other barrier defects. Addi- contact dermatitis is an example of this. Wet- tionally, there is a lack of evidence and standard- work exposure is a particularly significant factor, ized recommendations with regard to protecting and recent research within this area focuses on its and restoring skin barrier function. However, a influence on an interplay between work-related real breakthrough has taken place in the under- and domestic exposures. standing of barrier function, an important and Although the last decade has seen significant necessary step in the direction of improved treat- progress in the understanding of the skin barrier ment. Tove Agner , Copenhagen

VIII Agner Section I: Basic Parameters

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 1–7 (DOI: 10.1159/000441539)

Filaggrin and Skin Barrier Function

a b Sanja Kezic Ivone Jakasa a Coronel Institute of Occupational Health, Academic Medical Center, University of Amsterdam, Amsterdam , The Netherlands; b Laboratory for Analytical Chemistry, Department of Chemistry and Biochemistry, Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb , Croatia

Abstract of potentially hazardous chemicals, which might cause The skin barrier function is greatly dependent on the adverse effects in the skin, such as contact dermatitis, or structure and composition of the uppermost layer of the systemic toxicity after their passage into blood. In an- epidermis, the stratum corneum (SC), which is made up other direction, a leaky epidermal barrier will lead to en- of flattened anucleated cells surrounded by highly orga- hanced loss of water from the skin. A recent study has nized and continuous lipid matrix. The interior of the shown that even subtle increase in epidermal water loss corneocytes consists mainly of keratin filaments aggre- in newborns increases the risk for AD. Although there gated by filaggrin (FLG) protein. Next, together with sev- are multiple modes of action by which FLG might affect eral other proteins, FLG is cross-linked into a mechani- skin barrier it is still unclear whether and how FLG defi- cally robust cornified cell envelope providing a scaffold ciency leads to the reduced skin barrier function. This for the extracellular lipid matrix. In addition to its role for chapter summarizes the current knowledge in this field the SC structural and mechanical integrity, FLG degrada- obtained from clinical studies, and animal and in vitro tion products account in part for the water-holding ca- models of FLG deficiency. © 2016 S. Karger AG, Basel pacity and maintenance of acidic pH of the SC, both crucial for the epidermal barrier homoeostasis by regulating activity of multiple enzymes that control desquamation, lipid synthesis and inflammation. The major Filaggrin and the Skin Barrier determinant of FLG expression in the skin are loss-of- function mutations in FLG , the strongest genetic risk fac- The main physical barrier to excessive water loss tor for atopic dermatitis (AD), an inflammatory skin dis- and ingress of exogenous chemicals, pathogens ease characterized by a reduced skin barrier function. and UV radiation resides in the uppermost epi- The prevalence of FLG mutations varies greatly among dermal layer, the stratum corneum (SC). The SC different populations and ranges from about 10% in comprises the corneocytes, flattened skin cells de- Northern Europeans to less than 1% in the African pop- void of a nucleus which are surrounded by highly ulations. An impaired skin barrier facilitates absorption organized lipid lamellar matrix. The outflow of water from the water-rich epi- of FLG on the other side are involved in the main- dermis toward the skin surface as well as inflow of tenance of skin hydration and acidic milieu, both substances from outside can occur through inter- crucial for the optimal activity of enzymes in- cellular routes along lipid bilayers or through volved in skin inflammation, lipid synthesis and transcellular routes across the corneocytes. The desquamation. Recent studies in individuals with intercellular route is the main diffusional path- ichthyosis vulgaris (IV) with inherited deficiency way, although the corneocyte route may become in FLG and in FLG-null (Flg–/–) mice have in- relevant for small hydrophilic compounds such deed confirmed cytoskeletal abnormalities, im- as water [1]. The amount of a compound which paired lamellar body loading, disorganization of enters the skin is dependent on its solubility in the the lipid lamellar structure, and altered expres- SC, which is greatly influenced by the content and sion and localization of tight junction proteins [5, relative composition of the lipids and water in the 6]. It is obvious that deficiency in FLG can theo- SC. On the other side, the diffusion resistance of retically affect both, solubility properties of the the SC, which determines the rate by which a SC as well as diffusion resistance. Over the last compound diffuses across the SC, is largely de- decade, a large number of clinical and experimen- pendent on the organization of the lipid bilayers tal studies have been undertaken to elucidate the interacting with protein components of the cor- mechanisms by which FLG deficiency affects skin neocytes [1] and the path length for diffusion, barrier function. In most of these investigations, which depends on the thickness of the SC, num- skin barrier function has been assessed by mea- ber of layers of corneocytes, their size and their suring transepidermal water loss (TEWL). As wa- cohesion [2] . ter, being a small hydrophilic molecule, might not A heightened attention for the role of filaggrin be representative for the penetration of other sub- (FLG) in skin barrier function has been under- stances, in various studies percutaneous penetra- scored by a strong and robust association between tion of hydrophilic and hydrophobic model pen- loss-of-function mutations in FLG and atopic der- etrants have been determined. We review here re- matitis (AD), a common inflammatory skin dis- cent findings on the association between FLG and ease [3]. A skin barrier defect is one of the most skin barrier function obtained from clinical stud- distinctive hallmarks of AD. On a population lev- ies, and in vitro and animal models of FLG defi- el, approximately 50% of moderate-to-severe AD ciency. cases can be attributed to FLG mutations, whereas in the case of mild-to-moderate AD the attributable risk amounts to 15% [3] . In addition to high Clinical Studies penetrance, FLG mutations are prevalent in certain populations. Approximately 10% of Northern The effect of FLG on skin barrier function in vivo Europeans from the general population are FLG in humans has mainly been investigated in clini- mutation carriers. The prevalence of FLG muta- cal studies by comparing TEWL in AD patients tions in the Asian population varies from 3 to 6%, with (ADFLG ) and without (ADnon-FLG ) loss-of- while the prevalence of FLG mutations in the Af- function mutations in FLG . AD is a common in- rican population is less than 1% [4] . flammatory skin disease characterized by Th2- FLG may affect the skin barrier by multiple mediated immune aberrations and impaired skin mechanisms. As a component of the cornified cell barrier function [3] . FLG mutations, which are envelope and a filament-aggregating protein, common in European and Asian populations, are FLG is important for the structural and mechani- a strong risk factor for the development of AD cal integrity of the SC. The degradation products with the estimated attributable risk in patients

2 Kezic Jakasa

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 1–7 ( DOI: 10.1159/000441539) with moderate-to-severe AD ranging from 4.2 to for FLG mutations and AD, Winge et al. [16] 15.1% [3] . The complex interplay of atopic in- measured TEWL in patients with IV with or with- flammation and FLG makes the assessment of the out concomitant AD. As compared with healthy individual effect of FLG mutations on skin barrier controls, significantly higher TEWL values were failure in AD difficult. It is well known that the found only in the AD patients with FLG muta- impairment in skin barrier function correlates tions. Furthermore, there was a clear dose-related with the severity of skin inflammation regardless increase in TEWL according to genotype, with of FLG genotype status [7, 8]. Janssens et al. [8] the highest TEWL in the carriers of two FLG mu- showed that the organization of lipid bilayers and tations. Interestingly, in the studies by Perusquía- relative content of very long fatty acid chains in Ortiz et al. [17] , complete FLG deficiency showed ceramides, both contributing to the skin barrier only a moderate increase in TEWL as compared function, depend on disease severity. Notably, to healthy controls (7.54 ± 0.90 and 5.41 ± 0.32 these changes were independent of FLG muta- g/m 2/h, respectively; p < 0.03), which is in line tions, but they were correlated with the levels of with the findings of Gruber et al. [5] . Further- FLG degradation products, which are important more, in this study [5] , TEWL was only signifi- constituents of natural moisturizing factors cantly elevated in IV patients with double allele (NMF) in the SC. FLG mutations are the main mutations and not in patients with single allele determinants of NMF levels in the skin, and wild- mutations when compared with FLG wild-type type AD patients have reduced NMF levels [9] . A carriers. It has to be emphasized, however, that in recent study by Cole et al. [10] , in which the whole most studies in IV patients the sample size was transcriptome has been analyzed by RNA se- likely too low to detect small, but possibly physi- quencing, revealed two different patterns in AD ologically relevant differences between the sub- patients: patients with wild-type FLG showed groups regarding the number of FLG mutations dysregulation of genes involved in lipid metabo- and presence of AD. A recent study by Kelleher et lism, while in AD patients with FLG mutations a al. [18] showed that even moderate increase in type 1 interferon-mediated stress response was TEWL of approximately 2 g/m 2/h leads to a sig- dominant. Furthermore, the inflammation and nificantly increased risk of AD. Skin barrier im- Th2-dominant cytokine milieu itself downregu- pairment as assessed by increased TEWL at 2 days lates the expression of FLG, but also affects the and 2 months proved to be a strong predictor of composition and organization of SC lipid bilayers AD development at 1 year of life. In that large [8, 9, 11]. The differences in the disease pheno- birth cohort study, Kelleher et al. [18] assessed type of the included AD patients and often under- skin barrier function in the newborn period and powered studies might at least partly explain the early infancy. While at birth, there was no differ- discrepancies in the effects of FLG mutations re- ence in mean TEWL values between carriers and garding skin barrier function reported in the lit- noncarriers of FLG mutations (7.3 ± 3.38 and 7.33 erature. Angelova-Fischer et al. [12] and Junger- ± 3.62 g/m 2 /h, respectively), by 2 and 6 months, sted et al. [13] found significantly increased FLG mutation-carrying infants had a significantly TEWL in the AD patients with FLG mutations higher mean TEWL compared to FLG wild-type (AD FLG ) as compared to the healthy controls. infants. In a study by Flohr et al. [19] , increased However, no significant differences in the TEWL TEWL was significantly associated with the pres- values between ADFLG and ADnon-FLG groups ence of FLG mutations in healthy children as well were observed, which is in concordance with the as in children with AD at 3 months of age. findings of several other studies [9, 14, 15]. To in- Whereas most publications focused primarily vestigate the effect on the skin barrier separately on TEWL, the characterization of the epidermal

FLG and Skin Barrier Function 3

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 1–7 ( DOI: 10.1159/000441539) barrier properties by measuring percutaneous Mildner et al. [22], which might stimulate the ex- penetration is scarce. In the study by Jakasa et al. pression of FLG in atopic keratinocytes [26] . [14], uninvolved skin of AD patients showed However, Küchler [27], who also used normal fi- higher permeability for polyethylene glycol mol- broblasts, suggested that rather the shorter length ecules of 370 Da in comparison to healthy skin, of the cultivation period at the air-liquid interface irrespective of their FLG genotype. Recently, had a major impact on the outcome in the study Joensen et al. [20] investigated the levels of phthal- by Mildner et al. [22] . ate metabolites as a biomarker of dermal absorp- In a recent study by van Drongelen et al. [21] tion of phthalates. The study showed that the performed in N/TERT-based human skin equiva- carriers of FLG loss-of-function mutations had lent, no effect of FLG -KD on the SC lipid organi- significantly higher internal exposure to phthal- zation or composition has been observed, which ate metabolites than those with wild-type FLG is in line with findings of Mildner et al. [22] . Váv- genotypes, suggesting increased skin permeabili- rová et al. [24] reported similar CER profiles in ty due to FLG mutations. both, FLG-KD and normal skin models, although the FLG-KD skin model showed a 2-fold increase in free fatty acid levels. All FLG -KD skin models In vitro Models showed reduced levels of FLG degradation products, urocanic and/or pyrrolidone carboxylic acid Since single contributions of FLG mutations are [22, 23, 25] , compared to the normal skin model, difficult to assess in vivo [21] several in vitro FLG- which is in agreement with in vivo findings [15, deficient skin models have been developed in re- 28] . cent years. These skin models are based on FLG The skin barrier function of in vitro models is knockdown (KD) by small interfering RNA [22– typically investigated using dye penetration as- 24] or small hairpin RNA [21, 25] interference of says. To investigate the effect of FLG -KD on SC normal human keratinocytes. Mildner et al. [22] permeability, Mildner et al. [22] and Pendaries et were the first to describe the effect of reduced FLG al. [25] used hydrophilic dye, Lucifer yellow. In expression in an in vitro skin model by silencing both studies, increased permeability toward a hy- FLG with small interfering RNA interference of drophilic dye indicated altered skin barrier func- normal human keratinocytes used to establish the tion in an FLG-deficient skin model. Lucifer yel- epidermal component of organotypic skin cul- low penetrated into the SC and diffused further tures. Histological investigation showed that the down to the basal layer or polycarbonate filter of main morphological alteration in FLG -KD or- the FLG-deficient skin model, respectively, while ganotypic skin culture was the reduction in num- in normal control epidermis it was retained with- ber and size of keratohyalin granules and distur- in the SC. A similar finding was observed in the bance of lamellar body formation. FLG -KD did study by Küchler et al. [23], where the FLG -KD not influence keratinocyte differentiation, keratin skin model showed higher permeability toward solubility or the total SC lipid composition. In lipophilic testosterone in comparison to the nor- contrast to the study by Mildner et al. [22] , mal skin model, but, interestingly, there was no Pendaries et al. [25] , Küchler et al. [23] and Váv- significant increase in permeability to hydrophil- rová et al. [24] observed altered keratinocyte dif- ic caffeine. In contrast to the study by Küchler et ferentiation and SC morphology in the used FLG - al. [23] , van Drongelen et al. [21] showed that KD skin models. It was suggested [25] that this FLG-KD did not affect SC permeability of their difference might be explained by the presence of skin model for lipophilic butyl p -aminobenzoic normal fibroblasts in the collagen matrix used by acid.

4 Kezic Jakasa

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 1–7 ( DOI: 10.1159/000441539) Animal Models reduced skin barrier function. In particular, studies in healthy individuals with FLG mutations are The first transgenic animal model that had been lacking. However, studies in IV patients without used to study the functional consequences of FLG concomitant AD suggest that FLG deficiency deficiency was a flaky tail (ft) mouse carrying a leads to reduced skin barrier function in a dose- frameshift mutation in the murine FLG gene [29] . response fashion. The impact of complete FLG However, this mouse model has on the back- deficiency on the barrier permeability seems ground another recessive hair mutation, matted modest; in contrast, a recent birth cohort study (ma), causing development of spontaneous der- has shown that even changes of less than 2 TEWL matitis with increased IgE levels. The ft/ma mice units predisposes infants to develop AD. It is no- showed increased TEWL and reduced SC hydra- table that reduced skin barrier function assessed tion [29] . Recently, Kawasaki et al. [6] generated by TEWL has not been observed in the FLG–/– Flg– /– mice. These mice had dry scaly skin, loss of mouse model. In contrast to the permeability to keratin patterns and increased desquamation un- water, however, the skin of FLG–/– mice allowed der mechanical stress. Interestingly, SC hydration the penetration of both hapten and protein anti- and TEWL were normal although they had im- gens. Further studies in larger, well-character- paired lipid composition and decreased amount ized cohorts of AD patients and healthy controls of FLG degradation products, NMF. Further- of different FLG genotypes supported by studies more, neonatal and adult Flg– /– mice showed no in FLG-deficient models will be needed to better detectable change in the morphology or occlusive understand how FLG deficiency affects skin bar- functions of epidermal tight junctions [30] . How- rier function. ever, the skin of Flg– /– mice was more permeable to antigens, leading to enhanced responses in hapten-induced contact hypersensitivity and Acknowledgment higher serum levels of anti-ovalbumin IgG1 and IgE. This work was supported by H2020 COST Action TD1206 ‘StanDerm’.

Conclusion

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patients with atopic dermatitis. J Al- 2013; 27: 1552–1558. profile and altered acidification path-

lergy Clin Immunol 2014; 134: 82–91. 18 Kelleher M, Dunn-Galvin A, Hourihane ways in a 3D skin construct. J Invest

11 Howell MD, Kim BE, Gao P, Grant AV, JOB, Murray D, Campbell LE, McLean Dermatol 2014; 134: 746–753. Boguniewicz M, DeBenedetto A , WHI, Irvine AD: Skin barrier dysfunc- 25 Pendaries V, Malaisse J, Pellerin L, Le Schneider L, Beck LA, Barnes KC, tion measured by transepidermal water Lamer M, Nachat R, Kezic S, Schmitt Leung DYM: Cytokine modulation of loss at 2 days and 2 months predates AM, Paul C, Poumay Y, Serre G, Simon atopic dermatitis filaggrin skin expres- and predicts atopic dermatitis at 1 year. M: Knockdown of filaggrin in a three-

sion. J Allergy Clin Immunol 2009; J Allergy Clin Immunol 2015; 135: dimensional reconstructed human epi- 124(suppl 2):R7–R12. 930.e1–935.e1. dermis impairs keratinocyte differen-

tiation. J Invest Dermatol 2014; 134: 2938–2946.

6 Kezic Jakasa

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 1–7 ( DOI: 10.1159/000441539) 26 Berroth A, Kuhnl J, Kurschat N, 28 Kezic S, Kemperman PM, Koster ES, de 30 Yokouchi M, Kubo A, Kawasaki H, Yo- Schwarz A, Stäb F, Schwarz T, Wenck Jongh CM, Thio HB, Campbell LE, Ir- shida K, Ishii K, Furuse M, Amagai M: H, Fölster-Holst R, Neufang G: Role of vine AD, McLean WHI, Puppels GJ, Epidermal tight junction barrier func- fibroblasts in the pathogenesis of atopic Caspers PJ: Loss-of-function mutations tion is altered by skin inflammation, dermatitis. J Allergy Clin Immunol in the filaggrin gene lead to reduced but not by filaggrin-deficient stratum

2013; 131: 1547.e6–1554.e6. level of natural moisturizing factor in corneum. J Dermatol Sci 2015; 77: 28– 27 Küchler S: In vitro models of filaggrin- the stratum corneum. J Invest Derma- 36.

associated diseases; in Thyssen JP, Mai- tol 2008; 128: 2117–2119. bach HI (eds): Filaggrin: Basic Science, 29 Fallon PG, Sasaki T, Sandilands A, Epidemiology, Clinical Aspects and Campbell LE, Saunders SP, Mangan Management. Berlin, Springer, 2014, NE, Callanan JJ, Kawasaki H, Shiohama pp 75–81. A, Kubo A, Sundberg JP, Presland RB, Fleckman P, Shimizu N, Kudoh J, Ir- vine AD, Amagai M, McLean WHI: A homozygous frameshift mutation in the mouse Flg gene facilitates enhanced percutaneous allergen priming. Nat

Genet 2009; 41: 602–608.

Sanja Kezic Coronel Institute of Occupational Health, Academic Medical Center University of Amsterdam, Meibergdreef 15 NL–1105 AZ Amsterdam (The Netherlands)

E-Mail s.kezic @ amc.uva.nl

FLG and Skin Barrier Function 7

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 1–7 ( DOI: 10.1159/000441539) Section I: Basic Parameters

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 8–26 (DOI: 10.1159/000441540)

Stratum Corneum Lipids: Their Role for the Skin Barrier Function in Healthy Subjects and Atopic Dermatitis Patients

Jeroen van Smeden Joke A. Bouwstra Division of Drug Delivery Technology, Leiden Academic Centre for Drug Research, Leiden University, Leiden , The Netherlands

Abstract composition induce changes in lipid organization and Human skin acts as a primary barrier between the body subsequently correlate with an impaired skin barrier and its environment. Crucial for this skin barrier function function in both lesional and nonlesional skin of these is the lipid matrix in the outermost layer of the skin, the patients. Furthermore, the effect of filaggrin and muta- stratum corneum (SC). Two of its functions are (1) to pre- tions in the filaggrin gene on the SC lipid composition vent excessive water loss through the epidermis and (2) is critically discussed. Also, the breakdown products of to avoid that compounds from the environment perme- filaggrin, the natural moisturizing factor molecules and ate into the viable epidermal and dermal layers and its relation to SC-pH is described. Finally, the paper dis- thereby provoke an immune response. The composition cusses some major changes in epidermal lipid biosynthe- of the SC lipid matrix is dominated by three lipid classes: sis in patients with AD and other related skin diseases, cholesterol, free fatty acids and ceramides. These lipids and how inflammation has a deteriorating effect on the adopt a highly ordered, 3-dimensional structure of SC lipids and SC biosynthesis. The review ends with per- stacked densely packed lipid layers (lipid lamellae): the spectives on future studies in relation to other skin dis- lateral and lamellar lipid organization. The way in which eases. © 2016 S. Karger AG, Basel these lipids are ordered depends on the composition of the lipids. One very common skin disease in which the SC lipid barrier is affected is atopic dermatitis (AD). This review addresses the SC lipid composition and organiza- Atopic dermatitis (AD, also referred to as atopic tion in healthy skin, and elaborates on how these param- eczema) is a chronic, relapsing, inflammatory eters are changed in lesional and nonlesional skin of AD skin disease characterized by itch, xerosis and a patients. Concerning the lipid composition, the changes broad spectrum of clinical manifestations. There in the three main lipid classes and the importance of the is increasing evidence that the impaired skin bar- carbon chain lengths of the lipids are discussed. In addi- rier function is causative for AD. It is believed tion, this review addresses how these changes in lipid that a defect skin barrier facilitates the transport of allergens and irritants into the skin resulting in these and other studies suggest that FLG muta- skin inflammation [1]. The skin barrier is located tions may play a role in the skin barrier function, in the uppermost layer of the skin, the stratum but their contribution to reducing the skin barrier corneum (SC; fig. 1 ). Its function is to protect the as monitored by TEWL in AD patients remains a body from excessive transepidermal water loss point of debate. From these collective studies, it (TEWL), as well as to prevent the penetration of becomes apparent that other factors/components compounds into the body via the epidermis. The are important for the impaired skin barrier func- SC consists of corneocytes with lipids in the inter- tion in AD patients. This review will describe the cellular regions. In human SC, up to around 25 role of SC lipids with respect to the skin barrier corneocyte layers are present. The structure of the function in healthy skin as well as in patients with SC is often referred to as a ‘brick-and-mortar’ AD. structure, in which the corneocytes represent bricks and the lipids correspond to the mortar [2] . The corneocytes contain keratin filaments, a vari- Lipid Properties in Healthy Stratum Corneum ety of enzymes and water. The corneocyte outer layer is the cornified envelope, consisting of Lipid Composition in the Skin Barrier densely cross-linked proteins such as filaggrin, lo- The main lipid classes in human SC are ceramides ricrin and involucrin. Linked to this protein layer (CERs), cholesterol (CHOL) and free fatty acids is a monolayer of nonpolar lipids referred to as (FFAs). These lipid classes are present in an ap- the lipid envelope. These so-called bound lipids proximately equal molar ratio [19] . We will first may act as a template for the formation of the in- describe the most important observations report- tercellular lipid layers. Because the cornified ened until now. velope and bound lipids limit the uptake of sub- FFAs consist of a single carbon chain with a stances into the corneocytes, and the lipids form variation in chain length in human SC between the only continuous structure in the SC, the inter- around 14 carbon (C14) atoms and C34 atoms cellular lipid matrix acts as the main penetration (see fig. 2 a for the molecular structure). The most pathway for the diffusion of substances through abundant FFA chain lengths are those with 24 the skin and is therefore considered to be impor- and 26 atoms (fig. 3 a) [20–22] . In addition to sat- tant for the skin barrier. An important protein for urated FFAs that cover the majority of the total the structure of the cornified envelope is filaggrin amount of FFAs, hydroxy-FFAs, monounsaturat- (filament-associated protein) [3, 4] . Filaggrin is ed fatty acids (MUFAs) and small amounts of crucial for the alignment of keratin. Additionally, polyunsaturated fatty acids are present. The level metabolites of filaggrin are part of the natural of hydroxy-FFAs and MUFAs does not exceed moisturizing factor (NMF), necessary for proper 25% of the total FFA level [20, 23] . SC hydration. FLG -null mutations (and the intra- The lipid composition of CERs is much more genic copy number variation) are highly associ- complex. Each CER consists of at least one acyl ated with AD and identified as a major risk factor chain chemically linked to a sphingoid base. Both [5–11] . In a substantial subgroup of AD patients, chains do not have a strict molecular structure (as up to 50% of AD patients (of the Irish population) is reported for other organs), but show changes in are carriers of FLG gene mutations. However, specific positions of the standard CER structure studies on the role of FLG mutations for the im- ( fig. 2 b). The currently adapted (and since then paired skin barrier function in AD patients show expanded) nomenclature by Motta et al. [24] contradictory results, illustrating that this aspect therefore uses the molecular structure of both is not fully understood [12–18] . Collectively, chains to identify all CER subclasses reported in

SC Lipids 9

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 8–26 ( DOI: 10.1159/000441540) TEWL

Epidermis Stratum corneum Stratum granulosum Dermis

Stratum spinosum Subcutaneous tissue

Stratum basale a bcDermis

Lateral organization

Orthorhombic Hexagonal Liquid (dense) (less dense) (not ordered)

Lamellar organization

e SPP – 6 nm LPP – 13 nm d

Fig. 1. Explanation of the lipid organization in human SC. Cross-section of the skin ( a), zooming on the epidermal tissue (b ), in which the outermost skin layer, the SC, functions as a ‘brick-and-mortar’ barrier ( c ). It is suggested that pathogens penetrate into the deeper epidermal layers via the lipid matrix (intercellular; illustrated by the arrow). When focusing on this lipid matrix, one is able to distinguish the lipid lamellae ( d): stacked lipid layers in a highly ordered, 3-dimensional structure (e ). The lamellar organization is characterized by an SPP and an LPP. Besides, SC lipids adopt a lateral organization that can either be liquid, hexagonal, or orthorhombic. a, b Illustrations are licensed and adapted (© /istockphoto/j.van.smeden and © /shutterstock, respectively).

10 van Smeden Bouwstra

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 8–26 ( DOI: 10.1159/000441540) Fatty acid ɲ ʘ O

a HO

CER O Acyl chain

* NH OH Sphingoid base OH

O O O NH OH

b CER [E18:2O16S18]

Fig. 2. General structure of two main SC lipid classes. Changes in the molecular structure may occur (primarily) at the positions marked in black. In addition, the carbon chain length may vary (indicated by arrows). a Molecular structure of fatty acids. The α and ω positions may be hydroxylated (additional –OH group), and the alkyl chain may contain one or more double bonds (unsaturated fatty acids). This leads to several fatty acid classes: saturated, mono- and polyunsaturated, and α/ω-hydroxylated. b CER consists of an acyl chain attached via an amide bond to a sphingoid base. The sphingoid base may have optional double bonds or hydroxyl groups, leading to 5 different possibilities. When the 1-position is esterified (asterisk), this leads to the recently discovered subclass with 3 carbon chains [1-O- E…S]. The acyl chain can be hydroxylated at either the α or ω position. Hydroxylation at the ω position may trigger esterification, leading to CER with a very long acyl chain, the so-called acyl-CER, of which one example is depicted (CER-EOS). In total, 4 different acyl chain structures are currently known. In theory, 24 subclasses (6 × 4) may be present in human SC, but only 18 are identified so far in healthy human SC, and only 12 have been analyzed in AD skin.

human SC [25–28] . In addition, each of the two acyl chain. The acyl chain is either a nonhydroxy chains can vary in their number of carbon atoms, fatty acid (N), an α-hydroxy fatty acid (A) or an leading to literally hundreds of uniquely struc- esterified ω-hydroxy fatty acid (EO). The latter tured CER species. The knowledge of this impres- CER-EO subclass is often referred as acyl-CERs sive number of CERs identified in human SC and contains an additional fatty acid attached to gradually increased during the last 40 years. In the acyl chain and is usually a linoleate moiety early years, the discovery of CERs was mainly fo- (fig. 2 b). The introduction of liquid chromatogra- cused on the identification of subclasses using phy combined with mass spectrometry provided thin layer chromatography combined with NMR a rapid boost in identified CER classes, as Masu- and/or gas chromatography. By 2003, a total of kawa et al. [25] and van Smeden et al. [26] discov- nine CER subclasses were discovered [22, 29–32] , ered CER subclasses with a dihydrosphingosine including the CER subclasses with sphingoid bas- base (DS). In human SC, this fourth sphingoid es of either sphingosine (S), phytosphingosine (P) base has been identified with each of the three or 6-hydroxy-sphingosine base (H) coupled to an possible fatty acid subclasses (N, A and EO), lead-

SC Lipids 11

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 8–26 ( DOI: 10.1159/000441540) FFAs CERs 0.4 0.4 1.6 EODS >26OH OH 1.1 25 2.3 9.9 1.3 1.4 4.3 7.4 5.9 OH EOP Ǖ-Hydroxy- 26 16:0 EOH O... 20–23OH OH NS 24 2.0 6.5 CERs 2.6 17:0 EOS 19OH 4.5 7.4 1.6 :0 Acyl- 18OH 18 ADS 1.5 Hydroxy- CERs 17OH 22.1 FFA 2.9 NP 1.2 16OH 19–21:0 10.8 2.9 >20:1 AH 22:0 4.4 3.1 20:1

0.3 19:1 Saturated FFA :0 į-Hydroxy- MUFAs 23 2.9 3.2 18:1 CERs Nonhydroxy- CERs 17:1 2.7 8.8 AP

16:1 24:0 9.3 16.3 NH AS 14.5 9.6 >26:0 25:0 NT 5.3 26:0 NDS ab8.4 3.7 1.7 9.8

Fig. 3. Pie charts representing the human SC lipid distribution of FFA chain length and CER subclasses. a FFAs: saturated FFAs (notified by 0), MUFAs (notified by 1) and hydroxy-FFAs (notified by OH). b CERs: nonhydroxyl-, α-hydroxy- and acyl-CERs (esterified ω-hydroxy) and ω-hydroxy-CERs (notified ‘O…’ ). The ‘O…’ class consists of 0.7% CER-OS, 0.2% OP and 0.4% OH. Values in light gray represent the relative (%) abundance of that lipid subclass. Data of the FFA and CER profiles are adapted from van Smeden et al. [20] and t’Kindt et al. [27] , respectively.

ing to 12 different subclasses by 2010. Until now, carbon atoms of both the sphingoid base and acyl these 12 subclasses have been analyzed in AD pa- chain), the chain length varied between C32 and tients. Six additional CER subclasses have been C54 for nonacyl-CERs. The literature on acyl- reported in healthy human SC but not AD skin; CERs, however, reports an even longer total car- therefore, we do not elaborate on these six sub- bon chain length, with a variation between C64 classes in this review. When focusing on the and C78 (this includes the carbon atoms of the abundances of the various CER subclasses (fig. 3 , esterified fatty acid moiety) [20, 36] . 4 ), quantitatively determined liquid chromatography/mass spectrometry data by Masukawa et al. Lipid Organization in Healthy Stratum Corneum [33] and van Smeden et al. [20] demonstrate that Lipids in human SC form an exceptional lipid or- CER-NP is the most abundant CER in human SC ganization not observed in any other organ. This (∼ 25–30 mol%), and since then this has been became apparent in 1987: in between corneocytes, confirmed by several others [27, 34, 35]. The very a series of lipid lamellae was observed not equally long acyl-CERs (EOS, EODS, EOH and EOP) are in width, but adopting a broad-narrow-broad se- known to be essential for a proper SC barrier quence, indicating a trilayer repeat of lipid layers function (see Lipid Organization in Healthy Stra- [37]. As measured from the micrographs, the re- tum Corneum). However, the relative abundance peat distance of these repeating broad-narrow- is merely ∼ 8–13 mol%. When focusing on the to- broad units extent a space of approximately 12 tal chain length of the CER subclasses (that is the nm. This observation was considered a major

12 van Smeden Bouwstra

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 8–26 ( DOI: 10.1159/000441540) 30 Healthy Nonlesional Lesional 20

10 Relative amount Relative (% of total of CERs) (%

0 NS NP NH NDS AS AP AH ADS EOS EOP EOH EODS a CER subclass

6 g protein)

μ 4 (ng/ Absolute amount Absolute 2

ND 0 NS NP NH NDS AS AP AH ADS EOS EOP EOH EODS b CER subclass

Fig. 4. Bar plots showing the abundances of all CER subclasses in healthy subjects and AD patients. Comparison of the SC-CERs between two clinical studies in healthy subjects and nonlesional and lesional skin of AD patients. a Rela- tive data obtained from a study in Caucasian subjects [36] . b Absolute data from a study in Asians [49] . ND = Not determined. breakthrough. A few years after the visualization a correlation between the level of acyl-CERs in of the lipid lamellae, the first studies using small human SC and the dominance of the features in angle X-ray diffraction of human SC were per- the small angle X-ray diffraction pattern attrib- formed [38, 39]. The studies revealed that in hu- uted to the LPP. Furthermore, in human skin man SC, two lamellar phases are present with re- equivalents that have a high level of acyl-CERs in peat distances of either 13 or 6 nm (fig. 1 e), re- SC, only the LPP has been detected and no SPP ferred to as the long periodicity phase (LPP) and [41, 42] . Both studies suggest that acyl-CERs are the short periodicity phase (SPP), respectively. crucial for LPP formation. This is further demon- On the contrary, Garson et al. [39] reported a 6- strated by studies using lipid mixtures prepared and 4.5-nm lamellar phase. The reason for this with either isolated SC-CERs or synthetic CERs. anomaly was traced back to the different settings Not only the lamellar organization, but also of the equipment, thereby disabling the ability to the lateral organization is crucial for the skin detect the first-order reflection of the 13-nm LPP. barrier function. The lateral organization is the Additional studies by Schreiner et al. [40] revealed arrangement of the lipids within the plane of

SC Lipids 13

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 8–26 ( DOI: 10.1159/000441540) the lamellae, perpendicular to the lamellar lipid the SC of AD patients and controls, some publica- organization ( fig. 1 ). Here, we can distinguish tions report a decrease in CER levels [49, 50], three possible organizations: (i) the very densely whereas others do not report this in nonlesional packed, orthorhombic organization; (ii) the less AD skin [51, 52]. When focusing on the ratios be- dense, hexagonal organization, and (iii) the liquid tween the various classes of lipids in SC, Angelo- organization. Three methods have regularly been va-Fischer et al. [53] and Di Nardo et al. [54] have used to examine the lateral organization in hu- reported a reduced CER/CHOL ratio in nonle- man SC. The first studies were performed by Gay sional and lesional AD skin. Not only SC lipid et al. [43] and Mak et al. [44] using Fourier trans- classes, but also SC lipid subclasses were of high form infrared spectroscopy (FTIR). They report- interest to study. Therefore, studies also focused ed that human SC lipids assemble in a dense, or- in more detail on the lipids examining the indi- thorhombic, lateral packing. Simultaneously, this vidual lipid subclasses in AD. The first publica- was confirmed by wide angle X-ray diffraction tion on this subject reported that the level of CER- [39, 45]. In 2000, Pilgram et al. [46] used electron EOS was decreased in lesional as well as in nonle- diffraction and performed studies as a function of sional skin, while CER-NS, CER-AS and CER-AP depth. These studies state a very detailed analysis increased [50]. Additional studies confirmed the in which they demonstrate that besides an ortho- results of Imokawa et al. [50] and also showed a rhombic phase, also a hexagonal lateral packing reduction in CER-NP and CER-EOS levels in le- was present in human SC in vivo. Close to the SC sional skin [51, 54, 55] . In 2010, Jungersted et al. surface, the hexagonal lateral packing is relatively [56] reported similar changes in CER composi- more abundantly present than deeper in the SC. tion as reported earlier: an increase in CER-AP Similar but more detailed results were obtained and a decrease in CER-EOS, but, in addition, they using X-ray diffraction with a microfocus beam also noticed a decrease in CER-EOH, which was reporting that in the central part of the SC the or- unreported in previous studies. thorhombic lateral packing was predominantly All above-mentioned publications describing present [47]. By means of lipid model systems, it the changes in lipid composition in AD did not has recently been shown that an increase in the report on the lipid chain length distribution of hexagonal lateral packing indeed increases the CERs and FFAs. Farwanah et al. [52] compared permeability in lipid model systems mimicking the SC-CER profiles in nonlesional skin, but did the situation in human SC [48]. It is, therefore, not observe differences in the lipid profile. Ishika- expected that an increasing level of SC lipids wa et al. [49] determined the SC lipid profile in 8 adopting a hexagonal packing at the expense of AD patients in lesional and nonlesional skin and the orthorhombic packing results in an impaired in 7 control subjects, using liquid chromatogra- skin barrier function. phy combined with mass spectrometry (fig. 4 ). The absolute levels of the various subclasses of CERs as well as the chain length distribution of Lipid Properties in Atopic Dermatitis CER subclass CER-NS were reported. All acyl- CER levels in lesional skin were reduced com- Lipid Composition pared with control subjects. In addition, CER With respect to lipid composition in SC of AD subclasses CER-NS and CER-AS were signifi- patients, several changes have been observed cantly increased. For the first time, an increase in compared with control skin (summarized in ta- the level of particularly short chain CERs was re- ble 1 ). Concerning the absolute levels of the main ported. A significant increase in CER-NS with a lipid classes (FFAs, CERs and CHOL) between total chain length (sphingoid base and acyl chain)

14 van Smeden Bouwstra

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 8–26 ( DOI: 10.1159/000441540) Table 1. Overview of modulations in SC lipid parameters in AD

Changed parameter Observations in AD patients (compared with healthy subjects)

CER Increase in CER subclasses AS, AH, AP, ADS, NS Decrease in CER subclasses NP, NH, acyl-CERs (CER-EO) Increase in short chain CERs (C44) Fatty acids Increase in short chain FFAs (≤C18) Reduction in long chain FFAs (≥C24) Increase in MUFAs Decrease in hydroxy-FFAs Lateral lipid organization Less lipids adopt an orthorhombic packing Increased level of lipids adopting a hexagonal lipid packing Less conformational ordering of the lipids Lamellar lipid organization Reduction in repeat distance of lamellar phases

of C34 was shown (referred to as CER-NS C34) in showed an increase in the level of CERs with a total lesional skin. The increased level of CER-NS C34 chain length of 34 carbon atoms. The observations and the reduced level of acyl-CERs both corre- of an increase in the C34 CERs, as well as a reduc- lated highly with the skin barrier function moni- tion in the level of high-molecular-weight acyl- tored by TEWL. CERs, led to the hypothesis that the mean chain In a more recent study, the CER subclasses length of all CERs could be reduced in AD. Hence, were analyzed with respect to chain length distri- the mean CER chain length was calculated and bution and CER subclass composition in 28 AD subsequently compared between control subjects patients in nonlesional skin and 14 controls [57] . and AD patients. Indeed, the average chain length With respect to the level of subclasses, the results in nonlesional skin in patients with AD was re- were very consistent with those of previous stud- duced compared with controls (fig. 5 a), in line ies (fig. 4 ). Due to the larger cohort, they were able with the findings of Ishikawa et al. [49] for CER- to observe changes in CER subclass levels which NS. Besides the CER composition, the skin barrier were already significant in nonlesional skin. In function was monitored by measuring TEWL in line with previous studies, the total level of acyl- each of these patients and controls. In line with CERs (CER-EO) was reduced as well as the level many other reports, they showed that TEWL was of CER-NP. On the contrary, the levels of CER- significantly increased already in nonlesional skin. NS and CER-AS were increased. The level of the When plotting the mean CER chain length as a remaining CER subclasses also changed signifi- function of TEWL for each individual, they dem- cantly, but these changes were less abundant. It onstrated an excellent correlation (r = 0.73), indi- should be mentioned that all CER classes with an cating that CER chain length is an important fac- α-hydroxy fatty acid chain (CER-A) were in- tor for the impaired skin barrier function in AD. creased, whereas all acyl-CER classes proved to be In a subsequent paper, they investigated the decreased in SC of AD patients. CER composition of lesional skin of the same pa- In the same study, the chain length distribu- tients and combined those data with those for tion of each of the CER subclasses was examined nonlesional skin and the controls. This study in patients and controls as well. It was reported clearly showed that all changes in CER composi- that particularly CER-NS, CER-AS and CER-AH tion in nonlesional skin were also observed in le-

SC Lipids 15

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 8–26 ( DOI: 10.1159/000441540) Average CER chain length Average SC lipid chain length CER + FFA p = 0.012 p = 0.006 Average FFA chain length p < 0.0001 p < 0.0001 24 p < 0.001 p < 0.001 35 48 47.0 ± 0.7 34.2 ± 0.3 34 46.4 ± 0.7 22 46 21.4 ± 0.4 33.3 ± 0.7 33 20.3 ± 0.9 44.3 ± 2.3 20 44 32 31.1 ± 1.2 18.2 ± 0.6 31 (carbon atoms) (carbon atoms) (carbon atoms) 42 18

Average chain length 30 Total lipid chain length chain lipid Total Average CER chain length 40 16 29

Control Nonlesional Lesional Control Nonlesional Lesional Control Nonlesional Lesional a

50 p = 0.0078 50 r = –0.79 r = –0.78 35 40 40 34 /h) /h) 2 2 30 33 30 32 20 TEWL (g/m TEWL

20 (g/m TEWL

(carbon atoms) 31 Total lipid chain length chain lipid Total 10 10 30

29 0 0 29 3031 32 33 34 35 4.06.0 8.0 10.0 12.0 14.0 bcdMean lipid chain length (carbon atoms) Scissoring bandwidth (FTIR bandwidth, cm–1) Nonlesional Lesional

Fig. 5. Scatter dot plots of data on the SC lipid composition and organization in healthy subjects and AD patients [adapted from 36 ]. Noncarriers and carriers of an FLG mutation are represented by filled and open symbols, respectively. Control subjects are indicated by circles. Nonlesional skin and lesional skin of AD patients are indicated by diamonds and squares, respectively. a The average carbon chain length of CERs, FFAs and the combination of both. Horizontal lines represent the average ± SD. b , c Correlation dot plots in which TEWL values of all individual subjects are plotted versus the average lipid chain or the lateral lipid organization (i.e. FTIR CH2 scissoring bandwidth), respectively. The dashed line indicates the optimal linear fit through all data points. The r values in the upper right corners correspond to Spearman’s correlation coefficients. d Paired plot of the calculated total lipid chain length of a selected subset of patients from which both nonlesional and lesional skin was analyzed. sional skin, but the changes were more aggravat- larly as reported by the latter authors, although ed [36] . Especially the reduction in acyl-CERs was acyl-CERs were not included in their analysis. pronounced in lesional skin, but also the levels Almost no data are reported on the FFA com- of CER C34 were drastically increased. As the position in AD skin. There is one publication on changes were similar but more pronounced, a very long FFAs in AD skin (>C24). It was report- further significant reduction in CER chain length ed that this level of very long chain FFAs was re- was observed, and the data supported the earli- duced in both lesional and nonlesional skin of AD er observed correlation between average chain patients [59]. In 2014, more information became length and TEWL (fig. 5 b). Tawada et al. [58] available concerning the FFA composition in AD confirmed the findings of van Smeden et al. [36] patients [36] . Not only the saturated fatty acids, in AD patients. They noticed that the level of but also the hydroxy-FFAs and MUFAs were ex- CERs with a long chain length was reduced simi- amined. The relative level of the saturated FFA

16 van Smeden Bouwstra

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 8–26 ( DOI: 10.1159/000441540) fraction remained unchanged in AD patients lamellae were noticed. In another study, a much compared with healthy subjects. However, the larger group of patients was involved. Also, in this level of hydroxy-FFAs decreased, while the level extended group of patients and controls, a signif- of MUFAs increased, primarily in lesional AD icant increase in the level of lipids adopting the skin. The chain length distribution of the FFAs hexagonal lateral packing was observed [57] . As changed drastically. The level of very long chain the hexagonal packing is less dense than the or- FFAs (≥ 24 carbon atoms) was strongly reduced, thorhombic lateral packing, this change in lateral whereas shorter FFAs were increased. This was packing may result in a higher permeation more pronounced in lesional skin compared with through the lipid domains in the SC [48] . In a nonlesional skin. clinical study in nonlesional AD skin, both the From the reduced CER and FFA chain lengths, lateral and lamellar lipid organization was as- the reduction in average lipid chain length (that is sessed at the same skin spot of each patient. Re- CER and FFA chain length combined) per indi- garding the lateral lipid organization, FTIR mea- vidual was calculated. The results are shown in surements indicated a reduced presence of an or- figure 5 a. Very similar as in case of the CER chain thorhombic lateral packing in agreement with the length, the average lipid chain length in lesional electron diffraction measurements. In addition, and nonlesional skin was shorter than in control the conformational disordering of the lipids in skin, and the most drastic reduction in chain nonlesional skin was also increased. Both obser- length was observed in lesional skin. The relation vations indicate that the SC lipids in AD are in a between SC lipid chain length and skin barrier less ordered state than in control skin. Concern- function was also assessed. Mean SC lipid chain ing the lamellar organization (examined by X-ray length values were plotted against TEWL values: diffraction), a reduced repeat distance of the la- it became evident that chain length correlated ex- mellar phases was observed already in nonlesion- cellently with the impaired skin barrier function al skin. This may be explained by the reduced ( fig. 5 b). These studies strongly indicate that the chain length of the SC lipids and the reduced lev- chain length of lipids plays a key role in the im- el of acyl-CERs, as acyl-CERs are important for paired skin barrier in AD skin. This provides new LPP formation. The same publication also report- insights in the treatment of AD patients as nor- ed that the changes in lipid composition in the malization of the lipid metabolism may be an ef- diseased state correlated to a high extent with the fective treatment in skin barrier repair. modulation in lateral packing and the lamellar phases in nonlesional skin [57] . Furthermore, the Lipid Organization reduced orthorhombic lateral packing combined Fartasch et al. [60] reported an impaired extru- with the reduced repeat distance of the lamellar sion process of these lamellar bodies in patients phases correlated very well with the increased with AD. A disturbed lamellar body extrusion TEWL values. This was a first indication that not process may result in a lower level of lipids in the only lipid composition, but also lipid organiza- intercellular regions, which is indeed reported by tion plays a role in the impaired skin barrier func- several groups [61–63]. In 2001, the lateral pack- tion in AD patients. Moreover, it was the first ing was examined in 3 patients in nonlesional study to demonstrate that these modulations oc- skin using electron diffraction. This study indi- cur already in nonlesional AD skin. This is an im- cated that AD patients indeed have an increased portant observation as this may implicate that a level of lipids adopting a more hexagonal lateral normalization of the lipid composition in nonle- packing when compared with 3 control subjects sional skin may contribute to the restoration of [64] . Furthermore, some changes in the SC lipid the barrier function and thus reduce the reoccur-

SC Lipids 17

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 8–26 ( DOI: 10.1159/000441540) with an increased abundance of FFAs and CERs 2,851 with relatively short chain lengths exhibit an increased presence of lipids adopting a hexagonal

2,850 phase. This explains the increase in TEWL in AD )

–1 patients and certainly implies that the lipids play

(cm indeed a prominent role in the impaired barrier 2,849 function in AD skin, in both nonlesional and lesional skin. To examine whether only a correla- 2,848 FTIR stretching peak position peak FTIR stretching tion exists or that the barrier lipids are an under- 4 6 8 10 12 14 lying factor for the impaired skin barrier, addi- FTIR scissoring bandwidth (cm–1) tional studies were performed using model lipid membrane systems. Using these model systems, it is possible to examine the relationship between Fig. 6. Scatter dot plot illustrating the relation between lipid composition, lipid organization and perme- the density of the lipid packing (FTIR CH2 scissoring vibrations) and the conformational ordering (FTIR CH2 ability. These studies showed that both (i) a stretching vibrations). Noncarriers and carriers of a FLG broader carbon chain length distribution and (ii) mutation are represented by filled and open symbols, an increase in the level of unsaturation contribute respectively. Control subjects are indicated by circles. Nonlesional skin and lesional skin of AD patients are indi- to an increased presence of lipids adopting a hex- cated by diamonds and squares, respectively. The dashed agonal packing. In addition, a reduction in acyl- line indicates the optimal linear fit through all data CER reduces the formation of LPPs. Further- points. r = –0.84 (Spearman’s correlation coefficient). more, it was shown that these changes in lipid organization resulted in a higher permeability [48, 65, 66] . This does not only demonstrate an exist- rence of lesional skin in AD patients. In a following correlation between lipid properties and im- up study, the lipid organization in lesional skin paired skin barrier function in AD, but also shows was examined using both the CH 2 scissoring and that the lipids are a key underlying factor in the CH2 symmetric stretching vibrations of the lipids. impaired lipid barrier in these patients. It was reported that changes in lateral packing and conformational disordering were more pronounced in lesional skin compared with nonle- Enzymes Involved in Lipid Biosynthesis sional skin and controls: a higher level of lipids adopting the hexagonal phase (reduction in band- The expression of several enzymes involved in width of the scissoring vibrations in the FTIR lipid metabolism is expected to be modified in spectrum) coincided with a higher level of con- AD skin compared with control skin at both the formational disordering ( fig. 6 ) [unpubl. data]. genetic and the proteomic level. The most impor- Both the changes in the bandwidth of the scissor- tant enzymes involved in lipid biosynthesis re- ing bands and the CH 2 stretching frequencies ported to be involved in AD are briefly discussed. correlated excellently with an impaired skin barrier function in lesional and nonlesional skin Elongases measured by TEWL ( fig. 5 c) [36]. In addition, it Seven different enzymes make up the elongase fam- was demonstrated that the lipid chain length (i.e. ily, referred to as elongases 1–7 (ELOVL1–7). The a combination of FFA and CER chain length) was most relevant elongases for the fatty acid synthesis directly related to both the lipid organization and in the epidermis are ELOVL1, ELOVL4 and skin barrier function (TEWL). SC of AD patients ELOVL6. These enzymes elongate FFAs with chain

18 van Smeden Bouwstra

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 8–26 ( DOI: 10.1159/000441540) lengths between C20–C26, C26–C32 and C14– Ceramidases for the Breakdown of Ceramides in C18, respectively. In an AD murine model, it was a Sphingoid Base and a Fatty Acid Chain observed that at mRNA and protein level ELOVL1 It has been suggested that in AD the activity of and ELOVL4 are reduced [67] . These findings sug- these enzymes is increased. This results in a high- gest a reduced average chain length of CERs and er level of spingosine-1-phosphate, which may FFAs in SC of AD patients may be caused by a re- trigger the release of TNF-α and IL-8 from kerati- duced expression and/or activity of these elongases. nocytes and thus enhance skin inflammation [76] .

Serine Palmitoyl Transferase Sphingomyelin Deacylase Serine palmitoyl transferase is responsible for the The enzyme (glucosyl-CER) sphingomyelin de- first step in CER synthesis involved in linking ser- acylase stimulates the cleavage of the acyl chain ine to a fatty acid chain. Hardly any information from the glucosyl-CER and sphingomyelin [77] . is available concerning this enzyme in AD skin, Hara et al. [78] performed studies in AD patients but it has been shown that serine palmitoyl trans- demonstrating high expression of glucosyl sphin- ferase knockout results in inflammatory reactions gomyelin deacylase in these patients. It should be and high proliferation of keratinocytes in the skin noted, however, that the total amounts of these of a mouse model [68] . metabolites are far too little to fully explain the changes in SC lipids observed in these patients. Ceramide Synthases CER synthases (CerS) are involved in linking the very long fatty acid chain with the sphingoid base, Effect of FLG Mutations on Lipid Barrier a crucial step in synthesizing CERs. It has been Properties reported that a reduction in CerS3 results in the absence of all acyl-CERs demonstrating its im- The above-mentioned data demonstrate that a portance for the synthesis of this extraordinary modulation in lipid metabolism and subsequent class of CERs [69] . More recently, Eckl et al. [70] modulations in lipid composition and organiza- demonstrated in patients with congenital ichthy- tion differentiate the SC lipids of AD patients osis that synthesis of very long chain CERs by from control subjects. The underlying factors, CerS3 is important for the formation of a proper however, have not been fully identified so far. SC barrier, as missense mutations in these pa- Several studies report on the relationship between tients lead to defects in sphingolipid metabolism FLG mutations and the lipid composition in le- and abnormal SC lipid lamellae. sional and nonlesional skin. Jungersted et al. [56] did not observe a correlation between FLG muta- Sphingomyelinase and Glucosylcerebrosidase tions and SC lipid composition (they screened Two enzymes responsible for the final step in only for the 2 most prevalent FLG mutations). the CER synthesis are (acid) sphingomyelinase Another study of Angelova-Fischer et al. [53] ex- (aSMase) and glucosylcerebrosidase (GBA) [71] . amined the relation between FLG mutations and Both enzymes are affected by the pH of the micro- the level of lipid classes in AD nonlesional skin. environment and the activity of serine proteases Small but statistically significant differences were [72–74] . In 2004, it was already observed that reported: compared with control subjects and AD sphingomyelinase activity in lesional as well as patients without FLG mutations, patients with nonlesional skin was reduced [75] . This enzyme FLG mutations demonstrate (i) increased levels catalyzes the conversion of sphingomyelin to (absolute and relative) of CHOL in nonlesional CER-NS and CER-AS. skin, (ii) a reduction in the CER:CHOL ratio and

SC Lipids 19

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 8–26 ( DOI: 10.1159/000441540) (iii) an increase in the amount of triglycerides hibitors (LEKTI) involved in the degradation of [53] . In a recent study, we also examined the cor- corneodesmosomes have a different activity in relation between FLG mutations and changes in AD skin and are affected by environmental con- lipid composition compared with healthy conditions [83] . These serine proteases also influence trols, but neither the modulations in lipid compo- enzymes involved in the last step of CER synthesis sition nor modulations in lipid organization were [72]. It has been suggested that filaggrin may have associated with (any of the) FLG mutations [57] . an indirect effect on the activity of some of these It should be mentioned, however, that besides enzymes: metabolic products of filaggrin form a FLG mutations many other factors may result in major part of the NMF components. Therefore, a a modulation in lipid composition and organiza- reduction in filaggrin levels (or the presence of tion in AD patients, such as daily life influences, FLG-null mutations) will result in a reduction in and inflammation. For this reason, it is important SC-NMF levels. Indeed, reduced NMF levels have to study FLG mutations in human skin equiva- been demonstrated in AD patients [13, 84]. A re- lents mimicking several aspects of the skin barrier duction in SC-NMF may result in an increase in of AD patients. Two studies show no changes in pH, as Kezic et al. [85] show a negative correlation the lipid composition [79, 80] in human skin between NMF levels and pH. Studies on SC-pH in equivalents after FLG knockdown, whereas a AD patients demonstrate a higher surface pH third study reports that FLG mutations results in [85–87]. It is hypothesized that an increase in SC- a strong increase in FFA levels [81] . However, a pH may affect local enzymatic reactions, which strong increase in the level of FFAs does not cor- may explain the reduced activity of GBA and respond to the findings in AD patients [53, 59] . If aSMase [73, 88]. Overall, it is assumed that chang- a higher level of FFAs is present in human skin es in local SC-pH may have a detrimental effect equivalents with FLG knockdown, an increase on the SC barrier, as is well reviewed by Elias and in ordering of the SC lipids is expected, but the Steinhoff [89] . opposite result – a reduction in lipid chain ordering – has been reported. This indicates that in these cultures not only the FFA level increased, Effect of Inflammation on the Lipid but also the chain length and/or degree of unsatu- Metabolism and Lipid Barrier Properties rated fatty acids are also expected to be increased as this would result in a more hexagonal packing. Another key issue in AD is the effect of inflam- A very recent paper reports on the transcriptom- mation on lipid biosynthesis and skin barrier ic analysis of pediatric AD patients and demon- function. An indication that cytokines, such as strates the importance of lipid biosynthesis in IL-4, affect lipid metabolism in the epidermis has atopic skin pathology irrespective of FLG muta- been provided by Hatano et al. [90] . They fo- tions [82] . cused on the effect of IL-4, interferon (IFN)-γ and TNF-α on several enzymes such as aSMase, GBA and acid ceramidase. Using normal human Natural Moisturizing Factor and pH Levels keratinocytes and epidermal sheets, supplementation of TNF-α (proinflammatory cytokine) and AD skin is characterized by a dry and flaky ap- IFN-γ (Th1 cytokine) decreased the level of pearance. Dry skin is often traced back to reduced aSMase and GBA at mRNA level, whereas the levels of NMFs contributing to an optimal hydra- level of mRNA acid-ceramidase was increased. tion level in the SC. Furthermore, there are indi- IL-4 did counteract this affect in epidermal cations that serine proteases (kallikreins) and in- sheets, but not in keratinocytes. Supplementa-

20 van Smeden Bouwstra

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 8–26 ( DOI: 10.1159/000441540) tion of TNF-α and IFN-γ did also result in a re- FLG mutations and inflammatory aspects are duced level of CERs in epidermal sheets. This is a two important factors for the pathogenesis of AD, first clear indication that inflammation affects but their relation with the SC barrier function is lipid metabolism in the epidermis. In addition, not completely understood. Filaggrin is a mem- Tawada et al. [58] demonstrated that IFN-γ de- ber of the proteins in the cornified envelope and creased mRNA expression of ELOVLs and CerS, FLG mutations as such may affect the skin barrier which led to a reduced level of CERs with long function by changing the cornified envelope per- chain fatty acids in cultured human keratino- meability. However, FLG mutations do not cor- cytes and epidermal sheets. relate with the SC lipid composition and thus the Another indication that inflammation is an SC lipid organization [36, 56], whereas reduced important aspect for epidermal lipid metabolism levels of NMF – in part filaggrin metabolites – is the observation of more drastic changes in lipid correlate to a much higher extent with the SC lip- composition and organization in lesional skin in ids and the impaired skin barrier function in AD AD compared with nonlesional skin in the same [57] . The fact that SC lipids do not correlate with patient ( fig. 5 d) [36]. In AD patients, average FFA FLG at DNA level, but show a certain correlation and CER chain lengths were shorter in inflamed at metabolite level, may suggest that a common skin areas than in nonlesional skin areas. Recent- factor affects both NMF levels and lipid metabo- ly, two studies were performed to examine wheth- lism. Factors that may play a role are inflamma- er inflammation affects lipid metabolism in the tory regulators and an altered SC-pH gradient as epidermis of human skin equivalents. Tawada et we discussed above. A relation between filaggrin, al. [58] have shown that IFN-γ may affect lipid NMF levels, SC lipids and inflammation is occa- biosynthesis in the skin as IFN-γ reduces the ex- sionally depicted in clear schemes, providing a pression several enzymes of the ELOVL family at clear overview of different aspects that may affect gene and protein level. In another study, it was the SC barrier function in AD [92, 93] . However, shown that TNF-α and IL-31 reduce the total lev- although some changes in SC lipid composition el of acyl-CERs [91] . In addition, FFAs with a may indeed be due to direct changes in the SC-pH chain length of C24 and longer were reduced. gradient, other lipid modulations are highly un- These features were also observed in AD skin. likely to originate from changes in the SC-pH gra- When focusing on the enzymes involved in lipid dient, for example, the reduced SC lipid chain biosynthesis, the two cytokines reduced the ex- length. The synthesis and elongation of the FFAs pression of ELOVL1 and CerS3 [58, 91] . takes place in the keratinocytes located in the viable layers, thus not close to the interface between the stratum granulosum and SC. Changes in the Concluding Remarks SC-pH gradient are therefore unlikely to directly affect the lipid biosynthesis in the lower epider- SC lipids are an integral part for proper skin bar- mal layers. However, the conversion of CER pre- rier function. In AD, changes in lipid composi- cursors into their final constituents by GBA and tion negatively affect the organization of these aSMase does occur at the stratum granulosum-SC lipids, inflicting a reduced skin barrier function in interface and in the lower layers of the SC. The these patients. Although the magnitude of these reduced levels of acyl-CERs could therefore di- lipid changes relates to some extent to the sever- rectly originate from a higher SC-pH due to im- ity of the disease [57] , the particular role of each paired conversion from glucosyl-CERs to CERs individual lipid class in the SC remains to be elu- by GBA. This incomplete conversion has been cidated. demonstrated in Netherton’s syndrome, an in-

SC Lipids 21

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 8–26 ( DOI: 10.1159/000441540) flammatory skin disease with a severely deterio- Some of these changes in lipid composition rating SC barrier. A study on GBA in Netherton may be normalized by the application of formula- patients demonstrated reduced expression of tions supplemented with specific lipids. An ex- GBA and subsequently reduced levels of acyl- ample is the reduced level of acyl-CER and the CERs in relation to acyl-glucosyl-CERs (the pre- reduced level of saturated FFA with a long fatty cursors of acyl-CERs). The observed changes in acid chain, such as C22 and C24. However, other SC lipids in this disease are comparable to those deviations in SC lipids, such as increased levels of observed in patients with AD, but to a much high- CER-C34, CER-NS, CER-AS or MUFA, cannot er extent [94] . The fact that Netherton patients be compensated for by the treatment of formula- demonstrate chronically inflamed skin could tions supplemented with specific lipids. In these shed light on the importance of inflammation for cases, lipid biosynthesis needs to be normalized the impaired skin barrier function and altered and this requires further focused research: the epidermal lipid metabolism [95] . A study by identification of enzymes that play a key role in Bonnart et al. [96] highlights an important role the alterations in the SC lipid composition and for both SC lipids and filaggrin for the SC barrier the circumstances at which their expression and function: they demonstrate that the expression of activity will be altered. One important question epidermal elastase 2 in Netherton skin impairs that needs to be addressed is whether an altered the skin barrier function by filaggrin and lipid surface pH and inflammation are key triggers for misprocessing. The importance of inflammation a deviation in lipid biosynthesis or whether other, on SC barrier lipids has been demonstrated in yet unknown, factors play an important role, such other inflammatory skin diseases as well. In pso- as an altered skin microbiome. Future studies riatic plaques, for example, the CER subclass should shed light on these unknown factors with composition has been altered in the SC [24] and the final goal to treat these patients most efficient- differences are observed in lamellar SC lipid orga- ly. Nevertheless, it has been demonstrated over nization compared with the undisturbed and the three past decades that SC lipids play a key healthy skin [71] . Tawada et al. [58] showed a re- role in the impaired skin barrier function of AD duced chain length in several subclasses of CERs patients, not only in the lesional regions, but al- in psoriasis skin. ready in the nonlesional skin.

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Clin Immunol 2014; 134: 781.e1–791.e1.

Joke A. Bouwstra Division of Drug Delivery Technology Leiden Academic Centre for Drug Research, Leiden University Einsteinweg 55 NL–2333 CC Leiden (The Netherlands)

E-mail bouwstra @ lacdr.leidenuniv.nl

26 van Smeden Bouwstra

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 8–26 ( DOI: 10.1159/000441540) Section I: Basic Parameters

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 27–37 (DOI: 10.1159/000441541)

Importance of Tight Junctions in Relation to Skin Barrier Function

Johanna M. Brandner Department of Dermatology and Venerology, Laboratory for Cell and Molecular Biology, University Hospital Hamburg-Eppendorf, Hamburg , Germany

Abstract 76 [2] . While TJs have been investigated for de- Tight junctions (TJs) are complex cell-cell junctions that cades in simple epithelia and endothelia, in mam- form a barrier in the stratum granulosum of mammalian malian skin their intensive elucidation has only skin. Besides forming a barrier themselves, TJs influence started at the beginning of this century, resulting other skin barriers, e.g. the stratum corneum barrier, and in the discovery of a new barrier contributing to are influenced by other skin barriers, e.g. by the chemical, overall skin barrier function [3–11] . the microbiome, or the immunological barrier and likely by the basement membrane. This review summarizes the dynamic interaction of the TJ barrier with other barriers Tight Junctions and Their Proteins in in the skin and the central role of TJs in skin barrier func- Mammalian Skin tion. © 2016 S. Karger AG, Basel Several TJ proteins have been identified in human, but also in porcine, murine and canine skin [for human and mouse skin, see 12, 13, for pig Tight junctions (TJs) are complex cell-cell junc- skin, see 14, 15 and for dog skin, see 16 , 17 ]. The tions consisting of transmembrane proteins, e.g. localization patterns of these proteins in the epi- members of the families of claudins (Cldns), TJ- dermis are diverse: the cell-cell junction localiza- associated marvel proteins and junctional adhe- tion of some of them is restricted to the stratum sion molecules as well as TJ plaque proteins, such granulosum, e.g. occludin (Ocln) or cingulin, as zonula occludens (ZO) proteins 1–3, MUPP-1 whereas others are found in the stratum granulo- and cingulin [1]. TJs are well known to be con- sum and upper stratum spinosum, e.g. ZO-1 and nected to the actin filament cytoskeleton [1] . Re- Cldn-4, or in all layers including the stratum cor- cently, it was also shown that they are likely con- neum (SC), e.g. Cldn-1. All of the TJ proteins co- nected to intermediate filaments, namely keratin localize at cell-cell borders in the stratum granulosum [12, 13], being also the layer where typical cytes TJs form a barrier to Na+ , Cl – , Ca 2+ and wa- TJ structures are found [3, 4, 7, 10, 18] . Further, ter [28] , indicating a similar role in the epider- cytoplasmic staining of several TJ proteins is mis. However, further studies addressing the found [12, 13] . In addition to the interfollicular question of TJ permeability/barrier function to epidermis, TJ proteins are also found in hair fol- different solutes in the epidermis are definitely licles [19, 20] and in sweat glands [21] , again with needed. diverse localization patterns but also colocalizing Also, knowledge about alterations in TJ barri- at the areas where the outermost living layers face er function in skin diseases is limited. In psoriasis, the ‘outside’. Alterations in TJ protein expression TJ barrier function for a 557-Da tracer is still and localization have been observed in several present, but its localization changes from the skin diseases, including neonatal ichthyosis scle- granular cell layer in healthy skin to deeper layers rosing cholangitis (NISCH) syndrome (Cldn-1 in psoriatic skin [5] . In a mouse model for chron- knockout), atopic dermatitis, ichthyosis vulgaris ic allergic dermatitis, impaired barrier function and psoriasis [for reviews, see 12 , 13 ]. for tracers <5 but not >30 kDa was found [29] . A special kind of barrier may be provided by TJ remnants containing Cldn-1 and Ocln in the Barrier Function of Tight Junctions in the Skin SC. It was observed that the presence of these TJ remnants at the lateral plasma membranes coin- Localization of typical TJ structures goes along cides with a delayed degradation of lateral cor- with a barrier function of TJs in the granular cell neodesmosomes compared to apical or basal cor- layer. This barrier function was shown for a 557- neodesmosomes [30, 31] . It was thus hypothe- Da tracer and, after predigestion of the skin with sized that Cldn-1 or TJ remnants in the SC may exfoliative toxin, for a 32-kDa tracer [4, 5, 18] . protect corneodesmosomes from degradation Both tracers were applied from the dermis and [30, 31]. However, even though very attractive, it were stopped on their way from inside to outside. is difficult to bring this hypothesis in line with When these tracers are applied to the top of the the fact that Cldn-1-knockout mice have a more epidermis, they are already stopped by the SC and compact SC, which also seems to be the case for do not reach the TJs. However, TJs are bidirec- some humans with Cldn-1 null mutation (NISCH tional [22] , which means that if they are perme- syndrome) [4, 32] . able for a solute, the direction of permeation only depends on the gradient of the molecule at both sides. Consequently, if TJs form a barrier, they Nonbarrier Functions of Tight Junction form a barrier to both sides. Thus, if these mole- Proteins in the Skin cules would reach the TJ from outside to inside, which may be the case when the SC barrier is im- Nonbarrier functions of epidermal TJ proteins paired, they are likely to be stopped. have been reviewed recently [33–35] , and here the In addition to these intermediate-sized and focus will be on new data generated since these large molecules, a stop of the ion tracer lantha- reviews were published. Already the localization num was shown at TJs in the granular cell layer of patterns of the various TJ proteins hint at addi- the epidermis [23–26] , but this location was de- tional, TJ-structure/barrier-independent func- bated by others [27] . tions. For example, Cldn-1 is localized in all layers Barrier function of TJs for other molecules, of the epidermis. Thus, it is likely that, besides its including water or other ions, has not been shown barrier function in the granular cell layer, it has yet for the epidermis. But in cultured keratino- additional functions in the other layers. For the

28 Brandner

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 27–37 ( DOI: 10.1159/000441541) Microbiome barrier $ SC barrier Q W L TJ barrier P S L H F S Chemical barrier U W R L Skin barrier E G L L Immunological barrier H H D C V O

M N MC

Fig. 1. Schematic drawing of the various barriers in the skin and their interaction with the TJ barrier. Arrows with continuous lines: influence already shown, arrows with dashed lines: influence hypothesized. M = Macrophages; MC = mast cells; N = neutrophils.

basal cell layer, one could hypothesize that Cldn-1 role for Cldn-1 in differentiation and supports is involved in cell proliferation. It was shown pre- previous data [34] . viously that knockdown of Cldn-1 can increase For Ocln, we could show that its knockdown cell proliferation in keratinocyte cultures [36] . in keratinocytes results in decreased susceptibili- This goes in line with our observation that in an ty to the induction of apoptosis and decreased atopic dermatitis-like allergic dermatitis mouse cell-cell adhesion [38], thus demonstrating that model (AlD), absence of Cldn-1 at the cell-cell this TJ protein is also involved in a variety of borders in the lower epidermal layers significant- functions in addition to barrier function. ly correlates with an increase in cell proliferation in eczematous skin [37] . However, at scratch wound margins, there is a decrease in cell prolif- Interaction of Tight Junctions with Other eration after Cldn-1 knockdown [Volksdorf et Barriers of the Skin – Importance of Tight al., in preparation]. Thus, the physiological state Junctions for Skin Barrier Function (or cell culture conditions) seems to play an important role for the final consequences of the A variety of experimental data published in recent knockdown of Cldn-1 on proliferation. years support the hypothesis that TJs and other Absence of Cldn-1 in the lower epidermal lay- barriers in the skin, i.e. the SC barrier, the micro- ers in eczema of AlD mice also significantly cor- biome barrier, the chemical barrier (antimicro- related with alterations in the differentiation bial peptides, AMPs) and the immunological markers CK14 and CK10 [37] , which suggests a barrier, influence each other ( fig. 1 ). Because the

Tight Junctions and Skin Barrier Function 29

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 27–37 ( DOI: 10.1159/000441541) basement membrane (BM) is also to some extent sequences on the TJ barrier still has to be shown. a barrier and might influence TJs, it is also includ- The up-regulation is found in patients with and ed in this chapter. without FLG mutation. In general, it is tempting to speculate that there Tight Junctions ↔ Stratum Corneum is a compensatory mechanism by up-regulating The SC is the outermost layer of the skin which the TJ barrier when the SC barrier is compromised. provides a formidable barrier against external assaults such as pathogens, UV or irritants, as well Tight Junctions → Stratum Corneum as an inside-out barrier against the loss of water There are increasing data that alterations in TJs or and solutes [39] . TJ proteins result in SC alterations. Knockdown of Cldn-1 in cultured keratinocytes effects an al- Stratum Corneum → Tight Junctions teration in proteins important for SC formation, A closer look at the influence of the SC on TJs re- namely involucrin, loricrin, filaggrin and trans- vealed that the removal of SC by tape stripping glutaminase-1 [28, 37, 40] . Also, in Cldn-1-knock- results in an up-regulation of TJ proteins within out mice, alteration in filaggrin was found. Fur- 1 h [23, 40] , while lanthanum penetration seems ther, changes in lipid composition were observed to be unchanged at this time point [23] . However, in these mice and, consequently, it was shown this does not exclude that there are other, yet that the SC water barrier was impaired [43] . In unknown, functional changes which accompany total, this results in an impaired skin barrier, and this up-regulation. Impaired SC function due to the mice die on the first day of life due to in- the absence of filaggrin does not result in obvious creased water loss [4]. Also, as already mentioned, changes in TJ proteins and functionality in FLG SC morphology is changed in some patients with knockout (FLG–/–) mice [29] . But, interestingly, the NISCH syndrome (human Cldn-1 knockout), FLG knockout in humans, i.e. patients with ich- but functional studies have not been performed thyosis vulgaris, show a down-regulation of Ocln yet [32]. All of these data clearly show that ab- and ZO-1 and impaired barrier function for lan- sence of Cldn-1 influences the SC. However, it is thanum [41] . The cause of this discrepancy be- not clear whether this influence is due to the ab- tween mice and men is not clear yet. Of note, in sence of Cldn-1 in TJs and therefore impaired TJ FLG–/– mice also no spontaneous dermatitis was barrier function, or whether it may be a result of induced under normal humidity and specific a TJ-independent function of Cldn-1. We could pathogen-free conditions [29]; thus additional show that alterations in transepidermal water loss environmental factors may play a role in what is significantly correlate with a decrease in Cldn-1 seen in ichthyosis vulgaris patients. Further, even intensity in eczema of AlD mice in the upper, but though patients with no obvious signs of inflam- not in the lower layers, indicating indeed a TJ- mation were selected for the study, it might be related influence. This is further supported by the possible that subtle inflammation due to in- fact that Ocln knockdown [38] , addressing TJs by creased antigen uptake via the compromised bar- C-terminal Clostridium perfringens enterotoxin rier may result in a secondary down-regulation of [44] in reconstructed human skin, and overex- Ocln and ZO-1 (see also Tight Junctions ↔ Im- pression of Cldn-6 in mouse epidermis [9] result munological Barrier). in SC protein/lipid alterations. In nonlesional skin of patients with atopic dermatitis, which is known to exhibit impaired SC Tight Junctions ↔ Microbiome Barrier barrier function [42], an up-regulation of Cldn-4 The skin’s microbiome is known to play an im- is observed [37]. Whether this has functional con- portant role as a barrier against pathogens. This

30 Brandner

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 27–37 ( DOI: 10.1159/000441541) barrier function is provided by occupying space Tight Junctions → Antimicrobial Barrier and nutrients and thereby inhibiting pathogen To my knowledge, little is known up to now about growth, by the production of bactericidal com- the influence of TJs on the antimicrobial barrier. pounds and by the inhibition of Staphylococcus Future work may elucidate this interesting con- aureus biofilm formation. Further, interaction of nection. the innate and the adaptive immune system is known [45] . Tight Junctions ↔ Chemical Barrier The chemical barrier of the epidermis consists Microbiome Barrier → Tight Junctions of AMPs which are mainly produced by kerati- Concerning the influence of the microbiome on nocytes and immune cells. They are important TJs, we could show that the presence of Staphylo- for the protection of the body against infection coccus epidermidis, a common commensal on and also play a role in inflammation and wound human skin, results in an increase in TJ protein healing [49] . Some AMPs are constitutively ex- expression in ex vivo skin and in an increase in pressed in healthy skin; others are induced/up- transepithelial resistance (TER) in cultured kera- regulated in the skin from patients with psoria- tinocytes [15] . This effect is likely to be mediated, sis, atopic dermatitis or chronic wound infec- at least in part, by the activation of Toll-like re- tions [50] . ceptors (TLRs), as TLRs have been shown to influence TJs in cultured keratinocytes. Ligands Antimicrobial Peptides → Tight Junctions to TLR-1/TLR-2 [Pam 3 -Cys-Ser-(Lys)4 ], TLR-2 Treatment of cultured primary keratinocytes (peptidoglycan), TLR-3 (poly I:C), TLR-4 (lipo- with the AMPs human β-defensin-3, cathelicidin polysaccharide), TLR-5 (flagellin), TLR-2/TLR-6 (LL-37) and psoriasin results in an increase of (Malp-2) and TLR-9 (CpG oligonucleotide) have TER and a decrease in permeability for the 4-kDa been shown to influence TER (all TLRs investi- tracer FITC-dextran [51–53] . This is accompa- gated), and tracer permeability [fluorescein (332 nied by changes in mRNA and protein expression Da) or biotin-SH (557 Da); only TLR-1/TLR-2, in many TJ proteins, including sealing and pore- TLR-2, TLR-3 and TLR-2/TLR-6 have been in- forming Cldns (table 1 ). The role and relevance of vestigated] [46–48]. In general, TER reflects the the changes in the specific TJ proteins after AMP ion permeability of the epithelial layer, compris- treatment still has to be shown, but in general ing transcellular and paracellular (TJ-mediated) this finding suggests that the challenge in the skin ion permeability. The higher the TER, the lower barrier by pathogens or certain skin diseases may the permeability. Tracer permeability reflects the increase TJ barrier function via AMPs, therefore paracellular (TJ-mediated) permeability of the enforcing the innate immune system. Of note, molecules of the chosen size. Dependent on the this effect is not restricted to the skin. In the intes- duration of incubation and the time point of tine, a positive effect of AMPs on TJs was ob- investigation, the changes in TER/tracer perme- served, too [54] . ability are accompanied by changes in TJ protein levels, e.g. Cldn-1, Cldn-23, Ocln and ZO-1 for Tight Junctions → Antimicrobial Peptides TLR-2. In addition, for TLR-2 phosphorylation of To my knowledge, there is up to now no report Ocln by atypical protein kinase C was observed about the influence of TJs on AMPs. But due to [46–48] . In this context, it is interesting to note the interconnectedness of the various barriers in that TLR-2-knockout mice exhibit delayed and the epidermis, it is hypothesized that the connec- incomplete barrier recovery following tape strip- tion between TJs and AMPs is bidirectional. ping [47] .

Tight Junctions and Skin Barrier Function 31

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 27–37 ( DOI: 10.1159/000441541) Table 1. Influence of AMPs and cytokines on TJ protein expression and localization as well as TJ function

AMP/ Change in TJ protein mRNA or Change in TJ protein Change in TJ Ref. cytokine protein level localization functionality

Cathelicidin CK: increase in Cldn-1, -3, -4, Not investigated CK: increase in TER, 51 LL-37 -7, -9, -14, Ocln mRNA/protein, decrease in 4-kDa tracer increase in Cldn-5, -6, -12, -16, -17 permeability mRNA Human CK: increase in Cldn-1, -3, -4, -14, CK: increased localization of CK: increase in TER, 53 β-defensin-3 -23 mRNA/protein; increase in Cldn-1, -3, -4, -14, -23 at cell decrease in 4-kDA tracer Cldn-2, -5, -9, -11, -15, -16, -17, borders permeability -20,- 25 mRNA S100A7/ CK: increase in Cldn-1, -4, -14 and Not investigated CK: increase in TER, 52 psoriasin Ocln mRNA/protein, increase in decrease in 4-kDa tracer Cldn-3, -7, -9 mRNA permeability IL-1β Tissue: short term: increase in Tissue: short term: broader/ CK: short term: increase in 55 ZO-1 protein increased staining of Ocln, TER during TJ formation; CK: short term (24 h): increase in ZO-1, decreased staining of long term: decrease in TER Cldn-4 mRNA; long term (96 h): Cldn-1 in (uppermost) layers during TJ formation decrease in Cldn-1 protein/ CK: short term: increased mRNA, Ocln protein localization of Cldn-1, -4 at cell borders; long term: decreased localization of Cldn-1, Ocln, ZO-1 at cell borders IL-4 CK: increase in Cldn-1, no change Not investigated Donor-dependent TER 12, 36 in Ocln, ZO-1 protein increase or decrease IL-13 CK: increase in Cldn-1, no change Not investigated Slight increase in TER 36 in Ocln, ZO-1 protein IL-17 CK (HaCaT): decrease in Cldn-7, Not investigated Not investigated 72 ZO-2 mRNA TNF-α Tissue: increase in ZO-1 Not investigated CK: short term: increase in 55, 73 TER during TJ formation and in cells with preformed TJs; long term: decrease in TER Histamine RHS: decrease in Cldn-1, -4, Ocln, Not investigated RHS: increased 74 ZO-1 protein permeability for 557-Da tracer

CK = Cultured keratinocytes; RHS = reconstructed human skin.

Tight Junctions ↔ Immunological Barrier Immunological Barrier → Tight Junctions The immunological barrier comprises constitu- Several cytokines have been shown to influence ents of the cellular and the humoral immune sys- TJ proteins and TER (table 1 ), clearly indicating tem in the epidermis, e.g. Langerhans cells (LHCs) that TJs can be modulated by the immunological or cytokines. It plays an important role in the de- barrier. However, their influence seems to be defense against pathogens. pendent on (i) incubation time/time of readout

32 Brandner

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 27–37 ( DOI: 10.1159/000441541) and (ii) the individual donor. For example, it was [59] . Further studies concerning this interesting shown that the proinflammatory cytokine IL-1β connection are underway. results in an increase of TER during short-term incubation and a decrease after long-term incu- Tight Junctions ↔ Basement Membrane bation of cultured keratinocytes [55], and that The BM is a highly specialized structure that con- IL-4 can result in increased and decreased TER in nects the dermis and the epidermis. It is mainly cultured keratinocytes depending on the donor composed of laminins, nidogen, collagen types [12] . Thus, the specific effect of a cytokine in the IV and VII, and perlecan, but other proteins are tissue is likely to depend on the temporal and mi- also present [60, 61] . The BM is freely diffusible croenvironmental context. In addition, to date, for ions/molecules of different sizes, e.g. lantha- most influences of cytokines on TJs have only num [24] , horseradish peroxidase (40 kDa) [62, been shown by using TER measurements and/or 63] and cationic ferritins (450 kDa) [64]. How- changes in TJ proteins/mRNA. In-depth analyses ever, it provides a barrier for larger substances of changes, including tracers of different sizes, (Thorotrast, 70–90 Å) [65] or negatively charged still have to be done. macromolecules (native anionic ferritins) [64] Concerning cellular constituents of the im- even though this barrier is not absolute, and small mune system, it was shown that LHCs express TJ amounts of the substances enter the epidermis proteins [26, 55, 56] and that they can form TJs [64, 65]. Further, it is a barrier to herpes simplex with the surrounding keratinocytes when activat- virus [66] . The BM also seems to be a barrier to ed by tape stripping or in erythematous lesional cells. Keratinocytes and melanocytes are normal- skin of patients with atopic dermatitis [26, 57] . ly found above the BM, and a cardinal criterion This enables LHC to collect antigens from above for invasive carcinoma is disruption and active the TJ barrier in the stratum granulosum without destruction of the BM. Also, for cells entering the compromising this barrier [26] . Of note, this is epidermis from the dermis it is likely that active not the case in nonlesional skin of atopic derma- mechanisms allow their passage through the BM titis patients or in patients with ichthyosis vulgar- [67] . is and psoriasis [57] . A similar mechanism seems In general, BM defects result in skin fragility, to be used in nasal epithelium of patients with blistering and recurring wounds, as seen in skin allergic rhinitis, where also dendritic cells ex- BM disorders such as epidermolysis bullosa [61, pressing Cldn-1 were shown to penetrate beyond 68] . Therefore, impaired skin barrier function is Ocln-identified TJs, while this was not observed found because of destroyed integrity of the skin. in healthy controls [58] . TJ proteins are down-regulated near transmi- Basement Membrane → Tight Junctions grating granulocytes (CD15-positive cells) in pso- Not much is known about the influence of BM riasis, hinting at the necessity of their destruction on TJs. A deficiency in laminin or collagen IV is for neutrophil transmigration to the skin surface lethal in early murine embryonic development. [55] . Near macrophages, there is no change in TJ Perlecan knockout results in heart failure, which proteins [55] . In the AlD mouse model, inflamma- is lethal in the midgestational stage of mice. Thus, tory cell infiltration significantly correlates with the the effect on TJs could not be investigated in these down-regulation of Cldn-1 in the epidermis [37] . mice. Mice with knockout of nidogen 1 + 2 die perinatally due to lung and heart abnormalities. Tight Junctions → Immunological Barrier They do not show an alteration in the inside-out- We observed that the knockdown of ZO-1 induc- side (water loss) or the outside-inside (dye pene- es the release of IL-1β from primary keratinocytes tration) barrier at this time, but effects in adult

Tight Junctions and Skin Barrier Function 33

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 27–37 ( DOI: 10.1159/000441541) mice cannot be investigated due to perinatal le- Conclusion thality [for review see 60]. However, laminin coating results in reduced inulin permeability of In summary, the skin barrier consists of several HaCaT cells, which might be due to the stimula- barriers among which the TJ barrier plays an im- tion of TJ formation [69] . Also, in Sertoli cell portant role as it can influence and/or be influ- preparations, reconstituted BM induces TJ for- enced by the other barriers. Thus, epidermal bar- mation [70] . rier defects found in several skin diseases due to genetic or environmental influences are in all like- Tight Junctions → Basement Membrane lihood not restricted to one barrier, but are a con- To my knowledge, nothing is known up to now sequence of the interaction of the various barriers. about the influence of TJs on BM. In principle, there are two possible scenarios: (1) The impairment of one barrier can lead to a deterioration of other barriers, therefore resulting in an Tight Junctions as a Boundary between accumulation of negative effects or even a vicious Microbial and Immunological Barriers circle. (2) The impairment of one barrier can be partly compensated by the up-regulation of other Even though the microbial barrier on the surface barriers. Investigation of skin diseases regarding of the skin is protecting our body, it would be this specific interplay will enhance our knowledge harmful if these microbes unnecessarily activated and understanding of skin diseases and result in the immune system. One mechanism to avoid this improved treatment options and outcomes. is via the separation of the microbial and the immunologic barrier. As under nonpathogenic conditions the microbial barrier is found above and Acknowledgments the immunological barrier below TJs [26, 71], it is tempting to speculate that TJs may play an impor- I thank Katja Baesler and Christopher Ueck for their skil- tant role in this separation and thus are pivotal for ful help in designing the figure. This work was supported innate tolerance. For a more in-depth discussion by the Deutsche Forschungs gemeinschaft (BR 1982/4-1) of this topic see Brandner et al. [13] . and by H2020 COST Action TD1206 ‘StanDerm’.

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Johanna M. Brandner Department of Dermatology and Venerology Laboratory for Cell and Molecular Biology University Hospital Hamburg-Eppendorf, Martinistrasse 52 DE–20246 Hamburg (Germany)

E-Mail brandner @ uke.de

Tight Junctions and Skin Barrier Function 37

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 27–37 ( DOI: 10.1159/000441541) Section I: Basic Parameters

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 38–46 (DOI: 10.1159/000441543)

Antimicrobial Peptides, Infections and the Skin Barrier

Maja-Lisa Clausen Tove Agner Department of Dermatology, Bispebjerg Hospital, University of Copenhagen, Copenhagen , Denmark

Abstract Different skin components help to protect The skin serves as a strong barrier protecting us from in- against infections: the physical barrier of tightly vading pathogens and harmful organisms. An important bound keratinocytes (with constant shedding of part of this barrier comes from antimicrobial peptides the outermost layers); lipids with antimicrobial (AMPs), which are small peptides expressed abundantly activity and an acidic milieu creating an unfavor- in the skin. AMPs are produced in the deeper layers of able environment for pathogens; immune media- the epidermis and transported to the stratum corneum, tors and proinflammatory cytokines (produced where they play a vital role in the first line of defense by keratinocytes to recruit and signal immune against potential pathogens. Numerous AMPs exist, and cells), and the production of a constitutive level of they have a broad antibiotic-like activity against bacteria, antimicrobial peptides (AMPs), providing fur- fungi and viruses. They also act as multifunctional effec- ther microbial defense. Thus, keratinocytes play a tor molecules, linking innate and adaptive immune re- critical role in forming both a physical barrier and sponses. AMPs play an essential part in maintaining an an immunologic shield alerting the immune sys- optimal and functional skin barrier – not only by direct tem and producing proinflammatory mediators killing of pathogens, but also by balancing immune re- and AMPs, all to create a strong barrier and de- sponses and interfering in wound healing, cell differen- fense against harmful microorganisms. tiation, reepithelialization and their synergistic interplay with the skin microflora. © 2016 S. Karger AG, Basel Antimicrobial Peptides in the Epidermis

AMPs are small peptides abundantly expressed in The skin provides a strong barrier against invad- the skin with a broad antimicrobial activity ing microbes, protecting us from both our com- against bacteria, fungi and viruses [1, 2] . They are mensal skin microflora and potential pathogens. produced predominantly in the suprabasal layers Although it is in permanent contact with micro- and stratum basale of the epidermis and trans- organisms, severe skin infections are rare. ported by lamellar bodies to the stratum corneum, where they play a vital role in the skin barrier. responses, for example [13–18]. The regulation of They also act as multifunctional effector mole- AMPs is complex. Many factors induce AMP ex- cules, with influence on cell migration, prolifera- pression, among others proinflammatory cyto- tion and differentiation as well as cytokine pro- kines, bacterial components, injury and inflam- duction, playing an important role in linking in- mation; however, it varies greatly between the innate and adaptive immune responses [3–5] . dividual peptides [1, 19–27] . In healthy skin, the dominant source of AMP In the skin, the two most characterized fami- production is keratinocytes, providing a constitu- lies of AMPs are defensins and cathelicidins. tive level which serves as a first line of defense Defensins are small, 2- to 6-kDa, cationic pep- against invading pathogens. Upon infection or tides with cysteine-rich residues forming charac- injury, or if the constitutive level of AMPs fails to teristic disulfide bridges [7] . They are divided into clear an infection, AMP up-regulation will take two main classes, α- and β-defensins, while a place in keratinocytes, though infiltrating cells third class, θ-defensins, has so far not been identi- like neutrophils and mast cells will contribute fied in humans. α-Defensins are mainly produced with the majority of AMPs [1, 6–8] . in neutrophils, whereas β-defensins are produced A major role for AMPs is their direct antibiot- in a variety of cells, including neutrophils, kerati- ic-like inhibition of microbes and pathogens. nocytes and sebocytes [28, 29]. Defensins are Most AMPs have an overall positive charge, al- packed in the lamellar bodies within keratino- lowing them to interact with negatively charged cytes and released to the cell surface [26, 30] . Hu- phospholipids in the cell walls of microbial and man β-defensin (hBD)-2 has only bacteriostatic anionic components of fungi and viruses, result- activity against Staphylococcus aureus [11, 31, 32] , ing in transmembrane pore formation and cell but potent bactericidal activity against Escherich- lysis [7, 9, 10] . ia coli and Pseudomonas aeruginosa , whereas Individually, the antimicrobial activity of hBD-3 is very potent against S. aureus [11, 33] , AMPs varies greatly, but acting in synergy they including MRSA, and shows broad activity demonstrate much more potent function [11, 12] . against Candida albicans , E. coli , Streptococcus Co-expression is common in the skin, making pyogenes , P. aeruginosa and Enterococcus faecium synergistic activity very important for the in vivo [33–35]. hBD-2 and hBD-3 are induced by proin- relevance of AMPs in their protection against inflammatory cytokines, e.g. IL-1β and TNF-α/ fections. Studies on in vitro antibacterial activity IFN-γ, through STAT1 and NF-κB, but also by reveal that pH, reduced or oxidized forms of microbial stimuli, injury and UV-B [20, 36–39] . AMP, recombinant or natural AMP, bacterial hBD-1 is expressed constitutively and is not near- strain, cell differentiation and culturing condi- ly as potent as the other β-defensins [6, 22, 23, 31] . tions, all are factors with significant impact on an- Despite a more weak antimicrobial activity of tibacterial activity. Due to the influence of these hBD-1 on its own, several studies have shown a multiple factors, the in vivo effects of AMPs are greatly increased effect when hBD-1 acts in syn- difficult to predict and interpret. ergy with other defensins [11, 40] , and the re- In addition to their direct antimicrobial activ- duced form, in contrast to the oxidized form, has ity, AMPs have an important function in linking also shown increased activity [41] . innate and adaptive immune responses. Their Cathelicidins, also called LL-37, are small, 12- role as effector molecules is very broad: they stim- to 80-amino acid, cationic, amphipathic peptides. ulate the production of cytokines and chemo- They are encoded by the human cathelicidin kines, attract immune cells to the site of infection/ gene, and hCAP-18, a precursor, is processed to inflammation and modulate Toll-like receptor the active form of LL-37 by proteases in keratino-

AMPs, Infections and the Skin Barrier 39

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 38–46 ( DOI: 10.1159/000441543) cytes [42]. Like defensins, they are localized in la- P. acnes. Adrenomedullin has numerous physio- mellar bodies in keratinocytes [43] and demon- logical roles, including vasodilation, hormone strate broad antimicrobial activity towards S. au- regulation and wound repair [63–65] . ALP (anti- reus, as well as other Gram-positive and -negative leukoprotease)/SLPI (secretory leukoprotease in- bacteria [11, 44–49]. LL-37 also fights viruses and hibitor), a serine protease inhibitor, is constitu- is potent against vaccinia virus and herpes sim- tively expressed in keratinocytes and effective plex virus (HSV) at physiological concentrations against Gram-negative and -positive bacteria as [44, 47]. LL-37 demonstrates important ‘alarmin’ well as C. albicans and HIV-1 [66, 67] . Elafin, an- properties, recruits neutrophils, T cells, mast cells other serine protease inhibitor, also called skin- and monocytes to sites of infections as well as derived ALP or SKALP, has shown killing activity promotes angiogenesis [3, 50–52] . against P. aeruginosa and S. aureus [68–71] . Lyso- zyme is localized in the cytoplasm of keratino- Other Antimicrobial Peptides Resident in Skin cytes but not detected in stratum corneum or skin Cells Participate in the Defense Barrier washing fluid. It is active against both Gram-neg- Psoriasin, initially found in keratinocytes from ative and -positive bacteria [72–75] . psoriasis patients [53], is very potent against E. coli at low concentrations and active against S. aureus at higher concentrations [54] . Psoriasin Antimicrobial Peptides and Impaired Barrier acts as a strong modulator of neutrophil activa- Function tion [55, 56] . RNase7 is part of the RNaseA superfamily and Upon barrier disruption, increased levels of is produced in keratinocytes [57] . RNase7 seems AMPs are expressed in the skin (fig. 1 ). This is to play an important role in protecting healthy seen in healthy skin as well as nonlesional skin of skin from S. aureus infections [58, 59] due to its atopic dermatitis (AD) and psoriasis after barrier very potent activity against S. aureus [57] . It also disruption by tape stripping or injuries [76] . In- exhibits antimicrobial activity against E. coli , P. creased expression of psoriasin and RNase7 was aeruginosa, E. faecium , Propionibacterium acnes observed already after 1–2 h, and psoriasin levels as well as MRSA [57, 59, 60] . It is considered one remained elevated for as long as 7 days [77, 78] . of the most potent AMPs due to its strong activity In contrast to psoriasin and RNase7, where up- at low concentrations [57] . regulation was reduced by occlusion, hBD-2 was Dermcidin is produced constitutively from also induced by barrier disruption, but only after sweat glands, secreted into the sweat and trans- 24 h and occlusion. This could be due to different ported to the epidermal surface [61] . It shows ac- regulatory factors, since prolonged occlusion also tivity against S. aureus, E. coli and C. albicans in causes inflammation and increased cytokine pro- environment resembling human sweat condi- duction, which might be of importance for hBD-2 tions [61]. Dermcidin is not inducible, but rather induction, whereas barrier disruption alone was a part of the constitutive antimicrobial defense, sufficient for RNase7 and psoriasin up-regula- and it is expressed exclusively in sweat glands, not tion. Murine studies have shown similar results, in keratinocytes [62]. Unlike the other AMPs, where murine orthologs of hBD-2 and LL-37, dermcidin is negatively charged, and it is specu- i.e. murine BD3 and cathelicidin-related AMPs lated to use a different, yet unknown, mode of (CRAMPs), were up-regulated after barrier dis- action for antibacterial activity. Adrenomedullin ruption by tape stripping or acetone treatment is found in keratinocytes, hair follicles and sweat [79, 80]. An initial fast increase was seen in <1 h, glands, with activity against E. coli , S. aureus and and mRNA levels were increased after 1–4 h and

40 Clausen Agner

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 38–46 ( DOI: 10.1159/000441543) Bacteria Healthy skin Infected/injured skin Fungi Viruses

hBD-1 Dermcidin Wound repair Direct killing of microbes Stratum corneum RNase7 Psoriasin LL-37 Increased expression of AMPs from hBD-2,-3, RNase7, LL-37, psoriasin

Stratum resident and spinosum spinosum infiltrating cells

Chemotaxis Angiogenesis Basal expression of AMPs from resident cells Monocytes T cells

Mast cells Neutrophils

Fig. 1. In healthy skin, a constitutive expression of AMPs from resident cells provides a first line of defense against invading pathogens. Upon infection or injury of the skin barrier, infiltrating cells provide a high level of AMPs and resident keratinocytes increase the production of AMPs. AMPs are then involved in a cascade of functions, including direct killing of microbes, attracting inflammatory cells, stimulate chemokine production, angiogenesis and wound repair.

returned to normal levels after 24 h. The initial with increased barrier function determined by re- quick increase in AMPs is most likely the release duced transepidermal water loss [81] . This is in of preformed AMPs from lamellar bodies within contrast to previous studies, which clearly indi- keratinocytes. The up-regulation paralleled lipid cated a disrupted barrier to induce AMP expres- metabolic responses, probably due to the co-as- sion; however, since urea has previously been sembly of AMPs and lipid precursors in lamellar shown to be a skin irritant [82] , it might be that bodies. The normalization of AMP expression urea alone can induce AMP expression despite followed the normalization of the permeability improved permeability barrier. barrier function, indicating barrier properties to An imbalanced AMP response is linked to sev- be important in relation to AMPs. Further linking eral inflammatory skin diseases, in particular pso- the interaction between AMP and permeability riasis and AD [2, 8, 49] . High levels of AMPs in barrier function, the study showed that CRAMP- psoriasis are believed to protect against skin in- knockout mice displayed a significant delay in fections [31, 49, 83], and, in spite of the disturbed barrier recovery after tape stripping compared to barrier function, patients with psoriasis rarely mice with normal CRAMP gene (LL-37) function suffer from severe S. aureus skin infections [84] . [80] . On the other hand, it is hypothesized that in- In contrast to these studies, where barrier dis- creased levels of certain AMPs in psoriasis might ruption leads to induced AMP expression, urea contribute to inflammation and actively contrib- has shown to increase AMP expression together ute to the disease itself [85, 86] . In AD, reduced

AMPs, Infections and the Skin Barrier 41

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 38–46 ( DOI: 10.1159/000441543) expression of certain AMPs, compared to psoria- seen towards HSV, where LL-37 exhibited signif- sis, has long been believed to explain the frequent icant killing of HSV in vitro. Cathelicidin-knock- skin infections seen in AD [87–89] . However, a out mice had higher levels of HSV replication generally reduced level of AMPs is not evident, than wild-type mice, and lower expression of LL- and many AMPs are up-regulated in AD lesional 37 was found in the skin of AD patients with than skin compared to healthy control skin [77, 78, without previous HSV infection (eczema herpeti- 90–92] . cum) [47]. Hence, LL-37 seems to play an impor- Despite altered AMP expression in several tant role in optimal viral defense against vaccinia skin diseases, the clinical significance of the ex- virus and HSV. Further, the fungicidal effect of pression of AMP and their role in eliminating AMPs has been studied in vitro and confirmed a skin infections by direct antimicrobial activity role for these peptides also in the protection needs further investigation. The antibiotic-like against fungal skin infections [32, 96–98] . activity of AMPs is important in skin immune de- Despite clear evidence of the antimicrobial fense, but the discovery of their role as multifunc- properties of AMPs, many studies are performed tional effector molecules, linking and modulating in vitro, using different strains of bacteria, differ- immune response, indicates that other functions ent types of AMPs and varying differentiation may be even more significant. grades of keratinocytes, making comparison between studies challenging, and direct transfer of in vitro findings to in vivo significance is difficult. Antimicrobial Peptides and Infections Furthermore, although the individual antibacterial activity of each AMP might be of less impor- Numerous in vitro studies have shown antibacte- tance in vivo, synergism of several AMPs resulted rial effects of AMPs [31, 44, 54, 59, 60, 93, 94] , but in greatly increased activity [11, 12] . In vivo bio- the clinical significance of AMPs in skin infec- logical settings will provide an arsenal of different tions is still not completely established. With re- AMPs in response to microorganisms, creating spect to S. aureus skin infections, hBD-3 and optimal conditions for a synergistic and more RNase7, in particular, have been shown to be of complex immune response. importance in the protection and clearance of in- Another role for AMPs in the protection fections [34, 59, 93]. Studies in healthy individu- against infections is maintaining a balanced mi- als showed that low basal expression of RNase7 crobiota beneficial for skin health. Staphylococcus was associated with S. aureus skin infections, and epidermidis, a major component of the normal high hBD-3 levels led to a better recovery rate and skin microflora, induces the expression of AMPs, less severe symptoms [58, 95] . LL-37 also plays a leading to strengthened antimicrobial defense role in the inhibition of S. aureus , and neutrope- [23, 25, 99, 100]. The skin microflora also pro- nic mice with a deletion in the cathelicidin gene duces antimicrobial agents, some of which have showed greater susceptibility to group A strepto- shown selective bactericidal activity against skin coccal skin infections than mice able to produce pathogens, but not against commensal S. epider- cathelicidin [94] . In the protection against viral midis, thereby acting in synergy with host AMPs infections, LL-37 has shown activity against vac- to defend the skin [101–103]. The balance be- cinia virus. In vitro experiments showed that a tween S. epidermidis and S. aureus is also signifi- decreased level of LL-37 led to greater replication cant, and S. epidermidis has shown to inhibit bio- of vaccinia virus, and CRAMP-knockout mice film formation of S. aureus and even to eliminate were more susceptible to vaccinia pox formation S. aureus from the nasal cavity after inoculation than wild-type mice [44]. Similar activity was [101] . Future studies in the microbiome of dis-

42 Clausen Agner

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 38–46 ( DOI: 10.1159/000441543) eased skin and AMPs will deepen our under- play a role in angiogenesis, wound healing, reepi- standing of the important relationship between thelialization, cell migration and differentiation, commensal bacteria and innate antimicrobial de- and in the production of cytokines. fense. Adding to their importance in providing a healthy skin and barrier function, recent research has revealed a role for AMPs in the interplay with Conclusion the skin microflora. It seems that synergism between host commensals and AMPs might be yet AMPs are a group of small molecules with an another important function for AMPs in protect- essential role in the cutaneous defense and skin ing the skin, and they might even play a role in barrier function against infections and invading maintaining the homeostasis of the microflora pathogens. They possess direct antibiotic-like kill- present in the skin barrier [104] . The numerous ing activity against a variety of bacteria, viruses and functions described for AMPs highlight the im- fungi, thus serving as part of the first-line defense portance of a functional and well-regulated AMP to keep our skin free of infections. Upon injury to expression for maintaining an optimal skin bar- the skin barrier, inflammation and skin barrier rier. disruption, AMPs are up-regulated to create an even stronger antibacterial shield. In addition to their importance as a direct antimicrobial shield of Acknowledgment the skin, they also serve as strong immune modula- tors and multifunctional effector molecules, link- This work was supported by H2020 COST Action ing innate and adaptive immune responses. They TD1206 ‘StanDerm’.

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Tove Agner 2011; 131: 1974–1980. Department of Dermatology, Bispebjerg Hospital University of Copenhagen, Bispebjerg Bakke 23 DK–2400 Copenhagen (Denmark)

E-Mail Tove.Agner @ regionh.dk

46 Clausen Agner

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 38–46 ( DOI: 10.1159/000441543) Section I: Basic Parameters

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 47–60 (DOI: 10.1159/000441545)

Biological Variation in Skin Barrier Function: From A (Atopic Dermatitis) to X (Xerosis)

Simon G. Danby The Academic Unit of Dermatology Research, Department of Infection and Immunity, Faculty of Medicine, Dentistry and Health, The University of Sheffield Medical School, Sheffield , UK

Abstract Biological Variation of the Skin Barrier The skin barrier, formed by the stratum corneum, envel- with Age ops our bodies and provides an essential protective function. However, this barrier function differs between indi- Throughout our lives, the skin barrier undergoes viduals due to biological variation. This variation arises as a series of structural and functional changes that a result of inherited genetic variants, negative environ- have profound effects on our vulnerability to dry mental or extrinsic factors, and age. A multitude of ge- skin conditions. Following birth it takes more netic changes determine a person’s predisposition to a than 12 months for the structure and function of skin barrier defect and consequently their risk of develop- the skin barrier to reach adult levels, which can ing a dry skin condition, such as atopic dermatitis. Extrin- be viewed as a period of optimization ( fig. 1 ) [1] . sic factors, including the weather and detrimental skin How long this period of optimization takes, and care practices, interact with these genetic changes to de- what the ‘optimum’ level is for an individual is to termine the severity of the defect and additively increase some extent predetermined by our genetics, and the risk of developing dry skin conditions. How these dry individuals with a high risk of developing atopic skin conditions present clinically, and how they persist dermatitis (AD) already display a skin barrier de- and progress depends very much on a person’s age. Un- fect at birth [2]. Our environment then plays a derstanding how the skin barrier varies between individ- role in modifying the extent of this defect follow- uals, how it differs based on clinical presentation, and ing birth. At the other end of the age spectrum, how it alters with age is important in developing opti- intrinsic aging drives a steady decline in the met- mum therapies to maintain healthy skin that provides abolic activity of the skin and it begins to thin [3] . the best protection. © 2016 S. Karger AG, Basel As a result, the skin barrier condition of people over 60 years of age is profoundly different from younger adults, and from neonates and infants making it prone to xerosis and pruritus [4, 5] . Optimization Intrinsic and from birth extrinsic aging

Winter xerosis Fig. 1. The changing condition of Xerosis the skin barrier with age. The skin barrier undergoes a period of opti- AD Pruritus mization following birth. At the other end of the age spectrum, the barrier condition Skin Asteatotic Optimum eczema structure and function of the skin barrier barrier declines as a result of intrinsic and extrinsic (photo)aging. The prevalence of dry skin conditions (in 10 2030 40 50 60 70 80 90 boxes) alters during a person’s life Age (years) span.

Optimization of the Skin Barrier from Birth lular corneodesmosomal proteins is regulated by The epidermis reaches structural maturity in the SC-pH [10] . Adult skin surface pH is acidic under third trimester, somewhere between 30 and 37 normal conditions and helps to restrict the activ- weeks of gestation [6] . At this time, a well-defined ity of these proteases, which display optimum ac- stratum corneum (SC) is observed; however, the tivity at alkaline pH. At birth, skin surface pH is skin barrier does not achieve ‘optimum’ perfor- near neutral, and only reaches adult levels after mance for a number of months-years following the first 2–4 weeks following birth [7, 11, 12] . At birth [7]. This period of optimization is charac- some anatomical locations, including the but- terized by a number of differences in the struc- tocks and cheeks, normalization of skin surface tural and biophysical properties of the skin that pH can take much longer. distinguish it from adult skin (table 1 ). The composition and structure of SC lipids is In infants up to 24 months of age, the epider- altered in infants compared to adults as a result of mis is 20% thinner and the SC 30% thinner on dynamic changes in sebum production and re- average compared to adults [8] . The proliferation duced rates of lipid processing within the SC im- rate of epidermal keratinocytes is higher in neo- mediately following birth [9, 13–15]. In particu- nates compared to adults, and decelerates to- lar, a deficit in the essential ω-6 fatty acid linoleic wards adult levels over the course of about 12 acid as a component of ω-hydroxyceramides has months, indicative of higher metabolic activity been observed during this period [16]. Changes in [8] . Moreover, an increased rate of desquamation the composition of the lipids that make up the is evidenced by the reduced maturity of the up- lipid lamellae are known to affect both the struc- permost corneocytes. Observations in animals tural integrity of the SC and its permeability bar- confirm that there is an elevated proteolytic deg- rier function [17] . radation of the corneodesmosomal junctions that Following birth, composite levels of natural link the corneocytes of the SC together, thereby moisturizing factor (NMF), a collection of natu- facilitating more rapid shedding [9]. The activity ral humectants, appear elevated for the first few of SC serine proteases, including kallikrein (KLK) weeks, but then drop to levels below those in 5 and KLK7 for example, that cleave the extracel- adults for about 12 months before recovering [11,

48 Danby

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 47–60 ( DOI: 10.1159/000441545) Table 1. Age-related changes in skin barrier structure and function compared to young adult skin

Property Neonatal/infant skin Aged skin

Functional properties

Skin surface pH Elevated for 2 – 4 weeks following birth Elevated; women display (longer in the face and buttocks) elevated pH earlier than men TEWL Similar and elevated, with greater Decreased variation, depending on site and age Hydration Low at birth and then elevated with Decreased wide variation Repair rate Undergoing optimization Reduced Immune responsiveness Th2 bias immediately following birth Reduced Structural properties Epidermal thickness Thinner Thinner with a thinner papillary dermis SC thickness Thinner Thicker Surface corneocytes Smaller surface area Larger surface area Biological properties Protease activity and Elevated Decreased rate of desquamation Lipid lamellae Altered composition linked to reduced Decreased total levels and sebum production altered composition NMF Elevated at birth, but reduced during Reduced (specifically urea and lactate) the 1st year

12] . The level of lactate, a constituent of NMF de- utes. Irrespective of TEWL levels, infant skin dis- rived from sweat and the viable epidermis, displays an increased permeability to irritants, and is plays an inverse relationship with composite lev- more susceptible to cutaneous infections and the els. As a result of altered NMF levels and the development of dermatoses (such as irritant/al- changes in the lipid composition of the SC, infant lergic contact dermatitis and AD), compared to skin exhibits altered water holding and handling adult skin [23]. This increased susceptibility dur- properties relative to adults [18]. Skin hydration ing a period of optimization for the skin barrier (including SC water content) is low for up to 12 suggests that strategies to accelerate optimization weeks following birth, after which it improves could potentially minimize the risk of developing to become slightly elevated when compared to these conditions [24–26] . adults [12] . Transepidermal water loss (TEWL), a measure Deterioration in the Skin Barrier with Advancing of permeability barrier function, is either similar Age or elevated in infants compared to adults depend- At the other end of the age spectrum, the condi- ing on the findings of different studies [7, 11, 13, tion of the skin begins to decline. Exactly when 19–22]. Whilst a consensus has not been reached this decline starts is uncertain; however, it is clear with regard to TEWL levels during this period, that the skin of people over the age of 60 years is most studies agree that there is a greater variabil- very different from the skin of younger adults ( ta- ity in TEWL measurements, to which the hetero- ble 1). As a result of intrinsic, or chronological, geneity of the infant population likely contrib- aging, the metabolic activity of the skin slows

Biological Variation in Skin Barrier Function 49

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 47–60 ( DOI: 10.1159/000441545) down, resulting in slower epidermal turnover and a consequence of these same events during senes- skin atrophy [27] . The rate of desquamation also cence. Elevated ceramidase activity is also thought slows, in part as a result of reduced desquamatory to contribute to ceramide deficiency [44]. In ad- protease activity in the SC, leading to corneocyte dition to altered rates of ceramide synthesis and retention and thickening of the SC [28] . The degradation, a change in the composition of cer- thicker SC is associated with a steady reduction in amide esters is found in older women and most TEWL with advancing age [29, 30] . In agreement, likely contributes to the skin barrier defect [45] . SC permeability is decreased indicative of an im- The level of NMF in the skin is also reduced proved permeability barrier function. The skin’s with advancing age [46] . In particular lactate and responsiveness to irritants, such as sodium lauryl urea are specifically reduced compared to other sulfate (SLS), is decreased. Moreover, immune se- NMF components [42]. The reduction in skin hu- nescence, a gradual decline in immune function mectants, together with the lipid defect, leaves the with increasing age, reduces allergen responses. skin less able to hold onto and handle water, and In particular, the number of Langerhans cells in so it becomes more susceptible to dryness. A di- the dermis, which undertake immune surveil- rect relationship between the extent of the NMF lance roles, are decreased in aged skin [27] . As a deficiency and dryness of the skin in older people whole, aged skin, therefore, displays a significant- has been observed [47]. As a combined result of ly higher threshold for cutaneous inflammation the increased threshold for inflammation, the compared to young adult and infant skin [3] . increased permeability barrier function and the Whilst permeability barrier properties appear predisposition to dryness, noneczematous xerosis to improve, the ability of intrinsically aged skin to is more common in the elderly than the younger withstand damage, and repair itself after damage, adult [4, 5, 48–50] . is significantly decreased. The weakness of the skin is contributed to by the reduced thickness of the epidermis, particularly loss of the papillary Pathological Variations in Skin Barrier region which helps maintain dermal attachment Function under sheer stresses; reduced elasticity of the skin due to altered collagen structure; reduced struc- Aberrant variations in the structure and compo- tural integrity, and hydration of the SC [31–35] . sition of the skin barrier underpin a number of SC lipid levels are broadly reduced in people over skin conditions ( table 2 ). In the following sec- the age of 60 years compared to young adults [34, tions, some of the most common conditions aris- 36, 37]. Studies looking at the relative concentra- ing as a result of compromised skin barrier functions of ceramides, fatty acids and cholesterol in tion are discussed. the SC have produced conflicting results, owing at least in part to the differences between skin ag- Atopic Dermatitis ing in men and women. With advancing age, se- AD is a chronic, inflammatory disease of the skin, bum levels decline, ceramide levels drop off, and characterized by xerosis, pruritus and erythema- skin surface pH increases much earlier in women tous lesions [51] . The prevalence of AD is high, compared to men [36, 38–43] . Skin senescence affecting 15–30% of children and 2–10% of adults appears to suppress pH-induced protease activi- [52, 53]. Most cases of AD arise during the 1st ty, perhaps as a result of decreased protease ex- year of life, during the period of optimization for pression and delivery to the SC by lamellar bodies the new skin barrier [54]. Not only does AD pre- [3, 28, 34] . On the other hand, pH-mediated inhi- dominantly arise during a time when the skin bition of lipid synthesis is most likely amplified as barrier is immature, it also preferentially affects

50 Danby

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 47–60 ( DOI: 10.1159/000441545) Table 2. Pathological variation of skin barrier structure and function in xerotic skin conditions compared to healthy young adult skin

Property AD Xerosis (noneczematous)

Functional properties Skin surface pH Increased Increased TEWL Increased Increased Hydration Decreased Decreased Structural properties SC thickness Thinner Thicker Surface corneocytes Smaller Larger Biological properties Protease activity and Elevated resulting in premature Decreased leading to retention rate of desquamation desquamation hyperkeratosis Lipid lamellae Total lipid levels reduced, includes a Altered composition – may be linked to reduction in ceramides (specifically gender short-chain ceramides) NMF Reduced – associated with inherited Reduced – winter xerosis associated with and acquired filaggrin deficiency reduced levels of inorganic NMF constituents, urea and lactate

skin sites characterized by a thinner SC [51] . In agreement with this, late-onset AD, occurring at infants (infantile eczema) , lesions are generalized, a time when the skin barrier is fully matured and but most often affect the face first [55] . Facial skin immune responsiveness decreased, is often non- sites show a delayed acidification of the SC com- atopic (intrinsic) in nature and dependent on en- pared to other skin sites such as the forearms [56] . vironmental exposures [61] . In children, AD lesions become increasingly lo- The skin of patients with AD is characterized calized to the flexures, a shift that coincides with by a thinner epidermis [62, 63], less mature sur- the maturation of the skin barrier following birth. face corneocytes [64, 65], poor hydration, elevat- Large epidemiological studies demonstrate that ed TEWL and increased permeability to irritants AD is probably a life-long condition with persis- and allergens ( fig. 2 ; table 2 ) [66–69] . These tence into adulthood, especially in those who de- changes are observed in both lesional and nonle- velop the condition before the age of 2 years [57] . sional skin, and broadly result from abnormal ke- The persistence of AD into adulthood is signifi- ratinocyte differentiation and altered SC homeo- cantly associated with atopy at 3 months of age, stasis. An increased rate of desquamation is evi- highlighting both the role of the immune system dent in patients with AD compared to healthy and events early on in life [58, 59] . At birth, the controls. Increased mass levels and activity of ser- neonatal immune system is skewed towards ine proteases, including KLK5 and KLK7 with T-helper 2 (Th2)-cell mediated responses creat- chymotrypsin-like and trypsin-like activities, re- ing a bias for proallergic inflammation [60] . Dur- spectively, have been observed in AD skin, and ing the first months of life, therefore, the weak- coincide with altered homeostasis [66, 70, 71] . ened skin barrier, coupled with a propensity to- The activity of serine proteases in the SC is di- wards allergic inflammation, creates optimum rectly affected by pH, which is also increased in conditions for the development of true AD. In AD [10, 72, 73] . Increased skin surface pH is as-

Biological Variation in Skin Barrier Function 51

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 47–60 ( DOI: 10.1159/000441545) Stratum compactum Water gradient Irritant and allergen penetration Stratum disjunctum TEWL SC thickness SC Corneocyte maturation

Fig. 2. The structure of the skin barrier in AD skin compared to healthy Normal Dry Eczematous skin.

sociated with both elevated activity of degrada- The expression of differentiation-dependent tory proteases and also the inhibition of enzymes genes, including those encoding components of involved in the processing of lamellar lipids [74] . the cornified envelope, is altered and compromis- The result is reduced SC integrity/cohesion and es epidermal barrier structure [86–88] . The ex- defective lipid lamellae, with a consequent reduc- pression of filaggrin in particular is reduced and tion in epidermal barrier performance. results in a structural defect and reduced levels of In the lesional and nonlesional skin of AD pa- NMF [46, 89, 90] . A deficiency in filaggrin, and its tients, there is a reduction in the amount of total breakdown products, is associated with dry and lipids in the SC owing to a significant deficit of scaly skin, and correlates with clinical severity ceramides, especially long-chain ceramides [75, and the barrier impairment in AD [91–93] . Re- 76] . Ceramide insufficiency, and a shift from cently, it was found that allergen priming of den- long-chain to short-chain ceramides, results in dritic cells is enhanced in the absence of filaggrin, the defective formation of the lipid lamellae and demonstrating the importance of this protein to the corneocyte lipid envelope, and was found to the ability of the barrier to prevent allergen per- correlate with xerosis and reduced barrier func- meation [94, 95] . tion [76–78] . The pattern of fatty acids (free and Variants of the FLG gene that result in loss or as constituents of lipid esters) is also altered, with reduction of filaggrin function are the most wide- a notable reduction in ω-6 unsaturated fatty ac- ly replicated genetic risk factors for AD identified ids, including linoleic acid, and an increase in to date, and account for 15–50% of cases depend- monounsaturated fatty acids [79] . Monounsatu- ing on severity [96, 97] . Several environmental rated fatty acids such as oleic acid have been as- factors, including living with cats, attending a sociated with negative effects on epidermal bar- daycare nursery, and living in a hard-water area rier structure and function, whereas linoleic acid have been shown to modify FLG gene-associated has been associated with positive effects on epi- risk and highlight the role of environment [98] . dermal barrier repair [80–85] . FLG gene mutations, however, are just one exam-

52 Danby

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 47–60 ( DOI: 10.1159/000441545) ple of the multitude of gene variants associated (homozygous for mutations in the FLG gene) with AD risk. Our current knowledge of AD ge- [96] . NMF deficiency in the SC of IV patients, as netics explains only 14.4% of the heritability of a consequence of FLG gene mutations, is directly AD, so there is still much to learn [99] . From what related to the increased dryness of the skin [47, is known, variants affecting the skin barrier are 89, 93]. Patients with IV display a general skin prominent, and include variations in the FLG , barrier defect, characterized both by dryness (xe- SPINK5 , KLK7 , CLDN1, SPRR3 and CASP14 rosis) and elevated TEWL, and are predisposed to genes for example [51, 100, 101]. Yet, it is impor- the development of AD [73, 107] . tant to recognize that many variants associated with AD affect the immune system. Whilst skin Xerosis Cutis barrier disruption itself is sufficient to induce the Xerosis cutis, or dry skin, is both a condition itself release of proinflammatory cytokines [102, 103] , and a symptom of other conditions including AD the skin of AD patients exhibits a lower threshold [108] . In its simplest sense it results from a defi- for proallergic inflammation [95], thought to be ciency in water in the SC. The severity of xerosis conferred by variants affecting a number of im- cutis is dependent on the extent of the SC water mune system genes such as those encoding inter- insufficiency, or, put another way, the ability or leukin (IL)-4, IL-4 receptor, IL-13 and the high inability of the SC to trap and hold onto water. As affinity IgE receptor FcεR1 for example [101] . water levels decrease, corneocytes dry out and be- Moreover, the immune system and the skin bar- come brittle and rigid giving the skin surface a rier are interconnected. For example, the Th2 cy- rough texture. The uppermost corneocytes, where tokines IL-4 and IL-13, both mediators of proal- retained, ultimately desiccate and take on a pow- lergic inflammation in the skin, and the Th22 dery appearance. The SC loses its pliability and so cytokine IL-22 down-regulate the expression of cracks begin to form between the corneocytes, many skin barrier genes, including FLG [104– much like a river bed becomes cracked when de- 106]. As a result, proallergic immune responses prived of water. These cracks expose the body to triggered following disruption of the skin barrier the external environment, and its many irritants perpetuate cutaneous inflammation by further and allergens, negating the skin barrier altogeth- suppressing skin barrier function. er. Consistent with this is the finding that xerosis Both the skin barrier and the immune system is a risk factor for atopy independent of AD [109] . clearly play important roles in the development of Pruritus is a common consequence of xerosis, and AD. But for the prevention of AD, focus has been a symptom of AD that provokes scratching of the placed on the skin barrier because its disruption skin leading to further skin barrier damage [4] . appears to trigger AD onset and allow sensitiza- An escalating cycle, termed the itch-scratch cycle, tion to occur [69] . ensues both prolonging and exacerbating the xerotic and pruritic condition of the skin. In se- Ichthyosis vere cases, the skin becomes red and inflamed, Ichthyosis is a distinctive inherited, and in some and may progress to asteatotic eczema (eczema cases acquired, dry skin condition that arises as a craquelé); an inflammatory skin condition affect- result of abnormal cornification (formation of the ing the elderly that is characterized by ichthyosi- SC). Patients with ichthyosis have persistent dry, form scaling and fissuring. rough and thickened skin with distinctive scaling, Xerosis of the skin arises as a result of many likened to fish scales [73] . The common form of different changes in the biological composition of ichthyosis, ichthyosis vulgaris (IV), is an inherited the skin, some similar to AD and some distinct. In condition caused by loss of filaggrin function the context of AD, elevated skin surface pH and

Biological Variation in Skin Barrier Function 53

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 47–60 ( DOI: 10.1159/000441545) Healthy skin barrier Defective skin barrier

Corneocyte shedding Mature corneocyte Cornified envelope

Degraded Dehydrated corneodesmosome corneocyte Defective cornified Lipid lamellae envelope Corneodesmosome Degraded corneodesmosome High NMF Defective lipid Immature lamellae corneocyte Low NMF

Fig. 3. Skin barrier structure and function in healthy compared to xerotic skin with and without eczematous change. The noneczematous xerosis retention hyperkeratosis leads to increased SC dryness but reduced TEWL. In AD, the skin barrier defect, characterized by a thinner SC, reduces permeability barrier function and elevates TEWL, and results in poor SC hydration.

SC protease activity are associated with the devel- es skin hydration and elevates skin surface pH opment of xerosis [51] . Contrary to this, xerosis [110] . An alteration in the lipid composition of also appears to arise when protease activity is de- the lamellar membranes, which alters the order- creased [28]. In this scenario, the reduction in ing of the lipids, also decreases skin hydration and trypsin-like and chymotrypsin-like protease ac- additionally decreases skin barrier function mea- tivities involved in desquamation leads to a per- sured as TEWL [38, 111]. Repeated washing, win- sistence of corneodesmosomal junctions and the ter and advancing age are all common causes of retention of corneocytes. This biological varia- xerosis, and by association exacerbating factors tion is a key distinguishing feature between ec- for AD, and will be discussed in the following sec- zematous skin (i.e. AD skin) and noneczematous tions [5] . xerotic skin (fig. 3 ; table 2 ). The elevation of trypsin-like proteases in particular is a key mecha- Xerosis Induced by Frequent Washing nism that drives eczematous changes. Repeated washing, especially with harsh deter- Whilst retention hyperkeratosis likely contrib- gents and soap, extracts SC lipids and water-solutes to dryness, other changes in the composition uble NMF, and consequently reduces the ability of the SC are important in determining its hydra- of the SC to hold onto water [112, 113] . Harsh tion level, or conversely its dryness. These chang- surfactants in wash products, such as SLS, also de- es exhibit similarities with both neonatal and nature SC proteins and trigger the release of pro- aged skin, including decreased levels of NMF inflammatory cytokines, including IL-1 and IL-8, components and altered composition of the lipid and their repeated use can cause erythema and lamellae [51, 108]. A reduction in NMF levels in irritation [113, 114]. These negative effects of sur- the SC, especially lactate and potassium, decreas- factants are related to their ability to modify skin

54 Danby

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 47–60 ( DOI: 10.1159/000441545) surface pH, for instance synthetic detergents like gether these changes lead to decreased skin hy- SLS and alkyl carboxylates from traditional soaps dration and water holding capacity, and in- significantly increase the pH of the SC, with creased SC stiffness associated with cracking and knock-on effects on SC protease activity and lipid elevated SC pH [39, 110] . processing [72] . The effects of washing on protease activity are complex and dependent on the formulation of the wash product, and the fre- Conclusion quency and duration of washing [28] . SLS itself was found to amplify protease activity in the SC, The structure and function of the skin barrier var- whereas complex wash products can inhibit and ies greatly with age. Factor in the effect of inherit- promote the activities of different proteases de- able genetic differences, and the impact of our en- pending on their formulation and the chronicity vironment, the scale of biological variation is im- of washing. A common consequence, however, is mense. Nevertheless, when the skin barrier fails increased skin dryness unless moisturizers are to function properly these wide-ranging differ- used. Avoiding harsh detergents and soap is an ences appear clinically as dryness/ xerosis . Eczem- important strategy for the prevention and treat- atous changes, associated with elevated protease ment of xerosis and AD [115] . activity and subsequent skin barrier breakdown, may or may not be present. Similarly, atopy may Xerosis in Winter: Winter Xerosis or may not develop [121]. These clinical distinc- The development of xerosis is associated with tions give rise to a wide spectrum of ‘conditions’ the seasons, becoming more prevalent in the including, intrinsic and extrinsic AD , noneczema- winter months when the air is dry and the skin is tous atopic xerosis , nonatopic xerosis and astea- exposed to dramatic temperature shifts between totic eczema . But rather than being separate, these the cold outdoor and the warm, arid, centrally often represent different ‘stages’ of the same con- heated indoor environments [4]. The climat- dition. AD often begins as a ‘nonatopic’ condi- ic changes are matched by changes in the com- tion, progressing to true AD later following sen- position of the SC. During winter, the levels of sitization, and individuals with AD more often urea, lactate and inorganic constituents of NMF develop senile xerosis late in life [5, 122]. Our age such as potassium all decrease [39, 116] . The SC and environment plays a significant role in how level of each of these constituents directly corre- these conditions present clinically. Nevertheless, lates with SC hydration [110, 117] . Urea and despite similar, overlapping and evolving clinical α-hydroxy acids like lactic acid also exert control presentation, the causes of the skin barrier defect over desquamation and skin barrier homeostasis are diverse, as highlighted by the multifactorial by both enhancing desquamation rates and pro- gene-gene and gene-environment interactions moting expression of skin barrier components involved in the development of AD [123] . This [117–119] . On the other hand, filaggrin-derived has led to the suggestion that AD is not one con- NMF constituents are unchanged or elevated dition, but a spectrum of conditions with a shared during winter, possibly in an attempt to com- clinical phenotype [49, 124] . pensate for the loss of urea, lactate and potassi- Optimum therapeutics for the treatment and um [39, 110]. The lipid composition of the skin prevention of dry skin conditions will require an also appears to alter, with decreased levels of all understanding of the subclinical variation in the lipid classes [38] . As observed in aged skin and structure and function of the skin between indi- AD skin, there also appears to be a reduction in viduals. This variation arises as a result of genetic linoleate ester ceramide species [38, 120] . To- factors in combination with adverse environ-

Biological Variation in Skin Barrier Function 55

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 47–60 ( DOI: 10.1159/000441545) mental/extrinsic factors. As such, the biophysical ties early in life will inevitably help target general and biological assessment of the skin following preventative efforts to those at risk. In the future, birth offers the best opportunity for assessing the comprehensive skin barrier testing may enable condition of the skin barrier and determining the the use of tailored repair therapies based on the nature of repair required [2] . Early clinical trials nature of the skin barrier defect. Personalized have already demonstrated that prevention of AD treatment of dry skin conditions may be some is possible by ameliorating the skin barrier defect way off; however, tailoring treatment based on a [24–26] . The assessment of skin barrier proper- patient’s age is achievable now.

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Simon G. Danby The Academic Unit of Dermatology Research, Department of Infection and Immunity Faculty of Medicine, Dentistry and Health, The University of Sheffield Medical School Beech Hill Road Sheffield S10 2RX (UK)

E-Mail s.danby @ sheffield.ac.uk

60 Danby

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 47–60 ( DOI: 10.1159/000441545) Section II: External Factors Influencing Skin Barrier

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 61–70 (DOI: 10.1159/000441546)

Methods for the Assessment of Barrier Function

Dimitar Antonov Sibylle Schliemann Peter Elsner Department of Dermatology, University Hospital Jena, Jena , Germany

Abstract multitude of perspectives from which the skin Due to the ease of skin accessibility, a large variety of in- barrier has been studied. These include the per- vasive and noninvasive in vitro and in vivo methods have spective of skin physiology, i.e. studying the bar- been developed to study barrier function. The measure- rier function of the healthy skin; transdermal ment of the transepidermal water loss (TEWL) is most drug delivery, the skin as an obstacle to be passed widely used in clinical studies. The different methods of by substances to be delivered (with typical models determining TEWL, as well as skin hydration, skin pH, being diffusion cells [1, 2]); toxicological view/as- tape stripping and other modern less widely used meth- pects of irritant contact dermatitis (the develop- ods to assess skin barrier function, are reviewed, includ- ment of irritation in response to external agents), ing Raman spectroscopy and imaging methods such as and from the viewpoint of repairing or enhancing optical coherence tomography and laser scanning mi- the barrier function in health and disease, rang- croscopy. The modern imaging methods are important ing from wound healing to cosmetics. developments in the last decades which, however, deter- The many methods for studying the skin func- mine the structure and, hence, cannot replace the mea- tions in general may be roughly classified as non- surement of TEWL in questions related to function. invasive, invasive, in vitro, mathematical, spec- © 2016 S. Karger AG, Basel troscopic and other methods ( table 1 ). Many of those are applied to study skin barrier function.

The skin as the outmost organ of the human body serves as a barrier in many aspects, e.g. a mechan- Transepidermal Water Loss ical, water, chemical, thermal, radiation and immunological barrier, as well as a barrier against The skin as a mechanical barrier prevents the loss microorganisms. Due to the ease of skin accessi- of water, electrolytes and proteins from the body. bility, a large variety of invasive und noninvasive, The permeability barrier function is situated in in vitro and in vivo methods have been developed the epidermis, because the dermis stripped of the to study its functions. Such variety stems from the epidermis is almost entirely permeable. In the Table 1. Methods for studying skin functions in general [3, 4]

Noninvasive TEWL: open, closed, ventilated and condenser-type chamber methods bioengineering SC hydration: electrical capacitance, electrical conductance and impedance methods Skin color: colorimetry Skin blood flow with laser Doppler flowmetry and OCT/Doppler tomography Skin surface pH Skin roughness (replica methods – mechanical and in vivo optical profilometry) Other parameters of the skin (e.g. sebum production and skin elasticity) Invasive methods Skin biopsy Tape stripping (and other forms of horizontal sectioning) Suction blisters Microdialysis Other methods for harvesting material from the skin – scrapes to assess sebum and solvent extraction for lipid assessment In vitro models Diffusion cell Mathematical models Modelling of the diffusion and penetration through the skin Spectroscopic Attenuated total-reflectance/Fourier transform infrared spectroscopy methods and Direct and indirect fluorescence spectroscopy molecular imaging Remittance spectroscopy Photothermal spectroscopy Raman spectroscopy Skin ultrasound Visualizing skin thickness and density Autoradiographic Visualization and/or quantification of radiolabeled molecules in skin samples methods

epidermis, the permeability barrier is located pri- tion rate of an infant as an indicator of epidermal marily in the stratum corneum (SC) [5] . integrity and function [9] . The need for noninva- The quantification of transepidermal water sive methods suitable for use in human volun- loss (TEWL) with noninvasive bioengineering teers and in clinical studies has pushed the devel- methods has gained popularity as one of the most opment and the broad use of these methods [9] . reliable methods to assess the barrier function of Currently, the most widely used method is deter- the skin. There are published reports of attempts mining the water evaporation pressure gradient to measure the total water loss from the human in an open chamber, which was developed by body as early as the 17th century [6] . The water Nilsson [6] in Sweden. Its sensitivity was com- loss by means of insensible perspiration has been pared against that of other methods [10] . Nowa- estimated by measuring the weight loss of a per- days, published guidelines help provide stan- son on a balance [6] . This method has been em- dardization and unification of the TEWL mea- ployed to measure the insensible water loss in in- surement [11–13] . fants [7]. Studies on the insensible perspiration in Quantifying TEWL provides the most direct healthy skin and in different conditions such as measure of the barrier function. The designation psoriasis [8] have been published throughout the of the term ‘transepidermal water loss (TEWL)’ 20th century [reviewed in 6]. Measuring TEWL has been attributed to Rothman [14]. It denotes has evolved from a method to assess the dehydra- the amount of insensible perspiration which is

62 Antonov Schliemann Elsner

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 61–70 ( DOI: 10.1159/000441546) Table 2. TEWL measurement instruments [11]

Type Instruments Producer

Open chamber DermaLab Cortex Technology, Hadsund, Denmark Evaporimeter EP1 and EP2 ServoMed, Stockholm, Sweden Tewameter TM210 and TM300 Courage & Khazaka, Cologne, Germany Unventilated closed AS-CT1 Asahi Biomed Company Ltd, Yokohama, Japan chamber VapoMeter SWL3 Delfin Technologies, Kuopio, Finland Condenser-type Aquaflux Biox Systems Ltd, London, UK closed chamber

due to passive diffusion and not to active secre- technology is the open chamber method, where tion of the sweat glands [14]. By definition, only an open cylindrical probe is held vertically to the the body water originating from the deeper layers skin surface [6]. Two vertically aligned capaci- and passing the SC by passive diffusion represents tance sensors measure the relative humidity and the TEWL and reflects the condition of the per- two thermistors measure the temperature. From meability barrier [11, 13]. In practice, the water these measurements, the water pressure gradient originating from the sweat glands without active is calculated [6, 11–13] . Under ideal conditions sweating is also measured with TEWL, because it (room temperature 22–24 ° C and humidity 40– cannot be excluded [13] . 60%), TEWL can be measured between 0 and 250 Measuring the amount of water evaporating g/m2 /h with a variance of 5% [15] . The method is from the skin surface includes not only TEWL, limited to measuring horizontal surfaces. Contin- but also the water delivered to the skin surface uous measurements are possible, but usually a pe- by other means, such as sweating and to a lesser riod of 30 s is used [11] . Another technology is the extent from SC hydration (SC has usually a low ventilated chamber, which employs dry or moist- moisture content) and from sebum, as well as ened carrier gas and is capable of continuous from external factors such as occlusion or applied TEWL measurements [3], but it is not largely cosmetics. Actions are taken to reduce or stan- used due to concerns that it influences the skin dardize external influences in order that the mea- microclimate. The unventilated closed chamber sured evaporation of water from the skin surface methods use a cylindrical probe closed at the top really reflects TEWL. For example, inhibiting the [11, 16]. These probes are less prone to interfer- active sweat secretion has been achieved by sub- ence from air convection and can be used on noncutaneous atropine injections in studies from the horizontal surfaces. However, there are reports beginning of the 20th century [8] or nowadays on angular dependence from several studies [11] . by conducting the measurements in controlled In the condenser closed chamber method, the wa- conditions, with temperature under the thermal ter is condensed to ice and thus removed from the sweating threshold and after acclimatizing the chamber; therefore, continuous measurements subjects [13] . The method remains nevertheless are possible [16] . Drawbacks of both the closed sensitive and prone to interference. chamber and the ventilated chamber methods are There are a variety of instruments to measure the interference arising from occluding the skin TEWL on the market (listed in table 2 based on or from influencing the microclimate of the skin previous studies [11, 13] ). The most widely used [12, 13]. Other methods, such as determining the

Methods for the Assessment of Barrier Function 63

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 61–70 ( DOI: 10.1159/000441546) Table 3. Factors influencing TEWL measurements, which need to be controlled or accounted for in clinical studies [3, 11]

Endogenous (subject related) Exogenous Environmental Age Washing and detergents Air convection/movement Ethnicity (controversial) Wet work Ambient temperature Anatomical location Occlusion Relative humidity Skin temperature Use of emollients and external preparations Direct sunlight Sweating Smoking Season Circadian rhythm Skin condition/health

exact weight of the evaporated water through ab- of the skin. The conductance method gives infor- sorption in hygroscopic salt in an unventilated mation about the more superficial layers of the chamber, are not routinely used [13]. The results skin, while the capacitance method carries infor- obtained from different types of devices cannot mation from the deeper layers [17] . A multicell be compared. ‘SkinChip’® microsensor has been developed to For the most widely used method, the open provide a detailed capacitance mapping of the chamber, there are numerous studies exploring skin [18] . the influence of a variety of factors on the mea- The acidic skin surface pH is important for SC surements. Some of the most important factors homeostasis and the maintenance of the microbial influencing TEWL measurements (summarized flora. The skin surface pH is measured using glass in table 3 ), which need to be controlled for in clin- planar electrodes connected to a voltmeter. What ical studies, include the anatomical site and posi- is actually measured is the ‘apparent skin pH’, be- tion, the skin temperature, ambient temperature, cause the concept of pH relates to water solutions, sweating, air convection, humidity, direct sun- and on the skin surface there are lipids and other light, season and circadian rhythm [3, 11, 13] . compounds which release H+ ions into the water applied to the skin with the electrode [19] . The skin redness results from the increased Bioengineering Methods for Skin Barrier blood flow in the skin, and, apart from visual Assessment (Other than Transepidermal methods (i.e. redness by colorimetry), the skin Water Loss) blood flow could be directly assessed with laser Doppler flowmetry or modern methods such as Other frequently used noninvasive bioengineer- phase-resolved optical coherence tomography ing methods, e.g. SC hydration, colorimetry, skin and optical Doppler tomography. surface pH, sebometry and others, provide information on different characteristics of the skin and its condition, but are no direct measures of skin Tape Stripping and Other Invasive Methods barrier function. SC hydration is measured indirectly using the The removal of consecutive SC layers by means of electrical properties of the skin, which are depen- glues (usually, but not exclusively, on tapes [20, dent on the water content of SC [11] . The most 21] ) is a minimally invasive and largely used tech- commonly applied methods measure either the nique [20] . It has been used to evaluate the depth conductance, the capacitance or the impedance of penetration of pharmacological substances, to

64 Antonov Schliemann Elsner

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 61–70 ( DOI: 10.1159/000441546) evaluate the composition of the skin (lipids and Spectroscopic Methods, Molecular Imaging other components [22] ), and to study the develop- and Further Modern Methods ment of inflammatory mediators, wound healing and other processes. The removed tissue can be Modern techniques such as confocal laser mi- further examined with chemical, histochemical, croscopy and defining the water content by Ra- genetic, proteomic or other methods. For exam- man spectroscopy allow in vivo measurements, ple, a similar approach has been used in the past and contribute to the understanding of the struc- with dyes penetrating into the SC [21] . In this ture and function of the water barrier and the model, stripped layers of SC are analyzed by chro- water transport in the SC [26–28]. Such sophisti- mametry to quantify the amount of penetration of cated methods are not routinely used on a large the model dyes [21] . In another similar method, scale in clinical studies as is the measurement of the penetration of metal ions through the SC was TEWL, as they are also expensive and require examined by determining the quantity of the met- trained personnel to operate the instruments. al ions in stripped layers of the epidermis by means The method of Raman confocal spectroscopy of laser breakdown spectroscopy [23] . Addition- can be used to determine the water content at ally, a standardized tape stripping protocol has different depths of the skin, as well as the content been proposed for the need of patch testing [24] . of other substances [28, 29]. In Raman spectros- There are a variety of factors to be considered, copy, the sample is irradiated with low-power which can influence the amount of SC removed monochromatic laser light. The molecules are with each tape strip, and these need to be stan- excited to vibrational states and scatter light at dardized in order to provide reproducibility and wavelengths different from the inciting light comparability of the results. The anatomical site, (Raman spectra) [29]. The scattered light is cap- the season and the age of the subject affect the tured and analyzed, and thus noninvasive in vi- amount of corneocytes removed with each strip, vo measurements of the molecular composition as might other factors such as race, skin type, and concentrations in microscopic volumes at TEWL und pH of the skin [20] . Regarding the different depths are made possible [28–30]. In study protocol, the factors to be standardized in- vivo and in vitro studies with Raman spectros- clude the vehicle of the applied creams (if any), copy have confirmed the water gradient within the applied pressure, the type of tape and the ve- SC with a sharp drop in water content (from ap- locity of removal. proximately 70% in the viable epidermis to 15– Another technique is the induction of blisters 30% in SC) at the stratum granulosum-SC tran- through applying negative pressure to the skin sition. The role of aquaporins, tight junctions with special pumps [25]. This method is used to and other components involved in the water assess the penetration through the skin by mea- barrier function of the skin is reviewed in other suring the concentration in the blister fluid, or to chapters of this book. Raman spectroscopy stud- study models of inflammation and irritation by ies can be used to study SC hydration injury means of determining cells or inflammatory me- [28, 31] . diators from the blister fluid. Blisters of different Direct and indirect fluorescence spectroscopy sizes could be generated and various harvesting [4] track in vivo the skin penetration of model times could be used to allow for mediator release fluorescent substances and may be used to evalu- or inflammatory cell migration [25] . The disad- ate barrier creams [32] . Fluorescence lifetime im- vantages of the suction blister method include its aging may allow the observation of transepider- invasiveness and the influence of the inflamma- mal penetration without fluorescent model sub- tion by the suction trauma. stances [33] .

Methods for the Assessment of Barrier Function 65

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 61–70 ( DOI: 10.1159/000441546) Noninvasive methods for in vivo skin imag- horizontal sections (en face) [41] . Different in- ing have been greatly advanced in recent years, flammatory skin diseases, including experimen- including skin sonography, optical coherent to- tally induced irritant and allergic contact derma- mography (OCT) and laser scanning microscopy titis, have been described by means of both HD- (LSM) [34] . Sonography has a poor resolution, OCT [35, 43] and LSM [44]. It is still controver- the differentiation between inflammation and sial, but there are claims that the morphological edema is poor, and the gels eliminating the air be- data obtained by HD-OCT or LSM may help dif- tween the transducer and the skin interfere with ferentiate irritant from allergic morphology in skin physiology [35] . In OCT, the image is gener- doubtful patch test reactions [44–46] . It is a great ated similarly to the A-scans in sonography, using advantage of OCT and LSM methods that they low-coherence interferometry of infrared light provide in vivo noninvasive imaging without the and thus visualizing optical instead of acoustic in- alterations from fixation and extraction of water homogeneities; the image is a vertical slice and is or other components in conventional histology generated quickly [36–38] . The image resolution and electron microscopy [47] . Studies comparing of approximately 15 (10–30) μm is superior to TEWL and LSM note that the imaging methods ultrasound and inferior to LSM; the signal pene- provide more detailed information beyond that tration depth is approximately 1 mm to several of TEWL [48–50] . Such visual information is not millimeters [35, 39]. Individual cells cannot be numerical and would need to be expressed in a distinguished, but individual layers such as the score [49] . If the research question concerns the epidermis and dermis, adnexal structures and restoration of the epidermis or the wound healing blood vessel can [35] . OCT is useful for measur- process after suction blister wounds [49] or tape ing epidermal thickness [40] as well as other sig- stripping [48] , the visual methods have the ad- nal characteristics. Morphological changes in dif- vantage over TEWL to better determine the res- ferent conditions such as psoriasis and contact toration of normal morphology [48, 49]. In some dermatitis have been described [35] . experimental settings, where the standardization In high-definition OCT (HD-OCT), the reso- requirements for proper TEWL measurements lution is claimed to be 3 μm and the signal pene- are violated or difficult to comply with, the deter- tration depth up to 570 μm, thus enabling the vi- mination of the SC structure with LSM might be sualization of individual cells [41]. In LSM, the an alternative to determining barrier function by image is acquired point by point by laser scanning means of TEWL measurements [50] . Neverthe- in a particular plane at a particular depth in the less, the imaging methods determine the struc- sample and then reconstructed with a computer. ture and, hence, they cannot replace the measure- It takes usually longer, but modern devices gener- ment of TEWL for questions related to the func- ate images in 30 s [41] . There is a variety of LSM tion. methods to generate contrast in the image. The reflectance method uses variations in refractive indices of the tissue microstructure, while the Irritants: Methods Used for Challenging fluorescence method uses the fluorescence excita- Barrier Function tion of externally applied (including those inject- ed in the upper layers of the skin) or endogenous Multiple models used to study skin irritation fluorophores [42] . Image resolution is very good have been developed using visual irritation scores (approximately 1 μm) and signal depth is approx- and/or the various bioengineering methods ( ta- imately 200 μm. Image resolution is close to that ble 4). The models with a single application study of conventional histology, but the view is that of the acute irritation with application times be-

66 Antonov Schliemann Elsner

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 61–70 ( DOI: 10.1159/000441546) Table 4. Examples of common human in vivo models to study irritant effects on skin barrier

Single application models 4-hour occluded epicutaneous patch test To study acute irritation and skin tolerability [65] ≥24-hour occluded epicutaneous patch test To study acute irritation and skin tolerability [66, 67] Repetitive irritation models 21-day cumulative irritation test To study cumulative irritation [68] RIT Model cumulative irritation over 2 weeks [52] Repeated short-time occlusive irritation test Model cumulative irritation over 5 or 4 days [55] Tandem RIT Reveal synergistic, additive or antagonistic effects of irritants [58, 59] Modeling washing Soap chamber test Occluded forearm model to study cumulative irritation focused on products containing tensides [63] Forearm wash test Cumulative wash test in a setting that imitates washing procedures in everyday life [61] Occupational skin cleanser irritation potential test To evaluate occupational skin cleansers with an automated skin cleansing device in comparison to five established generic cleansers [64]

tween 4 and 24 h. To study the much more com- the measured parameters, the ranking of the test- mon cumulative irritation with weak irritants or ed protective creams was reproducible. Employ- from cosmetic preparations, repetitive occlusive ing a repeated short-time occlusive irritation test, models (over days to weeks) have been developed or a modification of it, a large variety of irritants [51] . Frosch and Kurte [52] introduced the re- has been studied, including lipophilic irritants petitive irritation test (RIT) with a cumulative ir- and sensorial irritation [56, 57] . A similar proce- ritation over a 2-week period by standard irri- dure, called tandem RIT, employs consecutive tants such as sodium lauryl sulfate, sodium hy- application of two irritants, for example 0.5% so- droxide, lactic acid and toluene. This model has dium lauryl sulfate and undiluted toluene [58] . been successfully used to study the efficacy of Tandem tests have been used to study the com- protective (barrier) creams against irritants [52, bined effects of multiple physical and chemical 53]. However, easier study protocols that provide irritants, and they reveal synergistic or antago- valid data in short time with less restrictions for nistic interactions [59, 60] . In the tandem RIT, the volunteers are preferred by both academia physical factors such as cold, airflow or occlusion and industry; therefore, short duration and easy have been studied in parallel with chemical irri- application given in a 1-week test using the fore- tants [59, 61, 62] . arm of healthy volunteers is highly desirable [54] . Because of their importance in the evaluation Such a protocol has been developed and validat- of hand dermatitis, many models of acute or cu- ed in a multicenter trial [55] . It was named re- mulative irritation have focused on hand washing peated short-time occlusive irritation test (ROIT) and the contact to water and detergents as irri- and involved the application of a standard irritants [63] . Some experimental settings also em- tant (0.5% sodium lauryl sulfate) under occlusion ployed mechanical devices to model the influence (for 30 min) twice daily with an interval of 3.5 h of rubbing [61] ; a standardized protocol to assess between the applications for 5 days. Although the irritant potential of occupational skin cleans- there were intercenter variations in the values of ers has also been validated [64] .

Methods for the Assessment of Barrier Function 67

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 61–70 ( DOI: 10.1159/000441546) Conclusion and costs. As TEWL remains the most direct method to quantify the changes in the permea- Multiple measuring methods are available to bility barrier function of the skin, it is still con- assess the barrier function quantitatively. Their sidered to be the gold standard and is most use is determined not only by the scientific widely used in skin physiology, cosmetology question studied, but also by their practicability and other fields.

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Dimitar Antonov, MD Department of Dermatology, University Hospital Jena Erfurter Strasse 35 DE–07743 Jena (Germany)

E-Mail dimitar.antonov @ med.uni-jena.de

70 Antonov Schliemann Elsner

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 61–70 ( DOI: 10.1159/000441546) Section II: External Factors Influencing Skin Barrier

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 71–79 (DOI: 10.1159/000441587)

In vivo Raman Confocal Spectroscopy in the Investigation of the Skin Barrier

a b Razvigor Darlenski Joachim W. Fluhr a b Department of Dermatology and Venereology, Tokuda Hospital, Sofia , Bulgaria; Department of Dermatology, Charité University Clinic, Berlin , Germany

Abstract dermal barrier and its respective functions are The epidermal barrier, predominantly attributed to the still developing [1, 2]. The most important part of stratum corneum (SC), is the outermost part of our body skin barrier function can be attributed to the stra- that comprises multiple defensive functions against ex- tum corneum (SC). The epidermal barrier pre- ogenous attacks and the loss of body substances, e.g. wa- vents the organism from loss of essential compo- ter. A novel investigative method, in vivo Raman confocal nents such as ions, water and serum proteins on spectroscopy (RCS), is employed to study the composi- the one hand, and on the other hand it protects tion of the epidermal barrier and compounds penetrat- against many external stressors, namely physical ing the epidermis both in a space-resolved manner. stress (mechanical, thermal and UV radiation), By using this method, a semiquantitative analysis of skin chemical stress (detergents, prolonged water ex- barrier constituents can be evaluated, namely SC lipids, posure, solvents and other chemicals) and envi- natural moisturizing factor components and sweat con- ronmental conditions [3] . stituents. The technique enables to examine epidermal Nowadays, the concept of SC structure and barrier impairment in experimental settings as well as the functions has evolved from a simple two-com- penetration of exogenous substances into the epidermis, partment system (‘brick-and-mortar’ model) to a e.g. retinol. RCS can reveal microcompositional changes regulated system with a metabolic activity. The in the skin barrier as a function of age. We also review the regulated system of the SC is linked to deeper use of RCS in studying antioxidant defense components. parts of the skin and serves ultimately as a biosen- This chapter discusses the application of in vivo RCS in sor for external factors to regulate different pro- the investigation of the epidermal barrier. cesses, e.g. proteolytic activity, DNA synthesis © 2016 S. Karger AG, Basel and lipid synthesis [4]. The regulated response is observed specifically during barrier insults after barrier disruption. The two major constituents of The skin separates the inner part of our body the SC, the corneocytes and lipids, comprise a against the potentially harmful environment. barrier with mechanical strength, elasticity and During fetal development and after birth, the epi- selective permeability. The different SC lipid classes are constituted the procedure and the lack of adverse events to by approximately 50% ceramides, 25% choles- the tested subjects. Furthermore, after assess- terol and 15% free fatty acids (in an equimolar ment of the compounds of interest in the mea- ratio) and some minor lipid components [5] . sured Raman spectrum, additional substances They originate from precursors, phospholipids, can be studied at later time points from the orig- glucoceramides, sphingomyelin and free sterols inal spectrum. delivered to SC by the lamellar bodies in the stratum granulosum (SG). The lamellar bodies also contain enzymes (hydrolases and proteases) Physical Basis of Raman Spectroscopy important for further lipid processing in the SC and involved in the regulation of desquamatory It was in 1928 when the Indian scientist Sir C.V. processes. Proteins such as keratins, loricrin, in- Raman first observed in practice the inelastic volucrin, filaggrin and corneodesmosine play an scattering of sunlight using a photographic filter important role in the structuring of the corneo- to create monochromatic light and a second cyte cytosol, as well as in the formation of the crossed filter to block this monochromatic light. cornified envelope and the intercorneocyte He found that a portion of the light has passed junctions [6] . Filaggrin, a protein formed by this filter and has changed its frequency. Sir Ra- the enzymatic transformation of profilaggrin man won the Noble Prize in physics in 1930 for packed in the keratohyalin granules of SG, ag- his discovery, and this effect was named after gregates keratin filaments in microfibrils. Ap- him. The Raman effect appears when electro- proaching the skin surface, filaggrin is mostly magnetic radiation hits a molecule and interacts degraded into free amino acids [pyrrolidone- with its electron density and the bonds in the 5-carboxylic acid (PCA) and urocanic acid molecule. When a molecule is excited by a pho- (UCA)], forming a major part of the highly hy- ton, it is in a virtual energy state, resulting into a groscopic complex – natural moisturizing factor vibrational mode of the molecule. Then a photon (NMF). The NMF is responsible for an equili- is scattered with lower (Stokes) or higher (anti- brated SC hydration. Stokes) energy than the incident photon. As a re- Since its introduction as a noninvasive tool, sult, the energy of the photon is being shifted up Raman confocal spectroscopy (RCS) has been or down. The energy needed for the exciting vi- employed in studying in vivo structural compo- brational mode is dependent on the molecular nents of the epidermal barrier (lipids, proteins, structure and the chemical bonds in each mole- NMFs and water gradient) together with its ma- cule. This means that measuring Raman spectra jor function to study the changes in the integrity is molecule specific. Consecutively, RCS can be and protective function of the epidermal barrier. used to detect molecular structures by registering Spectroscopic techniques such as infrared spec- Raman spectra from a selected skin layer [8] . troscopy have been used in studying skin micro- Mathematic equations and fitting procedures al- composition [7]. However, RCS has the advan- low the semiquantitative measurement of sub- tage of being water insensitive, which allows in stances that have a distinctive Raman profile (fin- vivo measurements of the water-enriched viable ger print) in the epidermis [11] . Figure 1 presents epidermis [8]. Other methods have been em- a schematic overview of the RCS measurement ployed in the in vivo investigation of skin barrier procedure. components and penetration profiles of exogenous substances, such as tape stripping [9, 10] . The advantage of RCS is the noninvasiveness of

72 Darlenski Fluhr

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 71–79 ( DOI: 10.1159/000441587) Monochromatic light source (laser)

Notch filter Lens

Charge-coupled

detector Window

Inelastic scattered light Elastic scattered light Absorbed light Absorbed Scattered light

Scattered light Transmitted light

Skin sample Raman intensityRaman

900 800 700 600 500 400 300 200 100 1,100 1,000 Wave numbers (cm–1)

Fig. 1. Schematic overview of RCS.

In vivo Raman Confocal Spectroscopy of the spectra of the skin were published in 1997 [12] . Epidermal Barrier Then, in 1998, in vivo spectra for NMF constituents and lipid composition of the SC were record- The confocal approach of RCS allows a spatial ed [8] . Further work by the latter group reported resolution of around 5 μm vertically and 2 μm the relative water concentration (in mass%) as a horizontally, which offers the possibility to gain a function of depth from the skin surface [11] . The detailed overview on skin barrier constituents. in vivo RCS allows semiquantitative measure- First in vivo study results obtained with Raman ments of the concentrations of NMF constituents

Raman Spectroscopy and Skin Barrier 73

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 71–79 ( DOI: 10.1159/000441587) thickness of 110 μm at the thenar of the palm ac- Water concentration profile cording to Caspers et al. [11] . The estimated values 70 were consistent with formerly published ultrastructural data [14] . In contrast to the arm, the SC * of the thenar showed only small variations in wa- 60 ter concentration profiles. Good correlation was reported between the pioneering in vivo data and the formerly estimated NMF profile concentra- 50 tions in vitro [15] . NMF is mainly generated in the lower parts of SC where filaggrin is degraded to amino acids (PCA and UCA) comprising NMF. 40 This is in agreement with the in vivo RCS data showing that the concentration of NMF constituents is almost not detectable 70 μm below the skin Water content (mass%) 30 surface with a steep increase up to approximately 50 μm from the surface (thenar skin) [11] . Sweat constituents potentially contributing to the water- 20 lipid acid environment of the skin could be docu- Standard water profile mented with highest concentrations at the skin surface and a subsequent decrease in deeper parts 1020 30 40 50 of the epidermis. Depth (μm) Lateral packing of the intercellular lipids of the SC has been reported to be similar when comparing in vivo RCS measurements with the conven- Fig. 2. A typical curve representing the water profile of SC obtained by in vivo confocal Raman microspectros- tional X-ray diffraction method [16] . The lipids at copy. * = Approximate water concentration/depth of the the forearm and the upper arm were more or- SC-SG border. dered than those at the cheek, which is indicative of a more resistant barrier. We studied alterations in the antioxidant net- (serine, glycine, PCA, arginine, ornithine, citrul- work in the SC induced by external stressors in line, alanine, histidine and UCA) as well as those vivo by using two different methods – resonance of sweat constituents – lactate and urea. The in and Raman spectroscopy [17]. Both methods vivo Raman water concentration profiles were were able to detect the infrared-induced deple- very similar to the ones obtained using in vitro tion of carotenoids. Only Raman microspectros- X-ray microanalysis [13]. A steep rise in water copy could reveal the carotenoid decrease after concentration from 15–25% in the SC to about topical disinfectant application. The carotenoid 40% at the SC-SG and a constant level of about depletion started at the surface and then extended 70% in the viable cells was revealed [11] . A typical further into deeper parts of the epidermis. After curve of water concentration as a function of 60 min, recovery began at the surface while deep- depth in the epidermis is shown in figure 2 . The er parts were still depleted. The disinfectant- and change from the slope to the plateau can be further infrared-induced carotenoid depletion in the epi- used to determine SC thickness in vivo. In healthy dermis recovers from outside to inside, and carot- human volunteers, the SC thickness at the volar enoids are probably delivered by sweat and seba- forearm is approximately 15 μm in contrast to a ceous glands. We showed that the Raman micro-

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Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 71–79 ( DOI: 10.1159/000441587) 30

10 AUC of NMF profiles (arbitrary units) (arbitrary profiles NMF of AUC 0 1–15 days 5–6 weeks 6 ± 1 months 1–2 years 4–5 years 20–35 years

Study group

Fig. 3. Semiquantitative NMF concentrations in different age groups.

scopic spectroscopy is suited to analyze carotenoid children, and adults aged 20–35 years [21] . A kinetics following stress and in recovery. In an- continuous increase in the water content was ob- other study, we showed that in untreated skin, the served as a function of epidermis depth for all age major fraction of the carotenoids is located in the groups. This increase was lower for the new- upper part of the SC [18]. The amount of carot- borns. With the exception of newborns, the per- enoid is lower in the upper part of the SC on the centage of water content revealed a saturation of forearm compared to forehead and palm with approximately 60% at a depth of 15–20 μm at the both methods. volar forearm. For all age groups, the area under the curve (AUC) of Raman depth profiles for Skin Barrier Properties Show Differences as a NMF were assessed at the skin surface (0–15 Function of Age μm). The mean AUC for NMF was higher in Infants aged 3–33 months showed a steeper wa- newborns in the depth range of 0–25 μm com- ter gradient in the SC and a higher water content pared to all other age groups. Clearly lower mean within the upper 20 μm of the epidermis com- AUCs were noted for the group aged 6 ± 1 months pared to adults [19] . Lower amounts of NMF for each depth as well as for the total depth (0–25 constituents were evidenced in infants in com- μm) when compared to the other age groups. parison to adults [19, 20] . Water absorption and This could be attributed to the effect of exoge- desorption properties were disturbed in infants, nous factors, such as washing (and depleting suggesting ongoing functional barrier adapta- NMF constituents), which is not yet observed be- tion after birth even in term-born neonates. We fore the age of 6 months. It may take almost a studied skin in vivo Raman profiles of healthy year for the complete adaptation to the dry envi- volunteers in six different age groups: full-term ronment, witnessed by the lowest NMF profiles newborns, 5-week-old babies, 6-month-old ba- in 6-month-old children and then the relatively bies, 1- to 2-year-old children, 4- to 5-year-old similar levels in older age groups (fig. 3 ).

Raman Spectroscopy and Skin Barrier 75

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 71–79 ( DOI: 10.1159/000441587) AD Healthy skin 70

40 Water (mass%) 30

05010 20 30 40 a Depth (μm)

Control 1.6 1.4 Healthy 1.2 AD in remission 1.0 0.8 0.6

Rel. content NMF 0.4 0.2 0 Fig. 4. Differences between healthy 04010 20 30 subjects and AD patients. a Water b Depth (μm) profiles. b NMF profiles.

No difference in the water profiles could be Raman Confocal Spectroscopy in Diseases found between young (mean age: 27 years) and Characterized by Epidermal Barrier Impairment old (mean age: 64 years) Asian subjects in the SC In atopic dermatitis (AD), a loss-of-function and the epidermis [reviewed in 22 ]. However, the mutation in the gene encoding for filaggrin has upper dermis of the older group was ‘more hy- been revealed [25] . Filaggrin is the precursor drated’ than the one of the younger group. An- protein for the amino-acid-derived components other study found thicker SC in older skin on the of the NMF. By measuring NMF with RCS in the forearm. However, no such difference could be SC of palm (thenar eminence) and forearm skin, observed at the cheek [23] . Small variations in the reduced levels of NMF constituents (produced lateral lipid packing were observed when com- by the degradation of filaggrin) have been wit- paring young and old volunteers [24] . nessed [26]. Carriers of FLG-null mutations

76 Darlenski Fluhr

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 71–79 ( DOI: 10.1159/000441587) have significantly reduced levels of NMF in the The penetration of hazardous substances SC on both skin locations and at all SC depths. has also been tracked by RCS. 2-Butoxyethanol, Carriers with a history of AD had significantly toluene and pyrene were applied in pure form, lower NMF levels than noncarriers with a his- or diluted in water or ethanol, on the skin of tory of AD. Figure 4 presents data on differences 3 healthy volunteers [34] . Good correlation in water and NMF concentration profiles be- with data from the literature was observed. In tween atopic and healthy individuals. addition, 2-butoxyethanol penetrated markedly Raman profiles have been obtained from pa- faster when dissolved in water as compared to tients with xerotic skin due to either HIV or AD ethanol. and in elderly subjects [27] . A decrease in lipids The critical point of assessing semiquantita- was shown for the elderly and HIV patients. De- tively substances in the epidermis is that it re- creases in lipids and increases in water concen- quires an adaptation of the Raman signal to the tration were witnessed in the dermis for HIV and optical properties of the skin [35–38] . In a con- atopic patients in comparison to healthy subjects. trolled study, a skin surrogate containing keratin, Psoriatic skin showed thicker SC and lower con- water and different lipid fractions was tested centrations of water than healthy skin [23] . Cho- [35]. A mathematical surrogate algorithm was lesterol, lactate and urea levels did not differ be- applied to correct the drug profiles in human tween both groups indicating normal sweat gland skin for signal attenuation facilitating reliable activity in psoriasis. drug quantification in human skin by RCS.

Penetration of Topical Substances and Their Effect on the Epidermis Assessed by Raman Conclusion Confocal Spectroscopy The use of moisturizers has been evaluated as RCS is a noninvasive and reliable tool for inves- useful in terms of enhancing skin hydration and tigating epidermal barrier properties. It is effec- SC thickness [28] . The hydration effect was first tive in the assessment of barrier constituents (lip- demonstrated by applying a wet towel on the ids, NMF and sweat components), epidermal skin, which resulted in an increased SC water water gradients and water-handling ability. concentration of almost 60%. Glycerol-based Monitoring the penetration of exogenous sub- creams increased the water concentration com- stance and their transformation in the epidermis pared to baseline at depths from 0 to 20 μm from unveils RCS as a useful method in studying skin the skin surface [29] . Paraffin and vegetable oils pharmacology and physiology in vivo without al- penetrated the SC in a similar manner [30] . In terations in epidermal properties. A major chal- contrast to petrolatum (control), SC swelling was lenge for this method is that it is still too expen- lower for the oils. Since these initial experiments, sive and requests trained personnel for conduct- a number of studies have been carried out showing the measurements. Fortunately, in the recent ing the beneficial effect of moisturizing formula- years, a wider use of RCS will impose the routine tions [31] . use of this method in dermatological research The penetration of retinol has been the subject and practice. of several studies [32, 33]. The substance could be traced in the epidermis of healthy subjects. The use of oleic acid, a lipid fluidizer, resulted in better retinol delivery into the skin in comparison to classic enhancers such as propylene glycol [33] .

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Joachim W. Fluhr, MD Department of Dermatology Charité University Clinic Charitéplatz 1 DE–10117 Berlin (Germany)

E-Mail Joachim.Fluhr @ charite.de

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Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 71–79 ( DOI: 10.1159/000441587) Section II: External Factors Influencing Skin Barrier

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 80–89 (DOI: 10.1159/000441547)

Irritants and Skin Barrier Function

Irena Angelova-Fischer Department of Dermatology, University of Lübeck, Lübeck , Germany

Abstract stratum corneum is a key determinant for the The barrier response to irritant challenge involves com- outcome of the interaction between the irritant plex biologic events and can be modulated by various and the skin. The permeability barrier formation environmental, exposure and host-related factors. Irri- results from a strictly regulated process of termi- tant damage to the epidermal barrier elicits a cascade of nal differentiation in which keratinocytes under- homeostatic or pathologic responses that could be inves- go sequential biochemical and structural trans- tigated by both in vitro and in vivo methods providing formations to form a layer of cornified cells in a different information at biochemical and functional lev- lipid-enriched extracellular matrix of approxi- el. The present chapter summarizes the changes in key mately equimolar ratios of ceramides, cholesterol barrier function parameters following irritant exposure and free fatty acids [2, 3] as a first line of defense with focus on experimental controlled in vivo human to the environment. Historically referred to as skin studies. © 2016 S. Karger AG, Basel biochemically inert, the stratum corneum is nowadays considered a sophisticated biosensor that regulates the skin barrier responses to various environmental insults, including chemical or me- Irritants and Skin Barrier Homeostasis chanical irritants. Abrogation of the skin barrier function independently of the type of irritant Irritants interact with different structural compo- damage initiates homeostatic responses in the nents of the skin and elicit a diverse spectrum of nucleated epidermal cell layers that aim at resto- reactions that extend from subclinical inflamma- ration of the skin barrier through a well-regulated tion or mere sensory responses to severe, wide- and coordinated cascade of metabolic events in- spread disease with systemic involvement [1] . volving the rapid secretion of a preformed pool of The barrier response to irritant challenge involves lamellar bodies, formation and secretion of new- complex biologic events and can be modulated ly synthesized lamellar bodies, enhanced choles- by various environmental, irritant-specific, expo- terol, fatty acid and ceramide synthesis and in- sure and host-related factors. The presence of a creased epidermal DNA synthesis [4–8] . The sig- competent epidermal barrier at the level of the naling mechanisms after an acute insult to the skin barrier have been extensively studied and ditions organic solvents such as undiluted tolu- shown to involve changes in ionic gradients [9– ene, octane, cumene or the short-chain aliphatic 11], nuclear hormone receptor activation [12–15] alcohol n-propanol lead to less pronounced alter- and release of numerous cytokines and growth ations in the skin barrier function or TEWL [24– factors from the skin residential cells that drive 27] . Compared to nonanoic acid or hydrochloric and further modulate the barrier response to ir- acid, SLS exposure was found to cause more se- ritants [4, 16–22] . vere barrier impairment even though the inflammatory responses were comparable [28] . Repeat- ed combined exposure to multiple chemicals may Non-Invasive Bioengineering Methods enhance the irritant-induced effects on TEWL and result in an additive or synergistic effect in A number of established biophysical methods al- comparison to repeated application of a single ir- low non-invasive in vivo investigation of the bar- ritant [29, 30] . Similarly, additive impairment of rier responses to irritant exposure in human skin. the skin barrier function following combined ex- Being objective and reproducible, the skin bioen- posure to SLS and mechanical irritation has been gineering methods have been largely employed reported [31]. Beyond the physicochemical prop- for studying the irritant potential of a specific erties of the irritant, the extent of damage to the chemical or a group of structurally related chem- skin barrier depends on the purity, concentra- icals, recognition of reaction patterns, predicting tion, duration and mode of exposure, and may be susceptibility and identification of populations at modified by both environmental and host-related risk for increased barrier damage through irri- factors [32–37]. Though differences in the instru- tants. Furthermore, due to their non-invasive- mental assessment of TEWL dependent on the ness, the skin bioengineering methods allow in- measurement principle (open, closed or condens- vestigation of the irritant interactions with the er-type chamber) have been observed, all current- epidermal barrier at multiple time points and ly available commercial devices have been shown have been shown to detect early, subclinical dam- to detect changes in the functional state of the age to the stratum corneum. skin barrier following irritant damage [38–40] . In addition to the instrumental variables and the Transepidermal Water Loss need for observation of the published guidelines Transepidermal water loss (TEWL) is one of the [41, 42], the time point of assessment may largely most important parameters for assessment of the influence the endpoints and needs to be taken functional state of the epidermal barrier in health into consideration for the correct interpretation and disease. The validity of TEWL as a measure of the readings [43] . to assess the permeability barrier function has Exposed to the same irritants under identical been confirmed by comparison of instrumental exposure conditions, some individuals display and gravimetric measurements in ex vivo and in pronounced inflammatory reactions whereas vivo animal as well as in vivo human skin models others may show no manifest signs of irritant [23] . The effects on TEWL exerted by different damage, consistent with the concept for the exis- chemical irritants and irritant classes in vivo have tence of substantial variations in the individual been shown to vary considerably. Irritants of the response to irritants [44] . Various tests based on corrosive type such as the anionic detergent so- experimental exposure to different classes of ir- dium lauryl sulfate (SLS) or alkaline agents such ritants such as alkaline agents, for example sodi- as sodium hydroxide cause pronounced increase um hydroxide or ammonium hydroxide [45–47] , in TEWL, whereas under the same exposure con- organic solvents such as dimethyl sulfoxide or de-

Irritants and Skin Barrier Function 81

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 80–89 ( DOI: 10.1159/000441547) tergents (SLS) have been introduced with the aim siderable variation, and, in addition to the differ- to identify individuals at increased risk for irri- ent exposure conditions and assessment time tant damage [48, 49] . The subjective component points that influence the results, the presence of in the case the outcomes rely on visual scoring, acute inflammatory lesions at the time of inves- however, may lead to inconsistencies, and, there- tigation may modify the barrier responses or fore, some of the earlier methods have been later TEWL readings before and after exposure [53, used in combination with or replaced by mea- 58]. As a single irritant challenge to the skin bar- surements of objective parameters of the skin rier reflects a momentary situation, the short- barrier function such as TEWL. The importance term irritation studies do not provide informa- of baseline TEWL for predicting the skin barrier tion on the cumulative irritant-induced effects on response to irritants, in particular detergents, has the barrier function or the skin repair capacity in been assessed in a number of studies and con- AD. Though, at present, there has been a limited troversially discussed. Several groups reported a number of studies on the changes in the skin bar- good correlation between the pre- and post-expo- rier function following cumulative exposure to ir- sure TEWL in experimentally induced irritation ritants in atopic skin, the so far published investi- models based on single as well as repeated expo- gations show consistent results and more pro- sure to SLS, whereas these observations could not nounced barrier function impairment or TEWL be confirmed in other studies, possibly due to increase after repeated single or tandem exposure methodological differences [38, 50–52] . Though to multiple irritants in AD compared to healthy, the relationship between baseline TEWL and en- non-atopic control subjects [25, 56] . hanced barrier damage by irritants is of consid- In 2006, Palmer et al. [59] identified loss-of- erable interest in particular in occupational set- function mutations in the gene encoding the epi- tings, the currently available knowledge has been dermal differentiation protein filaggrin ( FLG) as limited mostly to surfactant-induced irritation a significant predisposing factor for AD. These that may not predict the barrier responses to oth- findings have been subsequently confirmed in a er, chemically unrelated, primary irritants. number of studies in different populations, and, Impaired skin barrier function and properties, at present, the FLG mutations are considered the and increased baseline TEWL values even in clin- most important individual risk factor for AD ically uninvolved areas are important character- known so far [60] . FLG mutations have been istics of atopic skin disease [53–57] . The compro- shown to confer an early-onset, persistent disease mised barrier function and the epidemiological course with allergic sensitization and, in addi- evidence that atopic dermatitis (AD) predisposes tion, contribute to the risk for occupational irri- to an increased risk for contact dermatitis caused tant contact dermatitis [61–63] . FLG mutation by occupational exposure to irritants have stimu- carrier state adjusted for AD has been found to lated interest in studying the barrier function im- increase the risk 1.6-fold, whereas the adjusted pairment and the changes in TEWL as a measure risk for AD was 2.9-fold; individuals with AD car- to assess irritant susceptibility in atopic skin. rying FLG mutations have been identified to have Most published investigations on the barrier rean almost 5-fold risk to develop irritant dermati- sponse to irritants in AD rely on acute irritant tis and, therefore, defined as a highly susceptible challenge through short-term patch test applica- population. In addition to increased susceptibili- tion of model irritants [58] , with SLS and sodium ty, FLG mutation carrier state has been shown to hydroxide being by far the most commonly used contribute to the persistence of hand eczema and ones. The outcomes of the short-term irritant confer an unfavorable disease prognosis [64, 65] . challenge studies in atopic individuals show con- Despite the significance of these findings and the

82 Angelova-Fischer

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 80–89 ( DOI: 10.1159/000441547) role of filaggrin in the process of epidermal dif- values compared to younger age groups [72] . ferentiation, so far there have been few studies Experimental irritant-exposure studies in aged investigating the skin barrier responses to irri- individuals have shown delayed and less pro- tants in AD FLG mutation carriers. Two indepen- nounced changes in TEWL following occlusive dent studies investigated the outcomes of a 24- application of SLS in concentrations ranging hour patch test exposure to 1% SLS in relation to from 0.25% to 5.0% or repeated open application FLG mutation carrier state in AD individuals [66, of 7.5% SLS compared to the same exposure in 67] . Under identical exposure conditions, both young volunteers [68, 73–75] . Similarly, tandem studies found no significant differences in the se- repeated exposure to irritants with synergistic ef- verity of barrier function impairment or TEWL fects such as SLS and toluene has been shown to increase assessed by ΔTEWL (ΔTEWL = TEWL result in delayed and less pronounced responses, after exposure – baseline TEWL) between the AD i.e. significantly lower ΔTEWL in aged compared FLG mutation carriers and non-carriers. Further- to young individuals, providing evidence for a more, monitoring of the epidermal barrier recov- consistent pattern of irritant reactivity in aged ery through repeated measurements of TEWL up skin [24] . to 72 h after irritation showed no significant dif- An important aspect of the in vivo skin barrier ferences in the rate of barrier recovery between response to irritants is the capacity for accom- the AD FLG mutation carriers and non-carriers modation after prolonged exposure to chemical or the mutation carriers and the healthy controls irritants described as ‘hardening phenomenon’ [68]. These findings have been confirmed in a re- [76] . Though of considerable interest, the mecha- cent study using 3 different concentrations of the nisms of accommodation remain elusive. Earlier same irritant (0.25%, 0.5% and 1.0% SLS) and studies suggested that changes in the lipid com- monitoring of the barrier recovery up to 145 h af- position of the stratum corneum, in particular ter removal of the test chamber in AD FLG muta- up-regulation of ceramide 1 synthesis after re- tion carriers and non-carriers as well as in healthy peated SLS exposure, may provide a basis for ex- controls with and without FLG mutations [69] . planation; a recent study by an independent Host-related factors beyond AD that have group, however, found no changes in the ce- been investigated with respect to modification of ramide levels between accommodated and non- the skin barrier responses or barrier impairment accommodated skin [77, 78] . severity following irritant exposure include age, gender, complexion, hormonal influences, cyto- Skin Hydration kine polymorphisms, anatomic site and preexist- Within the stratum corneum, water plays a key ing skin disease [37, 44] . Numerous reviews on the role for the maintenance of the elasticity and ten- topic have previously been published; with respect sile properties along with influencing the barrier to the trend for increasingly aged population integrity, properties, and the overall appearance of worldwide, the modification of the barrier re- the skin [79] . The maintenance of the water bal- sponses to irritants as a function of age will be only ance in the stratum corneum is controlled through discussed. Advanced age leads to profound mor- the highly organized structure of the intercellular phologic and functional alterations in the skin lipid lamellae that restrict the transport of water barrier, including decreased lipid metabolism, im- and by the presence of a mixture of low-molecu- paired acidification, aberrant cytokine signaling lar-weight water-soluble hygroscopic compounds and delayed barrier repair following irritant or including amino and organic acids, urea and inor- mechanical damage to the skin [70, 71] . Elderly ganic ions, collectively described as natural mois- individuals tend to have lower baseline TEWL turizing factors (NMF). In addition to barrier per-

Irritants and Skin Barrier Function 83

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 80–89 ( DOI: 10.1159/000441547) turbation resulting in increased TEWL, irritants Removal of the skin surface lipids by organic influence the skin barrier function and water bal- solvents is an established model for studying the ance via interaction with the lipid components as skin barrier function under stress conditions. An well as changes in the levels of NMF [25, 80, 81] . in vivo human volunteer study of the skin barrier Compromised barrier function and reduced stra- function following acetone treatment at different tum corneum hydration may each lead to xerosis anatomic locations has shown consistently de- and scaling, and both symptoms are commonly creased skin hydration, assessed by capacitance observed following irritant contact with the skin. measurements [87] . Similarly, repeated exposure Most of the published instrumental studies on the to toluene as a single irritant was shown to de- effects of irritant exposure on skin hydration have crease capacitance, while combined exposure to been based on non-invasive assessment by electri- toluene and SLS in the same volunteers was found cal methods such as measuring conductance, ca- to enhance the effect [24, 88] . The findings for a pacitance or impedance [38] . Similarly to TEWL, more pronounced decrease in skin hydration af- the skin hydration measurements may be influ- ter tandem irritation have been further confirmed enced by individual, environmental, exposure and in an in vivo exposure study with octane or instrument-related variables [82]. Though differ- cumene applied sequentially with SLS [26] . In ent classes of chemical irritants, including deter- contrast to acetone and aromatic hydrocarbons, gents, organic solvents, alkaline agents or acids, the so far published in vivo bioengineering stud- have been investigated, surfactants and organic ies on the effects of short-chain aliphatic alcohols solvents have been of particular interest in view of on skin hydration in human volunteers show the frequent consumer or occupational exposure partly controversial results [89–92] . to these compounds and their known effects on the skin lipids. Decreased skin hydration after sin- Skin Surface pH gle as well as repeated exposure to surfactants has The acidic pH of the stratum corneum regulates been observed in numerous studies using a broad key protective functions of the skin, including range of concentrations and different durations of permeability barrier homeostasis, stratum corne- exposure [83, 84]. Comparison of the changes in um integrity, cohesion, antimicrobial defense and the skin hydration following 48-hour patch test primary cytokine release [93, 94] . The importance exposure to 8 different surfactants has shown dif- of the maintenance of an acidic pH has ferences in the outcomes with increase of capaci- been demonstrated in studies in hairless mice tance immediately after exposure to SLS, whereas showing that neutralization of the skin pH delays no such effect after simultaneous application of recovery after acute barrier perturbation by ace- disodium laureth sulfosuccinate, cocamidopro- tone treatment [95] . Key enzymes involved in the pyl betaine, cocamide DEA and lauryl glucoside in barrier formation and, in particular, ceramide the same volunteers was found [85] . Initially in- synthesis, are known to have pH optima within creased capacitance followed by decreased values the acidic range and, furthermore, acidic pH is re- has been found in other experimental studies of quired for the processing of lamellar bodies [5, SLS-induced irritation; though controversially 93] . Blocking of secretory phospholipase A2 or the discussed, these findings have been attributed to sodium proton exchanger NHE1, both involved in keratin swelling [86] . Within the group of anionic stratum corneum acidification, leads to increased surfactants, the carbon chain length was found to pH and compromised barrier integrity and cohe- influence the in vivo irritant potential and skin hy- sion [96–99] . Similarly, a shift towards a neutral dration with increasing number of carbon atoms pH may increase serine protease activity and im- enhancing the biological effects [83] . pact the barrier function and integrity [100–102] .

84 Angelova-Fischer

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 80–89 ( DOI: 10.1159/000441547) Exposure to common irritants such as soaps or de- cardinal sign of inflammation, the non-invasive tergents may result in increased pH and thus ad- assessment of the changes in the skin color or versely impact the skin barrier function. The rela- blood flow is an established and frequently used tionship between compromised barrier function approach for investigation of the skin response to and increased skin pH has been shown in in vivo irritants in vivo [37, 38]. Though the published lit- human skin studies in aged individuals [72, 103, erature allows little comparison between the stud- 104] , in pathologic skin conditions associated with ies based on the use of different measurement barrier abnormalities [53, 54, 93] as well as in dia- principles, irritants, exposure conditions, assess- per dermatitis, or in association with the use of ment time points and additional confounders cosmetic and cleansing products [105–108] . The such as pigmentation or desquamation, depen- changes in the epidermal barrier function and pH dent on the physicochemical properties, irritants following experimental exposure to different differ in their potential for inducing erythema classes of irritants in healthy individuals have been when applied epicutaneously. Applied simultane- investigated by Fluhr et al. [109] . The results of ously to the same volunteers and under identical the study showed increased skin surface pH after exposure conditions, the anionic detergent SLS cumulative exposure to 0.5% SLS applied as a sin- induces pronounced erythema whereas solvents gle irritant, as well as in combination with 0.04% such as toluene and n-propanol or weak organic sodium hydroxide, 1.0% ammonium hydroxide, acids such as fruit acids lead to minor changes in 1.0% dimethylamine or 1.5% trimethylamine ap- the instrumental readings or a * -values as a mea- plied under tandem repeated irritation conditions; sure for assessment of erythema [24, 26, 27, 29, simultaneous exposure to 0.2% acetic acid in the 87, 113]. The positive correlation between the ir- same volunteers induced no significant changes in ritant concentration, duration of exposure and the skin barrier function and skin pH. The chang- skin response has been shown in studies employ- es in pH after mechanical damage to the skin bar- ing a range of SLS concentrations from 0.125% to rier by occlusion or tape stripping have been inves- 2.0% applied under patch test conditions for dif- tigated by independent groups showing contro- ferent periods (3–48 h), followed by TEWL mea- versial results [110, 111] . surements and laser Doppler flowmetry [34] . In Irritants have mostly been studied in the con- addition to visual assessment and colorimetric text of potential negative impacts on skin barrier measurements, the use of non-invasive methods function and pH; the results of a study showing for measurements and imaging of the skin micro- decreased sensitivity to SLS after a 4-week appli- circulation may offer advantages for studying the cation of lactic acid are therefore interesting in skin irritant response suggested by studies provid- terms of protection and provide in vivo human ing evidence for detectable changes in the micro- skin evidence for the importance of the acidic pH circulation before the development of manifest for the maintenance of the skin barrier homeosta- erythema, and studies showing that the area of sis [112] . increased perfusion extends beyond the area of clinically manifest erythema [114, 115] . Erythema and Skin Microcirculation Beyond exerting a direct negative impact on the epidermal barrier function and properties, irri- Conclusions tant damage to the skin elicits a subclinical or clinically manifest inflammatory response of different Irritant damage to the epidermal barrier elicits a severity that could be influenced by exposure, ir- cascade of homeostatic or pathologic responses ritant- and host-related factors. As erythema is a that could be investigated by both in vitro and in

Irritants and Skin Barrier Function 85

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 80–89 ( DOI: 10.1159/000441547) vivo methods providing different information at As irritants interact with different components of biochemical and functional level. Though TEWL, the skin barrier, a combination of in vivo mea- stratum corneum hydration, skin surface pH and surements complemented by analytical methods erythema are still the most commonly used in may help to improve the understanding of the vivo parameters, modern technological develop- barrier responses to specific irritants and the ments beyond the spectrum of the present chap- identification of sensitive readout parameters for ter have become available for studying the dif- studying skin irritation in healthy and compro- ferent aspects of human skin irritation under mised conditions. experimental exposure or consumer conditions.

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degradation of lipid processing en- long-term application of synthetic de- matol Res 1996; 288: 383–390. zymes and profound alterations of bar- tergent preparations of pH 5.5 and pH 113 Fluhr JW, Bankova L, Fuchs S, et al: rier function and stratum corneum 7.0. Results of a crossover trial in Fruit acids and sodium hydroxide in

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Irena Angelova-Fischer, MD, PhD Department of Dermatology, University of Lübeck Ratzeburger Allee 160 DE–23538 Lübeck (Germany)

E-Mail irena.angelova-fischer @ uk-sh.de

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Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 80–89 ( DOI: 10.1159/000441547) Section II: External Factors Influencing Skin Barrier

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 90–102 (DOI: 10.1159/000441548)

Skin Barrier Function and Allergens

Kristiane Aasen Engebretsen Jacob Pontoppidan Thyssen National Allergy Research Centre, Department of Dermato-Allergology, Herlev and Gentofte Hospital, University of Copenhagen, Hellerup , Denmark

Abstract patients with ICD are, therefore, at great risk of devel- The skin is an important barrier protecting us from me- oping CA. Skin irritation leads to the release of IL-1 and chanical insults, microorganisms, chemicals and aller- TNF-α, which affects the function of antigen-presenting gens, but, importantly, also reducing water loss. A com- cells and promotes their migration to local lymph mon hallmark for many dermatoses is a compromised nodes, thus increasing the probability of CS and ulti- skin barrier function, and one could suspect an elevated mately the development of CA. risk of contact sensitization (CS) and allergy following © 2016 S. Karger AG, Basel increased penetration of potential allergens. However, the relationship between common dermatoses such as psoriasis, atopic dermatitis (AD) and irritant contact Normal Skin Barrier Function dermatitis (ICD) and the development of contact allergy (CA) is complex, and depends on immunologic re- The epidermis, and especially the outermost lay- sponses and skin barrier status. Psoriasis has tradition- er, the stratum corneum (SC), protects us from ally been regarded a Th1-dominated disease, but the the onslaught of microorganisms and the percu- discovery of Th17 cells and IL-17 provides new and in- taneous penetration of chemicals and allergens teresting information regarding the pathogenesis of (outside-inside barrier), but, importantly, also re- the disease. Research suggests an inverse relationship duces water loss (inside-outside barrier) [1] . The between psoriasis and CA, possibly due to increased epidermis is continuously regenerated. Prolifera- levels of Th17 cells and its associated cytokines. As for tive keratinocytes from the stratum basale move AD, a positive association to CS has been established in up to the stratum spinosum where intracellular epidemiological studies, but is still unresolved. Experi- lipids are synthesized and secreted into the inter- mental studies show, however, an inverse relationship cellular matrix. In the granular layer, important between AD and CS. The opposing and antagonistic in- proteins, including filaggrin, are produced. At fluences of Th1 (CS) and Th2 (AD) have been proposed last, keratinocytes reach the SC where they col- as an explanation. Finally, there is convincing evidence lapse and become anucleated corneocytes, which that exposure to irritants increases the risk of CS, and are shed off after a certain time [2] . Filaggrin is important as it presumably helps develops. There are two main phases: the induc- to aggregate keratin filaments into tight bundles tion phase, where the subject is sensitized, and an and attach to the cornified envelope, a rigid pro- elicitation phase, in which allergic contact derma- tein structure that surrounds corneocytes [3] . Lip- titis (ACD) is triggered [13] . Common contact al- ids in the intercellular matrix then get covalently lergens include metals, fragrance materials and bound to the cornified envelope, and the final preservatives found in commonly used products result is a strong, multilayered structure, which such as cosmetics and jewelry. The risk of CS does simplistically resembles bricks (the corneocytes not only depend on the integrity of the skin bar- and the cornified envelope) and mortar (the hy- rier, but also on the sensitizing potential of the drophilic lipids) [2, 4] . A reduced production of chemical, the frequency, duration and extent of filaggrin and its metabolites leads to decreased exposure, and if occlusion or local trauma/irrita- skin hydration, elevated pH and compromised tion is present. Differences in susceptibility from photoprotection, and can be primarily due to one person to another also play a minor role [14] . loss-of-function mutations in the filaggrin gene In this chapter, we will review the main com- (FLG) or secondarily due to exposure to exoge- ponents of three common dermatoses, namely nous factors or inflammation [3, 5–9] . psoriasis, atopic dermatitis (AD) and irritant con- The amount of passive water evaporation tact dermatitis (ICD), and discuss their relation- from the skin surface is known as transepidermal ship towards skin barrier function, skin sensitiza- water loss (TEWL) and is a marker of the inside- tion and development of contact allergy (CA). outside barrier [1]. Most often the inside-outside barrier correlates with the outside-inside barrier, but there are situations where this assumption Psoriasis does not apply. To get an estimate of the outside- inside barrier, penetration studies with tracer Psoriasis is a chronic condition that affects the compounds must be performed, but this can be skin and joints. The estimated prevalence varies challenging, as the tracer compound typically has between 0.5 and 4.6%, depending on race and to be detected by chemical analysis. northern residence [15]. Psoriatic skin is charac- Contact sensitization (CS) to chemicals is terized by epidermal thickening due to abnormal common and affects up to 20% of the general proliferation and impaired maturation of kerati- population [10] . It is characterized by the induc- nocytes, and clinically there are demarcated ery- tion of specific T-lymphocyte responses, primar- thematous patches or plaques on the skin covered ily T-cytotoxic (Tc)1/T-helper (Th)1 cells, but by thick and adherent silver scales [16] . Tradi- Th2 cells may also be involved [11] . The allergens tionally, psoriatic lesions have predominantly (haptens) differ in size and polarity, and can be been ascribed to Th1 cell activity with the produc- either water or lipid soluble. They penetrate the tion of IFN-γ and TNF-α as the main proinflam- SC in different ways depending on their charac- matory mediators. However, the discovery of teristics. The lipid-rich SC is resistant to water- Th17 cells and the cytokine IL-17 has dramati- soluble haptens, and they may instead penetrate cally changed this interpretation [17]. Hence, in- transcellularly through the eccrine glands or pilo- creased levels of Th17 cells have been found both sebaceous follicles. The primary pathway of pen- in psoriatic lesions [18] and in the circulation of etration for the lipid-soluble allergens is, howev- patients with psoriasis [19], and the amount of er, typically paracellular [12] . If topical exposure IL-17 mRNA correlates significantly with disease to the chemical allergens is repeated and pro- activity [20]. An important cytokine in the devel- longed, and exceeds the individual threshold, CS opment and maintenance of Th17 cells is IL-23,

Skin Barrier and Allergens 91

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 90–102 ( DOI: 10.1159/000441548) also known for its role in autoimmune diseases suggesting that primary barrier abnormality may [21, 22] . Elevated levels of IL-23 have indeed been also result in secondary immune activation [44] . found in both psoriatic lesions and in the serum It is also well known that psoriatic lesions can be of patients with psoriasis, and medical treatment caused by traumatization of the skin (Koebner blocking IL-23 reduce psoriasis severity, support- phenomenon) [45]. Occlusive dressings may on ing the pertinence of the Th17/IL-23 pathway in the other hand result in improvement or even psoriasis [23] . resolution of psoriatic lesions [46] . Furthermore, tape stripping resulted in rapid immune activa- Skin Barrier Function in Psoriasis tion within the epidermis and led to movement of The barrier function is decreased in lesional pso- inflammatory cells from the circulation into the riatic skin, and, accordingly, TEWL is increased dermis and epidermis in another experimental compared to normal skin [24–27] . Notably, study, again indicating that primary events may TEWL is found to be directly related to the clini- take place in the epidermis before secondary im- cal severity of the lesion, where TEWL is high in mune activation [47] . acute lesions and moderately increased in chronic lesions [1]. In a recent study, skin hydration, Psoriasis and Contact Allergy natural moisturizing factors and free fatty acids Clinical and epidemiological studies have clearly (FFAs) were all decreased in lesional psoriatic shown an inverse relationship between CA and skin compared to nonlesional and normal skin autoimmune diseases such diabetes type 1, rheu- [24] . A decrease in ceramide (CER) 1 in lesional matoid arthritis and inflammatory bowel diseases skin and severe structural alteration in intercel- [48–50] . lular lipid lamellae have been found in other It has been debated whether an inverse rela- studies [25, 28] . tionship also exists for psoriasis, as patch testing As filaggrin plays an important part in the final of patients with psoriasis has shown an overall differentiation of the epidermis and the forma- prevalence of CA to be around 20–25% [51–53] tion of the skin barrier, one could suspect an as- and even as high as 68% [54]. However, a large sociation between FLG mutations and psoriasis. epidemiologic study of psoriasis and concomitant However, several studies have rejected an associa- diseases found that CA was three times less fre- tion at least in Northern European descendants quent in patients with psoriasis than in a control [29–34] . Nonetheless, the expression of filaggrin group with nonpsoriatic skin diseases [55] . An is decreased or even absent in psoriatic skin le- inverse relationship between CA and psoriasis sions compared to uninvolved and normal skin was also found in a large patient- and population- [29, 35–37], likely due to down-regulation of fil- based study from Denmark. The odds ratio (OR) aggrin expression from blocking of the N-meth- for a person with psoriasis having a positive patch yl- D -aspartate receptor [38, 39] , overexpression test was 0.58 (95% confidence interval, CI, 0.49– of TNF-α [40, 41], lack of caspase 14 due to 0.68) in the patient-based study and 0.64 (95% CI IFN-γ [42] and increased levels of IL-17 [43] . 0.42–0.98) in the population-based study [56] . The potential role of a primary skin barrier ab- Supporting these observations, two experimental normality in the etiopathogenesis of psoriasis has studies found that patients with psoriasis were recently gained increased attention. There are less reactive after sensitization with the strong al- several indications supporting this assumption. lergen dinitrochlorobenzene (DNCB) when com- First of all, psoriasis was recently linked to the pared to healthy controls [57, 58] . The ability to deletion of LCE3B and LCE3C on the epidermal be sensitized was not investigated in these two differentiation complex on chromosome 1q21, studies, but a recent study showed that patients

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Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 90–102 ( DOI: 10.1159/000441548) with psoriasis and diabetes type 1 had a lower sen- sis typically displayed a maximum reaction after sitization ratio towards diphenylcyclopropenone 7 days, and the authors found that the clinically compared to healthy controls. Only 26% (3/23) of uninvolved skin of psoriatic patients had a differ- the patients with psoriasis and 36% (8/22) of the ent expression of numerous genes involved in patients with diabetes type 1 became sensitized metabolism and proliferation, possibly explain- compared to 65% (15/23) in the control group ing the delayed result. This study suggests that [59]. A reduced sensitization ratio to DNCB and false-negative patch test results due to early or nitrosodimethylamine has also been found in pa- premature reading might contribute to the low tients with rheumatoid arthritis, although the prevalence of CA in patients with psoriasis. How- difference was only significant for the latter [60] . ever, the inverse relationship between psoriasis These results suggest that an impaired reactivity and CA is not fully understood, and the exact towards allergens could be a common trait for mechanism remains to be elucidated. autoimmune diseases. It has been hypothesized that the inverse relationship between psoriasis and CA could relate Atopic Dermatitis to accelerated epidermal turnover [61] . However, other autoimmune diseases show a similar ten- AD is a chronic, relapsing inflammatory skin dency, and it seems more likely that the autoim- condition that is characterized by dry skin, pruri- mune diseases share an immunological milieu tus and dermatitis. Acute and chronic dermatitis which interferes with the mounting of a CA re- are located at distinct anatomical sites and typi- sponse [59]. The role of Th17 in autoimmune cally change with age. The prevalence has in- diseases has been well established [17] , and it has creased dramatically over the last three decades, been demonstrated that patients with autoim- and now affects between 15 and 30% of children mune diseases have higher systemic levels of and 2–10% of adults [64]. AD, as other atopic dis- Th17-associated cytokines (IL-17, IL-6, IL-21, IL- eases, is often associated with IgE-mediated sen- 22 and IL-23) than controls [19] . This might in- sitization to allergens, such as house dust mites, terfere with the regulation of antigen-presenting food (egg and milk protein), pollen and animal cells or with the maturation of naïve T cells, pre- dander, revealed by a positive skin prick test and/ venting the production of memory T cells needed or radioallergosorbent tests/IgE. for the allergic reaction [59] . Antigen-presenting The pathophysiology of AD is complex and in- cells play a crucial part in CS as they process, completely understood, but it is clear that there transport and present allergens to naïve T cells in exists both a strong genetic predisposition and skin draining lymph nodes. Cumberbatch et al. environmental triggers [65] . Immunologically, [62] found that the function of epidermal anti- AD is dominated by the Th2 phenotype in the gen-presenting cells (Langerhans cells) in unin- acute phase, with Th1, Th17 and Th22 cells con- volved skin of psoriasis patients was greatly im- tributing to the inflammatory response in chron- paired compared to those found in normal skin. ic lesions [66] . Traditionally, two different hy- Following patch testing, patients with psoria- potheses have been proposed. The first is an ‘in- sis sensitized to nickel developed a typical, but side-outside’ theory, where it is thought that the delayed reaction compared to nonpsoriatic pa- primary defect resides in the immune system, tients [63] . While nonpsoriatic patients usually de- causing excessive IgE sensitization, inflammation veloped positive patch test results after 48–72 h, and a secondary dysfunctional skin barrier. The many of the psoriatic patients still had a negative second is the ‘outside-inside’ theory, which pro- patch test at this time point. Patients with psoria- poses that the primary defect resides in the skin

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Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 90–102 ( DOI: 10.1159/000441548) barrier, causing increased allergen and pathogen tidine-rich protein, and a recent study showed that exposure, then leading to secondary excessive IgE nickel, electrophilic in nature, bound strongly to sensitization and inflammation [64, 67] . filaggrin [77] . It has also been proposed that cis - urocanic acid, a filaggrin metabolite, can be a nick- Skin Barrier Function in Atopic Dermatitis el-binding molecule in human skin [78] . In theory, In patients with AD, the skin barrier is deranged filaggrin deficiency might, therefore, facilitate in form of increased water loss (inside-outside) percutaneous penetration of metal allergens. In and enhanced percutaneous penetration of aller- German adults, a positive association between FLG gens and chemicals (outside-inside) in both le- mutations and subjectively reported intolerance sional and nonlesional skin. Compared to normal to nickel was found (OR 4.04; 95% CI 1.35–12.06) controls, TEWL has been found to be increased [79] . A Danish population study confirmed an as- twofold in nonlesional and fourfold in lesional sociation between nickel sensitization and FLG atopic skin [68] , albeit other studies have found mutations, but only in adults without ear-piercing less significant differences [69, 70] . Furthermore, [80] . Furthermore, ACD to nickel was reported at the increase in TEWL in nonlesional skin corre- a younger age for FLG mutation carriers and they lates with AD severity [71] . displayed stronger patch test reactivity than those Various causes of the impaired skin barrier without mutations [81] . In line with this, a signifi- function in AD have been explored, including cant association between CS to metals and AD has disturbed lipid composition, altered expression been found in both children and adults [82–85] . of proteins in the cornified envelope (involucrin However, remember that patch testing with metals and loricrin) and imbalance in structural proteins can sometimes be difficult and show various dif- in the epidermis [1, 72] . Sebaceous lipids on the ferent skin reactions that may mimic true allergic skin surface are significantly lower in patients ones. It has been investigated whether FLG muta- with AD than in healthy controls [73] . Further- tion carriers have an increased risk of CS to other more, atopic epidermis has shown a decrease in allergens than nickel [86] . A strong association was the total lipid amount, in particular CERs C1 and found, but only in FLG mutation carriers with self- C3, as well as an increase in FFAs and sterols [1, reported dermatitis. FLG mutations alone were 74]. A clinical study on the carbon length of FFAs not associated to an increased risk of sensitization. and CERs demonstrated a reduction in FFA and Experimental studies have shown that the percuta- CER chain length in lesional and nonlesional SC neous penetration of both lipophilic and hydro- of atopic patients compared to healthy controls philic chemicals was increased in clinically normal [75] . The changes correlated with a less dense lip- skin of AD patients compared with healthy sub- id organization and reduced skin barrier func- jects [87] . Moreover, the penetration rate of the tion, and were more prominent in lesional than hydrophilic chemicals tended to increase with in- nonlesional skin. Finally, loss-of-function muta- creasing AD severity and was significantly corre- tions in the FLG gene, which is present in approx- lated with total serum IgE. An increased diffusion imately 30% of AD patients, are known to cause has also been found for both polyethylene glycols reduced skin hydration and barrier function and [88] and sodium lauryl sulfate (SLS) [89] through strongly increase the risk of AD [64, 76] . the skin of AD patients compared to normal skin. The prevalence of CS in patients with AD could Atopic Dermatitis and Contact Allergy also be affected by the increased topical allergen Atopic skin is clinically characterized by xerosis exposure due to treatment. Moisturizers and topi- and intermittent or chronic dermatitis, and mo- cal agents such as corticosteroids or calcineurin lecularly by filaggrin deficiency. Filaggrin is a his- inhibitors are often prescribed for longer periods

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Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 90–102 ( DOI: 10.1159/000441548) to reduce xerosis and inflammation in atopic skin. skewing [102] . Acute AD is a Th2-polarized pro- Swedish children with moderate-to-severe AD cess, and, although speculative, there is the possi- used significantly more moisturizers and topical bility that positive patch results to fragrance and corticosteroids than children with mild AD in a rubber observed in AD patients in fact are acute recent survey [90] . Even though products are AD and not true allergic, but rather unspecific re- labeled as ‘hypoallergenic’, dermatologist recom- actions. Interestingly, several studies have found mended’ and ‘fragrance/paraben free’, a recent that patients with AD have a higher prevalence study proved that these products to a large extent of fragrance CS than controls [94, 96, 97] . None- [166/187 (88.8%) of tested products] contained theless, some studies have rejected an association one or more contact allergens and that many con- with fragrance CS [103, 104]. Furthermore, pen- tained potent allergens [91] . Taken together, atop- etration of metal ions via sweat ducts and hair fol- ic skin is heavily exposed to a variety of chemicals, licles, and changes in skin pH may lead to nonspe- often on a daily basis. This increased exposure cific inflammation [105] and reactions which are could, at least in theory, be expected to result in a not truly allergic in nature, but might be misread higher prevalence of CS towards chemicals found as positive if the patch testing is not performed in topical medicaments and personal care prod- correctly. Irritant reactions to chemicals and ucts due to the higher degree of exposure. When metals are common in patients with AD. In a the skin is inflamed, no danger signal is needed to study where 853 hard metalworkers were patch promote ACD when a weak hapten is introduced tested, pustular patch test reactions to nickel were into the skin, as it is already present due to the in- found in AD patients who were not sensitized to flammation. Several studies have found an asso- nickel when the patches were placed over dam- ciation between AD and an increased prevalence aged or inflamed skin [106] . German patients with of CS to topical chemicals such as corticosteroids, AD had more doubtful and irritant reactions on tixocortol pivalate, chlorhexidine and fragrances early readings when compared to controls, and [92–97] . A general population study found that the reactions on day 3 to fragrances and formalde- patients with AD had a higher prevalence of CS to hyde had a tendency to be stronger [107] . contact allergens found in topical products than Even though an association between AD and those without AD, and the association was stron- CS has been shown in epidemiological and clinical ger when FLG mutations were present [80]. In a studies, both experimental and clinical studies large US study, CS to lanolin, a well-known in- have also shown a significantly reduced risk of CS gredient in cosmetics, was higher among patients in patients with AD. One study investigated the with AD than in non-AD patients [98] , and an- percentage of AD patients who reacted to DNCB other study found that AD patients were more challenge and found that the reactivity depended likely to have positive patch test results to preser- on disease severity. Of those with mild disease, vatives than the control group [92] . AD has also 100% reacted, while 95% of those with moderate been associated with an increased risk of multiple and only 33% of those with severe disease reacted contact allergies (x > 3) in both Danish and Ger- [108] . AD patients with mild disease were also man patients [99–101] . found to be significantly less responsive to DNCB However, it is possible that the sometimes ob- than nonatopic controls [109]. Another experi- served increased prevalence of CS in AD patients mental study investigated the development of CS is at least in part due to false-positive patch test to Rhus in patients with AD and in healthy con- results. A recent study showed that the immune trols, and found that only 3% of 40 AD patients response in positive patch tests to fragrance and developed CS while 37% of 131 healthy controls to a lesser extent to rubber showed a strong Th2 were sensitized [110]. Results from a 15-year

Skin Barrier and Allergens 95

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 90–102 ( DOI: 10.1159/000441548) Hapten exposure

Th2 inflammation in AD decreases the Th1 Acute response in CS/ACD

CS/ACD (Th1/Tc1)

Topical therapy used to treat xerotic and Barrier abnormality in AD due to inherited inflamed skin in AD leads to elevated barrier deficiency, inflammation and exposure to ingredients in topical drugs and Chronic scratching increases the likelihood of emollients epidermal hapten penetration

AD (Th2)

Genetic predisposition such as Environmental exposures such as low mutations in genes encoding for filaggrin, humidity, alkaline soaps, house dust mites, tight junctions, serine protease inhibitors or urban residence, social class, maternal immunomodulatory factors alcohol intake and pollution

prospective study support the dose-dependent in- concentrations of allergens for sensitization [14] . verse relationship between AD and CS described This is supported by a recent study where de novo in the previously mentioned experimental studies. sensitization to DNCB in patients with AD was Patients with severe AD had a lower prevalence of compared to normal controls. Following sensitiza- CS than patients with moderate disease [95] . Sup- tion to DNCB, uninvolved skin of AD patients porting this, a recent register-based clinical study showed decreased hypersensitivity responses com- also found an inverse association between severe pared to normal controls when rechallenged after AD and CS [111] . The relationship between severe 1 month [113] . The degree of sensitization was as- AD and CS is, however, debated, and other stud- sociated with the type of the immune response, and ies have reported the opposite [101] . AD patients with a lower degree of sensitization One possible explanation for the observed had a skewing towards Th2 responses compared to inverse correlation between AD and CS in mainly the normal Th1 response in the nonatopic con- experimental studies is the opposing and mutually trols. However, the relationship between AD and antagonistic influences of Th1 and Th2 cells. The CS is clearly more complicated than a simple bal- dominant immune response in CS is a type 1 re- ance between Th1 and Th2 responses, and other sponse with the development of Th1 and Tc1 effec- subpopulations of T cells, including Th17, Th22 tor cells [112] . AD is a disease driven mainly by and regulatory T cells, may play an important role, Th2 inflammation, and the argument is, therefore, as well as barrier factors, but this is currently unre- that the Th1/Tc1 response will be repressed and the solved [114, 115]. The interplay between AD and sensitization less effective and/or require higher CS is summarized in figure 1.

96 Engebretsen Thyssen

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 90–102 ( DOI: 10.1159/000441548) Irritant Contact Dermatitis Skin Barrier Function in Irritant Contact Dermatitis Contact dermatitis (CD) can be divided into TEWL readings can be used to assess skin barrier ACD, ICD and protein CD, and is one of the disruption in ICD, and the recovery time after most frequent work-related diseases in indus- irritant insults can be more than 3 weeks [124] . trialized countries [116] . ICD is characterized Exposure to the known skin irritant SLS leads by local inflammation following single or re- to an alteration in skin lipid production and a peated exposure to an irritant [117] . This results disturbance in the extrusion of lamellar body lip- in inflammatory and cytotoxic effects that acti- ids [125]. In an experimental study in a mouse vate the innate but not the acquired immune model, disruption of the barrier with acetone or system. The irritant can be either a chemical tape stripping led to the release of cytokines such (e.g. detergents and cleansing agents, organic as IL-1α, IL-1β and TNF-α [126] . These findings solvents, cutting oil, disinfectants and water) or suggest a connection between the exposure to ir- a physical factor (mechanical friction, and cold ritants and barrier disruption, and the subsequent and dry environment) [118] . Acute ICD results activation of the innate immune system. from a relative major insult to the skin, often caused by an accidental exposure to a strong ir- Irritant Contact Dermatitis and Contact Allergy ritant such as an industrial or laboratory chem- The relationship between ICD and CA has been ical [119] . It is characterized by acute onset af- investigated in both animal and human studies. ter exposure with development of erythema, One of the first animal studies conducted on skin edema, vesicles and bullae. Chronic (cumula- irritation and skin sensitization showed that tive) ICD is usually due to repetitive exposure to guinea pigs could only be sensitized to nickel and weak irritants such as water and detergents, chromium salts in the presence of the known and/or various physical insults (e.g. friction, skin irritant SLS [127]. Since then, several animal microtrauma, low humidity and temperature) studies have confirmed that skin irritation en- [117]. Due to the repetitive nature of the expo- hances the response to skin allergens, and the sure, a complete restoration of the skin barrier most comprehensive work has been conducted function is prevented, and clinical dermatitis by Magnusson and Kligman [128] . When using arises even though the single exposure does not the contact allergen p-phenylene diamine, the exceed the individual elicitation threshold. presence of 5% SLS leads to an increase in skin Clinical signs include mild erythema, xerosis, sensitization from 38 to 78%; in another similar hyperkeratosis and fissuring, and develop slow- experiment, pretreatment with SLS increased the ly over weeks at the contact area. incidence of sensitization from 14 to 46%. They Subjects with an impaired skin barrier (e.g. concluded that a suitable level of skin irritation is sensitive skin and AD) have a decreased irritant necessary to obtain optimal development of skin threshold and/or require longer time to restore sensitization. Another clear example of the pro- barrier function, rendering them more suscep- found impact of skin irritation on the allergic re- tible to develop ICD. Experimental and epide- sponse was demonstrated in an experiment by miological data have shown that manifest AD or Basketter [129] where SLS was used as skin irri- even AD in childhood increases the risk of both tant and isoeugenol as contact allergen. Guinea acute and chronic ICD [120–122]. The risk of pigs already sensitized to isoeugenol were chal- chronic ICD appears to be increased in subjects lenged with the maximum nonirritant concen- with FLG mutations [122] , even in the absence tration of isoeugenol (5%), and 100% of the ani- of AD [123] . mals had a response. When lowering the concen-

Skin Barrier and Allergens 97

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 90–102 ( DOI: 10.1159/000441548) tration to 0.05%, only 15% of the animals re- which binds Langerhans cells to the epidermis, sponded. However, when the lowest concentra- thus promoting the migration of these cells to lotion of isoeugenol (0.05%) was tested in the pres- cal lymph nodes [136] . It has also been suggested ence of 0.05% SLS (which does not produce clini- that TNF-α increases the activity of a type-IV col- cal erythema), 90% of the animal responded. lagenase (MMP-9), making it easier for the Lan- McLelland et al. [130] conducted one of the gerhans cells to cross the basement membrane in most convincing studies on humans. They dem- the epidermis [137]. In draining lymph nodes, onstrated that doses of allergen that were not TNF-α is also believed to promote antigen pre- strong enough to elicit an allergic response in sen- sentation to naïve T cells [138] . sitized patients were capable to do so if SLS was added. In other studies the effect of irritant exposure on the threshold of reaction was investigated Conclusion in already sensitized subjects to metal allergens such as nickel, cobalt and chromium. Pretreat- In this chapter, we have explored common der- ment with 0.2% SLS for 24 h on the patch test matoses, namely psoriasis, AD and ICD, and dis- site had a substantial effect on the threshold elici- cussed their relationship towards skin barrier tation concentration in patients allergic to cobalt function and the development of CA. In all three [131] . On the SLS pretreated site, reaction was ob- conditions, the skin barrier function is compro- tained at approximately one order of magnitude mised, but the relationship between the dermato- lower than at the nontreated sites. For nickel, the ses and the development of CA is complex and threshold for reaction could be reduced by one to depends on immunologic responses and skin two orders of magnitude in a similar study setup barrier status. In general, research suggests an in- [132] . An experimental study by Agner et al. [133] verse relationship between psoriasis and CA, pos- supports these findings. They demonstrated that sibly due to increased levels of Th17 cells and its simultaneous exposure to SLS and nickel chloride associated cytokines. As for AD, epidemiological (NiCl 2) in patients with nickel allergy caused not studies suggest a positive association to CS, while only an additive, but also a synergistic effect on experimental studies show an inverse relation- the response, evaluated by clinical reading and ship. The opposing and antagonistic influences of colorimetry. Th1 (CS) and Th2 (AD) have been proposed as an The mechanistic explanation for these find- explanation for the results of experimental stud- ings is unresolved. Research has suggested that ies. Finally, there is convincing evidence that ex- the allergens (haptens) deliver both an irritant posure to irritants increases the risk of CS, and and an antigenic signal, and that the irritant sig- patients with ICD are, therefore, at great risk of nal is capable of stimulating cytokine release developing CA. from nonimmune skin cells such as keratinocytes [134] . Furthermore, Langerhans cells play an important part in the sensitization process, and their Acknowledgment ability to migrate to local lymph nodes appears to Jacob Pontoppidan Thyssen and Kristiane Aasen En- be dependent on the cytokines IL-1 and TNF-α gebretsen are financially supported by an unrestricted [135] . IL-1 is released by Langerhans cells and grant from the Lundbeck Foundation. In addition, this TNF-α mainly by keratinocytes. Skin irritation work was supported by H2020 COST Action TD1206 leads to the release of TNF-α, which has a range ‘StanDerm’. The funding sources did not play any role of effects promoting allergen sensitization. First in the writing of the paper. of all, there is down-regulation of E-cadherin,

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82 Hegewald J, Uter W, Pfahlberg A, et al: Contact Dermatitis 2011; 64: 325–329. Cutis 2000; 65: 49–53. A multifactorial analysis of concurrent 94 Thyssen JP, Linneberg A, Engkilde K, et 106 Uehara M, Takahashi C, Ofuji S: Pus- patch-test reactions to nickel, cobalt, al: Contact sensitization to common tular patch test reactions in atopic der-

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grams Th2 responses in individuals J Dermatol 2013; 168: 326–332. mation. Lancet 1991; 337: 211–214. with atopic dermatitis. J Invest Derma- 124 Lee JY, Effendy I, Maibach HI: Acute 135 Cumberbatch M, Dearman RJ, Grif-

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nol Allergy 2012; 96: 39–44. 125 Fartasch M, Schnetz E, Diepgen TL: 136 Schwarzenberger K, Udey MC: Contact 115 Agrawal R, Wisniewski JA, Woodfolk Characterization of detergent-induced allergens and epidermal proinflamma- JA: The role of regulatory T cells in barrier alterations – effect of barrier tory cytokines modulate Langerhans atopic dermatitis. Curr Probl Dermatol cream on irritation. J Investig Derma- cell E-cadherin expression in situ.

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Exp Dermatol 2002; 27: 138–146. test for contact hypersensitivity; in Dean JH, Luster MI, Munson AE, Kim- ber I (eds): Immunotoxicology and Immunopharmacology. London, Ra- ven, 1994, pp 693–702.

Kristiane Aasen Engebretsen National Allergy Research Centre, Department of Dermato-Allergology Herlev and Gentofte University Hospital Kildegårdsvej 28 DK–2900 Hellerup (Denmark)

E-Mail kristiane.aasen.engebretsen.02 @ regionh.dk

102 Engebretsen Thyssen

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 90–102 ( DOI: 10.1159/000441548) Section II: External Factors Influencing Skin Barrier

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 103–111 (DOI: 10.1159/000441549)

Penetration through the Skin Barrier

a b c Jesper Bo Nielsen Eva Benfeldt Rikke Holmgaard a b Research Unit for General Practice, Institute of Public Health, University of Southern Denmark, Odense , Regional Research c Unit, Zealand Region, Roskilde , and Department of Plastic Surgery, Breast Surgery and Burns Treatment, Rigshospitalet, Copenhagen , Denmark

Abstract decision making and treatment planning, as well as the The skin is a strong and flexible organ with barrier prop- evaluation of experimental studies of percutaneous pen- erties essential for maintaining homeostasis and thereby etration of chemicals. © 2016 S. Karger AG, Basel human life. Characterizing this barrier is the ability to prevent some chemicals from crossing the barrier while allowing others, including medicinal products, to pass at varying rates. During recent decades, the latter has re- Most chemicals penetrate the skin by passive dif- ceived increased attention as a route for intentionally de- fusion, whereas active transport plays a much livering drugs to patients. This has stimulated research in more limited role. Following skin exposure, methods for sampling, measuring and predicting percu- chemicals pass the upper skin structures, includ- taneous penetration. Previous chapters have described ing the stratum corneum (SC), enter the viable how different endogenous, genetic and exogenous fac- epidermis, continue passively through the basal tors may affect barrier characteristics. The present chap- membrane composed of glycoproteins and pro- ter introduces the theory for barrier penetration (Fick’s teoglycans and separating epidermis from der- law), and describes and discusses different methods for mis, to reach the vascularized dermis, where the measuring the kinetics of percutaneous penetration of blood vessels will enable systemic absorption. The chemicals, including in vitro methods (static and flow- skin is not a homogeneous layer, but includes a through diffusion cells) as well as in vivo methods (micro- number of appendages, including sweat glands dialysis and microperfusion). Then follows a discussion and hair follicles. In quantitative terms, these ap- with examples of how different characteristics of the skin pendages make up a very limited proportion of (age, site and integrity) and of the penetrants (size, solu- the total skin surface (<1%) and are of limited bility, ionization, logPow and vehicles) affect the kinetics quantitative importance for the percutaneous of percutaneous penetration. Finally, a short discussion penetration of most chemicals. They do, however, of the advantages and challenges of each method is pro- appear to be relevant pathways for some proteins vided, which will hopefully allow the reader to improve and larger particles [1] . SC Viable Skin 123epidermis Dermis Vascular surface circulation (4)

Fig. 1. Model of the absorption across the skin barrier. 1 = Penetration phase: passive diffusion into the lipophilic SC; 2 = permeation phase: the transport through the more aqueous, avascular, viable epidermis to the highly perfused dermis; 3 = resorption phase into the microcirculation and further to the systemic circulation or deeper into the tissue (regional penetration); (4) = affinity of the penetrant for the SC or the dermis (reservoir formation).

A kinetic model may be used to describe the layer and therefore the influence of chemical or pathway from absorption from the skin surface disease-related damage to this skin layer [3, 4] . It into the lipophilic SC and the subsequent perme- also gives support to the use of experimental ation through the more aqueous, avascular and models based on excised skin to study penetra- viable epidermis to the highly perfused dermis tion characteristics. ( fig. 1 ). Given that the diffusion is passive, the driving force is the concentration gradient combined Percutaneous Penetration with the chemical affinity for a chemical to ‘pre- fer’ a lipophilic or a hydrophilic environment. Mathematically, the passive diffusion through the Thus, a lipophilic compound will easily cross the skin has been described using Fick’s law of diffu- SC, but the penetration rate will slow down when sion from 1855 [5] . It is based on the fact that unit reaches the more hydrophilic epidermis. In bound molecules will move by passive diffusion these situations, we may observe a temporary res- towards equilibrium in response to a concentra- ervoir formation in skin compartments. Based tion gradient, and it proposes that the rate of this upon these observations, substances soluble in diffusion (the flux) going from one area with the lipophilic layer as well as in the more aqueous higher concentration to another area of lower structures will be expected to have the highest rate concentration is proportional to the concentra- of permeability through the skin barrier [2] . The tion gradient. This law serves only under very SC is generally recognized as the rate-determin- specific conditions. It does, however, give good ing barrier for percutaneous penetration. It con- approximations of flux rates related to dermal sists of a number of layers of corneocytes packed penetration [6] . in a lipid matrix. Thus, the main barrier is the out- Fick’s first law is described as: Jss = –D × ΔC/Δh, most 10–50 μm of ‘dead cells’, which emphasizes where Jss = flux of the penetrant under steady- the importance of the integrity of this very thin state conditions (penetration rate); D = diffusion

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Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 103–111 ( DOI: 10.1159/000441549) 120

100 Fig. 2. Determination of permeability coefficients. Cumulative amount 80 of the penetrating substance as a function of time illustrated graphi- 60 (mg) cally. The steady-state flux (Jss ) is the Steady-state slope slope of the linear part of the graph. 40 The lag time is the time intercept of 20 Lag time the linear part of the graph. Deter- mining of the steady-state flux from penetrated amount Cumulative 0 the slope of the graph will when di- 08020 40 60 vided by the concentration of the Time (h) applied penetrant give the permeability coefficient (kp ).

coefficient (the minus indicates that the flow is Methodologies for Studying Percutaneous from a higher to a lower concentration); ΔC = the Penetration concentration gradient across the membrane, and Δh = diffusion path length. Experimental studies of percutaneous penetra- The law may also be written as J ss = k p × ΔC, tion can be undertaken in vitro, ex vivo or in vivo. with kp being defined as the permeability coeffi- In 2000, different in vitro methods for studying cient of the penetrant through the membrane. percutaneous penetration were evaluated by the Thus kp is a penetrant-specific constant that may Percutaneous Penetration Subgroup of the EC be used to calculate the expected flux of a pene- Dermal Exposure Network as they studied stan- trant, given the knowledge of the size of the con- dardization and validation of different experi- centration gradient and path length, or to com- mental techniques. A few years later, an inter- pare the expected flux of different penetrants. For laboratory comparison of different penetration most exogenous penetrants presented to the skin techniques was made with great success and dem- surface, the concentration below the skin mem- onstrated comparability of the methods in nine brane will be insignificant during the initial phas- laboratories across Europe [7]. The same year, es of penetration, and ΔC can be approximated OECD issued a guideline for the use of in vitro to the concentration of the penetrant in the ap- techniques when testing percutaneous penetra- plied formulation/solution/vehicle. The perme- tion of chemicals. This guideline describes the use ability coefficient may, therefore, be calculated of the static diffusion and flow-through cells [8] , from experimental data presented as a graph of which both resemble the first in vitro technique the cumulative amount penetrated as a function developed back in 1940 during World War II for of time (fig. 2 ). The same graph may also be used evaluation of percutaneous penetration of chemi- to estimate the lag time by extrapolating back cal warfare agents [9] . from the steady-state slope to the time axis. Sev- The principle of the two diffusion cell types is eral experimental models allow for determina- that the substance of interest is applied to the tion of the cumulative amount penetrated as a surface of a piece of skin, which has been mount- function of time. ed so that it separates two chambers – the donor

Percutaneous Penetration: Methods and Variability 105

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 103–111 ( DOI: 10.1159/000441549) and the receptor chamber. Formulations of dif- Flow-Through Diffusion Cells ferent kinds (solutions, creams and patches) In the 1980s, the flow-through system was devel- may be applied to the skin surface as long as they oped, and, just as the static diffusion cells, the fit into the donor chamber. The skin is placed method is internationally validated and widely with the SC facing the donor chamber (up- used in percutaneous penetration research. The wards). The dermis in the receptor chamber is in flow-through system consists of multiple cells. In contact with the receptor fluid. The temperature this system, the receptor fluid is continuously re- around the cells is kept constant at 32 ° C in order placed and automatically collected at certain time to imitate the skin surface temperature, and a intervals in order to imitate the in vivo circulation magnetic stirrer in the bottom of the chamber of blood removing the penetrated substance from keeps the receptor fluid homogeneous in con- the dermal plexus into the systemic circulation. centration. The amount of substance penetrat- This constant replacement of receptor fluid opti- ing the skin is collected in the receptor fluid by mizes sink conditions and is an advantage when repeated sampling from the receptor fluid over a dealing with test substances of low solubility. prolonged period of time. The concentration of Compared to the static diffusion cells, the flow- the penetrant on the samples is determined by through system has a very small area for test sub- different analytical methods and subsequently stance application, and the design is a bit more plotted as a function of time. complicated due to the many tubes through which the samples flow into the sampling vials. The tube Static Diffusion Cell length has a tendency to influence the measured The static diffusion cell, also known as the Franz lag time especially when choosing low flow rates. diffusion cell, has been one of the most used in In vitro methods are known to almost com- vitro methods in percutaneous penetration re- pletely avoid all ethical questions and consider- search since 1975 [10] . The method is inexpensive ations as no lives are wasted or harmed during in use and has a simple design. The skin used may this kind of research and ‘left-over’ skin destined be either split skin or full-thickness skin, and may for destruction after plastic surgery, for example, be human or animal in origin. In the static diffu- can be used. The methods mentioned above are sion cell, the receptor fluid is manually sampled both inexpensive in use as soon as the system has at specific time intervals, dissimilar to the flow- been acquired. Both systems are easy to operate through system where the samples are collected and the design is simple in both cases, although automatically and where the receptor fluid is con- the static diffusion cells have a lower risk of mal- tinuously replaced. Therefore, the static cells need functioning due to fewer mechanical parts. The a bigger receptor chamber volume than the flow- larger application area in the donor chamber of through cells to avoid that the concentration of the static diffusion cell makes the absorption in- the test substance in the receptor chamber over dicator better than in the flow-through cells, but time increases and thereby significantly reduces the flow-through cells imitate real physiological ΔC. A reduced ΔC will reduce the rate of diffu- conditions better due to the continuous replace- sion across the skin barrier and hamper the as- ment of the receptor medium. sumption of sink conditions (i.e. the concentra- Exact guidelines have been developed for both tion of test substance in the receptor chamber is methods [8] , and for regulatory purposes both insignificant compared to the concentration in methods are acceptable, though each of them has the donor chamber, whereby ΔC equals the con- its advantages and limitations [11] . centration in the donor chamber) required when In vitro techniques have several advantages, estimating maximal flux and kp values. but in some situations in vivo experimentation

106 Nielsen Benfeldt Holmgaard

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 103–111 ( DOI: 10.1159/000441549) Fig. 3. Illustration of the microdialysis probe placed in the dermis, sampling increasing dermal drug concentrations following topical drug penetration. ⚫ = The penetrating molecule.

may be needed, e.g. the development of drugs for Similar for both methods is that a permeable topical application in humans or risk assessment probe is inserted into the dermis, where it imitates in occupational settings. In these instances, the the function of a small blood vessel. This will allow most accurately transferrable research is con- sampling of those chemicals that penetrate the ducted by in vivo human studies. There will al- epidermis and upper dermis, and reach the tissue ways be ethical considerations when choosing an around the probe. Passive diffusion, again accord- in vivo experimental method. Prolonged sam- ing to Fick’s law, will determine the transport of pling periods will be uncomfortable for the hu- the drug or chemical into the lumen of the probe, man volunteers, and a wide range of chemicals/ which is perfused with a tissue-compatible fluid. toxicants will for ethical reasons not be eligible for The probe is connected to pumps that assure a human in vivo studies. Whenever the chemicals steady flow of this isotonic fluid through the studied are metabolized in the skin, in vivo ex- probe, and samples can continuously be collected perimentation has an obvious advantage, since from the resulting dialysate (at the outlet end of skin penetration and sampling takes place in liv- the probe). Besides different technical require- ing tissue with full metabolic capacity. ments, the two experimental setups primarily dif- Two well-established in vivo methods for the fer in the characteristics of the probe types. study of percutaneous penetration exist, each with its specific advantages and challenges. Microdial- Microdialysis ysis ( fig. 3 ) was introduced in human studies in In microdialysis, the probes consist of semiper- the early 1990s [12] and institutionalized through meable structures. The molecules will enter the an FDA white paper in 2007 [13] , whereas micro- lumen by passive diffusion and thus be present perfusion was introduced in the late 1990s [14] . in the perfusate, which is now a dialysate, in the

Percutaneous Penetration: Methods and Variability 107

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 103–111 ( DOI: 10.1159/000441549) probe. The microdialysis probes have specific ed in the sample fluid collected from the probe, pore sizes, which set upper limits (cutoff value) which may jeopardize analytical procedures un- for the molecular size that can be sampled, but less relevant steps are taken. Thus, the resulting also exclude larger molecules and proteins from sample requires technically more demanding entering the sampling fluid. Unless protein has preanalytical steps before analysis of the sample been added to the perfusate, which is only done if fluid [16] . it enhances recovery of the substance of interest, dermal microdialysis sampling delivers protein- and enzyme-free samples, which make the pre- Biological Factors Affecting Percutaneous analytical steps relatively uncomplicated. The Penetration method does, however, require thorough prior considerations of the suitability of the substance The percutaneous penetration of exogenous com- of interest for microdialysis sampling, as the typi- pounds varies with the anatomical site. A more cal combination for many topical medical treat- than 40-fold difference in the skin penetration ments, i.e. a high or very high lipophilicity of the rate for hydrocortisone through plantar and scro- drug and a low drug concentration in the topical tum skin has been demonstrated [17] . Apparent- product, both render dermal microdialysis sam- ly, the variation in the penetration rate between pling challenging as the resulting samples may be skin areas is not directly related to the thickness of very low concentration [15] . of the skin at the particular site [18] , rather factors such as the number of follicles, thickness of the Microperfusion SC, the sebum composition as well as the distance Microperfusion – or open flow microperfusion – between capillaries and the surface of the skin all is also designed as a continuous tissue-specific appear to be of influence [19] . sampling method. Where the microdialysis probe Skin-related factors change as a function of has a semipermeable membrane, the microperfu- increasing age. These factors include blood flow, sion sampling probe has a membrane-free mac- pH, skin thickness, hair and pore density, and the roscopically perforated area with unrestricted ac- content and structure of proteins, glycosamino- cess to and exchange of solutes with the periprobe glycans, water and lipids. A recent review con- tissue. Therefore, the microperfusion technique cludes that these age-related changes may directly does not have the same limitations regarding or indirectly affect the percutaneous penetration sampling efficacy towards large and/or protein- rate of drugs in both directions [20]. Though re- bound penetrants as the microdialysis technique. pair mechanisms may become less effective with The method is, due to the open exchange area, re- age, causing barrier damage to remain for longer levant for sampling of large and/or lipophilic pen- with the potential for increased penetration, anal- etrants, which is where the microdialysis sam- yses of the influence of several other biological pling methodology is often challenged. However, factors on the penetration rate indicate that per- microperfusion is a more demanding method, cutaneous penetration may be slower in older in- both technically and labor-wise, since the method dividuals [20] . This is supported by observations needs to have a push as well as a pull pump func- of decreased transepidermal water loss among tion connected to the probe in order to counteract people aged 65 years or more [21, 22] as well as the tendency to induce edema in the tissue sur- the observation that fentanyl permeates the skin rounding the probe due to the open exchange of young individuals in greater amounts and at a area. As a consequence of the open exchange area, higher absorption rate than in middle-aged and proteins, enzymes and some cells may be includ- old individuals in vitro [23] .

108 Nielsen Benfeldt Holmgaard

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 103–111 ( DOI: 10.1159/000441549) Skin integrity also significantly affects the rate cals (e.g. ethanol, DMSO, sodium lauryl sulfate, of percutaneous penetration. The barrier func- glycols and glycol ethers) that may act as penetration depends mainly on the integrity of the SC. tion enhancers. Therefore, exposure conditions Changing or damaging the skin structure increas- need to be considered when planning experimen- es the permeability of chemicals through reduced tal studies or when evaluating experimental data lag time and increased permeation rates, and has on percutaneous penetration of neat chemicals or been observed following chemical (detergents/ chemicals in solutions with different vehicles. solvents), physical (weather, occlusion, sunburn and mechanical damage through abrasions) or pathological (skin disease as described in previ- The Best Experimental Model? ous chapters) effects on the barrier integrity [24–26] . The best experimental approach depends on the Thus, substantial interindividual variability research question asked. No single method will can be expected in vivo as well as in experimental fit all questions. Each model has advantages and studies, and for this reason we recommend broad challenges, as also discussed in a recent paper consideration of the above when choosing and on methodological considerations [16] . All four evaluating skin for experimental studies and models presented will allow for multiple applica- when evaluating results from experimental stud- tion sites, good reproducibility and continuous ies. sampling, the latter enabling characterization of the kinetics of the percutaneous penetration process. The in vitro models are generally seen as Physicochemical Characteristics Affecting having a simpler design and lower costs, and re- Percutaneous Penetration quiring less time and ethical considerations than do the in vivo methods. These features make the The passive diffusion that characterizes the per- in vitro models suitable for screening/comparing cutaneous penetration of most chemicals follows series of compounds. However, the in vivo mod- Fick’s law, and the chemical-specific permeability els are required when proceeding to a clinical sit- coefficient, kp , integrates a number of physico- uation, as this requires a physiological and meta- chemical properties affecting permeation. Thus, bolically active system to reach the most reliable kp depends mainly on the molecular weight, the results in drug development or skin absorption/ solubility characteristics expressed by the octanol- skin toxicity issues, for example. water partition coefficient (logPow) and the mo- When transferring experimental observations lecular size (stereochemistry). Generally (recog- into the real world, it is essential to consider the nizing that exemptions exist), optimal permeation experimental setup. Often chemicals are tested rates are obtained with smaller molecular weight under conditions prescribed by regulatory au- and logPow values between –2 and +2 [27] . Be- thorities or guidelines that are good for consis- sides these determinants, the vapor pressure, ion- tency and variability, but are not comparable with ization (which is pH dependent) and affinity for in-use conditions. This may be sufficient for protein binding will affect the concentration of making relative comparisons, but may not reflect unbound chemicals available for percutaneous real-life conditions where chemicals are often penetration at the surface of the skin barrier. mixed with vehicles/solubilizers or other chemi- Furthermore, outside the laboratory setting, cals (mixed exposure), and skin characteristics skin exposure is seldom to neat chemicals but to may be far from the attempted ideal situation in mixtures including solubilizers and other chemi- the laboratory.

Percutaneous Penetration: Methods and Variability 109

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 103–111 ( DOI: 10.1159/000441549) Based on the available published experimental Thus, experimental models for studying pene- data on percutaneous penetration, a number of tration through the skin barrier are generally a mathematical models have been developed for good and validated approach, enabling compari- the prediction of skin penetration (in silico mod- sons of the percutaneous penetration between dif- els). Their validity has primarily been demon- ferent chemicals and formulations, but quantitative strated for comparisons of specific groups of transfer of experimental results to real-life expo- chemicals (i.e. aliphatic alcohols), but the out- sure situations requires more delicate evaluations. come cannot be generalized for in vivo risk as- Experimental data are, therefore, better suited as an sessment, as they do not identify outliers and, just initial screening tool for hazard estimations than like the experimental models from which their for risk assessment following human topical expo- data originate, do not reflect the heterogeneity sure to chemicals, and as a tool to compare penetra- observed in real-life scenarios. tion characteristics between chemicals.

References

1 Jacobi U, Engel K, Patzelt A, Worm M, 10 Franz TJ: Percutaneous absorption on 16 Holmgaard R, Benfeldt E, Nielsen JB: Sterry W, Lademann J: Penetration of the relevance of in vitro data. J Invest Percutaneous penetration – method-

pollen proteins into the skin. Skin Phar- Dermatol 1975; 64: 190–195. ological considerations. Basic Clin Phar-

macol Physiol 2007; 20: 297–304. 11 Holmgaard R, Nielsen JB: Dermal Pen- macol Toxicol 2014; 115: 101–109. 2 Guy RH, Hadgraft J, Bucks DA: Trans- etration of Pesticides – Evaluation of 17 Feldmann RJ, Maibach HI: Regional dermal drug delivery and cutaneous me- Variability and Prevention. Danish Min- variation in percutaneous penetration of 14 tabolism. Xenobiotica 1987; 17: 325–343. istry of the Environment. Pesticides Re- C cortisol in man. J Invest Dermatol

3 Nielsen JB, Nielsen F, Sorensen JA: De- search No. 124 2009. Copenhagen, Dan- 1967; 48: 181–186. fense against dermal exposures is only ish Environmental Protection Agency, 18 Elias PM, Cooper ER, Korc A, Brown skin deep: significantly increased pen- 2009. BE: Percutaneous transport in relation etration through slightly damaged skin. 12 Anderson C, Andersson T, Molander M: to stratum corneum structure and lipid

Arch Dermatol Res 2007; 299: 423–431. Ethanol absorption across human skin composition. J Invest Dermatol 1981; 76: 4 Kezic S, Nielsen JB: Absorption of chem- measured by in vivo microdialysis tech- 297–301.

icals through compromised skin. Int nique. Acta Derm Venereol 1991; 71: 19 Rougier A, Dupuis D, Lotte C, Roguet R,

Arch Occup Environ Health 2009; 82: 389–393. Wester RC, Maibach HI: Regional varia- 677–688. 13 Chaurasia CS, Muller M, Bashaw ED, tion in percutaneous absorption in man:

5 Fick A: Über Diffusion. Ann Phys 1855; Benfeldt E, Bolinder J, Bullock R, et al: measurement by the stripping method.

170: 59–86. AAPS-FDA workshop white paper: mi- Arch Dermatol Res 1986; 278: 465–469. 6 Grandjean P: Skin Penetration – Haz- crodialysis principles, application and 20 Konda S, Meier-Davis SR, Cayme B, ardous Chemicals at Work. London, regulatory perspectives. Pharm Res Shudo J, Maibach HI: Age-related percu-

Taylor & Francis, 1990. 2007; 24: 1014–1025. taneous penetration. Part 1. Skin factors.

7 van de Sandt JJ, van Burgsteden JA, 14 Trajanoski Z, Brunner GA, Schaupp L, Skin Therapy Lett 2012; 17: 1–5. Cage S, Carmichael PL, Dick I, Kenyon Ellmerer M, Wach P, Pieber TR, et al: 21 Kottner J, Lichterfeld A, Blume-Peytavi S, et al: In vitro predictions of skin ab- Open-flow microperfusion of subcuta- U: Transepidermal water loss in young sorption of caffeine, testosterone, and neous adipose tissue for on-line contin- and aged healthy humans: a systematic benzoic acid: a multi-centre comparison uous ex vivo measurement of glucose review and meta-analysis. Arch Derma-

study. Regul Toxicol Pharmacol 2004; concentration. Diabetes Care 1997; 20: tol Res 2013; 305: 315–323.

39: 271–281. 1114–1121. 22 Seyfarth F, Schliemann S, Antonov D, 8 OECD: Test 428: Skin Absorption: In 15 Holmgaard R, Nielsen JB, Benfeldt E: Elsner P: Dry skin, barrier function, and vitro Method, OECD Guidelines for the Microdialysis sampling for investiga- irritant contact dermatitis in the elderly.

Testing of Chemicals. Paris, OECD, tions of bioavailability and bioequiva- Clin Dermatol 2011; 29: 31–36. 2004. lence of topically administered drugs: 23 Holmgaard R, Benfeldt E, Sorensen JA, 9 Pendlington R: In vitro percutaneous current state and future perspectives. Nielsen J: Chronological age affects the

absorption measurements; in Chilcott Skin Pharmacol Physiol 2010; 23: 225– permeation of fentanyl in human skin in

RP, Price S (eds): Principles and Practice 243. vitro. Skin Pharmacol Physiol 2013; 26: of Skin Toxicology. Chichester, Wiley, 155–159. 2008, pp 129–148.

110 Nielsen Benfeldt Holmgaard

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 103–111 ( DOI: 10.1159/000441549) 24 Kezic S, Nielsen JB: Absorption of chem- 26 Bodenlenz M, Höfferer C, Magnes C, 27 Nielsen JB, Sorensen JA, Nielsen F: The icals through compromised skin. Int Schaller-Ammann R, Schaupp L, usual suspects – influence of physico-

Arch Occup Environ Health 2009; 82: Feichtner F, Ratzer M, Pickl K, Sinner chemical properties on lag time, skin 677–688. F, Wutte A, Korsatko S, Köhler G, deposition, and percutaneous penetra- 25 Benfeldt E, Serup J, Menne T: Effect of Legat FJ, Benfeldt E, Wright AM, tion of nine model compounds. J Toxi-

barrier perturbation on cutaneous sali- Nedermann D, Jung T, Pieber TR: col Environ Health A 2009; 72: 315–323. cylic acid penetration in human skin: in Dermal PK/PD of a lipophilic topical vivo pharmacokinetics using microdi- drug in psoriatic patients by continu- alysis and non-invasive quantification of ous intradermal membrane-free sam-

barrier function. Br J Dermatol 1999; pling. Eur J Pharm Biopharm 2012; 81:

140: 739–748. 635–641.

Jesper Bo Nielsen Research Unit for General Practice, Institute of Public Health University of Southern Denmark J.B. Winsløwsvej 9a DK–5000 Odense (Denmark)

E-Mail jbnielsen @ health.sdu.dk

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Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 103–111 ( DOI: 10.1159/000441549) Section III: Optimizing Skin Barrier Function

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 112–122 (DOI: 10.1159/000441586)

Treatments Improving Skin Barrier Function

Marie Lodén Eviderm Institute AB, Solna , Sweden

Abstract dence from randomized studies also showed that Moisturizers affect the stratum corneum architecture and a moisturizer with barrier-improving properties; barrier homeostasis, i.e. topically applied ingredients are i.e. a moisturizer lowering transepidermal water not as inert to the skin as one might expect. A number of loss (TEWL) and reducing the susceptibility to ir- different mechanisms behind the barrier-influencing ef- ritation, also delays relapse of eczema in patients fects of moisturizers have been suggested, such as simple with AD and hand eczema. In a worst-case sce- deposition of lipid material outside the skin. Ingredients nario, treatment with moisturizing creams could in the moisturizers may also change the lamellar organi- increase the risks for eczema and asthma. Mois- zation and the packing of the lipid matrix and thereby turizing creams suitable to atopic skin are expect- skin permeability. Topically applied substances may also ed to demonstrate absence of barrier-deteriorat- penetrate deeper into the skin and interfere with the pro- ing properties. duction of barrier lipids and the maturation of corneocytes. Furthermore, moisturizing creams may influence the desquamatory proteases and alter the thickness of Skin Barrier Function the stratum corneum. © 2016 S. Karger AG, Basel In summary, the increased understanding of the interactions between topically applied substances and the epidermal biochemistry will enhance the Clinical consequences of potential differences in possibilities to tailor proper skin care. the efficacy of moisturizers include differences in The skin has several barrier functions, for ex- hydrating properties, effects on visible dryness ample against ultraviolet exposure, microbes and symptoms and, even more importantly, the likeli- diffusion of chemicals. The term ‘improvement in hood of reduced risks of eczema outbreak in pa- skin barrier function’ has grown in importance tients with atopic dermatitis (AD). Restoring skin during the last decades among consumers, pa- barrier function, e.g. in people with defects in the tients, dermatologists and those involved in the filaggrin gene, may therefore help to prevent the development of topical formulations. The im- development of atopic eczema, and halt the devel- provement in skin barrier function is recognized opment and progression of allergic disease. Evi- as a more healthy-looking and less sensitive skin. chronic anxiety, rheumatoid arthritis or multiple sclerosis [3] . Emollients and moisturizing creams belong to the most widely used preparations to relieve symptoms of dryness and improve skin barrier function. The term ‘emollient’ implies (from the Latin derivation) a material designed to soften the skin, i.e. a material that ‘smoothens’ the surface to the touch and makes it look smoother to the eye. The term ‘moisturizer’ is often used synony- mously with emollient, but moisturizers usually contain water and humectants, aimed at facilitating the treatment and to increase the hydration of the stratum corneum (SC; fig. 1 ). For example, low-molecular-weight natural moisturizing factors (e.g. urea, lactic acid, pyrrolidone carboxylic acid and amino acids) and lipids (e.g. fatty acids Fig. 1. Simple schematic representation of a moisturizer as a typical oil-in-water emulsion, where the big circles and ceramides) are components of the SC which (yellow; see online version for colors) denote fats/oils can also be found in moisturizers. Their role in (10–30%) whose surfaces are covered with emulsifiers moisturizers can be to replenish substances iden- (2–10%). In the water phase (50–80%; blue), the dots tified as low in xerotic skin. For example, the con- (red) represent the preservatives (0.3–2%), the long black tent of urea [4] and ceramides [5, 6] are reduced threads represent polymers used as thickeners (0.2–2%) and the drops (blue) represent the humectants (0.5– in dry SC of patients with AD. Furthermore, dry 10%). Other typical additives are stabilizers (antioxi- SC samples from old people and patients with dants/chelators), fragrances and botanical ingredients ichthyosis vulgaris have an altered amino acid (usually <1% each). composition [7, 8] . The type of emulsion and the selection of humectants, as well as other excipients, such as The improvement may be observed at different emulsifiers, lipids, chelators and preservatives, sites of the skin with different sustainability. For influence the skin [9, 10]. Like the permeability example, covering of the surface with emollients barrier, the antimicrobial barrier is compromised will temporarily reduce signs of dryness and im- in AD, where colonization by Staphylococcus au- prove the appearance, whereas deeper effects on reus is a common feature of AD. Not surprisingly, the intercellular penetration pathways may have differences in the effect of moisturizers on the a more long-standing effect on the risks for ecze- skin permeability barrier have been identified. ma. The findings that permeability barrier abnor- Formulations may fail to improve skin barrier malities drive disease activity in inflammatory function [11–14] and, even worse, sustain or ag- dermatoses have also grown the interest for treat- gravate an existing barrier disease [15]. In addi- ments which improve skin barrier function [1] . In tion, normal skin may react differently to envi- patients with AD, where the barrier is significant- ronmental stimuli depending on previous treat- ly impaired, the Dermatology Life Quality Index ment (fig. 2 ) [16–19] . is low [2] and the willingness to pay for complete Finding the most suitable moisturizer for the healing is comparable to that for relief of other individual patient is currently a matter of trial and serious medical conditions, e.g. angina pectoris, error. The majority of moisturizing creams on the

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140

120

100

40 Skin susceptibility (%) SLS to

0 Membrane 10% 5% 20% Lipid rich 40% 40% veg. urea urea glycerin mineral oil oil

Fig. 2. Skin susceptibility to an experimental challenge test of the skin with SLS, measured as TEWL, after daily use with different products for up to 7 weeks compared to nontreated skin [16, 17, 24, 45, 86] . The susceptibility to SLS is measured as TEWL and compared to the untreated control area, where the arrows denote significant differences compared to controls. Values are presented as percentage of untreated control skin, serving as 100% (dotted line).

market are regulated as cosmetics, but they may cation of a cream to the skin, the composition of also be classified as pharmaceuticals (equivalent to the cream will change, as volatile ingredients (e.g. medicinal products) or as medical devices. When water) evaporate and other ingredients interact they are regulated as pharmaceuticals or medical with the skin. In due course, the applied ingredi- devices they can also be marketed for treatment or ents have penetrated into the epidermis, been me- prevention of diseases, such as AD, psoriasis, ich- tabolized or have disappeared from the surface thyosis and other hyperkeratotic skin diseases [20] . due to contact with other surfaces and continuous During recent years, there has been an increase in desquamation. formulations certified as medical devices in Eu- Water in the moisturizing cream will give a rope for the treatment of skin diseases [21] . temporary increase in skin hydration by absorp- The present chapter will give an overview of tion into the epidermis, as proven by measure- the influence of moisturizing treatment on the ment of water loss from the skin after removal of barrier function of normal and dry skin. the moisturizer residue from the surface [23] . The formed layer of fats on the skin surface increases skin hydration [23] . The amount of product ap- Changes in the Skin Surface plied, and the content and types of fatty materials in the formulation determine the reduction in Emollients and moisturizers are applied to the water loss. A thick layer (3 mg/cm 2) of pure pet- skin with the aim of changing tactile [22] and vi- rolatum would give a similar reduction in TEWL sual characteristics of the surface. During appli- [23] as a semiocclusive silicone membrane [24] .

114 Lodén

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 112–122 ( DOI: 10.1159/000441586) A ten-times-thicker layer of petrolatum (30 mg/ sensitivity to nickel was also found when nickel- cm2 ) induces swelling of the corneocytes located sensitive humans treated their skin with a mois- centrally in the SC [25]. Topically applied lipids turizer without humectant compared to treat- may also enter the epidermis [26–32] , increase ment with a moisturizer with glycerin as humec- cell differentiation [33] and reduce skin permea- tant [18] . Recently, higher TEWL and a thinner bility [34, 35] . SC were observed following treatment with Aque- ous Cream BP, probably due to the fact that this cream contains SLS [43]. Clinically, interesting Techniques to Measure Changes in Skin differences between the impact of olive oil and Permeability sunflower oil on SC have also been reported, where treatment with olive oil for 4 weeks caused Noninvasive bioengineering techniques can be a significant reduction in SC integrity and in- used to evaluate treatment effects on skin barrier duced mild erythema in volunteers with and function [36] . Quantification of TEWL is a useful without a history of AD, whereas sunflower seed tool for monitoring the kinetics in the repair of a oil preserved SC integrity and did not cause ery- deteriorated barrier function. The level of TEWL thema in the same volunteers [44] . Olive oil was may also serve as an indicator of the permeability suggested to be able to exacerbate existing AD of the skin to topically applied substances [37, 38] . [44] . No differences in TEWL and skin suscepti- However, TEWL may not necessarily reflect per- bility were found between long-term treatment of meability to substances other than water. There- normal skin with 40% mineral oil and vegetable fore, changes in skin barrier function can also be oil [45] , but both formulations appear to weaken further explored by application of substances that the barrier function compared to no treatment. cause a biological response of the skin [11, 16, 17, However, repeated applications of urea-contain- 19, 39–42] . Substances used to assess skin barrier ing moisturizers have been noted to reduce TEWL function are those inducing vasodilatation (e.g. and make skin less susceptible to SLS-induced ir- nicotinates), irritation (surfactants such as sodi- ritation (fig. 2 ) [17, 46–48] . um lauryl sulfate, SLS; fig. 2 ), erosion (sodium hy- The mechanism for the improvement in skin droxide), wheel-and-flare reactions (dimethyl sulf- barrier function is not fully understood. Covering oxide), burning (chloroform:methanol), stinging the skin with a semiocclusive membrane has been (lactic acid) and reactions to allergens. shown to improve skin barrier [24] , but the composition of creams seems more important as two creams with similar occlusivity influenced skin Effects in Healthy Skin barrier function differently [24] . Furthermore, pH appears not to be crucial, as there was no differ- Treatment with moisturizers may influence the ence in the impact on skin barrier recovery be- barrier properties of healthy skin. Increased skin tween two creams with the same 5% urea composi- susceptibility to a surfactant (fig. 2 ) [16] , nickel tion, where one of the creams was pH adjusted to [41] and a vasodilating substance [19] has been 4.0 and the other to pH 7.5, neither in the early nor reported after treatment with a lipid-rich cream. in the late stages of the recovery [49] . However, in In addition, the time to induce vasodilatation was another study in healthy volunteers, TEWL and shorter for the lipid-rich cream than for a mois- skin responses to SLS irritation were increased in turizer containing 5% urea [19]. A more rapid a pH 8 site compared to areas with pH 3 and 5 after onset of vasodilation reflects a more rapid pene- a 5-week treatment with a moisturizer pH adjust- tration, i.e. weakened barrier function. Increased ed with glycolic acid and triethanolamine [50] .

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Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 112–122 ( DOI: 10.1159/000441586) Effects in Experimentally Damaged Skin eases, the visible symptoms of dryness are dimin- ished and the thickness of the hyperkeratotic In experimental models of dryness, moisturizers layer may become normal following treatment are usually reported to promote normalization of with moisturizers [48, 72]. However, the elevated the skin [46, 51–53] . The models include barrier TEWL may not always decrease to normal levels damage by successive tape stripping, or by expo- after treatment. Therefore, the use of barrier- sure to acetone or SLS. The treated skin abnor- deteriorating products on sensitive and already mality and the composition of the treatment may compromised eczematous skin may be counter- be crucial for the effects [13, 26, 52, 54, 55] . For active [43] . example, the humectant glycerin has been found Three different TEWL patterns are distin- to stimulate barrier repair in SLS-damaged hu- guished in skin barrier diseases: (1) abnormally man skin [51] . It has also been shown that the use high TEWL has been noted to remain unchanged of bath oils in the water reduces TEWL in per- in patients with AD, psoriasis and those working turbed skin [56]. Petrolatum has also been proven as cleaners and kitchen assistants after repeated to penetrate into the outer layer of delipidized SC use of a moisturizer [14, 48, 73, 74]; (2) abnor- and reduce TEWL [57]. Lipids have also been mally high TEWL may increase further (i.e. fur- suggested to influence cutaneous inflammation ther weakening of the skin barrier function) as [58, 59]. In a double-blind study, a physiological noted in patients with ichthyosis or in xerotic skin lipid mixture was found to promote barrier re- of elderly people [11, 12], and (3) TEWL decreas- covery in SLS-irritated and tape-stripped human es towards normal values (i.e. improvement in skin compared to the untreated control area [53] . the skin barrier) in ichthyosis, and childhood and However, the barrier recovery was not superior adult AD [15, 63, 75] . to its placebo (petrolatum) [53, 60] . The different effects of various moisturizers In addition, not only lipids but also nonionic are not fully understood, but certain ingredients, emulsifiers [10] and the humectants glycerin [51] for example emulsifiers, may induce subclinical and dexpanthenol [61] have been reported to in- irritation and barrier defects. For example, olive fluence barrier repair in experimentally damaged oil was recently suggested to potentially exacer- human skin. Furthermore, the 5% urea moistur- bate existing AD [76] . Furthermore, the elevation izer has repeatedly been shown to reduce TEWL of skin pH caused by some moisturizers has been and skin susceptibility to irritation [40, 45, 62–64] suggested to impair the epidermal barrier [77] . versus no treatment. Other moisturizers may also Improvement in skin barrier function has re- reduce TEWL, but measurements need to be done peatedly been found after treatment with urea, after careful removal of cream residues in order to even though not all urea creams improve skin facilitate conclusion of what actually is being mea- barrier function [45]. Urea is a component of nat- sured. The creams may well have acted as nonvis- ural moisturizing factors, which is derived from ible gloves, but the results may also have revealed filaggrin degradation [78] . Mutations in the filag- important and more sustainable SC changes. grin gene and the level of filaggrin degradation determine the content of urea in the SC [4, 79– 81]. In AD patients, the reduced filaggrin expres- Hyperkeratotic and Barrier-Diseased Skin sion of heterozygous null allele carriers has been proposed to be improved by topical application of Dry, scaly and hyperkeratotic skin is usually as- urea to the skin [82] . In a murine model of AD, sociated with a defect in barrier function [65–71] . topically applied urea improves barrier function In clinical studies in patients with barrier dis- by increasing the expression of antimicrobial

116 Lodén

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 112–122 ( DOI: 10.1159/000441586) 1.0 Censored Event Total Test cream 1 62 85 Reference cream 2 73 82 0.8

0.6

0.4

0.2 Probability of relapse-freeProbability maintenance of 0

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 Time (days)

1 85 46 26 26 24 24 24 23 22 16 4 1 1 1 0 2 82 30 16 14 13 11 9 8 8 7 0

Fig. 3. Kaplan-Meier plot of time to relapse of atopic eczema in the groups treated with a barrier- strengthening urea cream (1 = test cream) and a barrier-neutral reference cream (2), with the number of subjects at risk tabulated under the horizontal axis [reproduced from 84 , with permission]. peptides [82]. The defective barrier function in patients [85] and normal healthy individuals AD [67, 68] is suggested to have consequences showing no measurable effects on skin barrier not only on the development of eczema but also function [86] . on other conditions, such as asthma [80] . In the maintenance phase, the median number Since the barrier abnormality is considered a of eczema-free days was more than 26 weeks in critical trigger of dermatitis, treatment with a bar- the urea group compared to 4 weeks in the con- rier-improving urea moisturizer, i.e. a moistur- trol group using no moisturizer [83]. The proba- izer reducing TEWL, has been shown to delay bility of not having a relapse during the 26-week relapse of AD compared to no treatment [83] as period was 68% in the moisturizer group and well as urea-free cream neutral to the barrier 32% for those not using a moisturizer, which re- function ( fig. 3 ) [84] . The urea-free cream neutral sulted in a 53% relative risk reduction [83] . In the to the barrier-function [84] was a placebo to a latest study, 26% of the patients did not relapse glycerin-containing moisturizer used in the clear- during 26 weeks when using the urea cream com- ing phase of the study, where the patients cleared pared to 10% in the group using the urea-free ref- their eczema with topical corticosteroid prior to erence cream, i.e. a 37% risk reduction [84] . The being randomized to moisturizer treatment [84] . delay in eczema relapse has also been shown in The glycerin-containing cream and its placebo patients with hand eczema following treatment had previously been studied clinically in atopic with the 5% urea moisturizer, where the median

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Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 112–122 ( DOI: 10.1159/000441586) Moisturizer Barrier function Eczema Asthma

Fig. 4. The composition of the moisturizer determines the changes in skin barrier function, and the risk for eczema and potentially also asthma.

time to relapse showed a tenfold difference be- tive risk reduction of 50% in the cumulative inci- tween the urea moisturizer and no treatment (20 dence of AD was reported [93]. The contents of vs. 2 days, respectively) [87] . the moisturizers were not discussed in the pediat- The results from the barrier-strengthening ric trials, but since the composition may affect the urea cream study [83] can be compared to results clinical outcome, the need to also differentiate from similar studies focusing on long-term dis- moisturizers based on their mechanism of action ease control using anti-inflammatory agents. Al- is emphasized [95] . though these studies have different designs, the results suggest that a barrier-strengthening moisturizer may prevent the relapse of eczema to a Adverse Reactions comparable extent as intermittent treatment with anti-inflammatory agents on controlled atopic Moisturizers are rarely associated with health eczema [83, 88–92]. The similarity in the relapse risks, although they may be used on large body rates of the 5% urea cream and the reported anti- areas over a large part of the human life span. inflammatory treatments suggests that the use of However, some case reports on poisoning in chil- barrier-improving treatments is effective in the dren are noted due to topical treatment with, prevention of eczema. for example, lactic acid [96] and propylene glycol The findings demonstrated that skin barrier [97] . More commonly encountered adverse reac- dysfunction is recognized as central to AD initia- tions are various forms of skin discomfort from tion and progression; it has also been hypothe- moisturizers, since virtually any substance can sized that enhancement of a defective skin barrier cause skin reactions in sensitive areas in some in- early in life might prevent or delay AD onset. Re- dividuals. Patients with impaired barrier func- sults from recent trials also demonstrate that tion, such as atopics, are particularly at risk for emollient therapy from birth represents an effec- adverse skin reactions. The most common ad- tive approach to AD prevention [93, 94] , as in one verse reactions to moisturizers are sensory re- of the studies approximately 32% fewer neonates actions or subjective sensations (no signs of in- who received moisturizer had AD by week 32 flammation) immediately after application. Hu- than control subjects [94] and in the other a rela- mectants, such as lactic acid [98] , urea [99, 100]

118 Lodén

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 112–122 ( DOI: 10.1159/000441586) and pyrrolidone carboxylic acid [101] , and preser- remain high or even to increase under certain reg- vatives, like benzoic acid [101] and sorbic acid imens, whereas other moisturizers improve skin [100], are known to be able to cause such subjective barrier function. Different outcomes have also sensations. In addition, repeated exposure of sen- been reported in healthy skin, with some mois- sitive areas to mildly irritating preparations may turizers producing deterioration in skin barrier cause dermatitis. Furthermore, weakening of a function, while others improve the skin. healthy skin barrier function is also possible, with Moisturizers with barrier-improving proper- increased risk for outbreak of eczema due to trig- ties have been proven to delay the relapse of ec- gering of inflammation and disease activity [1] . zema, and one urea-containing moisturizer was Fragrances and preservatives are identified as also proven superior to a cream neutral to the the major sensitizers in topical formulations. Al- barrier function regarding the time to relapse of most all moisturizers in the supermarket contain atopic eczema. Certain moisturizers have also fragrances and over 100 fragrance ingredients been found to reduce the cumulative incidence of have been identified as allergens [102] . Humec- atopic eczema in childhood. In a worst-case sce- tants, emulsifiers and oils hardly ever cause con- nario, treatment with a moisturizing cream may tact allergy [102]. Lanolins are sometimes pro- increase the risk for eczema and asthma (fig. 4 ). posed to be a frequent cause of contact allergy, In the European guidance document for cos- but this is believed to be due to inappropriate test- metics, it has been detailed that moisturizers pre- ing conditions leading to false-positive reactions sented as having ‘properties to treat or prevent [102]. Adverse reactions to herbal extracts are atopy/atopic skin cannot be qualified as cosmetic rare, probably a manifestation of the usually triv- products’ [103] . Thus, it is anticipated that profes- ial amounts present in the finished product. sionals should be careful in recommending moisturizers without having evidence of their suitability for restoring or keeping the barrier function in Conclusions a good condition. Moisturizing cosmetics marketed to be ‘appropriate for/suitable to skins with One might expect that a patient’s impaired skin atopic tendency/atopic skin’ [103] are expected barrier function should improve in association to demonstrate absence of barrier-deteriorating with a reduction in the clinical signs of dryness. properties. An evidence-based approach is always However, despite visible relief of the dryness recommended for selecting moisturizers, as not symptoms, abnormal TEWL has been reported to all cream formulations are the same.

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Marie Lodén Eviderm Institute AB Bergshamra Allé 9 SE–170 77 Solna (Sweden)

E-Mail marie.loden @ eviderm.se

122 Lodén

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 112–122 ( DOI: 10.1159/000441586) Section IV: Clinical Aspects of Skin Barrier Function

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 123–134 (DOI: 10.1159/000441588)

Standards for the Protection of Skin Barrier Function

Ana Giménez-Arnau Department of Dermatology, Hospital del Mar, Institut Hospital del Mar d’Investigacions Mediques (IMIM), Universitat Autònoma de Barcelona, Barcelona , Spain

Abstract mechanical properties. Xerosis and the disrup- The skin is a vital organ, and through our skin we are in close tion of normal skin function can occur when the contact with the entire environment. If we lose our skin we epidermal water level falls below 10% [1] . The lose our life. The barrier function of the skin is mainly driven stratum corneum maintains a water gradient be- by the sophisticated epidermis in close relationship with tween its innermost and its outer surface, which the dermis. The epidermal epithelium is a mechanically, is in direct contact with the atmosphere. The lev- chemically, biologically and immunologically active barrier el of hydration is modified by water diffusion submitted to continuous turnover. The barrier function of from the dermis to the epidermis, water diffusion the skin needs to be protected and restored. Its own phys- within the stratum corneum and water loss iology allows its recovery, but many times this is not suffi- through superficial evaporation. The skin’s water cient. This chapter is focused on the standards to restore, content consists of transepidermal water and re- treat and prevent barrier function disruption. These stan- tained water. Transepidermal water originates dards were developed from a scientific, academic and clin- from the circulating blood, migrates through the ical point of view. There is a lack of standardized adminis- dermis into the epidermis, and eventually evapo- trative recommendations. Still, there is a walk to do that rates from the surface of the skin. This movement will help to reduce the social and economic burden of dis- of water plays a key role in the supply of nutrients eases characterized by an abnormal skin barrier function. to the epidermis. The water retained in the stra- © 2016 S. Karger AG, Basel tum corneum is located between the lipid bilayers and inside the corneocytes. This static water content maintains the mechanical properties of the Dry Skin: The Need to Restore Barrier cornified layer, increases the plasticity of the epi- Function dermis and enhances the hydrophilic properties of keratin. Dry skin or ‘xerosis’ is common in the general The stratum corneum should prevent the loss population. The disorder is characterized by the of fluids and electrolytes from the skin through presence of rough, flaky skin losing its normal the structure and function of its lipids, proteins and cells. The recovery of the barrier function af- volved in the desquamation are chymotryptic ter an acute depletion of the stratum corneum and tryptic enzymes (also called kallikrein 7 and lipid content is based on the reconstitution of 5, respectively) in the stratum corneum. Excess the lipid-enriched intercellular substance, a pro- protease activity can lead to stratum corneum cess involving two distinct stages: an initial short thinning, while reduced protease activity can phase and a second longer phase. At onset, the lead to stratum corneum thickening. The accu- response is fast and the content of the lamellar mulation of corneocytes on the surface of the bodies is released in the stratum granulosum. The stratum corneum leads to the condition termed synthesis of new lipids (ceramides, cholesterol ‘dry skin’. Reductions in enzyme activities to- and fatty acids) is slower and involves enzymes gether with retention of corneodesmosomes in that govern lipid formation. the upper layers of the stratum corneum contrib- The mixture of the required lipids in the cor- ute to dry skin [4] . rect proportions is essential for the reconstitution The water content of a healthy stratum cor- of the epidermal lipid bilayers. Topical applica- neum under normal conditions is 15–20%. When tion of only 1 or 2 of the 3 lipids required to main- the water content of the cornified layer falls below tain the intercellular barrier (ceramides, choles- 10%, the skin acquires a rough dry appearance. terol and fatty acids) could be detrimental to the To prevent this happening, the epidermis con- quality of the bilayers [2]. However, the topical tains a number of water-retaining substances, the application of a mixture of the 3 lipids in the cor- most important of which is natural moisturizing rect proportions accelerates the repair of the epi- factor, a compound composed of a mixture of dermal barrier. It has been reported that physio- amino acids, amino acid derivatives and salts gen- logical lipids applied to the skin pass through the erated by filaggrin hydrolysis. The water absorbed stratum corneum and are taken up by the lamellar by natural moisturizing factor from the environ- bodies in the stratum granulosum. This implies in ment and from inside the skin acts as intracellular theory that topical application of more physiolog- plasticizers in the stratum corneum. In animal ical lipids would contribute better to the repair of studies, environmental factors such as a decrease the epidermal barrier, as they would not only in air humidity has been shown to reduce the gen- form an occlusive layer on the skin but would also eration of free amino acids and filaggrin expres- deliver the raw materials required to create new sion in the stratum corneum, thus increasing skin lamellar bodies. Considering the important role dryness [5] . of free fatty acids, for example, in skin barrier Disruption of the epidermal barrier triggers a function in certain pathological conditions as metabolic response directed towards recovering atopic eczema [3] , clinical trials were developed epithelial homeostasis and reestablishing normal to compare the effect of different types of emol- corneocyte differentiation. The main response is lients restoring barrier function to study patho- an increase in the biosynthesis of lipids, such as logical or physiological conditions. cholesterol, ceramides and fatty acids, as men- Desquamation is a specific consequence of tioned above [2] . Slight disturbances in barrier the epidermal turnover. Correct desquamation is function usually only affect the superficial epider- as important as the physiological cornification mis, but repeated or severe damage gives rise to process. The appearance of dry skin can be the an inflammatory response that involves the deep- consequence of an abnormal desquamation. The er epidermal layers and even the endothelium [6, cells detach from the epidermis as a result of the 7] . These phenomena result in abnormal kerati- degradation of the corneodesmosomes induced nization and close the cycle that perpetuates le- by proteases. The most important enzymes in- sion formation.

124 Giménez-Arnau

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 123–134 ( DOI: 10.1159/000441588) Skin Barrier Disorders with Focus on Xerosis, Table 1. Etiologic factors involved in dry skin or xerosis Atopic Dermatitis, Ichthyosis and Hand appearance Eczema Inherited predisposition Age Xerosis Comorbid diseases It is estimated that generalized or diffuse xerosis Atopic dermatitis affects 75% of individuals over 75 years of age Psoriasis Hypothyroidism and is the most common cause of itch in this age Intestinal malabsorption group. While dry skin in older people is usually Related to environmental conditions first noted on the lower limbs, the disorder may Temperature spread to other areas of the body. Itch is often Humidity Exposure to sunlight more intense at night and after a hot bath. Air conditioning Changes in temperature, low air humidity or ex- Heating posure to solvents or detergents may accelerate Related to chemical agents the development of xerosis. While there are Soaps and bath gels many possible causes of dry skin in older people, Lotions and perfumes Detergents the most common mechanism involved is a low- Pharmacotherapy er rate of epidermal proliferation compared Related to physical insults with normal skin ( table 1 ) [8] . Skin aging in- Friction volves other physiological changes that may in- Abrasion duce xerosis. Age-related changes in collagen Radiation synthesis and degradation decrease skin elasticity and increase dryness sensation. The decline in gonadal and adrenal androgens is associated with decreased synthesis of sebum and cutane- position to atopy also favors overexpression of ous ceramides. Levels of filaggrin, the protein stratum corneum chymotryptic enzyme and the from which the components of natural moistur- consequent disruption of the epidermal barrier as izing factor are derived, are also lower in aged a result of premature corneodesmolysis. Deter- skin [9] . gents and topical corticosteroids increase the expression of this protease and contribute to the Atopic Dermatitis chronicity of the disease [14]. Filaggrin-null mu- Atopic dermatitis is a chronic inflammatory skin tations (e.g. R501X and 2282del14) have been disorder characterized by recurrent outbreaks of identified in up to 50% of patients with moderate- symmetrical eczema, the location of which de- to-severe atopic eczema in the European pop- pends on age, accompanied by severe pruritus. ulation and 20% in the Asian population. These The skin of patients with atopic eczema is charac- mutations significantly decrease filaggrin expres- terized by low ceramide levels, increased trans- sion in the skin, and development of atopic ec- epidermal water loss (TEWL) [10] and decreased zema is common in filaggrin mutation carriers. water-binding capacity [11] . The unaffected skin Since ‘natural moisturizing factor’ is a degrada- of patients with atopic dermatitis has also been tion product of filaggrin, it is significantly re- reported to have abnormally low levels of cera- duced in patients with atopic dermatitis with fil- mides 1 and 3, molecules rich in polyunsaturated aggrin mutations versus those without [15] . Pa- fatty acids, and particularly linoleic acid [12, 13] . tients suffering from severe atopic dermatitis do It has recently been shown that the genetic predis- not necessarily carry filaggrin mutations, but the

Standards for the Protection of Skin Barrier Function 125

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 00–00 ( DOI: 10.1159/000441588) atopic response itself (Th2 cytokines) leads to de- creasing keratinocyte expression of the filaggrin, and to filaggrin deficiency independent of the genetic status. Patients with filaggrin null mutations show increased TEWL. This fact supports a barrier dysfunction induced by poor filaggrin function rather than inflammation-causing barrier defects. Treating barrier dysfunction of atopic dermatitis proactively with effective emollients will benefit the prognosis of atopy [16–21] . a

Ichthyosis Ichthyosis is a family of genetic skin disorders, all characterized by xerosis or dry skin. Different epidermal defects are responsible for the different types of ichthyosis. Ichthyosis vulgaris is characterized by abnormalities in the formation of keratohyalin granules and consequently filaggrin. Im- munostaining of ichthyosis vulgaris skin biopsies showed reductions in filaggrin protein expression and profilaggrin mRNA within keratinocytes [19, 22] . Filaggrin mutations are observed approxi- b mately in 7.7% of the Europeans (general popula- Hand eczema. Acute course with itchy blisters tion) and 3% of Asians, being infrequent in black Fig. 1. a showing epidermal detachment. b Hand eczema with people. Two common filaggrin null mutations acute epidermolysis. (p.R501X and c.2282del4) cause ichthyosis vulgaris and predispose to eczema and secondary allergic diseases [19] . Different variants were described, including 7 that are prevalent, which are eczema and endogenous eczema types (fig. 1, 2 ) nonsense or frameshift mutations and resulted in [26]. With respect to irritant contact dermatitis, loss of filaggrin production in the epidermis. X- anyone can be affected, but, it is particularly not- linked recessive ichthyosis is characterized by the ed in individuals with constitutionally impaired presence of large scales. This condition is caused barrier function, such as patients with an atopic by a steroid sulfatase deficiency that leads to an dermatitis, especially those showing filaggrin null accumulation of cholesterol sulfate and a reduc- mutations [27–31] . In a study involving twins tion in cholesterol levels in the stratum corneum with hand eczema controlled for age and atopic [23, 24]. Treatment with moisturizers does not dermatitis, the effect of genetic risk factors ex- have any major impact on the skin barrier prop- plained 41% of the variance in the susceptibil- erties [25] . ity to develop hand eczema, leaving 59% of the variance to be caused by environmental factors Hand Eczema [32]. Environmental factors are thus important, Dermatitis of the hands (hand eczema) is a mul- and wet work is a risk factor by itself that can be tifactorial disease, comprising allergic and irritant responsible for epidermal barrier disturbance in contact dermatitis, contact urticaria, atopic hand everyone.

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Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 123–134 ( DOI: 10.1159/000441588) tive ingredients that rapidly penetrate the epidermis to stimulate the production pathways of intercellular lipids. This ‘inside-out’ approach, compared to the traditional ‘outside-inside’ approach, appears to produce more effective therapeutic outcomes. The topical preparations designed to treat dry skin are emollient or hydrating substances in preparations such as lotions or creams, i.e. oil-in-water emulsions (higher concentration of oil than water) or water-in-oil emul- a sions (higher concentration of water than oil). The mechanisms of action of the different active principles are discussed in the following. The main objective will be to restore epidermal homeostasis. Lipids are the essential ingredients in formulations used to restore barrier function. The delivery of water alone will not repair the lipid barrier, natural physiological lipids (cholesterol, ceramides and fatty acids) must also be supplied. Ac- cording to the opinion of some experts, nonphys- b iological lipids are not recommended because they do not contribute to the reconstitution of the Contact allergy. Desquamative fingertips of a Fig. 2. a fatty bilayers [33] . The main lipid components cosmetician due to acrylate allergy. b Positive patch test to acrylates responsible for contact allergy. found in the epidermis are ceramides (50%) and cholesterol derivatives (25%). The physiological lipids are speculated to have several advantages over nonphysiological molecules: they are not oc- Standards to Restore Barrier Function clusive, they penetrate the stratum corneum more easily, they gain better acceptance from patients Restoration of Active Barrier Function because they are natural, and they restore proper Restoration of barrier function in xerotic or atop- epidermal differentiation. In short, physiological ic skin (e.g. ichthyosis or hand dermatitis) is first lipids, such as ceramides, act as structural ele- based on the fast and effective standardized med- ments in the epidermal barrier and mediate the ical treatment of the disease if available. The man- stimuli that trigger epidermal repair. However, agement of these diseases will also benefit from human studies showing the superiority of these the topical application of the components re- preparations are lacking. quired by the epidermis to reestablish normal ke- We should differentiate between ‘humectant’ ratinocyte differentiation for the treatment of xe- and ‘hydrating’ molecules. Humectants are sub- rotic and ichthyosiform skin, especially during stances that attract and retain water; they play a the maintenance phase of the treatment of atopic passive role from the outside. A hydrating sub- dermatitis and any type of hand eczema. stance, however, is one that plays an active role in Some specialists currently advocate the useful- supplying and restoring water to the skin. Humec- ness of applying topical treatments containing ac- tants are generally hygroscopic substances, such

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Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 00–00 ( DOI: 10.1159/000441588) as glycerin or propylene glycol, while hydrating cholesterol), a physiological humectant (glycer- agents or moisturizers are complex mixtures of ol), an anti-itching agent (glycerol) and a com- active ingredients or special combinations of ami- ponent that enhances epidermal differentiation no acids. The inclusion of a humectant, such as (dexpanthenol). glycerol or urea, as an active ingredient in topical Humectants are natural oily substances that treatment for dry skin is based on the scientific do not intervene in the metabolic processes of the evidence that humectants are capable of correct- skin but rather act passively by preventing exces- ing defects in skin elasticity and barrier function sive water loss. They can be classified according to when these deficiencies are not related to lipid general chemical categories: hydrocarbons, oils loss. Glycerol plays a crucial role in keeping the and fatty alcohols, colloid substances and sili- stratum corneum hydrated: changes in aquaporin cones. 3, an epidermal water/glycerol transporter, lead to Moisturizers or hydrating agents play an ac- decreased hydration and loss of skin elasticity that tive role in the process of maintaining the water can be corrected by the topical application of glyc- balance of the stratum corneum and are listed in erol. It is, therefore, recommended that topical table 2 . moisturizers include glycerol [34–36] . Active relipidating agents supply the compo- The sensation of itch induces scratching, espe- nents the skin needs to balance the composition cially in xerosis, atopic dermatitis and hand ec- of the interlamellar lipid bilayers. In dry skin, zema, and it represents a physical aggression that the fatty acid content of these layers is low, and damages the epidermis. When the patient stops ceramide content is impaired and should be re- scratching, the epidermal restoration can start. stored. Topical application of certain natural agents, Some topical formulations for dry skin include such as glycine, blocks the release of histamine an active ingredient that stimulates cell prolifera- from mast cells, and thereby helps to break the tion and lipid synthesis. One example of this type self-perpetuating cycle of itching-scratching. Gly- of component is dexpanthenol, a precursor of cine blocks the release of histamine by mast cells pantothenic acid and a constituent of coenzyme [35]. Other products, in particular corticoste- A. It has been observed that pantothenic acid in- roids, are also used to treat itching. creases the proliferation and migration of fibro- As the skin and its different layers are struc- blasts [36] and stimulates intracellular protein tures that undergo continual renewal, dry skin synthesis [37] . can be treated by delivering components, such as Table 2 lists other active ingredients that play dexpanthenol, that stimulate and accelerate the a role in restoring the xerotic stratum corneum process of epidermal regeneration. Dexpanthenol [38, 39] . promotes fibroblast proliferation and migration, The concept of barrier creams has been widely and stimulates intracellular protein synthesis, discussed. Barrier creams are devices aiming to while hydroxy acids facilitate desquamation and prevent contact with exogenous hazardous sub- improve lipid biosynthesis [37] . stances and thus penetration and absorption of Topical preparations for barrier function re- contaminants. The objective is to avoid the risk of covery should contain molecules that activate the contact dermatitis, e.g. irritant contact dermatitis; epidermal regeneration process and restore the emollients can help to restore epidermal barrier lipid content of the horny layer. It is essential to function. It can be considered as a prophylactic choose the most suitable excipient for the area of measure to reduce contact dermatitis of the hands, the skin to be treated. The ideal formulation for example. Barrier creams are recommended in would contain physiological lipids (ceramides/ the presence of low-grade irritants to improve the

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Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 123–134 ( DOI: 10.1159/000441588) Table 2. Substances included in topical preparations for the treatment of xerosis

Type Compound Characteristic

Humectants Hydrocarbons Mineral oils, such as paraffin and Vaseline Fatty oils and alcohols Colloid substances Some are hygroscopic, e.g. cellulose derivatives (ethyl cellulose), natural polymers (xanthan gum) and synthetic polymers (carbopol) Silicones No strong smell Not comedogenic Excellent tolerance Nongreasy formulations Hydrating Polyols Highly hydrating agents Restore the flexibility of the cornified layer Prevent crystallization of lipids Promote corneodesmolysis Examples: glycerol, sorbitol and propylene glycol Urea A component of natural moisturizing factor Highly hygroscopic and good exfoliating qualities Reconstituted natural Mixture of amino acids, sodium lactate, lactate acid, citrate and others moisturizing factor Repairs the upper layers of the stratum corneum with a hydrating action similar to that of natural moisturizing factor Hyaluronic acid Creates a barrier layer High capacity to hydrate the stratum corneum Restores the flexibility and elasticity of the skin Well tolerated by the skin Active Ceramides Facilitate epidermal differentiation by reestablishing components of cellular lipids relipidating Cholesterol Ensures the availability of this natural lipid in the stratum corneum to facilitate ingredients regeneration and epidermal differentiation Essential fatty acids Provide consistency and cohesion in the stratum corneum Anti-inflammatory, immunogenic and antimicrobial activity Principal fatty acids: linoleic, γ-linoleic and arachidonic acids Fatty acids are found in vegetable oils, such as evening primrose, shea, jojoba, borage, olive, wheat germ and sunflower Other active Oats Complex composition: very rich in water, proteins, glucides, lipids, mineral salts and ingredients vitamins Hydrating, restructuring, antipruritic and anti-inflammatory Improves the compatibility between the components in the preparation Allantoin Conditioning, hydrating and keratoplastic α-bisabolol Anti-inflammatory, emollient and bactericidal Aloe vera Soothing and emollient Glycyrrhetinic acid Anti-inflammatory and emollient

Published with permission from Barco and Giménez-Arnau [38] and Elsevier.

barrier function against certain chemicals. Barrier Standards to Protect Barrier Function creams are useful if used regularly, but their effectiveness remains controversial. The barrier function, especially when we consider The use of active treatments that actively help hands suffering from hand dermatitis, can be pro- to restore the barrier function include topical cor- tected via the use of protective gloves as well as a ticosteroids and calcineurin inhibitors, for exam- safe cleaning methodology. Attempts to quantify ple (fig. 3 ). the effects of gloves on manual performance have

Standards for the Protection of Skin Barrier Function 129

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 00–00 ( DOI: 10.1159/000441588) tained using a good protective glove. The research in gloves shows conflicting results because of the lack of trials studying individual glove properties such as thickness, material composition, grip pattern or fit and their effects on real clinical performance. It is difficult to make a recommendation regarding the best glove. The current state of knowledge and the test methods available do not give glove designers adequate tools to improve glove performance using an evidence-based design process. There is, therefore, a need for tests that are repeatable, quantifiable and realistic, which could help to find new materials and manufacturing techniques to develop efficacious a gloves [40] . The correct use of gloves is crucial in order to achieve a correct barrier function protection. How the occlusion affects irritant contact dermatitis has been studied previously. The use of either antibacterial cleanser or gloves induced only mi- nor increases in TEWL. When combined, the two showed a tandem effect, as the TEWL increase was significantly higher [41] . Alcoholic disinfection of the hands was recommended instead of the continuous use of water based on less irritant skin properties and better b antimicrobial effects. This suggestion involves especially employees in the so-called wet-work set- Fig. 3. Leg dermatitis or eczema. a Limited contact dermatitis showing epidermal detachment or vesicles due ting. Alcoholic disinfection causes less irritation to the spongiotic inflammatory infiltrates in the epider- than water; nevertheless, it seems that some work- mis. b Microbial dermatitis secondary to venous stasis in ers, e.g. nurses, perceive it as more damaging and the distal part of the leg. the consequence can be a low compliance. Educa- tional programs are desirable [42] . Few systematic reviews were done regarding been in evidence since the Second World War, gloves and its appropriate use, but there is at least when the US Armed Forces commissioned Har- one entitled ‘Gloves, extra gloves or special types vard University to look at the effects of hand wear of gloves for preventing percutaneous exposure on manual dexterity. Since then, the work has injuries in health care personnel’. The main ob- spread and now encompasses a wide variety of jective of this Cochrane collaboration is to deter- products and considers many different aspects of mine the best glove to avoid percutaneous expo- manual performance, including tactile sensation, sure incidents in an occupation at risk of con- grip strength and friction. In every occupational tracting viral infections, e.g. health personnel. practice, grasping, dexterity, and tactile and ac- How the gloves can help or do not help maintain tive haptic sensing are properties to be main- epidermal barrier function is not addressed [43] .

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Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 123–134 ( DOI: 10.1159/000441588) Table 3. Skin protection program based on evidence from clinical and experimental studies

Use gloves when performing wet work Protective gloves should be used appropriately but for as short as possible Protective gloves should be intact, clean and dry inside When protective gloves are used for >10 min, cotton gloves should be worn underneath Wash hands in lukewarm (not hot) water; rinse and dry hands thoroughly after washing Hand washing with soaps should be substituted with alcohol disinfection when hands are not visibly dirty Do not wear finger rings at work Apply moisturizers on your hands during the working day, but especially after work and before bedtime; it may be reasonable to use a lighter moisturizing lotion during the day and a greasier fragrance-free, lipid-rich moisturizer before bedtime Moisturizers should be applied all over the hands, including the webs, finger tips and dorsal aspects Take care when doing domestic work; use protective gloves for dish washing and insulating gloves in the winter

Modified from Agner and Held [45].

Recently, a standardized in vivo test procedure The aim of primary prevention is to decrease was developed to assess cleansing efficacy espe- the incidence of barrier function disorders target- cially in the occupational setting where repeated ing the healthy population. Regulation of allergen hand washing procedures are done at short inter- exposure (e.g. chromate in cement or nickel) ei- vals. An automated cleansing device (ACiD) was ther by legislation on threshold values or regula- designed and evaluated. ACiD allowed an auto- tions on precautions in handling of allergenic mated, standardized skin washing procedure. Felt products reduces allergen exposure and therefore covered with nitrile as washing surface of the the incidence of allergic contact dermatitis. Previ- rotating washing units leads to a homogeneous ous or current atopic dermatitis is, as already cleansing result and does not cause skin irritation, mentioned, a significant endogenous risk factor which was confirmed clinically and by skin bio- for the development of barrier disorders. General engineering methods. However, this method is practicioners as well as health care personnel at still under investigation and cannot be recom- schools should actively participate in prevention mended as a standard method [44] . Table 3 sum- programs. Exposure to wet work is a particular marizes the recommendations based on evidence risk factor for the development of barrier function from clinical and experimental studies in order to disorders, and to achieve the optimal effect of pre- follow a complete skin protection program [45] . ventive efforts the focus should be on reducing wet exposure. Educational programs directed at the general population are attractive, but have Standards to Prevent Barrier Function never been scientifically evaluated. Protection of Disorders the hands is essential for the prevention of hand eczema and is a fundamental aspect in its treat- Barrier function disorders can involve different ment. The effectiveness of protective measures, cutaneous disorders, but chronic hand dermatitis such as the use of moisturizers and gloves, has is the condition with the greatest burden on the mostly been documented in laboratory studies patient and the society. Prevention of barrier dys- with experimentally damaged skin. Use of gloves function involves primary, secondary and tertiary in wet work has generally been recommended and strategies [45] . accepted as an important preventive measure.

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Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 00–00 ( DOI: 10.1159/000441588) Secondary prevention strategies are indicated more sophisticated research programs with re- whenever skin manifestations following barrier spect to primary prevention [46] . Recently, some disorders are already present. The objective of educational programs were described and were secondary prevention is to spot early skin chang- implemented in different countries with success es in order to rapidly implement corrective mea- [47–50] . But still, there is not a common consen- sures. Outpatient skin protection seminars for sus defining the minimum requirements of these workers in at-risk professions have been demon- educational programs in Europe. Regarding the strated to be useful. Skin protection seminars are cutaneous diseases involved, the occupational based on the methodological principles and pro- target population, the methodology and the as- cedures of adult education, and provide theoreti- sessment of efficacy of such educational interven- cal background knowledge and ‘hands-on’ train- tions, a consensus is still needed. ing in the selection and use of adequate skin protection strategies. Strategies for tertiary prevention are basically Conclusion the same as for secondary prevention, but the focus for tertiary prevention strategies are patients The skin is a vital organ that needs to be protected with severe and/or chronic hand eczema in whom and restored. Although standards were developed outpatient secondary prevention strategies have from the scientific and academic field in order to been inadequate. Tertiary prevention strategies restore, treat, protect and prevent barrier func- in (occupational) barrier disorders comprise con- tion impairment, especially in certain skin condi- certed (in-/outpatient) and interdisciplinary (oc- tions, standardized official recommendations are cupational dermatology, industrial health, health lacking. Still, there is work to be done that will educational, occupational therapeutic, psycho- help to reduce the social and economic burden of logical and trade association/administrative) in- diseases characterized by an abnormal skin bar- terventions with the aim of improving the affect- rier function. ed individual’s clinical condition and, where possible, allowing them to keep working in their occupation in the long run. References 1 Rycroft RJG: Low humidity and micro- In order to prevent different cutaneous dis- trauma. Am J Ind Med 1985; 8: 371–373. eases were the epidermal barrier function is im- 2 Man MQ M, Feingold KR, Thornfeldt paired, education has been suggested as the best CR, Elias PM: Optimization of physiological lipid mixtures for barrier repair. method. The most common cutaneous disorder J Invest Dermatol 1996; 106: 1096–1101. approached is chronic hand eczema (irritant as 3 van Smeden J, Janssens M, Kaye EC, well as allergic). A systematic review has been Caspers PJ, Lavrijsen AP, Vreeken RJ, Bouwstra JA: The importance of free conducted considering occupational irritant fatty acid chain length for the skin bar- hand eczema. The author suggests that some pos- rier function in atopic eczema patients. itive benefit is obtained using barrier creams, Exp Dermatol 2014; 23: 45–52. 4 Rawlings AV, Voegeli R: Stratum cor- moisturizers, after-work creams and complex ed- neum proteases and dry skin conditions. ucational interventions preventing occupational Cell Tissue Res 2013; 351: 217–235. 5 Katagiri C, Sato J, Nomura J, Denda M: irritant contact eczema in the short and long Changes in environmental humidity term. Nevertheless, in conclusion, the currently affect the water-holding property of the published studies do not reach statistically signif- stratum corneum and its free amino acid content, and the expression of filag- icant differences, and, therefore, further studies grin in the epidermis of hairless mice. are required to provide new evidence through J Dermatol Sci 2003; 31: 29–35.

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el. Contact Dermatitis 2013; 68: 239–299. tasch M, Gimenez-Arnau A, Nixon R, Thomsen SF, Agner T: Skin care educa- 42 Mischke C, Verbeck JH, Saarto A, Lavoie Sasseville D, Agner T: Guidelines for tion and individual counselling versus MC, Pahwa M, Ijaz S: Gloves, extra diagnosis, prevention and treatment of treatment as usual in healthcare workers gloves or special types of gloves for pre- hand eczema – short version. J Dtsch with hand eczema: randomised clinical

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Prof. Ana Giménez-Arnau Department of Dermatology, Hospital del Mar Institut Hospital del Mar d’Investigacions Mediques (IMIM) Universitat Autònoma de Barcelona, Passeig Marítim 25–29 ES–08003 Barcelona (Spain)

E-Mail 22505aga @ comb.cat

134 Giménez-Arnau

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 123–134 ( DOI: 10.1159/000441588) Section IV: Clinical Aspects of Skin Barrier Function

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 135–143 (DOI: 10.1159/000441589)

The Role of the Skin Barrier in Occupational Skin Diseases

a, b a a Pranee Kasemsarn Joanna Bosco Rosemary L. Nixon a Occupational Dermatology Research and Education Centre, Skin and Cancer Foundation Inc., Carlton, Vic. , Australia; b Department of Dermatology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok , Thailand

Abstract tiple contributing causes to OCD, as workers are exposed Occupational skin diseases (OSDs) are the second most to both irritants and allergens. AD is also associated with common occupational diseases worldwide. Occupation- skin barrier disruption and plays an important role in al contact dermatitis (OCD) is the most frequent OSD, and OCD. ICD often precedes and facilitates the development comprises irritant contact dermatitis (ICD), allergic con- of ACD, with impairment of the skin barrier contributing tact dermatitis (ACD), contact urticaria and protein con- to the concurrence of ICD and ACD in many workers with tact dermatitis. There are many endogenous and exoge- OCD. © 2016 S. Karger AG, Basel nous factors which affect the development of OCD, including age, sex, ethnicity, atopic skin diathesis, certain occupations and environmental factors. One of the most important contributing causes is skin barrier dysfunction. Occupational skin diseases (OSDs) are the second The skin provides a first-line defense from environmental most common occupational diseases presenting assaults and incorporates physical, chemical and biolog- to general practitioners worldwide [1]. There are ical protection. Skin barrier disturbance plays a crucial many endogenous and exogenous factors which role in various skin diseases such as atopic dermatitis affect the development of OSD, such as age, sex, (AD), ichthyosis, ICD and ACD. Genetic factors, such as ethnicity, atopic skin diathesis, occupations at filaggrin gene (FLG) mutations, and external factors, such risk and environmental factors [2] . OSDs are an as skin irritants interfering with stratum corneum struc- important problem causing quality-of-life im- ture and composition, may lead to abnormalities in skin pairment, embarrassment, financial hardship and barrier function and increased vulnerability to skin dis- work productivity loss [3]. Occupational contact eases. FLG encodes the cornified envelope protein, filag- dermatitis (OCD) is the most frequent cause of grin, which is involved in skin barrier function. FLG muta- OSD accounting for 77–95% of cases [4–6] and is tion is associated with the development of OCD. High-risk further classified as irritant contact dermatitis occupations for OCD include health care workers, hair- (ICD), allergic contact dermatitis (ACD), contact dressers and construction workers. There are often mul- urticaria (CU) and protein contact dermatitis (PCD), with ICD generally being more common and biological protection. Antimicrobial peptides, than ACD and CU/PCD [7] . However, this is not and humoral and cellular immune systems are in- always the case, as in some occupations, such as volved in the protection from microbes [16] . the construction industry and hairdressing, exposure to allergens predominates [8–11] . Moreover, Bioengineering Techniques for the Measurement in clinical practice, there are many patients who of Skin Barrier Function have multiple factors contributing to their OCD Measurement of TEWL is a marker for skin bar- [12, 13] . While the diagnostic criteria for ACD are rier function, since TEWL depends on skin hy- more clearly defined than for ICD, it is apparent dration, skin blood flow and skin temperature that females performing wet work have an in- [17, 18]. Increased TEWL was associated with increased likelihood of experiencing concurrent creased skin permeability and chemical absorp- ACD and ICD. Contact allergy and skin irritancy tion [19] . Other measurements of skin barrier may be correlated at an immunological level [14] . function include SC hydration [18, 20, 21] , SC co- In addition, skin barrier disruption plays an im- hesion, surface pH and paracellular permeability portant role in the development of both diseases. of the water-soluble tracer lanthanum [20] . Other skin diseases which contribute to impaired barrier function include atopic dermatitis (AD), ichthyosis and psoriasis [15] . Occupational Skin Diseases

Definition Skin Barrier OSD are skin disorders which are caused or aggravated by occupational factors. The most im- One of the most important functions of the skin is portant disorder is OCD, principally comprising to provide physical, chemical and biological pro- ICD, ACD and also CU/PCD. Other OSD include tection against the external environment (outside- folliculitis and acneiform eruptions (from oils inside barrier). Meanwhile, the skin also acts as an and greases), miliaria, chronic actinic damage inside-outside barrier to prevent excessive water and skin cancers [22] . OCD involves the hands in loss and desiccation by regulating transepidermal about 70–80% of cases [2, 8] . water loss (TEWL). The main physical barrier component is located in the stratum corneum Irritant Contact Dermatitis (SC), which is composed of hydrophilic corneo- ICD is an inflammatory skin condition caused by cytes acting as ‘bricks’ and lipophilic intercellular skin barrier disruption combined with the toxic lipid bilayers acting as ‘mortar’. Corneocytes are effects of predominantly chemical stimuli on epi- enclosed with a cornified envelope, comprised of dermal keratinocytes, together with activation of various structural proteins, with the major pro- innate immune responses [15, 23]. Both endoge- teins being loricrin, involucrin, small proline-rich nous and exogenous factors contribute to the protein, cystatin S and filaggrin, cross-linked by pathogenesis of ICD [23] . Irritants such as water, transglutaminase enzymes [15, 16] . However, soaps and detergents disturb skin barrier func- through tight junctions and desmosomes in the tion by removing lipid or impairing intercellular deeper skin layers, the nucleated epidermis also bilayer lipid organization of the SC. Moreover, contributes to skin protection against mechanical these irritants also penetrate to living epidermis, assaults. Intercellular lipids also play a crucial role damage the keratinocyte plasma membrane and in regulating skin permeability barrier function, interfere with the extrusion of lamellar body-de- preserving skin hydration and providing chemical rived lipids into intercellular lipid bilayers [24] .

136 Kasemsarn Bosco Nixon

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 135–143 ( DOI: 10.1159/000441589) Proinflammatory cytokines, including IL-1α, IL- Contact Urticaria 1β, TNF-α, GM-CSF and IL-8 are released from CU was first described by Fisher in 1973 as a keratinocytes [15, 25] . wheal-and-flare reaction following contact with a Clinically, ICD is indistinguishable from ACD, substance, usually clearing within hours; howev- and there is no readily available diagnostic tool. er, repeated episodes of CU may result in PCD The clinical diagnosis of ICD is often a default [29] . This particularly occurs in food handlers diagnosis, requiring exclusion of ACD through [29–31] . negative patch tests, a history of skin exposure to The most frequent clinical manifestation of irritants and a correlating clinical course of der- PCD is chronic or recurrent eczema. PCD is matitis [14, 23]. It is important to consider all caused by an IgE-mediated hypersensitivity reac- contributing factors, and there may be multiple tion; hence, skin prick testing with fresh material causes of dermatitis in one patient [12, 13, 26] . or commercial extracts is required to make the ICD is most commonly caused by wet work, but diagnosis [29, 31] . Serum specific IgE measure- other irritants include exposure to soaps and de- ment is useful for some known protein allergens tergents, oils, organic solvents, metalworking flu- [29] . ids, mechanical insults, such as pressure and friction, and environmental conditions such as heat, causing sweating [13, 22, 23] . Skin Barrier and Occupational Contact Dermatitis Allergic Contact Dermatitis ACD is a delayed-type hypersensitivity reaction There are many factors which determine the de- caused by antigen-specific T-cell activation in velopment of OCD; however, skin barrier im- sensitized individuals. In the induction phase, pairment is the key step in the pathogenesis of there is activation of innate immunity by specific occupational dermatitis [17, 32] . Emollients have hapten(s), and then Langerhans cells and dermal an important role in both the prevention and dendritic cells migrate to draining lymph nodes healing of OCD by normalizing the abnormal leading to the proliferation of hapten-specific T epidermal barrier. Furthermore, a randomized, cells, including Th1, Th2, Th17 and regulatory T controlled trial showed that regular use of skin cells. In the elicitation phase, subsequent expo- moisturizers protects against irritants [21] . Ge- sure to the same hapten leads to the rapid re- netic factors also influence the acquisition of sponse of effector T cells and other inflammatory OCD, particularly genes involved in skin barrier cells in the skin, initiating cytokine release and function, such as the filaggrin gene (FLG). FLG inducing skin inflammation [15] . In addition, im- encodes the cornified envelope protein filaggrin, pairment of the epidermal barrier may lead to which has a role in skin barrier function and SC allergen penetration and sensitization causing hydration [20] . However, in a prospective cohort ACD [15] while the interaction of irritants and study, Visser et al. [33] found that FLG mutations allergens will be considered below. were not associated with the development of The development of ACD is based on the in- hand eczema in trainee nurses. Risk factors interaction between genetic and environmental fac- cluded a history of AD, a history of hand eczema tors [27] . The environmental factors include the and exposure to wet work. Novak et al. [34] re- nature and concentration of allergens, duration ported the association between FLG mutations and frequency of allergen exposure, and skin bar- and nickel contact allergy, combined with intol- rier status [27, 28]. The gold standard for making erance to costume jewelry, but not with other the diagnosis of ACD is patch testing. contact allergens. The two most frequent null

Skin Barrier in Occupational Skin Diseases 137

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 135–143 ( DOI: 10.1159/000441589) Table 1. Common causes of occupational ACD and ICD in various occupations

Occupations ICD ACD

HCWs [36 – 39] Wet work Thiuram mix, carba mix, dithiocarbamate Soap MCI/MI Skin cleansers Antiseptics (triclosan, chlorhexidine, Antiseptics glutaraldehyde, benzalkonium chloride) Alcohol rubs Sweating from occlusive gloves Construction Alkalinity of cement Potassium dichromate workers [8, 43] Water Epoxy resin Cobalt chloride Hairdressers Wet work p-Phenylenediamine [10, 11, 50] Shampoo Toluene-2,5-diamine Cleaning products Ammonium persulfate Hydrogen peroxide Glycerol monothioglycolate 3-/4-Aminophenol Food handlers Wet work Thiuram mix and chefs Soap Carba mix

[29, 51 – 53] Detergents Nickel sulfate Foods Cobalt chloride Quaternium-15 Food proteins1

MCI/MI = Methylchloroisothiazolinone/methylisothiazolinone. 1 Associated with PCD.

mutations in FLG , R501X and 2282del4, were [13] . An interventional study found increased found to be positively associated with nickel sen- TEWL and clinical signs of chronic ICD involv- sitization and allergic nickel dermatitis in women ing the hands in nurses, as compared to controls without ear piercings [35] . [36] . In 2002, Nettis et al. [37] reported greater These studies support the significant role of prevalence of ICD (44.4%) than ACD (16.5%) in the skin barrier in the development of OCD, both HCWs, with 20% experiencing both conditions. ICD and ACD. Not surprisingly, occupations They found that patients with occupational ICD with exposure to both irritants and allergens gen- had more exposure to soaps and antiseptics, and erally have high rates of OCD. Following are oc- had the highest duration of daily glove usage. Ir- cupations with high prevalence of OCD, and the ritants include exposure to wet work, skin cleans- most common irritants and allergens are shown ers and antiseptics, and physical irritants such as in table 1 . heat and sweating from use of occlusive gloves. Glove powder may irritate: occlusion, friction Health Care Workers and maceration may increase the skin damage OCD is common in health care workers (HCWs) from other irritants [37] . Rubber accelerators, es- as a result of exposure to both wet work and vari- pecially thiurams, have been the most common ous allergens, especially those found in gloves occupational allergens in HCWs [38, 39] . Thiu-

138 Kasemsarn Bosco Nixon

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 135–143 ( DOI: 10.1159/000441589) rams were the most common rubber accelerators cern [46, 47]. A small in vivo study in nonatopic used in disposable latex and nitrile gloves [39] . hairdressers found that subjects with a lower ir- Immediate hypersensitivity reactions to natu- ritant threshold to sodium lauryl sulfate were ral rubber latex were previously one of the most more likely to develop hand dermatitis [48] . This important causes of skin problems in HCWs, emphasizes the role of easily irritated skin and with reported prevalence rates of up to 14.7% [37, presumably skin barrier impairment in the devel- 40, 41] . Symptoms included urticarial skin rashes opment of OCD. and PCD as well as noncutaneous symptoms, in- Occupational ACD was found to be more cluding rhinoconjunctivitis, asthma and anaphy- common than ICD in hairdressing, and appren- laxis. The latex allergy epidemic caused a shift in tices experienced more ACD than qualified hair- the use of initially powder-free latex gloves to the dressers [10]. The most common occupational al- subsequently nonlatex disposable nitrile gloves, lergens were hair dyes, and bleaching and perm- consequently reducing the rate of latex allergy in ing agents (table 1 ). Recent emerging allergens in HCWs [42] . hairdressers included cysteamine hydrochloride, a preservative in cosmetic and chloroacetamide, Construction Workers used as a glycerol monothioglycolate substitute Cement and construction workers experience [11] . AD is a known risk factor for the develop- high rates of OCD, especially tile settlers and ter- ment of OCD in hairdressers, particularly ICD razzo workers [8] . ACD was more prevalent than [49] . Nevertheless, it is recommended to patch ICD in German construction workers, with the test all hairdressers with dermatitis, regardless of three most common allergens being potassium their atopic status [50] . dichromate, epoxy resin and cobalt chloride [8] . The alkalinity of cement and exposure to water Chefs and Food Handlers are causes of skin irritation and barrier disruption High rates of OCD also occur in food handlers, [43]. In addition, hexavalent chromium in wet including chefs, bakers, butchers and caterers. cement is an important allergen. A recent study Frequent hand washing is common in these oc- of Taiwanese cement workers revealed that high cupations, as well as contact with proteins in chromium exposure was significantly related to meat, seafood, fruits and vegetables. Disruption increased TEWL. They also found that workers of the epidermal barrier by ICD and AD may fa- who smoked and had high chromium exposure cilitate the penetration of high-molecular-weight had increased TEWL compared to nonsmokers proteins, leading to PCD [29, 51] . with low chromium exposure. They concluded ICD was reported to be the most common di- that chromium might induce skin barrier damage agnosis in food handlers in Denmark, followed by and smoking may potentiate its penetration [44] . CU/PCD and ACD [31]. However, in a recent United States study, ACD occurred more fre- Hairdressers quently than ICD in food service workers [52] . Skin barrier impairment in hairdressers often Risk factors for the development of ICD were at- starts early in apprentices, who perform frequent opy, frequent hand washing (>20 times per day) hair washing: a third of them developed hand ec- and contact with squid [53] . The most common zema 1 year after starting training [45] . However, allergens in the US were related to gloves, then hairdressers are additionally in contact with aller- nickel and cobalt [52]. PCD should be considered gens used in hair coloring, bleaching and perm- in the differential diagnosis of hand eczema in ing. The penetration of chemical substances as a food handlers, with skin prick testing being per- result of inappropriate gloves usage is also of con- formed with both fresh food and commercial

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Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 135–143 ( DOI: 10.1159/000441589) food extracts. Vester et al. [31] found that fresh The Interaction of Irritant Contact Dermatitis food yielded more positive results than commer- and Allergic Contact Dermatitis in cial agents. Occupational Diseases

There are often multiple contributing causes to Atopy, Occupational Contact Dermatitis and OCD, and workers are often exposed to both ir- the Skin Barrier ritants and allergens, yet there are few real-life studies on how allergens and irritants may inter- Previous studies have shown that skin atopy is not act. ACD often develops after ICD, which is sup- only a risk factor for OCD, but is related to an un- ported by both in vitro and in vivo studies. A study favorable disease outcome, reflecting the signifi- of DNFB (2,4-dinitrofluorobenzene) in mice by cant role of the skin barrier in OCD [2, 53, 54] . A Bonneville et al. [62] revealed that the intensity of large population-based study by Dickel et al. [54] ACD after the ICD response was proportional to reported that 37% of workers with OSD had an the magnitude of ICD. It was concluded that hap- atopic skin diathesis compared to 20% in the nor- ten-induced irritation conditioned the develop- mal population, particularly those in the food in- ment and severity of ACD. It had previously been dustry, florists and HCWs. An atopic skin diathe- suggested that there was augmentation of skin resis, as indicated by previous or present AD, was sponses in vivo from a combination of exposure to the most relevant predisposing factor to ICD [32, irritants and allergens [63]. Smith et al. [25] pro- 37] . The risk of developing hand dermatitis, par- posed that sensitization in ACD occurred only in ticularly ICD, was increased 3-fold in atopic indi- the presence of a ‘danger signal’ or irritancy. The viduals [49] . irritant substances induced cytokines, which were FLG loss-of-function mutations were identi- released mainly from keratinocytes, and may be fied as a major predisposing factor for AD, espe- required for contact sensitization. It was suggest- cially in a white European population [55] . Natu- ed that activation of innate immunity by irritants ral moisturizing factor in the SC was decreased in reduced the threshold for ACD [15] . carriers of these mutations [18]. While it previ- There are numerous case reports of occupa- ously appeared that FLG mutations and AD to- tional ACD following ICD, usually caused by gether increased the risk of developing ICD, a re- chemicals but occasionally also by mechanical cent cohort study identified that in fact AD was factors [64–66] . Sensitization occurring after a the most important factor and that the additional single exposure has been linked to strong sensi- effect of FLG mutations was not significant [33, tizers, including methylchloroisothiazolinone/ 56] . Skin barrier impairment in AD patients in- methylisothiazolinone, epoxy resin and acrylates, creases the likelihood of epidermal hapten pene- and has occurred simultaneously with severe tration [57] . Repeated exposure to chemicals, es- ICD. It has been proposed that these be termed pecially skin irritants, may cause reactivation of ‘superallergens’ [67] . AD [58, 59]. However, the rate of contact sensitization to all chemical groups except fragrance was inversely associated with the severity of AD com- The Effect of Multiple Irritants pared to controls [60] . Th2 inflammation in AD seems to decrease Th1 response in contact sensi- In real life, just as multiple factors contribute to tization [57] . Guidelines have been developed for OCD, there is exposure to multiple irritants, and the employment of people with AD in high-risk their impact and potential interactions are not occupations [61] . well understood [68] . There may be effects from

140 Kasemsarn Bosco Nixon

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 135–143 ( DOI: 10.1159/000441589) sequential subclinical exposures to irritants as training programs on glove use and education well, influencing the recovery time of the skin [47, 49] . The regular use of emollients has been barrier [69] . beneficial in the treatment of OCD, particularly with regard to repair of the skin barrier, and may also prevent the development of hand dermatitis Prevention of Occupational Contact [21, 70–72]. Workers with severe AD should Dermatitis avoid high-risk occupations, and all workers should be educated to protect their skin, to pre- Various measures have been investigated to vent the development of OCD [54, 61, 73] . Res- prevent the development of OCD, especially toration of skin barrier function is crucial for the in high-risk occupations. These have included recovery of OCD [17] .

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Rosemary L. Nixon, BSc (Hons), MBBS, MPH, FAFOEM, FACD Occupational Research and Education Centre Skin and Cancer Foundation Inc. Level 1/80 Drummond Street Carlton, VIC 3053 (Australia)

E-Mail rnixon @ occderm.asn.au

Skin Barrier in Occupational Skin Diseases 143

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 135–143 ( DOI: 10.1159/000441589) Section IV: Clinical Aspects of Skin Barrier Function

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 144–151 (DOI: 10.1159/000441590)

Wet Work and Barrier Function

Manigé Fartasch Department of Clinical and Experimental Occupational Dermatology, Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Institute of the Ruhr University Bochum (IPA), Bochum , Germany

Abstract substances seemed not to affect the skin negatively. But Wet work defined as unprotected exposure to humid regarding the effect of occlusion, there is experimental environments/water; high frequencies of hand washing evidence that previously occluded skin challenged with procedures or prolonged glove occlusion is believed to sodium lauryl sulfate leads to an increased susceptibility cause irritant contact dermatitis in a variety of occupa- to the irritant with an aggravation of the irritant reaction. tions. This review considers the recent studies on wet- These findings might have relevance for the real-life situ- work exposure and focuses on its influence on barrier ation in so far as after occupational glove wearing, the function. There are different methods to study the effect skin is more susceptible to potential hazards to the skin of wet work on barrier function. On the one hand, occu- even during leisure hours. © 2016 S. Karger AG, Basel pational cohorts at risk can be monitored prospectively by skin bioengineering technology and clinical visual scoring systems; on the other hand, experimental test procedures with defined application of water, occlusion Wet work is one of the most important culprits and detergents are performed in healthy volunteers. harming skin barrier function [1] . Epidemio- Both epidemiological studies and the results of experi- logical studies [2–5] have shown that this type of mental procedures are compared and discussed. A vari- exposure characterizes most occupations where ety of epidemiological studies analyze occupational co- cases of occupational contact eczema of the hands horts at risk. The measurement of transepidermal water occur. It has been recognized as the main risk fac- loss, an indicator of the integrity of the epidermal barrier, tor in a variety of occupations like hairdressing, and clinical inspection of the skin have shown that espe- health care [6], catering, cleaning and housework cially the frequencies of hand washing and water con- [7] , cement workers and construction industry, tact/contact to aqueous mixtures seem to be the main agriculture, wood work, rubber industry and en- factors for the occurrence of barrier alterations. On the gineering [for a review see 8 ]. The ‘repetitive’ ex- other hand, in a single cross-sectional study, prolonged posure to wet work, its duration, intensity and glove wearing (e.g. occlusion for 6 h per shift in clean- frequency are essential determinants for the de- room workers) without exposure to additional hazardous velopment of irritant contact eczema (ICE). Ec- zema occurs when the sequence of events that ir- of glove use by preventing exposure to water ex- ritate the skin rises above a certain frequency for ceeds the believed ‘skin-irritating’ effect of oc- a certain period of time [9] . The main pathogenic clusion-induced perspiration [30] and, further, mechanisms involved here are the damage to the whether its benefit is linked to the duration of skin barrier. exposure and to what extent [20] . The development of occupational ICE and alterations in the skin barrier might facilitate the occurrence (induction) of allergic type IV contact Quantification of Wet-Work Exposure and eczema, type I (immediate-type reaction) reac- Definition tions or even aggravate allergic reactions [10] . Re- garding pathomechanisms, not only enhanced To date, there seems to be no specific and vali- penetration through the skin barrier is discussed dated objective instrument that measures dermal but also simultaneously occurring immunologi- exposure to wet work. None of the current direct cal effects (danger signal) [11–17] caused by the and indirect methods assessing dermal exposure inflammatory reaction. These effects initiate and is suitable for the measurement of the duration modulate cutaneous immune responses to chem- and frequency of wet-work exposure, which ical substances resulting in type IV skin sensitiza- seems to be important for the development of tion or the augmentation of sensitization [18, 19] . ICE. All recognized methods evaluating dermal A number of questions that are important for exposure (discussed by Behroozy and Keegel [8] ) occupational health and safety are still unan- only measure mass or concentration of the con- swered when dealing with exposure to humidity taminant on the skin or surfaces, and none of (e.g. water contact) and/or occlusion regarding them is designed to measure wet-work exposure. the barrier function. The most commonly used methods to assess the quantity of wet-work exposure are by self- reported questionnaires administered to exposed Different Forms of Wet Work individuals [31–34] or by direct observation to see how often and for how long an individual is Experimental studies could demonstrate that exposed to wet work [35–37] . The observation prolonged, intense or frequent contact to mois- method appears to provide more reliable data ture (water) or/and occlusion alone cause ‘irri- than questionnaires, as demonstrated by Jung- tant’ contact eczema, even without the influence bauer et al. [7] , who reported an overestimation of other irritant substances [20–24] . Activities of the duration and an underestimation of the that involve exposure to water, detergents and frequency of wet work during a shift. other skin-irritating substances, frequency of Since there is no clear scientific definition for hand washing [7, 25, 26] and activities that need wet work; some study groups choose definitions to be done with moisture-resistant occlusive which are used in regulatory guidelines. In Ger- gloves are considered to be harmful for the epi- many, regulation of wet work has been proposed dermal barrier – the latter possibly due to ‘skin- and guidelines were introduced by the German irritating’ effects of occlusion-induced perspira- Federal Ministry of Labor and Social Affairs (Ger- tion [7]. On the other hand, wearing protective man technical standards for hazardous substanc- gloves is a worldwide proposed measure [7, 27– es) [38] – wet work is defined as having wet hands 29] to prevent exposure to water and other haz- for more than 2 h per regular work day (per shift), ardous substances. It remains to be determined hand cleansing more than 20 times per day or whether the benefit of the ‘skin-protective’ effect wearing of occlusive gloves for 2 h per day [5, 30,

Wet Work and Barrier Function 145

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 144–151 ( DOI: 10.1159/000441590) 31, 39–41]. Similar guidelines and definitions or clinical inflammatory reactions, was applied. were also published in Australia in 2005 as the These biophysical parameters were usually cor- Australian ‘ASCC guidance on the prevention of related with clinical visual inspections (clinical dermatitis caused by wet work’ (www.safework- score) of the hands. Especially when studying ap- australia.gov.au). Of course, one should be aware prentices, it was demonstrated that an increased that the definition of wet work is a simplified def- basal TEWL (before exposure) did not indicate an inition, which is also used to regulate the duration elevated risk for developing hand eczema [46] . of wet-work exposure in Germany. The exposure On the other hand, some of the studies stated that to humidity and the wearing of occlusive gloves repetitive monitoring of TEWL in the occupa- are here regarded as similar hazards and the dura- tionally primarily exposed area (dorsum of the tions of both forms of exposure are added up [20, hand) may be important for early detection of 42]. The German basic definition does not take subclinical skin irritation [46] . Instead, Kütting et into account that under occupational conditions al. [47] demonstrated that the relevance of TEWL wet work is not ‘water only’ but a combined expo- monitoring in a prospectively followed cohort of sure to water, water-soluble irritants and moist 800 metal workers for up to 1 year was rather low. hands due to glove use. No difference in TEWL (ΔTEWL) of the dominant hand over the study period was detected, and no significant correlation between TEWL Barrier Function and Wet Work: Cohort and the dermatological examination (global skin Studies and Experimental Studies score values) was seen, too. In wet-work occupations, both forms of expo- There are different possibilities to study wet work sures, i.e. humid exposure and occlusion, mostly and its effect on barrier function. One method is occur simultaneously. Recently, in a cross-sec- to monitor cohorts prospectively by bioengineer- tional study, 177 employees of a semiconductor ing methods and by clinical inspection. The other production company were studied. The clean- common method is to develop experimental in room workers were wearing occlusive gloves dur- vivo test procedures in healthy volunteers. Both ing the whole shift. Thus, the study allowed for study types and their outcome are compared and the analyses of the isolated effects of occlusion discussed. [42] . The skin condition of both hands was in- spected and barrier function was monitored via Cohort Studies TEWL and stratum corneum hydration measure- There are studies on barrier function during wet ments. The authors came to the conclusion that work where cohorts at risk, e.g. metal workers prolonged wearing of occlusive gloves with clean [43]; hairdressers [44, 45] and nurses [46] , were hands and without exposure to additional haz- studied prospectively for a certain period of time ardous substances did not seem to affect the skin in field and/or cross-sectional studies [42, 43, 45– negatively. 47]. The evaluation of the epidermal barrier function and the grading of irritation of the skin were Experimental Studies on Water Exposure and mainly performed by noninvasive bioengineering Occlusion and Their Effects on Barrier Function methods: measurements of transepidermal water Unprotected exposure to water [20, 48–51] and loss (TEWL), an indicator of the integrity of the prolonged occlusion [52] are both known to in- epidermal water diffusion barrier, and the hy- duce a variety of skin changes which seem to af- dration of the horny layer. Additionally, quanti- fect morphology [53–55] and function of the epi- fication of erythemas, which reflect subclinical dermal barrier [22, 23, 27, 56]. The effects of di-

146 Fartasch

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 144–151 ( DOI: 10.1159/000441590) rect water exposure [21] on the skin or the effect soaked cotton patches and compared this expo- of occlusion-induced perspiration [52] are com- sure intraindividually with occlusion choosing plex. It is known that water exposure can modify different daily exposure periods (2, 3 and 4 h) for physiologic functions of the skin. Part of the wa- 7 days. Whereas occlusion did not induce mea- ter-associated irritancy may result mainly from sureable alterations in skin physiology, water ex- occlusion or from occlusion as an additive factor posure for more than 3 h daily caused a signifi- [for a review see 21 ]. The changes induced in the cant increase in TEWL. All the mentioned studies epidermal barrier by water are not believed to be could document that water exposure either by a direct effect but rather due to secondary altera- immersion or water-soaked patches is able to in- tions in the horny layer [55] caused by hydration duce barrier alterations with increases in TEWL. with 3- to 4-fold expansion of stratum corneum Previous experimental studies, which had thickness, induction of large pools of water in the mainly focused on occlusion, demonstrated that intercellular spaces and disruption of intercellu- short-term occlusion of healthy skin (4, 6 or 8 h lar lipid structures [54] . Furthermore, the influ- for 7 consecutive days and occlusion for 72 con- ence on epidermal DNA synthesis [57] and other secutive hours) did not seem to induce measur- immunological mechanisms, e.g. the release of able alterations in skin physiology [20, 22, 23, 29, cytokines, may also play a role in the irritancy of 49, 56, 62] or lipid profiles of the epidermal bar- water [58–60] . rier [56] . Only in studies with long-term experi- In experimental settings, different techniques mental exposure, it was proven that occlusion via are applied to study water exposure. One is to closed chambers [63] , a tandem application of study water under occlusion either by water cup cleanser followed by overnight occlusion [24] or occlusion or using large Finn chambers [61] on by prolonged glove occlusion for 6 h/day for 14 the skin or pieces of vinyl gloves fixed with adhe- days [22, 23], induced an elevation in TEWL, a sive tape [20, 24] . It is well known that occlusive sign of a negative effect on skin barrier function. studies have produced most of the experimental On the other hand, when pre-irritation of the skin data on water irritancy [21]. However, using these of the hand or back [22, 23, 49, 64, 65] was fol- procedures, the influence of occlusion cannot be lowed by occlusion [65] or water immersion [49] , separated from the influence of water [21] . The changes in TEWL [61] were the main differences other methods applied to study the effect of wa- in skin physiology, with decreased healing of so- ter on the skin are either immersion of the skin dium lauryl sulfate (SLS)-damaged skin [56] . Far- [49–51] or using water-soaked patches [20, 53] . tasch et al. [20] detected no alteration in barrier Ramsing and Agner [49] studied the effect of function, but 24 h after the 1-week occlusion pro- water on experimentally irritated skin showing cedure, the skin still demonstrated higher suscep- that immersion for 15 min twice daily for 2 weeks tibility to SLS irritation than the control areas, as caused a significant increase in skin blood flow illustrated by amplified barrier disturbance. while clinical evaluation did not reveal a difference. In 2014, Firooz et al. [51] demonstrated that Experimental Comparison of Water Exposure immersion in water for 30 min/day for 5 days al- versus Occlusion and Combination Treatment ready seemed to induce an increase in TEWL. Only a few experimental data are yet available In a study by Kligman [53] , normal skin oc- which compare the skin hazards due to water excluded with water-soaked patches for 2 weeks posure with those induced by occlusion alone (exchanged every 2 days) induced inflammatory [20]. The comparison of water exposure versus reactions on the skin. Fartasch et al. [20] studied occlusion showed that 1 week of water exposure the response to water exposure also via water- (for 3 or 4 h per day) already induces measurable

Wet Work and Barrier Function 147

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 144–151 ( DOI: 10.1159/000441590) barrier alterations with a mild but significant in- irritant reaction with a higher increase in TEWL crease in TEWL values. When the wet-exposed and clinical scores compared to the nontreated and occluded areas were subjected to 24-hour SLS areas. The increase did not show a clear linear irritation, the irritant reaction to SLS was ampli- dose-response relationship since 4 and 6 h of wet fied when wet work (either occlusion or water ex- work did not necessarily result in a stronger in- posure) had been performed in advance with dis- crease in TEWL levels than 3 h. An explanation tinct patterns of response regarding the different for this might lie in the well-known interindi- exposure forms. When healthy skin was occluded, vidual variation in responses of the skin to irri- no significant disruption of the permeability bar- tation and interindividual susceptibility to SLS rier (at least following occlusion for 2–6 h daily) and wet work. Thus, the biological diversity could be detected. This finding is in accordance might be responsible for the lack of clear dose with most of the published bioengineering studies (time)-response relationships. [22, 23, 29, 49, 56, 62] . However, previously oc- Since is not clear in how far the barrier-dis- cluded skin challenged with SLS showed an aug- turbing effects act even additive by the combined mentation of the irritant reaction to the anionic effect of ‘glove occlusion followed by work in wet detergent SLS, which was demonstrated by the in- environments’ in comparison to ‘occlusion alone’ crease in TEWL and visual scores. Possible rea- for the same exposure period, in an additional ex- sons for the increased irritability by occlusion re- perimental approach, in a small number of sub- flected by TEWL and clinical scores might be due jects, a tandem application consisting of a 3-hour to immunological reasons. Occlusion may induce occlusion followed by a 3-hour water exposure subtle subclinical inflammatory reactions leading had been compared with a 6-hour permanent oc- to the release of preformed cytokines, which then clusion [20]. The skin sites which were either ex- aggravate the reaction to SLS or might result in posed to the long-term occlusion or to the tan- increased SLS penetration due to slight structural dem exposure showed significant differences in alterations in the barrier [66] , a mechanism which TEWL compared to the irritated control sites. was shown previously in diseased skin [67] . In an- However, a comparison of the TEWL increase be- other study [56], the effect of SLS on previously tween the two procedures disclosed no statistical- occluded skin (8 h for 7 consecutive days and oc- ly significant differences. The present study sug- clusion for 72 consecutive hours) was also ana- gests that occlusion followed by water exposure lyzed. No significant differences regarding the compared to occlusion alone seemed not to lead susceptibility to SLS irritation were found in the to more pronounced inflammatory reactions un- occluded areas compared to the nonoccluded der the given circumstances. control area. The difference in the results may be Of course, this approach had its limitations due to the fact that the application of SLS had al- since the duration of occlusion of the tandem ap- ready been performed 4 h after the last occlusion plication [20] had been in the range of 3 h, which (and not after 24 h like in other regimens [20] ). was previously found to influence barrier proper- Furthermore, a higher concentration of SLS had ties only slightly, and it might well be that a longer been used (1 vs. 0.5%) to induce an irritation. One duration of occlusion followed by water exposure could speculate that the induction of a stronger might increase the susceptibility of the skin to clinical irritation may suppress subtle differences chemical substances; furthermore, only 12 sub- regarding increased SLS penetration or already jects participated in this group. Thus, this expo- existing subclinical local inflammatory responses. sure procedure requires further study, especially In a study by Fartasch et al. [20] , the wet- since the risk of hand eczema from wet work work-pretreated areas demonstrated a stronger might be related to the frequency of glove use and

148 Fartasch

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 144–151 ( DOI: 10.1159/000441590) wetting/washing cycles rather than the total dura- gloves), the irritant effect of detergent might be tion of wet work. aggravated, and the skin seems to be more prone In experimental studies comparing the differ- to react to chemical stress. This might have impli- ent forms of wet-work-induced barrier disrup- cations for the ‘domestic exposure after work’ tion, skin hydration by occlusion had a different since the skin seems to be more prone to react to biological impact on the skin than water expo- stress, e.g. domestic wet work with irritants. This sure. This study further provides experimental might be of particular relevance for the occur- data that the ‘skin-protective’ effect of glove use rence of ICE in female employees, since due to a by preventing water exposure might be greater higher susceptibility of the skin after wet work than the believed ‘skin-irritating’ effect of occlu- (even occlusion by gloves), even minimal domes- sion-induced perspiration. tic wet work exposure and house cleaning proce- Further, there is experimental evidence that dures during leisure hours [1, 32, 33] might in- after water exposure or occlusion (the use of crease the risk to acquire ICE.

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Prof. Dr. med. Manigé Fartasch Department of Clinical and Experimental Occupational Dermatology Institute for Prevention and Occupational Medicine of the German Social Accident Insurance Institute of the Ruhr University Bochum (IPA) Bürkle-de-la-Camp Platz 1 DE–44789 Bochum (Germany)

E-Mail fartasch @ ipa.ruhr-uni-bochum.de

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Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 144–151 ( DOI: 10.1159/000441590) Section IV: Clinical Aspects of Skin Barrier Function

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 152–158 (DOI: 10.1159/000441592)

Saving the Barrier by Prevention

Elke Weisshaar Department of Clinical Social Medicine, Occupational and Environmental Dermatology, University Hospital Heidelberg, Ruprecht Karls University, Heidelberg , Germany

Abstract search in Germany has shown that the different measures One third of all occupation-related diseases are diseases of prevention in occupational dermatology are very ef- of the skin, and in most of these cases the skin barrier is fective. This integrated concept of an in-/outpatient dis- involved. Professions such as metalworkers, hairdressers, ease management reveals remarkable pertinent efficacy and health care and construction workers are mainly af- for patients with severe occupational dermatoses in at- fected. Among them, contact dermatitis is the leading risk professions. © 2016 S. Karger AG, Basel skin disease. It usually presents as hand eczema caused by or leading to impaired barrier function. All this significantly impacts the function of the hands, reduces the ability to work and especially impairs the patient’s qual- Measures of prevention have been developed and ity of life. Diagnostics and therapy are of great impor- implemented in the field of occupational derma- tance; in addition, prevention programs are meanwhile tology in Germany, and evidence for their effica- an important mainstay of the overall therapeutic con- cy has recently been evaluated. Their aim is to cept. They comprise measures of secondary (outpatient) contribute permanently to the prevention of oc- and tertiary (inpatient) prevention. Secondary preven- cupational dermatoses and to amend the medical tion measures include occupation-tailored teaching and care of patients with occupational skin diseases prevention programs, and the dermatologist’s examina- (OSDs) [1–6] . These concepts are mainly based tion and report. In severe cases or if therapy is not suc- on the idea of an integrative medical and educa- cessful in the long term, or if the diagnosis is not clear, tional health promotion [3] . All measures of pre- measures of tertiary prevention may come into action. vention require a team of well-trained staff in- They are offered as an inpatient treatment and preven- cluding especially dermatologists trained in oc- tion program. The aims are prevention of the job loss, but cupational dermatology. especially to reach a long-term healing up and getting There are measures of primary prevention to back to normal occupational and leisure life in the sense reduce the risk of occupational dermatoses (e.g. of attaining full quality of life. During the last years, re- by laws on work protection and health educational instructions) and measures of secondary pre- and moisturizers. These aspects are presented vention that have been developed as individual elsewhere in this book and will not be discussed prevention concepts. Depending on the severity in this chapter. Such measures obey national legal of the skin disease, they can be realized as out- regulations, and follow national and European patient dermatological care, seminars for outpa- standards. A very recent example is a research tients on skin protection and advisory services to project called ‘SafeHair’, which runs in the frame- companies [2, 5–15] . Tertiary individual pre- work of an EADV (European Academy of Der- vention (TIP) includes integrated in-/outpatient matology) campaign and aims to prevent OSD by health educational and medical care interven- defining common standards of safety and health tions for severe occupational dermatoses [16–19] . in hairdressing [1] . In Germany, analogous to this concept, the workers’ statutory insurance introduced the so-called administrative procedure ‘Skin’ in 2004 [1, 6] . Measures of Secondary Individual Prevention Administrative supervision is well organized in Germany using a hierarchical multistep interven- Measures of SIP (outpatient) are applied if a tion advanced by the German social security sys- person suffers from a skin disease that is most tem including a compulsory statutory accident likely caused by the occupation and usually insurance. It must provide measures of preven- presents with initial signs [1, 6]. Secondary pre- tion including all suitable means because this in- vention aims at early detection and diagnostics surance aims to prevent work-related risks and of OSD and increasing opportunities for inter- occupational diseases. ventions to prevent chronification or progres- There is no unique term for skin protection sion of the skin disease. Measures of secondary measures used in secondary and tertiary preven- prevention include diagnostics and treatment, tion. They are called educational training pro- teaching offers and psychological understand- gram, skin protection program, skin protection ing as offered in a skin protection seminar and seminar, educational seminar, skin care educa- improvement of working conditions [1, 6]. The tion, secondary individual prevention (SIP) sem- aims of such a skin protection seminar are pre- inar or interdisciplinary prevention program [5– sented in table 1 . In Germany, one important 15] . In this chapter, the term ‘skin protection measure is to start the dermatologist’s report seminar’ is used. procedure (so-called ‘Hautarztbericht’). Fur- thermore, a skin protection seminar is offered to affected patients, usually in the form of a so- Measures of Primary Prevention called SIP seminar. In the mid-90s, clinics specialized in occupational dermatology initiated Primary interventions to prevent skin barrier the first skin protection seminars for hairdress- dysfunction aim to avoid OSD in healthy individ- ers in cooperation with the statutory accident uals and to keep an intact barrier function, espe- insurance in charge. In the course of the subse- cially in workplaces with hazardous exposures to quent years, similar seminars were developed the skin. Measures of primary prevention com- for other professions, such as health care profes- prise for example the risk assessment of the work- sions, kitchen workers, cleaners and metal- place, technical means, safety regulations, avoid- working professions [7–14] . In the context of ance of direct skin contact with irritants and indi- those seminars, the statutory accident insurance vidual skin protection, e.g. the use of protective can induce additional preventive measures gloves and the application of skin barrier creams ( table 2 ). When attending a skin protection se-

Saving the Barrier by Prevention 153

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 152–158 ( DOI: 10.1159/000441592) Table 1. Objectives of the skin protection seminars for SIP (outpatient) as well as within TIP [modified according to ref. 6, 7]

Knowledge enhancement about skin diseases, allergies and their causes Generating strategies to protect the skin during working procedures and to reduce risk behavior Improving skin-protective measures (technical, organizational and personal) and instructions for the application of effective skin protection (including gloves, skin cleaning and skin care) Enhancement of health awareness, motivation for the transfer of discussed measures into the work situation Instructions for coping with disease episodes Knowledge enhancement in terms of insurance aspects regarding the skin disease, e.g. dermatologist’s procedure (If applicable, proposals to) completion of diagnostics (If applicable, proposals to) more efficient therapy Better, faster and more effective medical and occupational rehabilitation Avoiding aggravation of the skin disease and job loss Reducing follow-up costs So-called side effects, e.g. distribution of acquired knowledge to colleagues, family and friends If applicable, inspection of the workplace If applicable, assessment of further skin diseases and initiation of suitable therapy

Table 2. Potential measures of SIP programs of the workers’ accident insurances after the seminar according to the procedure Skin of the German statutory accident insurance [modified according to ref. 6, 7]

Pass on proposed diagnostic and therapeutic measures to the dermatologist in charge Initiation of an outpatient treatment measure (treatment order according to §3 BKV) Initiation of inpatient treatment measures (TIP) Optimization of individual skin protection measures (e.g. gloves and skin protection, care and cleaning) Measures directly including the workplace (e.g. workplace inspection, information of the company physician, modification of workplace conditions, job change within the company and replacement of an occupational substance/allergen) Initiation of a disease assessment process (‘Feststellungsverfahren’) including an expert’s report

minar, the patients receive a thorough dermato- ports and studies showed that the participating logical examination by a dermatologist and patients assessed very positively that there was teaching units in health education, which usu- sufficient time to share experiences with other ally last 1–2 days [1, 7]. This is also very impor- patients and to ask questions during the semi- tant for the reason that many affected patients nars [1, 6–11] . More than one third of the par- who are working in professions or environ- ticipants showed severe skin lesions when at- ments that may be hazardous to the skin do not tending the seminar [7] . Further studies con- apply any measures of skin protection and skin firmed the positive effects of the skin protection care in a sufficient manner and are neither seminars. For example, 1 year after participa- aware of the harmful effects on the skin nor of tion, 72% of the participants showed a signifi- the high importance of prevention measures cant improvement in skin disease and quality of [7–11] . All this also aims to support the pa- life (QOL), and skin protection measures were tient’s dermatologist at home [7–11] . Several re- applied significantly more frequently [10–12] .

154 Weisshaar

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 152–158 ( DOI: 10.1159/000441592) There are only limited controlled clinical stud- dressers showed that even after 5 years, signifi- ies dealing with the efficacy of skin protection cantly more hairdressers of the intervention seminars as a measure of SIP. In Germany, this is group had continued their profession than in the due to the fact that this kind of studies can hardly control group, which consisted of 85 patients who be realized, as the accident insurances, according had received dermatological therapy but no skin to the German Code of Social Law (SGB VII, §14), protection seminar, and the result was confirmed are liable to grant prevention measures against by another 10-year follow-up study [5] . work-related health hazards and occupational The efficacy of a secondary prevention pro- diseases. It would be illegal to ban patients from gram with education in skin care and individual preventive measures. Thus, control groups pre- counseling including allergy tests and assessment senting these problems, but not receiving preven- of work- and domestic-related exposures has re- tion measures are difficult to be found in Ger- cently been shown in comparison to a control many. A systematic review on the effectiveness of group receiving exclusively dermatological thera- prevention programs for hand dermatitis taking py in a randomized clinical trial in Denmark [22] . into account all studies until January 2010 could Health care employees (n = 255) with self-report- only retrieve 7 controlled studies, most of which ed hand eczema were included and followed-up were not conducted in Germany [20] . Moderate for 5 months. The primary objective of the study evidence for the effect of prevention programs on was clinical severity of the disease, and secondary lowering OSD occurrence and improving adher- outcomes were severity of hand eczema, QOL, ence to preventive measures, and low evidence self-evaluated severity of hand eczema, skin-pro- for a beneficial effect on clinical and self-reported tective behaviors and knowledge of hand eczema outcomes were found. No studies reporting on from onset to follow-up. Results showed that the costs were identified. It can be concluded that intervention decreased hand eczema severity and there is moderate evidence for the effectiveness of improved QOL, and had positive effects on self- prevention programs of hand eczema versus usu- evaluated severity and skin protection behaviors, al care or no intervention [20] . However, mean- including knowledge [22] . while new studies on the effectiveness of skin protection and skin care, for example, were not included. Recent German studies showed that Measures of Tertiary Individual Prevention structured skin protection seminars improve the knowledge on OSD (an effect which was still evi- The intervention strategies comprise a program dent after 3 months [21] ) and behavioral param- of measures stepwise increasing in intensity. If eters such as measures of skin care and skin pro- the above-mentioned measures of primary pre- tection 1 year after attendance [11]. A long-term vention and SIP are not sufficient, or if patients study among elderly care nurses showed that even suffer from particularly severe and therapy-re- 6 years later, parameters such as remaining in the fractory OSD, a further module will be applied: a occupation, severity of hand eczema and skin TIP measure in the form of an interdisciplinary, protection behavior were improved when the modified inpatient treatment and prevention nurses had taken part in a skin protection semi- program. This program includes several phases nar and a refresher seminar [14] . Similar success which are described in the following. was reported in a study including 134 patients The first phase usually consists of an inpatient with work-related hand eczema, presenting better treatment measure lasting 3 weeks in an institu- effects in mild hand eczema and worse effects in tion specialized in occupational dermatology severe ones [15] . A 5-year follow-up in 215 hair- [16] . In the course of the interdisciplinary inter-

Saving the Barrier by Prevention 155

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 152–158 ( DOI: 10.1159/000441592) vention program, allergological and skin physio- continuation of the therapeutic and preventive logical diagnostics, as well as phase-adapted der- measures initiated. This inpatient phase is fol- matological therapy are conducted, in which ste- lowed by another 3-week absence of work under roid-free treatment is favored. Dermatological outpatient dermatological care in the patient’s treatment aims to achieve complete healing of the hometown in order to make sure that the treat- skin disease and to restore the skin barrier func- ment success achieved in the inpatient program is tion [this vol., pp. 123–134; 144–151]. Besides the stabilized without occupational skin burden in the intensification and optimization of the dermato- patient’s private environment. This phase mainly logical therapy, intensive health educational and aims to completely restore the barrier function of health psychological interventions (including the skin because it is known that this time is need- health-related pedagogic and psychological coun- ed after healing of hand eczema to restore the nor- seling) are undertaken to increase motivation and mal skin barrier function. A third phase consists knowledge, and to effect modifications in attitude of the resumption of work under optimized skin and behavior in terms of an adequate application protection measures and continuation of the out- of skin protection measures, always keeping con- patient dermatological treatment by the local der- ditions of professional and individual workplaces matologist according to §3 BKV. in mind [12] . The recommended skin protection This package of treatment measures was evalu- measures can be tested and practiced in workplace ated in the framework of a prospective multicenter simulation models. The skin protection items, German cohort study [16–19] . It was funded by the such as gloves and skin barrier creams, are later German Statutory Accident Insurance (Deutsche handed over to the patient for further use at the Gesetzliche Unfallversicherung) from 2005 to 2013 workplace. During this phase, employees of the [Multicenter study ‘Medical-occupational in-pa- workers’ accident insurances offer regular (most- tient treatment program skin-optimization and ly weekly) consulting hours during TIP measures quality assurance of the treatment measure to answer questions of TIP participants concern- (ROQ)’]. The long-term evaluation was extended ing employers’ responsibilities, reimbursement of over 3 years from the beginning of TIP (inpatient workers’ accident insurances and long-term pro- treatment program). The study included 1,788 pa- vision of skin protection items, for example. Tech- tients with a mean age of 43.2 years (range 17–67 nical or workplace-related prevention issues in- years). They had the following professions (poten- cluding the occurrence of an identified allergen tially hazardous to the skin): 29.4% health care can be discussed with the insurance prevention professionals, 27.4% metalworkers, 10.1% hair- service. An essential part of the treatment concept dressers, 8.8% construction workers, 6.2% food forms the coordination of the ensuing treatment handlers, 4.5% cleaners and 3.5% workers in chem- by the local dermatologist after the inpatient treat- ical industry (10.0% others); 82.5% of the pa- ment measures [in Germany according to §3 BKV tients could be followed up for 3 years. No signifi- (German Occupational Disease Act provided as a cant difference surfaced in comparison to the drop- so-called treatment agreement by the workers’ ac- out cases (intention-to-treat analysis) [17] . cident insurance)]. The local dermatologist is in- The majority of the patients (n = 1,670; 93.4%) formed about the fact that the patient attends a suffered from hand eczema, and most presented TIP procedure before the inpatient measure starts with mixed forms (several etiologies): 81.3% and a treatment report is promptly sent usually on showed an irritative, 55.0% an atopic and 39.6% the day of discharge. It contains precise recom- an allergic component [17] . A fundamental aim of mendations concerning dermatological therapy the inpatient measure was the reduction in thera- and disease prevention to guarantee a seamless pies potentially rich in side effects in terms of the

156 Weisshaar

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 152–158 ( DOI: 10.1159/000441592) integrity of epidermal barrier function. Most pa- In summary, these effects remained stable for tients (89.4%) reported to have applied topical the whole follow-up period of 1–3 years after the glucocorticoids prior to the inpatient treatment, inpatient measure [6, 18, 19]. In total, 97% of the with 52.5% of patients using highly potent topical participants were able to resume work within a class III and IV ones. In the course of the inpatient 3-year period after completing the inpatient treat- treatment and during the following 3 weeks, a ste- ment measure. Of the participants, 83% contin- roid-free therapy could be performed in 93.2% of ued to pursue an occupation, most of whom the patients [17]. A reduction in glucocorticoid (70.6%) managed to remain in their original pro- use could be achieved even in the long term, i.e. 3 fession. Analysis of a subgroup indicates that this years after the inpatient treatment, 60% of all pa- effect is fundamentally linked to the educational tients still did not require glucocorticoids. Fur- and motivational measures of the prevention pro- thermore, the percentage of patients applying gram [6, 19] . class III and IV glucocorticoids could be halved to 24.6% (vs. 52.5% prior to the inpatient treatment measure) [17] . During the inpatient phase, a sig- Conclusion nificant improvement in OSD severity [measured using OHSI (Osnabrück Hand Eczema Severity It can be concluded that the intensified efforts of Index)] and a significant increase in QOL accord- the SIP combined with the TIP program are suc- ing to the Dermatology Life Quality Index could cessful. This also includes patients with advanced be noted [17]. Even 1 year later, we still observed OSD and therapy-refractory disease courses. Due a significant reduction in OSDs, use of topical glu- to this concept, most patients can resume their cocorticoids and the number of days of absence profession with a significantly improved skin sta- from work [18] . Very impressively, QOL was sig- tus and increased QOL. All this demonstrates the nificantly improved [18] . long-term benefit of TIP.

References

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Elke Weisshaar, MD, PhD Department of Clinical Social Medicine, Occupational and Environmental Dermatology University Hospital Heidelberg, Ruprecht Karls University Thibautstrasse 3 DE–69115 Heidelberg (Germany)

E-Mail elke.weisshaar @ med.uni-heidelberg.de

158 Weisshaar

Agner T (ed): Skin Barrier Function. Curr Probl Dermatol. Basel, Karger, 2016, vol 49, pp 152–158 ( DOI: 10.1159/000441592) Author Index

Agner, T. 38 Elsner, P. 61 Kasemsarn, P. 135 Angelova-Fischer, I. 80 Engebretsen, K.A. 90 Kezic, S. 1 Antonov, D. 61 Fartasch, M. 144 Lodén, M. 112 Benfeldt, E. 103 Fluhr, J.W. 71 Bosco, J. 135 Nielsen, J.B. 103 Bouwstra, J.A. 8 Giménez-Arnau, A. 123 Nixon R.L. 135 Brandner, J.M. 27 Holmgaard, R. 103 Schliemann, S. 61 Clausen, M.-L. 38 Jakasa, I. 1 Thyssen, J.P. 90 Danby, S.G. 47 van Smeden, J. 8 Darlenski R. 71 Weisshaar, E. 152

159 Subject Index

ACD, see Allergic contact dermatitis Barrier cream 128, 129 AD, see Atopic dermatitis Basement membrane (BM), tight junction interactions Aging 33, 34 Raman confocal spectroscopy findings 75, 76 BM, see Basement membrane skin barrier function effects 49, 50 Allergic contact dermatitis (ACD), see also Occupational Cathelicidins, see Antimicrobial peptides skin diseases Ceramidase, atopic dermatitis findings 19 irritant contact dermatitis interactions 140 Ceramide synthase, atopic dermatitis findings 19 overview 137 Claudin, see Tight junction Antimicrobial peptides (AMPs) Contact allergy, see Atopic dermatitis; Irritant contact barrier disruption effects on expression 40–42 dermatitis; Psoriasis cathelicidins 39, 40 Contact dermatitis, see Allergic contact dermatitis; defensins 39 Irritant contact dermatitis; Occupational skin dermicidin 40 diseases infection response 42, 43 Contact urticaria (CU), overview 137 RNase7 40 CU, see Contact urticaria skin expression 38–40 tight junction interactions 31, 32 Defensins, see Antimicrobial peptides Atopic dermatitis (AD) Dermicidin, see Antimicrobial peptides contact allergy 94–96 Desquamation, dry skin 124 filaggrin mutations 2–4, 9, 52, 125, 126, 140 Dry skin, see Xerosis immune response 52, 53 moisturizer studies 118 Eczema, see Hand eczema; Irritant contact dermatitis pathophysiology 93, 94 Emollient, see Moisturizer Raman confocal spectroscopy findings 76 skin barrier function 50–53, 94 Filaggrin (FLG) stratum corneum lipids atopic dermatitis mutations 2–4, 9, 52, 125, 126, 140 ceramidase dysfunction 19 knockdown mouse studies of barrier function 4 ceramide synthase dysfunction 19 knockout mouse 2, 5 composition 14–17 mutation effects on lipid barrier properties 19, 20 elongase dysfunction 18, 19 occupational skin disease mutations 137, 138, 140 glucosylcerebrosidase dysfunction 19 skin barrier function 1, 2 organization of lipids 17, 18 FLG, see Filaggrin serine palmitoyl transferase dysfunction 19 Fluorescence spectroscopy, skin barrier function sphingomyelinase dysfunction 19 assessment principles 65 sphingomyelin deacetylase dysfunction 19

160 Glucosylcerebrosidase, atopic dermatitis findings 19 Occupational skin diseases (OSDs), see also Allergic contact dermatitis; Contact urticaria; Irritant contact Hand eczema, overview 126 dermatitis Humectants, barrier function restoration 128, 129 irritant contact dermatitis-allergic contact dermatitis interactions 140 ICD, see Irritant contact dermatitis multiple irritants in development 140, 141 Ichthyosis overview 135, 136 overview 126 prevention, see Prevention, skin barrier dysfunction skin barrier function effects 53 skin barrier studies Inflammation atopy effects 140 atopic dermatitis 53 construction workers 139 stratum corneum lipid metabolism and barrier filaggrin mutations 137, 138, 140 properties 20, 21 food handlers 139, 140 Irritant contact dermatitis (ICD), see also Occupational functional overview 136 skin diseases hairdressers 139 allergic contact dermatitis interactions 140 health care workers 138, 139 contact allergy 97, 98 types 136, 137 overview 97, 136, 137 wet work, see Wet work skin barrier function 97 OCT, see Optical coherence tomography Irritants Optical coherence tomography (OCT), skin barrier erythema and skin microcirculation 85 function assessment principles 66 skin barrier function assessment OSDs, see Occupational skin diseases repeated short-time occlusive irritation test 67 repetitive irritation test 67 Penetration, see Percutaneous penetration skin barrier function effects Percutaneous penetration homeostasis 80, 81 experimental models 109, 110 skin hydration studies 83, 84 factors affecting skin surface pH studies 84, 85 biological factors 108, 109 transepidermal water loss studies 81, 82 physicochemical factors 109 Fick’s law of diffusion 104, 105 Laser scanning microscopy (LSM), skin barrier function modeling 103, 104 assessment principles 66 techniques for study Lipids, see Stratum corneum flow-through diffusion cell 106, 107 LSM, see Laser scanning microscopy microdialysis 107, 108 microperfusion 108 Microdialysis, percutaneous penetration studies 107, overview 105, 106 108 static diffusion cell 106 Moisturizer Prevention, skin barrier dysfunction adverse reactions 118, 119 overview 141, 152–153 barrier function restoration 127, 128 primary prevention measures 153 effects on skin secondary individual prevention measures 153–155 barrier-diseased skin 117, 118 tertiary individual prevention measures 155–157 experimentally damaged skin 116 xerosis 131 healthy skin 115 Primary prevention, see Prevention, skin barrier hyperkeratotic skin 116, 117 dysfunction emulsion properties 113 Psoriasis selection 113, 114 contact allergy 92, 93 skin interactions 114, 115 immune response 91, 92 skin permeability measurement 115–117 skin barrier function 92

Natural moisturizing factor (NMF) Raman confocal spectroscopy (RCS), skin barrier atopic dermatitis 20 function assessment Raman confocal spectroscopy 72, 75–77 aging studies 75, 76 NMF, see Natural moisturizing factor atopic dermatitis studies 76

Subject Index 161 interpretation 73–75 Tape stripping, skin barrier function assessment natural moisturizing factor 72, 75–77 principles 64, 65 physical basis 72 Tertiary individual prevention, see Prevention, skin principles 65 barrier dysfunction schematic overview 73 TEWL, see Transepidermal water loss topical substance penetration effects 77 Tight junction (TJ) RCS, see Raman confocal spectroscopy antimicrobial peptide interactions 31, 32 Repeated short-time occlusive irritation test (ROIT), barrier function 28 skin barrier function assessment principles 67 basement membrane interactions 33, 34 Repetitive irritation test (RIT), skin barrier function boundary between microbial and immunological assessment principles 67 barriers 34 RIT, see Repetitive irritation test chemical barrier interactions 31 RNase7, see Antimicrobial peptides functional overview 27 ROIT, see Repeated short-time occlusive irritation test immunological barrier interactions 32, 33 microbiome barrier interactions 30, 31 SC, see Stratum corneum nonbarrier functions of proteins 28, 29 Secondary individual prevention, see Prevention, skin proteins 27, 28 barrier dysfunction stratum corneum interactions 29, 30 Serine palmitoyl transferase dysfunction 19 TJ, see Tight junction Skin surface pH Transepidermal water loss (TEWL) irritant studies 84, 85 irritant studies 81–83 skin barrier function assessment principles 64 skin barrier function assessment principles 61–64, Sphingomyelinase, atopic dermatitis findings 19 136 Sphingomyelin deacetylase, atopic dermatitis findings 19 Wet work Stratum corneum (SC) barrier function studies developmental changes 40, 41 cohort studies 146 functional overview 1, 2, 9, 90, 91 experimental studies 146–149 lipids definitions 145, 146 atopic dermatitis forms 145 ceramidase dysfunction 19 overview 144, 145 ceramide synthase dysfunction 19 quantification 145 composition 14–17 elongase dysfunction 18, 19 Xerosis glucosylcerebrosidase dysfunction 19 barrier function organization of lipids 17, 18 overview 123, 124 serine palmitoyl transferase dysfunction 19 prevention of disorders 131 sphingomyelinase dysfunction 19 protection standards 129–131, 132 sphingomyelin deacetylase dysfunction 19 restoration standards 127–129 composition in healthy individuals 9–14, 72 overview 125 filaggrin mutation effects on lipid barrier skin barrier function effects properties 19, 20 frequent washing-induced xerosis 54, 55 inflammation effects on metabolism and barrier winter xerosis 55 properties 20, 21 xerosis cutis 53, 54 natural moisturizing factor and pH 20 treatment 128, 129 prospects for study 21, 22 penetration, see Percutaneous penetration tight junction interactions 29, 30 Stratum corneum hydration, skin barrier function assessment principles 64

162 Subject Index Current Problems in Dermatology Editors: P. Itin, G.B.E. Jemec ISSN 1421–5721

46 Actinic Keratosis Editors: H.P. Soyer, T.W. Prow, Brisbane, Qld.; G.B.E. Jemec, Roskilde VIII + 154 p., 33 fig., 29 in color, 4 tab., hard cover, 2015. ISBN 978–3–318–02762–4

47 Alopecias – Practical Evaluation and Management Editors: D. Ioannides, Thessaloniki; A. Tosti, Miami, Fla. VIII + 164 p., 85 fig., 80 in color, 13 tab., hard cover, 2015. ISBN 978–3–318–02774–7

48 Tattooed Skin and Health Editors: J. Serup, Copenhagen; N. Kluger, Helsinki; W. Bäumler, Regensburg X + 258 p., 110 fig., 85 in color, 25 tab., hard cover, 2015. ISBN 978–3–318–02776–1

49 Skin Barrier Function Editor: T. Agner (Copenhagen) VIII + 164 p., 26 fig., 6 in color, 14 tab., hard cover, 2016. ISBN 978–3–318–05585–6

Although a very fragile structure, the skin barrier is probably one of the most important organs of the body. Inward/out it is responsible for body integrity and outward/ in for keeping microbes, chemicals, and allergens from penetrating the skin. Since the role of barrier integrity in atopic dermatitis and the relationship to filaggrin mutations was discovered a decade ago, research focus has been on the skin barrier, and numerous new publications have become available. This book is an interdisciplinary update offering a wide range of information on the subject. It covers new basic research on skin markers, including results on filaggrin and on methods for the assessment of the barrier function. Biological variation and aspects of skin barrier function restoration are discussed as well. Further sections are dedicated to clinical implications of skin barrier integrity, factors influencing the penetration of the skin, influence of wet work, and guidance for prevention and saving the barrier. Distinguished researchers have contributed to this book, providing a comprehensive and thorough overview of the skin barrier function. Researchers in the field, dermatologists, occupational physicians, and related industry will find this publication an essential source of information.