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Didier Pin Dermatology and dermatopathology Unit LONG-TERM PARTNERS PRE-CONGRESS SYMPOSIUM / ESVD - ECVD 2011 ICF session Skin barrier function and stratum corneum hydration A major function of the skin is to provide a protective barrier at the interface between the hostile external environment, the “outside”, and the organism, the “inside” (Elias et al., 2003; Proksch et al., 2008). This protective barrier is mainly represented by the epidermis which comprises the physical, the chemical or biochemical (antimicrobial, innate immunity) and the adaptive immunological barriers. Table 1 shows the multiple protective functions of mammalian stratum corneum (SC) and their principal compartment, structural and chemical bases, and known regulatory signals. Here, we are focusing on the permeability barrier, since the formation of a permeability barrier that impedes transcutaneous loss of water is of major importance, because it is required for life in a desiccating, terrestrial environment. While each protective function of the skin can be considered as a discrete process, these individual functions are often kinked and even co-regulated. Under normal circumstances, epidermis must be as impermeable as possible except for a small amount of water loss to hydrate the outer layers of the SC. Although all living cells require water to function, some “dead” cells also require water to be metabolically active. As it is the case of corneocytes, epidermis must retain sufficient water to (a) hydrate the outer layers of the SC to maintain its flexibility and deformability and (b) to provide enough water to allow enzyme reactions that facilitate SC maturation events, together with corneodesmolysis and ultimately desquamation (Rawlings, 2006). Where is the barrier and how does it form? Although the deep nucleated epidermis play a role, particularly in the generation of the SC, most of the critical protective functions against external insults localize to the outermost, anucleated layers of the epidermis, the SC and are mediated by either the corneocytes or the extracellular matrix (Elias, 2007). The corneocytes are keratinocyte-derived anucleated cells providing structural support for the SC. They lack intracellular organelles that are degraded during the final differentiation of keratinocytes. During the final stages of normal differentiation, keratins are aligned into highly ordered and condensed arrays through interactions with filaggrin, which is the VetAgro Sup, Campus Vétérinaire de Lyon – France 2011 1 Didier Pin Dermatology and dermatopathology Unit proteolytic product of profilaggrin, synthesized in the keratinocytes of the stratum granulosum (SG) and contained in the keratohyalin granules. Filaggrin functions as an intermediate filament-associated protein (IFAP) to aggregate keratin filaments into macrofibrils which compose the matrix filling the cytosol of the corneocytes. The corneocytes are 0.2 to 0.3 µm thick and their diameter is 30 to 50 µm (Pouillot et al., 2008). They are surrounded by a cell envelope composed of an inner cornified cell envelope and an outer corneocyte-bound lipid envelope, which is a plasma membrane-like structure and replaces the plasma membrane on the external aspect of mammalian corneocytes. The cornified envelope which is 0.015 to 0.02 µm thick, results of cross-linking of specialized cornified envelope structural proteins, including loricrin, involucrin, trichohyalin and the class of small prolinrich proteins, by both disulphide bonds and N-epsilon-(gamma-glutamyl)lysine isopeptide bonds formed by transglutaminases. The corneocyte lipid envelope is a 0.05-µm-thick structure of ω-hydroxyceramides with very long chain N-acyl fatty acids covalently attached by ester linkage to the proteins of the cornified envelope (Proksch et al., 2008). These lipids also interdigitate with the intercellular lipid lamellae. Corneocytes are interconnected by corneodesmosomes and tight junctions which both play a role in the barrier function and physiological desquamation process (Haftek et al., 2011). The intercellular lipid matrix, located between corneocytes, constitutes about 10 to 15% of the dry weight of SC. The lipid species of the SC are composed of 50% ceramides, 10% fatty acids, 25% cholesterol, and 15% cholesterol and glucosylceramide derivatives. The majority of lipids of the SC are synthesized by the keratinocytes in the upper layer of the stratum spinosum (SS) and the SG. At the SS-SG interface, fatty acids, sphingolipids, and other lipid precursors are extruded to reside in the intercellular lipid matrix where they form extracellular, nonpolar, lipid-enriched lamellar membranes that are impermeable to water (Elias et al., 2003). In fact, there are two distinct states of the intercellular lipid matrix: the non permeable (gel) and the permeable (liquid crystalline) matrix, the latter being permeable to water and electrons. In contrast, the cornified envelope, the corneocyte lipid envelope and the intercellular lipid matrix create a dense and impermeable network (Pouillot et al., 2008). The lamellar bodies play a central role in the barrier edification. The epidermal lamellar body (LB, often called Odland body) is an ovoid, 0.25 to 0.3 µm membrane bilayer-encircled, secretory organelle that is unique to mammalian epidermis and some other keratinizing epithelia. They contain not only pro-barrier lipids and their respective lipid processing enzymes, but also additional structure proteins (e.g. corneodesmosin), antimicrobial peptides (e.g. hBD2 and LL-37), proteases (stratum corneum chymotryptic enzyme, SCCE, stratum corneum tryptic enzyme, SCTE, and probably aspartate or cysteine proteases) and their inhibitors (elafin or skin-derived antileukoprotease, SKALP, secretory leucocyte protease inhibitor, SLPI, lymphoepithelial kazal-type protease inhibitor, LEKTI, and the cysteine protease inhibitor, cystatin C/K) that participate in cohesion, desquamation, and antimicrobial function (Elias, 2003; Elias, 2007). VetAgro Sup, Campus Vétérinaire de Lyon – France 2011 2 Didier Pin Dermatology and dermatopathology Unit Sources of stratum corneum hydration The state of SC hydration depends on: - the rate at which water reaches the SC from the tissue below, - the rate at which water leaves the skin surface by evaporation, - the ability of the SC to retain water. The SC uses three main mechanisms to hold onto water: - the intercellular lamellar lipids, whose physical conformation, predominantly an orthorhombic laterally-packed gel and 13 nm long periodicity lamellar phase, mixed with the corneocyte-bound lipid envelope, provide a tight and semi-permeable barrier to the passage of water through the tissue; - the presence of fully matured corneodesmosome-bound interdigitating corneocytes which influence the tortuosity of the SC and thereby the diffusion path length of water; - the presence of both intracellular and extracellular hygroscopic materials called ‘natural moisturizing factors” (NMF). Much of the NMF is represented by amino acids (glutamine, histidine, and arginine) and their deiminated derivatives (pyrrolidone carboxylic acid, urocanic acid, and ornithine/citrulline/aspartic acid, respectively) derived from the breakdown of filaggrin. As NMF compounds are present in high concentrations within corneocytes and represent up to 20% to 30% of the dry weight of the SC, they can trap water within the corneocyte cytosol. Other components found within but also external to the corneocytes include lactates, urea, and electrolytes (Table 2). Other natural hygroscopic agents are important for SC hydration. Although glycerol is a well-known cosmetic ingredient, its role as natural endogenous humectant has been elucidated recently (Fluhr et al., 2003; Hara et al., 2002; Choi et al., 2005). The endogenous glycerol is derived from the sebaceous gland (sebum triglycerides) and also from the circulation, transported to the epidermis by aquaporin 3, a member of the aquaglyceroporin family, through water/glycerol channels (Hara et al., 2002). Hyaluronic acid is a major component of and provides hydration and structural integrity to the dermis but this hygroscopic polymer of sugar molecule is also naturally present in the epidermis (Sakai et al., 2000), binds to the extracellular space via CD44, and plays a role in epidermal barrier function (regulating both epidermal differentiation and lipid synthesis and secretion), and probably in SC hydration (Bourguignon et al., 2006). Then, the water-retaining capacity of the SC is highly dependent upon the thickness of the SC, the precise phenotype of the corneocytes, their volume, and their organization, the precise composition and physical packing state of barrier lipids, and finally the presence of highly hygroscopic compounds largely founds within the corneocytes (Rawlings, 2006). For examples, first, for the same volume of SC, the diffusion path length of water diffusing VetAgro Sup, Campus Vétérinaire de Lyon – France 2011 3 Didier Pin Dermatology and dermatopathology Unit through the SC lipids will be less if the corneocytes are smaller, second, the tight layering of the corneocytes provides them the ability to retain water while their membrane is permeable to water but impermeable to proteins. Naturally, as water is being constantly lost from the skin surface, water gradient are established within the different layers of the SC. It was established that the natural hydration levels in the SC are between 15% and 40%-45% (in comparison, the whole body contains a mean of 65% water), from 15% at the skin surface to 40% at the innermost layer compared with 70%-80% within the granular layer. These data suggest that a barrier to water loss begins prior to the formation of the SC and is present at the SC-SG interface and that a selective retention of water in the different SC cellular layers is required to explain the apparent discontinuity in hydration between different corneocyte layers (Rawlings, 2006). This function of the SC is believed to be largely dependent on the presence of the so-called NMF. All of the NMF components generated from filaggrin decrease in concentration toward the surface of the SC (Rawlings, 2006). A fraction of the water is tightly bound to hygroscopic molecules (the NMF) and lipids in the skin (Verdier-Sévrain and Bonté, 2007). This fraction of water content is proportional to external relative humidity. Under normal circumstances only a very small amount of water must be present in the intercellular lipid lamellae (Rawlings, 2006). The remaining fraction of water is bound within the intracellular keratin and usually does not change in nonpathological conditions (Verdier-Sévrain and Bonté, 2007). Method for assessing skin hydration A lot of techniques have been developed for measuring water in skin such as water flux analysis, electrical measurements (resistance, capacitance, and impedance), heat conductivity, photoacoustic spectroscopy viscoelastic properties, microwave propagation, dye fluorescence, topography, infrared spectroscopy, and electron probe analysis (Warner et al., 1988), all of these were ex vivo methods. Recently, the ex vivo findings have been proven noninvasively in vivo using the in vivo confocal Raman microscopy (Caspers et al., 2001). Other innovative methods have been developed to measure skin hydration such as silicon image sensor technology that provides sensitive imaging of the skin capacitance, near infrared (NIR) multispectral imaging method that measures the absorption of NIR light by water in living tissue from its reflectance spectrum, and optical coherence tomography, nuclear magnetic resonance spectroscopy, and transient thermal transfer (Verdier-Sévrain and Bouté, 2007). An easier in vivo method is the measurement of TEWL. In fact, the TEWL is a marker of the inside-outside barrier only. The outside-inside barrier often correlates with the inside-outside barrier, but not always. Nevertheless, an inverse relationship between TEWL and SC hydration is well known. High TEWL values, as a marker of disturbed skin barrier function, are frequently correlated with low hydration of the SC as shown in experimental settings after skin cleansing with soaps and detergents or in diseased skin (Proksch et al., 2008). VetAgro Sup, Campus Vétérinaire de Lyon – France 2011 4 Didier Pin Dermatology and dermatopathology Unit A model to better understand the barrier function Disruption of the permeability barrier by a variety of insults, including mechanical trauma such as tape stripping or contact with solvent such as acetone, stimulates a vigorous homeostatic repair response in the underlying viable epidermis that leads to the rapid restoration of permeability barrier function (Proksch et al., 1993). Function Permeability* Hydration* Principal compartment Extracellular matrix Structural basis Chemical basis Lamellar bilayers Corneocyte Cytosol Ceramides, chol, non essential FA in proper ratio Filaggrin proteolytic products, glycerol (AQP3), HA, xylose Intercellular DSG1/ pH, Ca++ (TRPV) DSC1 et CDSN, claudin1 and 4, occludin, JAM-1 AMPs, FFA, Sph 1,25(OH)2D3, IL-1α Cohesion (integrity) Extracellular and desquamation* matrix CD, tight junctions Antimicrobial* Lamellar bilayers Mechanical* Antioxidant* Extracellular matrix Corneocyte Extracellular matrix Extracellular matrix Corneocyte Chemical (Ag exclusion) Initiation of inflammation* (1st cytokine activation) Psychosensory Extracellular interface* matrix UV light Corneocyte Cornified envelope, keratin filaments Lamellar bilayers Regulatory signals (receptors) IL-1α, Ca++, pH, liposensors, SP through PAR2, TRPV1 and 4 Relative humidity (TRPV4) γ-Glutamyl isopeptide bonds Chol, FFAs, secreted vitE, redox grandient Hydrophilic products of CD Proteolytic activation of pro-IL-1 α/β Ca++, CholSO4, liposensors ? Lamellar bilayers Barrier lipids GCs, heat (TRPV3) Cytosol Trans-urocanic acid (histidase activity) Extracellular lacunae Cytosol Same as for permeability function pH, serine proteases activation * Abnormal in atopic dermatitis, SP serine protease, AMPs antimicrobial peptides, Sph sphingosine, DSG1/ DSC1 desmogleïn/desmocollin, CDSN corneodesmosin, TRPV transient receptor potential vanilloid, HA hyaluronic acid, CD corneodesmosome, AQP3 aquaporin-3, PAR2 proteinase-associated receptor 2, FFA free fatty acid, JAM-1 junctional adhesion molecule Table1 Multiple protective functions of mammalian SC (Elias, 2007; Elias et al., 2008; Verdier-Sévrain and Bonté, 2006) Chemical Composition (%) VetAgro Sup, Campus Vétérinaire de Lyon – France 2011 5 Didier Pin Free amino acids Pyrrolidone carboxylic acid Lactate Sugars Urea Chloride Sodium Potassium Ammonia, uric acid, glucosamine, creatine Calcium Magnesium Phosphate Citrate and formate Dermatology and dermatopathology Unit 40 12 12 8.5 7 6 5 4 1.5 1.5 1.5 0.5 0.5 Table 2 Chemical composition of NMF (Verdier-Sévrain and Bonté, 2007) Microphotograph Epidermis of canine skin: 1 stratum basale, 2 stratum spinosum, 3 stratum granulosum, 4 stratum corneum compactum, 5 stratum corneum disjunctum, rectangular area indicates the location of the skin barrier, the hydration gradient through the epidermis is noted. 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Barrier function regulates epidermal lipid and DNA synthesis. Br J Dermatol 1993; 128: 473-82. Rawlings AV. Sources and role of stratum corneum hydration. In: Skin Barrier. Elias PM, Feingold KR, eds, Taylor & Francis Group: New York, 2006, 399-425. Sakai S, Tasuda R, Sayo T, Ishikawa O, Inoue S. Hyaluronan exists in the normal stratum corneum. J Invest Dermatol 2000; 114: 1184-7. Verdier-Sévrain S, Bonté F. Skin hydration: e review on its molecular mechanisms. J Cosmet Dermatol 2006; 6: 75-82. VetAgro Sup, Campus Vétérinaire de Lyon – France 2011 7