Dry skin and impairment of barrier function associated with itch - new insights

Itch is a common symptom in dry skin related to inflammatory skin diseases, normal aging, and systemic diseases such as chronic renal failure, and HIV. However, correlations between itch and objective measures of barrier function and skin dryness such as skin hydration and transepidermal water loss have been rarely found. Recent experimental evidence indicates that damage to the stratum corneum with acetone/ether and water elicits a scratching response in mice and rats. These responses correlate to the number of PGP 9.5 immunoreactive fibers in the epidermis and to FOS-like immunoreactivity in the spinal cord. Other neuromediators involved in the pathogenesis of itch in dry skin are nerve growth factor (NGF), muscarinic acetylcholine receptors, and opiates. Serine proteases such as tryptase and their respective proteinase-activating receptor 2 (PAR2), recently found in both skin and nerves of patients with atopic eczema, suggest that these molecules may have a role in itch in dry skin. This has also been exemplified in the itchy and hyperkeratotic phenotype of the stratum corneum chymotryptic enzyme (SCCE) transgenic mouse model, which is over-expressing a serine protease. Developing inhibitors to these neuropeptides and mediators may be an attractive strategy for anti-itch treatment. The significant progress made in development of moisturizers may have an additional benefit in reducing the itch associated with dry skin. Formulating topical combination therapies containing moisturizers and anti-pruritics can significantly reduce the itch associated with dry skin. This paper will review the current clinical knowledge on the association between dry skin and itch and the recent advances in understanding the pathophysiology of this problem.     

Amino acid composition,including key derivatives of eccrine sweat: potential biomarkers of certain atopic skin conditions

The free amino acid (AA) composition of eccrine sweat is different from other biological fluids, for reasons which are not properly understood. We undertook the detailed analysis of the AA composition of freshly isolated pure human eccrine sweat, including some of the key derivatives of AA metabolism, to better understand the key biological mechanisms governing its composition. Eccrine sweat was collected from the axillae of 12 healthy subjects immediately upon formation. Free AA analysis was performed using an automatic AA analyser after ninhydrin derivatization. Pyrrolidine‐5‐carboxylic acid (PCA) and urocanic acid (UCA) levels were determined using GC/MS. The free AA composition of sweat was dominated by the presence of serine accounting for just over one‐fifth of the total free AA composition. Glycine was the next most abundant followed by PCA, alanine, citrulline and threonine, respectively. The data obtained indicate that the AA content of sweat bears a remarkable similarity to the AA composition of the epidermal protein profilaggrin. This protein is the key source of free AAs and their derivatives that form a major part of the natural moisturizing factor (NMF) within the stratum corneum (SC) and plays a major role in maintaining the barrier integrity of human skin. As perturbations in the production of NMF can lead to abnormal barrier function and can arise as a consequence of filaggrin genotype, we propose the quantification of AAs in sweat may serve as a non‐invasive diagnostic biomarker for certain atopic skin conditions, that is, atopic dermatitis (AD).     

Increased mass levels of certain serine proteases in the stratum corneum in acute eczematous atopic skin

Acute eczematous atopic dermatitis (AD) is associated with increases in stratum corneum (SC) serine protease activity. The purpose of this study was to examine whether the increased SC protease activities in acute eczematous atopic dermatitis were associated with increased mass levels of SC proteases. Six subjects with healthy skin and six patients with AD each with non-lesional skin or lesional acute eczematous skin had the mass levels of their extractable SC kallikreins (KLK), plasmin and urokinase quantified using Luminex multiplex bead-based assays from SC tape strippings. The mass levels of KLK5 and KLK14 together with urokinase were not elevated in the SC in atopic skin. However, the mass levels of KLK7 and KLK11 together with plasmin were greatly elevated compared with the extracts from the non-lesional and the healthy skin and correlated with the corresponding enzymatic activities.     

Profiling of serine protease activities in human stratum corneum and detection of a stratum corneum tryptase-like enzyme

Seasonal variation in stratum corneum (SC) biophysical and biological characteristics has been described previously. In particular, the winter season has been shown to affect more severely the properties of facial skin compared with forearm skin. Moreover, when compromised, such as in dry skin conditions, facial SC has been shown to contain increased inflammatory cytokines and proteases. Nevertheless, there have been no comparative studies of the activities and depth activity of several proteases in the SC on different body sites and at different times of the year. In this study, we examined the distribution of key serine protease activities (kallikrein 5, kallikrein 7, urokinase, plasmin and a tryptase-like enzyme) in different layers of the SC on the cheek and the forearm by analysis of consecutive tape strippings of healthy Caucasian subjects during winter and summer. The protein content of the tape strippings was quantified by absorption measurements with a recently developed and novel infrared densitometer SquameScan 850A while the SC enzyme activities were determined using fluorogenic peptide substrates. Transepidermal water loss (TEWL), skin pH and skin hydration were higher on the cheek than on the forearm. In the same way, the activity of the inflammatory-related proteases plasmin, urokinase and tryptase was approximately five to eight times and the activity of the desquamatory-related proteases kallikrein 5 and kallikrein 7 approximately two to four times higher on the cheek than on the forearm. There were no gender-related differences in these enzyme activities except for the increased kallikrein 7 in the forearm skin of the female subjects in winter. Reduced kallikrein 5 was associated with increased SC cohesion, as judged by increased protein removal, in forearm skin in the winter months of the year although the skin was clinically normal. It can be concluded that (i) protected skin areas show lower TEWL, skin pH and skin hydration and less protease activities than skin areas that are exposed to the environment, possibly indicating subclinical inflammation on these body sites, (ii) in normal healthy forearm skin, the outer SC exhibits greater serine protease activity than its deeper layers, (iii) compared with the forearm, urokinase- and plasmin-like activities are elevated on SC strippings from the cheek, confirming activation of the plasminogen cascade, and (iv) tryptase-like activity in the SC is also elevated in samples from the cheek, possibly indicating involvement of mast cells in these barrier-compromised body sites or the synthesis of a novel tryptase-like enzyme by keratinocytes. Although elevation of the activities of urokinase, plasmin, kallikrein 5, kallikrein 7 and now a tryptase-like enzyme was observed on SC derived from skin of clinically normal cheeks, we anticipate even higher activities in skin conditions where the epidermal barrier is further impaired.     

Investigation of interactions between silicones and stratum corneum lipids

Ingredients of topically applied skin care formulations have not only positive effects on the appearance of human skin but can also disturb the Stratum corneum (SC) lipid barrier. In the present study, the influence of silicones (PDMS), as often used cosmetic ingredients, on the microstructure of SC lipids was investigated. For this purpose the interactions of four different PDMS with excised human SC were examined first using differential scanning calorimetry (DSC) and wide angle X-ray diffraction for physical characterization. Because the physical properties of human stratum corneum strongly depend on the lipid composition, showing inter-and intra-individual differences, the interactions with an in vitro model lipid system containing SC fatty acids were also studied, using polarized light microscopy, transmission electron microscopy, small angle X-ray diffraction and DSC. The results revealed that the investigated PDMS do not change either the microstructure of excised human SC or the biphasic lamellar/inverse hexagonal structure of the in vitro model. We concluded that PDMS will not cause any side-effects when topically applied and that our simplified in vitro model could be helpful for estimating interactions between cosmetic ingredients and other topically applied substances and the skin barrier at an early moment of formulation development.     

Studies on isolated corneocytes

The function of the stratum corneum is dependent on the functions of its constituent parts. This paper focusses on corneocyte dimensions and the implications of change in dimension for function. A technique in which it is possible to obtain a value for corneocyte thickness is described. The changes in corneocyte dimensions with age and site and after topical applications are documented.
Techniques which measure the rate of desquamation are also described. The rate of passive corneocyte loss has been estimated by counting the numbers of corneocytes shed into chambers fixed to the skin surface. In addition the corneocyte loss after a standardized rotational stimulus to the skin surface has been measured. Scanning and transmission electron microscope examination of corneocytes obtained by a scrub procedure is also described. A preliminary report on an immunological approach to the study of the biochemistry of corneocytes and stratum corneum is also included.
Etude de cornéocytes isolés     

Skin barrier structure, function and formation - learning from cryo-electron microscopy of vitreous, fully hydrated native human epidermis

The hydration level of the stratum corneum largely depends on the physical state and molecular organization of the stratum corneum intercellular lipid matrix. A better understanding of stratum corneum lipid organization may thus aid the development of more rational cosmetic formulations. Several experimental and theoretical problems of a fundamental character remain, however, unresolved. These are, e.g. that precise quantitative skin barrier compositional data are difficult to obtain and that in vitro experimental skin barrier models usually are of limited value because of the prevailing non-equilibrium conditions in vivo. However, new experimental methods have recently been developed, which may help to overcome some of these limitations. These are, e.g. direct electrospray ionization-mass spectrometry (ESI-MS) of intact long-chain skin ceramides and direct high-resolution cryo-transmission electron microscopy (cryo-TEM) of vitreous sections of native, fully hydrated epidermis. Here, we show that cryo-transmission electron micrographs of vitreous normal human skin often dramatically differ from those obtained by conventional electron microscopy of resin-embedded skin. Our epidermal cryo-TEM data are subsequently discussed in relation to central problems of present conceptions of skin barrier structure, function and formation.     

Location and nature of the epidermal permeability barrier

The lowest region of human and pig stratum corneum has been isolated as an integral layer, referred to as the stratum compactum. This preparation is resistant to disruption by enzymes, 6M urea, Triton X-100 and organic solvents. Our evidence suggests that penetration of materials to and through the corneum depend on the state of cohesion between cells, and of the organization of intercellular lipid species. As the cells move up towards the outside of the stratum corneum, the cohesive forces are reduced due to desmosome degradation and lipid modifications, with ultimate dyshesion and sloughing of individual cells.
A number of linoleate-rich lipids have been identified within human and pig epidermis which could contribute to the formation or maintenance of an epidermal permeability barrier. Our current belief is that the molecule key to barrier function is a hydroxylated linoleate derivative which appears to be formed in the stratum compactum region.
Situation et nature de la barrière de perméabilitéépidermique     

Topical proteolytic enzymes affect epidermal and dermal properties

Although proteolytic enzymes have a history of use in skin care products it is not known whether they simply induce superficial exfoliation or with continued use can alter epidermal and dermal skin properties. We examined herein whether enhanced exfoliation resulting from treatment with an aspartyl dependent acid protease produces appearance improvements and over time, changes in the epidermis and dermis. Test participants applied 15% enzyme containing serum and a simple moisturizer twice daily for 3 months; a matched control group applied the same serum (without enzyme) and moisturizer. Changes in skin smoothness and texture, the depth and number of lines and wrinkles, and epidermal and dermal firmness and thickness were examined in a double-blind fashion. Treatment with 15% enzyme product(s) resulted in significant improvement in epidermal properties after 1 month and both epidermal and dermal properties after 3 months. The control group showed modest improvements in surface properties only. These results demonstrate that significant appearance benefits can be derived from use of exfoliative proteolytic enzymes. Such improvements are the result of changes to the epidermis and dermis and are strikingly similar to results observed with higher concentrations of AHAs (alpha hydroxy acids).     

Moisturization processes in living human skin studied by magnetic resonance imaging microscopy

Magnetic resonance imaging is a non-invasive, non-destructive and chemically specific imaging method widely used in medicine to reveal information about both tissue structure and function. It can measure water in tissue, but it has been difficult to achieve the necessary sensitivity and resolution when applying it to studies of the dry, thin stratum corneum. In this paper the use of magnetic resonance imaging to image the outer layers of the skin with a resolution of 0.06 mm is reported. Configuring the magnetic resonance imaging method in this way has made it possible for the first time to actually 'see'directly the moisturization in the stratum corneum. It is no longer necessary to rely upon methods which can only show side-effects of moisturization, such as changes in the appearance of the skin cells. As magnetic resonance imaging is harmless, it can be used repeatedly on the same skin and so produce a series of stills, or a time-lapse video, clearly showing the actual process of moisturization and related phenomena. The behaviour of skin has been observed during both hydration and dehydration; the two processes follow different time courses. Two layers have been observed in the stratum corneum, which appear to be different when the skin is hydrated.
For the first time the actual surface of normal skin has been revealed on magnetic resonance images.     

Increased basal transepidermal water loss leads to elevation of some but not all stratum corneum serine proteases

There are indications of elevation of some inflammatory serine proteases in barrier damaged skin (e.g. plasmin and urokinase). Moreover, many other serine protease activities are present such as desquamatory enzymes as well as a newly detected tryptase‐like serine protease. However, the activities of these proteases have never been correlated with stratum corneum (SC) barrier function. The activity of extractable key serine proteases (SC trypsin‐like kallikreins, SC chymotrypsin‐like kallikreins, SC tryptase‐like serine protease, urokinase and plasmin) was measured from the outermost layers of SC obtained from facial tape strippings in clinically normal subjects. The protein content of the tape strippings was quantified by absorption measurements with the novel infrared densitometer SquameScan^TM 850A and the protease activities by the use of fluorogenic peptide substrates. SC barrier function, SC hydration and skin surface pH were measured using AquaFlux^TM, NOVA dermal phase meter and Skin‐pH‐Meter^®, respectively. As expected, SC hydration was reduced with increased transepidermal water loss (TEWL) values indicative of barrier impairment. Surprisingly, SC chymotrypsin‐like activity showed no correlation with hydration or TEWL, whereas all other enzymes positively correlated with impaired barrier function and some were statistically significant: SC trypsin‐like kallikreins (R² = 0.66, P < 0.01), SC tryptase‐like enzyme (R² = 0.95, P < 0.001), plasmin (R² = 0.86, P < 0.001) and urokinase (R² = 0.50, P < 0.05). All enzymes except urokinase also negatively correlated with SC hydration. Elevated levels of SC serine proteases have been associated with some dermatological disorders, such as atopic dermatitis, psoriasis and rosacea but these results indicate that these enzymes are also elevated with milder forms of barrier disruption, which is not clinically evident as irritated skin. As these proteases are elevated in the SC, they will also be elevated in the epidermis where they can be involved in neurogenic inflammation and epidermal barrier impairment via activation of the protease‐activated receptors. These results highlight the need for using serine protease inhibitors especially for urokinase and plasmin, SC tryptase‐like serine protease and possibly SC trypsin‐like kallikreins even in milder forms of barrier damage.     

Involvement of transglutaminase in ex vivo maturation of cornified envelopes in the stratum corneum

The cornified envelope (CE) is a thin insoluble structure enveloping corneocytes, and is essential for the barrier function of the stratum corneum (SC). Our previous studies revealed that immature CEs are detected in the outermost layer of SC of barrier-impaired epidermis including the face and in various inflammatory disorders, using a non-invasive method to evaluate CE maturity. However, factors attributable for immaturity of CEs are still unclear. The aim of the present study is to clarify whether immature CEs in the SC have the potential to mature. SC samples, in which immature CEs abundantly exist, were collected from the cheek of healthy volunteers by tape-stripping, and were incubated ex vivo under the humidified air at 37 degrees C. Then, CE maturity was evaluated by staining with a combination of anti-involucrin and Nile red to detect involucrin antigenicity in the immature CEs and hydrophobicity in the mature CEs, respectively. Ex vivo incubation of the SC resulted in the conversion of immature CEs into mature CEs in terms of loss of involucrin antigenicity and acquisition of hydrophobicity. Application of buffer solutions of various pH onto the SC prior to incubation revealed that maturation of CEs was proceeded at range of pH 5-7, corresponding to intrinsic pH range within the SC. Chelating agents, ethylenediamine-N, N, N', N',-tetraacetic acid (EDTA) and ethyleneglycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), and thiol alkylating agents, N-ethylmaleimide and iodoacetamide, inhibited the maturation. Labelled cadaverine as an exogenous substrate for transglutaminase (TGase) could be incorporated into CEs during maturation. Extractable involucrin-like protein detected in the SC samples before incubation concomitantly disappeared with CE maturation, suggesting incorporation of endogenous substrates into the CEs. These results obviously demonstrate that maturation of CEs was mediated by TGase activity in the SC, and that immature CEs found in the outermost face SC have potential to mature by cross-linking of endogenous CE precursors present in the SC. Reduction of environmental humidity during ex vivo incubation of the SC resulted in marked suppression of maturation of CEs, and application of a moisturizer, glycerine, onto the SC replenished the suppression of maturation, suggesting that water content in the SC may affect the TGase reaction in the SC. Therefore, various factors, including a decrease in the water content in the SC, may account for impaired maturation of CEs in the face SC.     

Trends in stratum corneum research and the management of dry skin conditions

The structure, composition, formation and function of the stratum corneum have been the subject of intense research over the last few decades. As has become apparent, stratum corneum barrier function is not only dependent on one single component but also on its total architecture. Recent developments in understanding lipid composition have led to a new ceramide nomenclature system, a new proposal for a molecular model of the interactions between ceramides, cholesterol and fatty acids, and the demonstration of the presence of crystalline orthorhombic and gel hexagonal lipid phases in the stratum corneum. Linoleate-containing ceramide one, now known as CER EOS, have been shown to be essential for the formation of the 13 nm long periodicity phase (LPP) observed by electron microscopy and X-ray diffraction studies, whereas long-chain fatty acids are important for the formation of the crystalline lipid phases essential for barrier function. The role of the corneocyte envelope, its constituent proteins and its transglutaminase-mediated maturation processes have been shown to be essential for good skin condition. Several proteases may have a role in corneodesmolysis, particularly serine and cathepsin-like enzymes. Novel filaggrin polymorphisms have been identified that may be involved in the expression of a dry skin phenotype. Disturbances in lipid packing states, reduction in ceramide levels (particularly the phytosphingosine-containing ceramides), reductions in the levels of long-chain fatty acids and loss of the LPP largely account for the perturbations in lipid structure that occur in dry skin. The reduced corneodesmolysis that occurs in this xerotic skin disorder is now well accepted and is caused by reductions in the levels and activities of stratum corneum proteases together with elevated levels of corneodesmosomal glycoproteins in the superficial layers of the stratum corneum. Additionally, increased levels of fragile corneocytes are associated with reduced transglutaminase activity and corneocyte envelope cross-linking events. However, in comparison with the advances in our understanding of the textural changes that occur in dry skin, the somatosensory changes are poorly understood and the itching associated with dry skin is still an under-researched area. The unique biosensor role of the stratum corneum essential for a competent natural moisturizing barrier may also have a role to play in the action of anti-ageing technologies by controlling the expression and secretion of epidermal cytokines and growth factors. Technologies to treat the surface textural skin problems, enhance the differentiation process, particularly lipid biosynthesis, and to control the somatosensory problems in dry skin have received much attention in the last decade. This paper will review the state of the art of stratum corneum biology and the trends in the management of dry skin.     

Bioengineering and subjective approaches to the clinical evaluation of dry skin

Dry skin (also known as xerosis) is a cutaneous reaction pattern indicative of abnormal desquamation, which has not only cosmetic considerations, but can also lead to the penetration of irritants and allergens through the stratum corneum (SC). Over the last few decades, our understanding of the structure, composition, formation and function of the SC has advanced tremendously; however, despite these advancements, the occurrence of dry skin remains prevalent in the adult population. The clinical evaluation of dry skin is therefore of significant importance to the cosmetic industry not only for understanding the condition but also for measuring the effects of treatment. Traditionally, dry skin has been evaluated by visual inspection, however, recently a variety of bioengineering techniques have emerged enabling the investigator to objectively assess the extent of xerotic conditions. The most frequently employed methods for the evaluation of dry skin are discussed in this review, including regression testing, squametry, measurement of transepidermal water loss, epidermal hydration, profilometry, confocal Raman spectroscopy, optical coherence tomography, in vivo confocal microscopy and magnetic resonance imaging.     

Approche osmotique de l'hydratation du stratum corneum

Afin de reprendre l'hypothèse selon laquelle l'absorption de l'eau par le stratum corneum est un phénomène osmotique et afin de d'effectuer des études, in vitro , de la biophysique de la diffusion de l'eau par osmose, nous avons mis au point un Modèle Osmotique de Cornéocyte (MOC), qui est constitué par de la kératine isolée par une membrane semi-perméable.
Immeré dans de l'eau, le MOC voit sa masse augmenter. En effet, bien qu'insoluble, la kératine est hydrophile et en accord avec le Deuxième principe de la thermodynamique, elle fait diffuser par osmose, de l'eau vers l'intérieur du MOC.
A saturation, l'eau absorbée représente 13 fois la masse de kératine.
La diffusion s'effectue selon une cinétique classique: la masse de l'eau qui pénètre est proportionelle à la racine carrée du temps.
Les éléments constitutifs du Facteur Naturel d'Hydratation comme l'urée, l'acide lactique, l'acide pyrrolidone carboxylique, accroissent la vitesse de diffusion et la quantité totale d'eau absorbée. Un humectant comme la glycérine produit un effet semblable. L'urée est le composé qui donne les meilleures performances.
L'accroissement de la vitesse de diffusion par osmose dans le MOC en présence de composés hydratants peut s'interpréter comme un accroissement de l'hydrophilie de la kératine induite par ces composés.
Le MOC semble pouvoir permettre, outre une étude théorique sur le phénomène osmotique dans le stratum corneum , des études préliminaires in vitro des produits hydratants hydrosolubles utilisables en cosmétologie.     

Eucalyptus increases ceramide levels in keratinocytes and improves stratum corneum function

The objectives of this study were to identify a plant extract that would improve stratum corneum functions and to elucidate the mechanism(s) involved. Based on the information that stratum corneum functions depend on the level of ceramide in the stratum corneum, we identified a Eucalyptus extract that was able to increase the level of ceramide in human keratinocytes in culture and in human stratum corneum and that improves the stratum corneum water holding and barrier functions. Addition of the Eucalyptus extract to human keratinocytes in culture increased the level of ceramide in a dose-dependent manner and also increased the biosynthesis of ceramide, glucosylceramide and sphingomyelin. Topical application of the Eucalyptus extract on the dry skin of human subjects induced by acetone and diethylether treatment resulted in a significant increase in ceramide level in the stratum corneum, a significant improvement in its water-holding function and an improvement in its barrier function. The addition of macrocarpal A, one of the main components of the Eucalyptus extract, to human keratinocytes in culture increased the level of ceramide and the mRNA expression of serine palmitoyltransferase, acid sphingomyelinase, neutral sphingomyelinase, glucosylceramide synthase and glucocerebrosidase in a dose-dependent manner. Our results indicate that the increased content of ceramides in the stratum corneum may underlie the therapeutic effect of the Eucalyptus extract. Our results also indicate the possibility that macrocarpal A is the key component that stimulates the synthesis of ceramide in the stratum corneum.