Cytokines at different stratum corneum levels in normal and sodium lauryl sulphate-irritated skin

BACKGROUND/PURPOSE: Cytokines play an important role in inflammatory and repair processes occurring in the skin. The objectives of this study were to determine the amounts of cytokines and protein isolated by tape stripping in the different layers of the stratum corneum (SC), and to compare normal skin with skin exposed in vivo to the irritant sodium lauryl sulphate (SLS). METHODS: In eight volunteers, we determined the amount of total and soluble protein and also interleukin-1alpha (IL-1alpha) in pooled tape strips obtained from the upper, intermediate and lower parts of the SC. Three different types of tape were compared (Diamond , D-squame or Sentega tape). In a separate study, 20 volunteers were repeatedly exposed to 0.1% SLS over a 3-week period. The amounts of IL-1alpha, IL-1RA and IL-8 in strips obtained from the three different SC levels of SLS-exposed skin were compared with an unexposed site. RESULTS: For normal skin, the amounts of soluble protein and IL-1alpha were similar for the three tapes. Diamond tape showed the highest yield of total protein. The total protein yield per strip decreased to lower SC levels, whereas soluble protein and IL-1alpha normalized by soluble protein did not change across the SC. After SLS induced skin irritation, IL-1alpha decreased and IL-1RA and IL-8 increased at increasing depth into the SC. CONCLUSIONS: Tape stripping is a suitable method to determine SC cytokine concentrations in human skin. With this technique, it is possible to study changes in cytokine concentrations at different SC layers after skin irritation.     

Effect of synthetic vernix biofilms on barrier recovery of damaged mouse skin

Abstract:  The aim of this work was to investigate whether topical application of synthetic biofilms supports and accelerates the recovery of the murine skin barrier, disrupted by sequential tape stripping. Therefore, various biofilms were applied topically on disrupted mouse skin to determine which formulation could improve barrier function, as was observed previously for the natural biofilm vernix caseosa (VC). The biofilms [i.e. particles (synthetic corneocytes) embedded in a synthetic lipid matrix] mimic closely the physicochemical properties and structure of VC. Various formulations were prepared using different particle:lipid ratios, particles with different initial water content and uncoated or lipid-coated particles. It was observed that application of all tested formulations improved the skin barrier recovery rate and reduced crust formation and epidermal hyperproliferation. However, only one of the biofilms [i.e. B1; composed of uncoated particles with 50% (w/w) initial water content and particle:lipid ratio of 2:1] mimicked the effects of native VC most closely. This indicates the importance of the presence of individual components, i.e. barrier lipids and water, as well as the ratio of these components. Consequently, these observations suggest the potential use of this biofilm treatment clinically.     

Variation in barrier impairment and inflammation of human skin as determined by sodium lauryl sulphate penetration rate

BACKGROUND: Skin irritability after a brief exposure to the model skin irritant, sodium lauryl sulphate (SLS), is known to vary considerably between individuals. A difference in the skin barrier to SLS may contribute to this variation. To date, no human in vivo data have been available on SLS penetration into the skin. OBJECTIVES: We studied whether the SLS penetration rate into the stratum corneum (SC) is related to impairment of the water barrier function and inflammation of the skin. METHODS: The penetration of SLS into the SC was assessed using a noninvasive tape-stripping procedure in 20 volunteers after a 4-h exposure to 1% SLS. Additionally, the effect of a 24-h exposure to 1% SLS on the skin water barrier function was assessed by measuring the transepidermal water loss (TEWL). The accompanying inflammation was quantified by measuring erythema. RESULTS: The mean +/- SD diffusivity of SLS (D) and the SLS permeability coefficient (Kp) were 1.4 +/- 0.6 x 10(-8) cm2 h(-1) and 1.5 +/- 0.7 x 10(-3) cm h(-1), respectively. A multiple regression analysis showed that the baseline TEWL, SC thickness and SLS penetration parameters K (SC/water partition coefficient) and D clearly influenced the increase in TEWL after the 24-h irritation test (explained variance: r2 = 0.80). Change in erythema was mainly influenced by SC thickness. CONCLUSIONS: We found that variation in the barrier impairment and inflammation of human skin depends on the SLS penetration rate, which was mainly determined by SC thickness.     

Barrier repair in chronic plaque-type psoriasis

Purpose: To investigate barrier repair after mild trauma in lesional skin of psoriasis patients with chronic plaque-type disease and to compare this with non-involved psoriatic skin and normal controls.
Methods: Transepidermal water loss (TEWL) readings were taken from involved psoriatic skin and non-involved skin of psoriasis patients as well as the skin of normal controls. Three readings were performed at each site: the basal state, immediately after 20 tape strippings and 1 week post stripping.
Results: Higher baseline, post-stripping and 1-week recovery TEWL readings in psoriatic-involved skin compared to non-involved and normal control skin. No significant difference in barrier recovery rate in psoriatic-involved skin compared to non-involved and normal control.
Conclusion: Although there appears to be a derangement of barrier function in lesional skin of psoriasis patients compared to non-lesional skin and the skin of healthy controls, the barrier recovery function of lesional psoriatic skin is still fully operational.     

Transcutaneous penetration of toluene in rat skin a microdialysis study

Percutaneous absorption of lipophilic substances has major implications for therapeutical use or toxicological effects. We, therefore, using dermal microdialysis, measured local toluene concentrations and assessed the effects of duration of exposure, skin barrier disruption and the use of skin-care products. Three microdialysis membranes (3000 kDa) were inserted intradermally at a length of 2 cm in the abdominal skin of 82 anaesthetized male Wistar rats. They were perfused with albumin solution (5%) at 10 microl/min. A skin area of 1.5 x 0.6 cm above the membranes was exposed to toluene (100%, 200 microl) for 15 or 240 min. Dialysate was sampled at 20-min intervals. Using GC-FPD (gas charomotography flame photometric detector), it was analysed for toluene. In addition, the effects of tape stripping and pretreatment with topical products were assessed. In each of the 12 permutations of exposure time, pretreatments and tape stripping, five to eight animals were investigated. Maximum toluene concentrations were reached at 60 min after exposure (3.07 +/- 0.40 microg/ml, 15 min; 5.38 +/- 0.92 microg/ml, 240 min). In 15-min exposure experiments, dermal toluene concentrations decreased slowly to reach baseline values after 240 min. After 240-min exposure, a plateau of approximately 6 microg/ml was reached after 60 min. Neither tape stripping nor the pretreatment with barrier cream induced a significant change on dermal toluene concentrations. The slow kinetics of toluene penetration results in a steep concentration gradient in the skin with very-high local toluene concentrations and a delayed wash out, which might be relevant not only toxicologically, but also therapeutically.     

Extraction and quantification of amino acids in human stratum corneum in vivo

Background Amino acid (AA) levels in stratum corneum (SC) are potential biomarkers of skin health while their systemic levels may be used to diagnose inherited metabolic diseases. Objectives To examine reverse iontophoresis, in human volunteers, as a minimally invasive tool to analyse AAs within the skin and subdermally. Methods In four volunteers, the amounts of iontophoretically extracted AAs were compared with those determined in the SC following repetitive tape stripping and with the plasma concentrations. Glucose levels, evaluated in the different compartments, were used as a control. Results SC concentrations of 13 essentially zwitterionic AAs were ∼100‐fold higher than the respective plasma levels. Passive and reverse iontophoretic extraction for 4 h did not deplete the SC depot of AAs, a fact reinforced by postextraction tape stripping, which revealed that AAs remained in the SC at this time. In contrast, glucose was much less abundant in the SC and was fully and relatively quickly extracted by reverse iontophoresis. Conclusions It follows that reverse iontophoresis is useful for quantifying AAs in the SC and these data are highly correlated with levels obtained by tape stripping. However, reverse iontophoresis is impractical for the routine monitoring of AA plasma concentrations (unlike the situation for glucose, the skin reservoir of which is much smaller).     

An ex vivo human skin model for studying skin barrier repair

In the studies described in this study, we introduce a novel ex vivo human skin barrier repair model. To develop this, we removed the upper layer of the skin, the stratum corneum (SC) by a reproducible cyanoacrylate stripping technique. After stripping the explants, they were cultured in vitro to allow the regeneration of the SC. We selected two culture temperatures 32°C and 37°C and a period of either 4 or 8 days. After 8 days of culture, the explant generated SC at a similar thickness compared to native human SC. At 37°C, the early and late epidermal differentiation programmes were executed comparably to native human skin with the exception of the barrier protein involucrin. At 32°C, early differentiation was delayed, but the terminal differentiation proteins were expressed as in stripped explants cultured at 37°C. Regarding the barrier properties, the SC lateral lipid organization was mainly hexagonal in the regenerated SC, whereas the lipids in native human SC adopt a more dense orthorhombic organization. In addition, the ceramide levels were higher in the cultured explants at 32°C and 37°C than in native human SC. In conclusion, we selected the stripped ex vivo skin model cultured at 37°C as a candidate model to study skin barrier repair because epidermal and SC characteristics mimic more closely the native human skin than the ex vivo skin model cultured at 32°C. Potentially, this model can be used for testing formulations for skin barrier repair.     

The skin: an indispensable barrier

Abstract: The skin forms an effective barrier between the organism and the environment preventing invasion of pathogens and fending off chemical and physical assaults, as well as the unregulated loss of water and solutes. In this review we provide an overview of several components of the physical barrier, explaining how barrier function is regulated and altered in dermatoses. The physical barrier is mainly localized in the stratum corneum (SC) and consists of protein‐enriched cells (corneocytes with cornified envelope and cytoskeletal elements, as well as corneodesmosomes) and lipid‐enriched intercellular domains. The nucleated epidermis also contributes to the barrier through tight, gap and adherens junctions, as well as through desmosomes and cytoskeletal elements. During epidermal differentiation lipids are synthesized in the keratinocytes and extruded into the extracellular domains, where they form extracellular lipid‐enriched layers. The cornified cell envelope, a tough protein/lipid polymer structure, resides below the cytoplasmic membrane on the exterior of the corneocytes. Ceramides A and B are covalently bound to cornified envelope proteins and form the backbone for the subsequent addition of free ceramides, free fatty acids and cholesterol in the SC. Filaggrin is cross‐linked to the cornified envelope and aggregates keratin filaments into macrofibrils. Formation and maintenance of barrier function is influenced by cytokines, 3′,5′‐cyclic adenosine monophosphate and calcium. Changes in epidermal differentiation and lipid composition lead to a disturbed skin barrier, which allows the entry of environmental allergens, immunological reaction and inflammation in atopic dermatitis. A disturbed skin barrier is important for the pathogenesis of contact dermatitis, ichthyosis, psoriasis and atopic dermatitis.     

Polymorphisms in the interleukin-1 gene influence the stratum corneum interleukin-1 alpha concentration in uninvolved skin of patients with chronic irritant contact dermatitis

Background:  Interleukin (IL)-1α and its receptor antagonist IL-1ra play a role in skin inflammation. Several polymorphisms in the IL1 gene cluster, coding for IL-1α, IL-1ra, and IL-1β, influence their protein expression. Within this cluster, strong linkage disequilibrium has been shown.
Objective:  We studied the association between the polymorphisms IL1A -889 (C→T) and IL1B -31 (T→C) and the concentration of IL-1α and IL-1ra in the stratum corneum (SC).
Method:  In 124 patients with chronic irritant contact dermatitis, we genotyped the IL1A -889 and IL1B -31 polymorphisms and determined the amount of IL-1α and IL-1ra on tape strips obtained from uninvolved skin of the volar forearm.
Results:  The SC IL-1α concentration was 23% and 47% lower in subjects with IL1A -889 C/T genotype and T/T genotype, respectively, compared with wild-type genotype. In subjects with IL1B -31 C/C genotype, the IL-1α concentration was 51% lower compared with C/T and T/T genotypes. The ratio IL-1ra/IL-1α increased twofold in IL1A -889 C/T genotype and threefold in T/T genotype compared with wild type.
Conclusions:  We have shown a clear effect of IL1 genotype on protein expression in the SC. This altered expression may be responsible for the interindividual differences in the inflammatory response of the skin.     

Knock‐down of filaggrin does not affect lipid organization and composition in stratum corneum of reconstructed human skin equivalents

Human skin mainly functions as an effective barrier against unwanted environmental influences. The barrier function strongly relies on the outermost layer of the skin, the stratum corneum (SC), which is composed of corneocytes embedded in an extracellular lipid matrix. The importance of a proper barrier function is shown in various skin disorders such as atopic dermatitis (AD), a complex human skin disorder strongly associated with filaggrin (FLG) null mutations, but their role in barrier function is yet unclear. To study the role of FLG in SC barrier properties in terms of SC lipid organization and lipid composition, we generated an N/TERT‐based 3D‐skin equivalent (NSE) after knock‐down of FLG with shRNA. In these NSEs, we examined epidermal morphogenesis by evaluating the expression of differentiation markers keratin 10, FLG, loricrin and the proliferation marker ki67. Furthermore, the SC was extensively analysed for lipid organization, lipid composition and SC permeability. Our results demonstrate that FLG knock‐down (FLG‐KD) did not affect epidermal morphogenesis, SC lipid organization, lipid composition and SC permeability for a lipophilic compound in NSEs. Therefore, our findings indicate that FLG‐KD alone does not necessarily affect the functionality of a proper barrier function.     

Calmodulin‐like skin protein level increases in the differentiated epidermal layers in atopic dermatitis

In atopic dermatitis (AD), the skin barrier is disturbed, and the expression of calcium‐dependent S100 proteins and the calcium gradient is also altered in the epidermis. The calmodulin‐like skin protein (CLSP), which is expressed in the differentiated epidermis, is believed to modulate the function of calcium‐dependent proteins involved in barrier formation and is significantly increased in the epidermis of psoriatic patients. We, therefore, investigated the CLSP level in skin biopsies taken from patients with acute exacerbated and non‐exacerbated AD as well as from healthy control subjects. Immunohistochemical, Western blot and ELISA analyses showed significant increases (P < 0.03) in CLSP level in the epidermis from patients with acute exacerbated AD as compared to that from patients with non‐exacerbated AD and from control subjects. Such increased expression of CLSP may help re‐establish a functional epidermal barrier in acute AD.     

Stratum corneum cytokines and skin irritation response to sodium lauryl sulfate

Little is known about cytokines involved in chronic irritant contact dermatitis. Individual cytokine profiles might explain at least part of the differences in the individual response to irritation. Our objective was to investigate the relation between baseline stratum corneum (SC) cytokine levels and the skin response to a single and a repeated irritation test. This study also aimed to determine changes in SC cytokine levels after repeated irritation. Transepidermal water loss (TEWL) and erythema were measured in 20 volunteers after single 24-hr exposure to 1% sodium lauryl sulfate (SLS), and during and after repeated exposure to 0.1% SLS over a 3-week period. SC cytokine levels were measured from an unexposed skin site and from the repeatedly exposed site. Interleukin (IL)-1alpha decreased by 30% after repeated exposure, while IL-1RA increased 10-fold and IL-8 increased fourfold. Baseline IL-1RA and IL-8 values were predictors of TEWL and erythema after single exposure (r = 0.55-0.61). 6 subjects showed barrier recovery during repeated exposure. Baseline IL-1RA and IL-8 levels are likely to be indicators of higher skin irritability after single exposure to SLS. Barrier repair in some of the subjects might explain the lack of agreement between the TEWL response after single and repeated irritation.     

Molecular interactions of plant oil components with stratum corneum lipids correlate with clinical measures of skin barrier function

Plant‐derived oils consisting of triglycerides and small amounts of free fatty acids (FFAs) are commonly used in skincare regimens. FFAs are known to disrupt skin barrier function. The objective of this study was to mechanistically study the effects of FFAs, triglycerides and their mixtures on skin barrier function. The effects of oleic acid (OA), glyceryl trioleate (GT) and OA/GT mixtures on skin barrier were assessed in vivo through measurement of transepidermal water loss (TEWL) and fluorescein dye penetration before and after a single application. OA's effects on stratum corneum (SC) lipid order in vivo were measured with infrared spectroscopy through application of perdeuterated OA (OA‐d₃₄). Studies of the interaction of OA and GT with skin lipids included imaging the distribution of OA‐d₃₄ and GT ex vivo with IR microspectroscopy and thermodynamic analysis of mixtures in aqueous monolayers. The oil mixtures increased both TEWL and fluorescein penetration 24 h after a single application in an OA dose‐dependent manner, with the highest increase from treatment with pure OA. OA‐d₃₄ penetrated into skin and disordered SC lipids. Furthermore, the ex vivo IR imaging studies showed that OA‐d₃₄ permeated to the dermal/epidermal junction while GT remained in the SC. The monolayer experiments showed preferential interspecies interactions between OA and SC lipids, while the mixing between GT and SC lipids was not thermodynamically preferred. The FFA component of plant oils may disrupt skin barrier function. The affinity between plant oil components and SC lipids likely determines the extent of their penetration and clinically measurable effects on skin barrier functions.     

Repair of acetone- and sodium lauryl sulphate-damaged human skin barrier function using topically applied emulsions containing barrier lipids

BACKGROUND: It is generally acknowledged that well-formulated moisturizing skin care products can restore disturbed barrier function that can be assessed by transepidermal water loss (TEWL) measurements. When ceramides and/or other barrier lipids are incorporated, it is, however, not always clearly demonstrated which ingredients of the formulation exert the beneficial effects. OBJECTIVES: In this study the effects of topically applied ceramide-containing mixtures on the barrier repair of sodium lauryl sulphate (SLS)- and acetone-induced skin damage have been studied in human volunteers. TEWL and stratum corneum hydration measurements were carried out. The emulsions applied contained either a mixture of two types of ceramides, CerIII and CerIIIB (emulsion 1) or a complete mixture of ceramides III, IIIB and VI together with phytosphingosine, cholesterol and the free fatty acid linoleic acid (emulsion 2). RESULTS: After SLS damage, it was observed that barrier recovery was significantly accelerated by topical application (14 days, 2 x/d) of emulsion 2 compared with the results obtained with emulsion 1. Corneometrical results were not relevant due to the occurrence of scaly fissured skin, failing to provide a good skin/probe contact. Although no effect on TEWL could be observed, the improvement of skin hydration after acetone treatment and a single application of the emulsions, was significantly more positive for emulsion 2 than for emulsion 1. CONCLUSIONS: The investigative methods used in this study show that ceramides combined with other skin lipids can improve barrier repair after damage.