Occupational Exposure to Factory\u2010Derived Particulate Matter 2.5 Induces Cutaneous Toxicity in Workers and HaCaT Keratinocytes Through Oxidative Stress and Inflammation

Disorders of skin barriers: clinical implications.

The maturity of the corneocyte envelope (CE) provides information about the barrier functionality of the stratum corneum (SC). Corneocytes are enclosed by the CE, a protein-lipid matrix, contributing to mechanical resistance and hydrophobicity of the SC. The aim of the work was to develop a novel and robust approach to characterize CE maturity based on rigidity, hydrophobicity and surface area. This offers an alternative approach to the Nile red staining and antigenicity of involucrin to characterize the CE. The photoexposed (PE) cheek and photoprotected (PP) post-auricular sites were selected for investigation. Nine tape strips were obtained from the cheek and post-auricular sites of healthy Caucasians. CEs on the first and last tape strip were subjected to sonication to assess rigidity, and Nile red staining to determine hydrophobicity per unit surface area. In addition, the presence of involucrin and lipids was assessed to determine CE maturity by examination of the red/green pixel ratio, percentage of involucrin expressing CEs and alternatively the ratio of fluorescence density. The CE rigidity was lower in the deeper SC layers of the cheek, whereas post-auricular CEs were mechanically more resistant. Post-auricular CEs from the superficial SC had a larger surface area with a stronger fluorescence signal than those from the cheek. Interestingly, those CEs from the deeper SC layers had similar surface areas in both anatomical sites but were significantly different in hydrophobicity. These three parameters can be summarized as a relative CE maturity index that expresses CE maturity more precisely with a higher sensitivity than the conventional involucrin and Nile red staining approach. CEs of the cheek surface are more mature than CEs in the deeper SC layer, whereas CEs obtained from the post-auricular surface are more mature than those from the cheek surface. The combined method developed allows characterization of CE maturity based on hydrophobicity per unit surface area and rigidity rather than a simple ratio of lipid to involucrin. A more robust and sensitive measurement has therefore been developed addressing the limitations of earlier protocols.

Tape stripping of human stratum corneum has been performed to measure stratum corneum mass, barrier function, drug reservoir and percutaneous penetration. However, the technique itself requires further development to facilitate interpretation. In this study we quantified stratum comeum (SC) tape stripping and water kinetic parameters utilizing three types of adhesive tapes, in an in vivo randomized clinical trial. Stratum corneum was tape stripped, and the mass of SC removed by each tape was quantified utilizing a protein assay. Transepidermal water loss (TEWL) was measured and barrier disruption and SC water kinetics calculated. Three commonly utilized acrylate adhesive tapes were utilized and a comparison made between them. Each type of tape successfully stripped the stratum corneum, but the rayon tape did not induce SC barrier disruption. Neither the type of tape nor the site stripped significantly influenced the mass of SC removed. Water kinetic parameters did not differ significantly for the tapes that did induce barrier disruption. Individual variation in barrier disruption to water following tape stripping was demonstrated. The tapes utilized removed a similar amount of SC. The tapes have a different propensity to cause barrier disruption. Some individuals do not demonstrate increased TEWL despite an equivalent mass of SC being removed compared to those who do show a response.

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.

Stratum corneum is the outer most part of skin for barrier function. Disorder in stratum corneum is related with many skin diseases including acne, atopic dermatitis and psoriasis. In developed countries, about 20% of the population has disorder in the barrier function of stratum corneum. Adhesive tape stripping is a method to disrupt skin barrier function in studying disorder in stratum corneum. In this study, we obtained NIR (Near-Infrared) spectrum of human skin after tape stripping. Changes in skin spectra after barrier disruption were investigated through principal component analysis (PCA) of spectrum. PCA analysis revealed that peaks for -NH stretching and -CH vibration mainly contributed to the spectral variation caused by barrier disruption. Furthermore, second derivative of spectrum revealed that acute barrier disruption contributes to spectral changes in the region related with secondary structure of protein, lipid and water associated with lipid in stratum corneum. We demonstrated that acute barrier disruption affected features in NIR spectrum. These spectral changes revealed that acute barrier disruption affected keratin protein and ceramide in human stratum corneum. These results suggest that NIR spectroscopy can be used to monitor changes in filamentous network and lamellar structure in stratum corneum. NIR spectroscopy can provide non-invasive method to investigate skin disease related with barrier disruption by monitoring disturbance in protein and lipid structure in stratum corneum.

Deletion of the epidermal water/glycerol transporter aquaporin-3 (AQP3) in mice reduced superficial skin conductance by approximately 2-fold (Ma, T., Hara, M., Sougrat, R., Verbavatz, J. M., and Verkman, A. S. (2002) J. Biol. Chem. 277, 17147-17153), suggesting defective stratum corneum (SC) hydration. Here, we demonstrate significant impairment of skin hydration, elasticity, barrier recovery, and wound healing in AQP3 null mice in a hairless (SKH1) genetic background and investigate the cause of the functional defects by analysis of SC morphology and composition. Utilizing a novel (3)H(2)O distribution method, SC water content was reduced by approximately 50% in AQP3 null mice. Skin elasticity measured by cutometry was significantly reduced in AQP3 null mice with approximately 50% reductions in elasticity parameters Uf, Ue, and Ur. Although basal skin barrier function was not impaired, AQP3 deletion produced an approximately 2-fold delay in recovery of barrier function as measured by transepidermal water loss after tape stripping. Another biosynthetic skin function, wound healing, was also approximately 2-fold delayed by AQP3 deletion. By electron microscopy AQP3 deletion did not affect the structure of the unperturbed SC. The SC content of ions (Na(+), K(+), Ca(2+), Mg(2+)) and small solutes (urea, lactic acid, glucose) was not affected by AQP3 deletion nor was the absolute amount or profile of lipids and free amino acids. However, AQP3 deletion produced significant reductions in glycerol content in SC and epidermis (in nmol/microg protein: 5.5 +/- 0.4 versus 2.3 +/- 0.7 in SC; 0.037 +/- 0.007 versus 0.022 +/- 0.005 in epidermis) but not in dermis or blood. These results establish hydration, mechanical, and biosynthetic defects in skin of AQP3-deficient mice. The selective reduction in epidermal and SC glycerol content in AQP3 null mice may account for these defects, providing the first functional evidence for physiologically important glycerol transport by an aquaporin.

Ceramides (CERs) in the upper layer of the skin, the stratum corneum (SC), play a key role in the skin barrier function. In human SC, the literature currently reports 11 CER subclasses that have been identified. In this paper, a novel quick and robust LC/MS method is presented that allows the separation and analysis of all known human SC CER subclasses using only limited sample preparation. Besides all 11 known and identified subclasses, a 3D multi-mass chromatogram shows the presence of other lipid subclasses. Using LC/MS/MS with an ion trap (IT) system, a Fourier transform-ion cyclotron resonance system, and a triple quadrupole system, we were able to identify one of these lipid subclasses as a new CER subclass: the ester-linked ω-hydroxy fatty acid with a dihydrosphingosine base (CER [EOdS]). Besides the identification of a new CER subclass, this paper also describes the applicability and robustness of the developed LC/MS method by analyzing three (biological) SC samples: SC from human dermatomed skin, human SC obtained by tape stripping, and SC from full-thickness skin explants. All three biological samples showed all known CER subclasses and slight differences were observed in CER profile.

Barrier-disrupted skin: Quantitative analysis of tape and cyanoacrylate stripping efficiency by multiphoton tomography.

Atopic dermatitis: Therapeutic concepts evolving from new pathophysiologic insights

For terrestrial creatures, the water-impermeable barrier function of the skin is essential to maintain life in the face of environmental dryness. Stratum corneum, which plays a crucial role in the barrier function, is composed of two components, i.e., protein-rich nonviable cells and intercellular lipid domains. When the barrier function is damaged by a surfactant, organic solvent or tape stripping, a series of homeostatic systems operates to restore the barrier function to its original level. At the first stage of the barrier repair process, exocytosis of lipid-containing granules, lamellar bodies, is accelerated and the internal lipid is secreted into the intercellular domain between the stratum granulosum and stratum corneum, forming a water-impermeable membrane. The barrier function is strongly associated with skin pathology. Abnormality of the barrier function is observed in a variety of skin diseases, such as atopic dermatitis. Although the barrier function of healthy skin can recover automatically after damage, the recovery is delayed by emotional stress or by aging. Moreover, under low environmental humidity, barrier damage induces epidermal hyperplasia and inflammation. On the other hand, acceleration of the barrier recovery prevents epidermal hyperplasia induced by barrier disruption in a dry environment. Thus, methods to improve the barrier function are very important for clinical dermatology. In the last two decades, various chemical and physical factors that accelerate the barrier recovery process have been reported. In this chapter, I will describe those findings and discuss some new biological aspects of epidermal barrier function.

Oxidative stress (OS) is common in inflammatory conditions and may be important in atopic dermatitis (AD) etiology. The aim of this project was to study the involvement of oxidation in FSL-1 (deacylated lipoprotein)-triggered signaling pathways leading to AD-typical cytokine expression in HaCaT keratinocytes. HaCaT keratinocytes, pretreated with the inhibitor to OS N-acetylcysteine (NAC), were exposed to FSL-1, a stimulator of AD-related cytokines. Cytokines expression was studied by real time polymerase chain reaction (PCR); nuclear factor-kappa B (NF-κB) and p38 mitogen activated protein kinase (MAPK) activities were studied by western blotting; and the oxidative state of cells was determined by the dichlorofluorescein (DCF) assay. We found that endogenous OS in keratinocytes appeared 4 h after FSL-1 administration. OS activated NF-κB, but not p38 MAPK, and the inhibition of OS reduced FSL-1 induced interleukin (IL) 33, thymic stromal lymphopoietin (TSLP) and TNFα mRNA expression. We conclude that FSL-1 triggers an OS reaction in HaCaT keratinocytes, which is probably a secondary event affecting the expression of specific AD typical cytokines, possibly through the NF-κB pathways. This role of OS in the inflammatory response in AD is worth further investigating.

It has been reported that artificial restoration of barrier function by a water vapor-impermeable membrane after tape stripping induces barrier abrogation in hairless mice, impeding rather than enhancing barrier recovery. To address this issue, we examined the morphologic changes in the epidermis after tape stripping and occlusion with a water vapor-impermeable membrane in murine skin. Male hairless mice were used for all studies of barrier perturbation and occlusion. Barrier disruption was achieved by repeated application of cellophane tape. Immediately after tape stripping the animals were wrapped in a tightly fitting water vapor-impermeable membrane. Transepidermal water loss (TEWL) was measured 20 min after tape stripping and 14, 24, 36, 48 and 60 h after occlusion. For electron microscopy the samples were treated with osmium tetroxide (OsO4) or ruthenium tetroxide (RuO4). When tape-stripped animals were wrapped in a water vapor-impermeable membrane, thereby preventing water flux, barrier function did not recover normally. These results demonstrate that an artificial block to TEWL with an impermeable membrane did not enhance barrier recovery. By electron microscopy many transitional cells and lacunae of various sizes were seen within the intercellular spaces of the stratum corneum after occlusion following tape stripping. Occlusion also caused alterations in both lipid lamellar membrane structures in the stratum corneum interstices and the lamellar bodies in the cytosol of granulocytes and transitional cells. Secreted lamellar body contents also appeared to be abnormal in the stratum corneum-stratum granulosum junction.

We have previously shown that alpha-melanocyte-stimulating hormone (alpha-MSH) can oppose tumor necrosis factor alpha activation of NF-kappaB (1-2 h) and intercellular adhesion molecule 1 up-regulation (mRNA by 3 h and protein by 24 h) in melanocytes and melanoma cells. The present study reports on the ability of four MSH peptides to control intracellular peroxide levels and glutathione peroxidase (GPx) activity in pigmentary and nonpigmentary cells. In human HBL melanoma and HaCaT keratinocytes tumor necrosis factor alpha and H(2)O(2) both activated GPx in a time- and concentration-dependent manner (by 30-45 min). alpha-MSH peptides were found to inhibit the stimulated GPx activity and had biphasic dose-response curves. MSH 1-13 and MSH [Nle(4)-d-Phe(7)] achieved maximum inhibition at 10(-10) and 10(-12) m, respectively. Higher concentrations (10-100 fold) of MSH 4-10 and MSH 11-13 were required to produce equivalent levels of inhibition. alpha-MSH was also capable of reducing peroxide accumulation within 15 min, and again this inhibition was biphasic. The data support a role of alpha-MSH in acute protection of cells to oxidative/cytokine action that precedes NF-kappaB and GPx activation. The rapidity and potency of the response to alpha-MSH in pigmentary and nonpigmentary cells suggest this to be a central role of this peptide in cutaneous cells.

TCF/Lef1-Mediated Control of Lipid Metabolism Regulates Skin Barrier Function

It is not known whether distinct anatomical locations will respond with different recovery rates following acute barrier challenges. To investigate whether barrier parameters differ at five body sites during recovery from acute disruption. Acute barrier disruption was achieved by tape stripping and by acetone extraction of stratum corneum lipids. Transepidermal water loss (to assess barrier function), capacitance (for stratum corneum hydration) and skin surface pH were measured at each of five different body sites in 14 human volunteers. Individual measurements were obtained every 24 h for 96 h. Lipid-rich skin areas (e.g. the forehead) were the most vulnerable to barrier disruption by either method. While acetone treatment affected barrier function and decreased stratum corneum hydration, tape stripping similarly altered barrier function but increased capacitance values. Although the effect of barrier disruption on surface pH appeared to vary with location, no significant pattern of variation emerged. Independent of the method used for barrier disruption, the pH normalized within 96 h. Skin at different body sites shows distinct patterns of barrier recovery that are likely to be related to structural and physiological differences. Therefore, 'anatomically specific' regimens appear possible and relevant for the treatment of cutaneous disorders. In addition, adequate statistical analyses are essential to detect real differences in barrier recovery parameters.