Mao-Qiang Man

Barrier recovery is impeded at neutral pH, independent of ionic effects: implications for extracellular lipid processing

Abstract Epidermal permeability barrier homeostasis requires the postsecretory processing of polar lipid precursors into nonpolar lipid products within the stratum corneum (SC) interstices by a family of lipid hydrolases. A specific requirement forβ-glucocerebrosidase (β-GlcCer’ase), which exhibits a distinct acidic pH optimum, is particularly well documented. Therefore, we sought to determine whether the recovery of the barrier after acute insults requires acidification of the SC. We examined permeability barrier recovery by assessing changes in transepidermal water loss (TEWL), SC membrane ultrastructure utilizing ruthenium tetroxide (RuO4) postfixation, and β-GlcCer’ase activity by in situ zymography at an acidic vs neutral pH. Barrier recovery proceeded normally when acetone-treated skin was exposed to solutions buffered to an acidic pH. In contrast, the initiation of barrier recovery was slowed when treated skin was exposed to neutral or alkaline pH, regardless of buffer composition. In addition, enhancement of the alkaline buffer-induced delay in barrier recovery occurred with Ca2+ and K+ inclusion in the buffer. Moreover, the pH-dependent alteration in barrier recovery appeared to occur through a mechanism that was independent of Ca2+- or K+-controlled lamellar body secretion, since both the formation and secretion of lamellar bodies proceeded comparably at pH 5.5 and pH 7.4. In contrast, exposure to pH 7.4 (but not pH 5.5) resulted in both the persistence of immature, extracellular lamellar membrane structures, and a marked decrease in the in situ activity of β-GlcCer’ase. These results suggest first that an acidic extracellular pH is necessary for the initiation of barrier recovery, and second that the delay in barrier recovery is a consequence of inhibition of postsecretory lipid processing.

FILAGGRIN DEFICIENCY CONFERS A PARACELLULAR BARRIER ABNORMALITY THAT REDUCES INFLAMMATORY THRESHOLDS TO IRRITANTS AND HAPTENS

BACKGROUND Mutations in the human filaggrin gene (FLG) are associated with atopic dermatitis (AD) and are presumed to provoke a barrier abnormality. Yet additional acquired stressors might be necessary because the same mutations can result in a noninflammatory disorder, ichthyosis vulgaris. OBJECTIVE We examined here whether FLG deficiency alone suffices to produce a barrier abnormality, the basis for the putative abnormality, and its proinflammatory consequences. METHODS By using the flaky-tail mouse, which lacks processed murine filaggrin because of a frameshift mutation in the gene encoding profilaggrin that mimics some mutations in human AD, we assessed whether FLG deficiency provokes a barrier abnormality, further localized the defect, identified its subcellular basis, and assessed thresholds to irritant- and hapten-induced dermatitis. RESULTS Flaky-tail mice exhibit low-grade inflammation with increased bidirectional, paracellular permeability of water-soluble xenobiotes caused by impaired lamellar body secretion and altered stratum corneum extracellular membranes. This barrier abnormality correlates with reduced inflammatory thresholds to both topical irritants and haptens. Moreover, when exposed repeatedly to topical haptens at doses that produce no inflammation in wild-type mice, flaky-tail mice experience a severe AD-like dermatosis with a further deterioration in barrier function and features of a T(H)2 immunophenotype (increased CRTH levels plus inflammation, increased serum IgE levels, and reduced antimicrobial peptide [mBD3] expression). CONCLUSIONS FLG deficiency alone provokes a paracellular barrier abnormality in mice that reduces inflammatory thresholds to topical irritants/haptens, likely accounting for enhanced antigen penetration in FLG-associated AD.

Variation of Skin Surface pH, Sebum Content and Stratum Corneum Hydration with Age and Gender in a Large Chinese Population

Background and Objectives: Evidence suggests the importance of skin biophysical properties in predicting diseases and in developing appropriate skin care. The results to date of studies on skin surface pH, stratum corneum (SC) hydration and sebum content in both genders and at various ages have been inconclusive, which was in part due to small sample size. Additionally, little is known about the skin physical properties of Asian, especially Chinese, subjects. In the present study, we assess the difference in skin surface pH, sebum content and SC hydration at various ages and in both genders in a large Chinese population without skin diseases. Methods: 713 subjects (328 males and 385 females) aged 0.5–94 years were enrolled in this study. The subjects were divided by age into 5 groups, i.e., 0–12, 13–35, 36–50, 51–70 and over 70 years old. A multifunctional skin physiology monitor was used to measure SC hydration, skin surface pH and sebum content on both the forehead and the forearms. Results: In males, the highest sebum content was found on the forearm and the forehead in the age groups 36–50 (93.47 ± 10.01 μg/cm2) and 51–70 years (9.16 ± 1.95 μg/cm2), while in females, the highest sebum content was found on the forearm and the forehead in the age groups 13–35 (61.91 ± 6.12 μg/cm2) and 51–70 years (7.54 ± 2.55 μg/cm2). The forehead sebum content was higher in males aged 13–70 years than in age-matched females; the sebum content on the forehead in both males and females was higher than that on the forearm. Skin surface pH on the forehead of both males and females over the age of 70 years was higher than that in younger groups. SC hydration on the forehead in both males and females was lower above the age of 70, and the one in males aged 13–35 was higher than that in females (43.99 ± 1.88 vs. 36.38 ± 1.67 AU, p < 0.01). SC hydration on the forehead in both males and females did not significantly differ from that on the forearm. Conclusions: In a large Chinese cohort, the skin surface pH, sebum content and SC hydration vary with age, gender and body site.

Topical treatment with thiazolidinediones, activators of peroxisome proliferator-activated receptor-γ, normalizes epidermal homeostasis in a murine hyperproliferative disease model

Abstract:  In a murine model of epidermal hyperplasia reproducing some of the abnormalities of several common skin disorders, we previously demonstrated the antiproliferative and pro‐differentiating effects of peroxisome proliferator‐activated receptor (PPAR)α, PPARβ/δ, and liver X receptor activators. Unlike other subgroups of PPAR activators, thiazolidinediones (TZDs), a family of PPARγ ligands, did not inhibit keratinocyte proliferation in normal murine skin. Here, we studied the effects of two TZDs, namely ciglitazone (10 mM) and troglitazone (1 mM), in the same murine model where epidermal hyperproliferation was reproduced by repeated barrier abrogation with tape stripping. Topical treatment with ciglitazone and troglitazone resulted in a marked and significant decrease in epidermal thickness. Furthermore, in all TZD‐treated groups, we observed a significant decrease in keratinocyte proliferation using proliferating cell nuclear antigen, 5‐bromo‐2′‐deoxyuridine, and tritiated thymidine incorporation. However, using the terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling assay, we found no difference in apoptosis between different treatments, emphasizing that it is the antiproliferative role of these activators that accounts for the decrease of epidermal thickness. Finally, using immunohistochemical methods, we determined the effects of ciglitazone on keratinocyte differentiation in this hyperproliferative model. We observed an increased expression of involucrin and filaggrin following ciglitazone treatment, suggesting a pro‐differentiating action of TZDs in this model. In summary, topical TZDs significantly reduce epidermal keratinocyte proliferation while promoting differentiation in a murine model of hyperproliferative epidermis. Together, these results suggest that in addition to their metabolic effects currently in use in the treatment of type 2 diabetes, topical TZDs could be considered as potential alternative therapeutic agents in hyperproliferative skin diseases such as psoriasis.

Murine atopic dermatitis responds to peroxisome proliferator-activated receptors α and β/δ (but not γ) and liver X receptor activators

BACKGROUND Atopic dermatitis (AD) is a chronic inflammatory dermatosis now increasingly linked to mutations that alter the structure and function of the stratum corneum. Activators of peroxisome proliferator-activated receptors (PPARs) alpha, beta/delta, and gamma and liver X receptor (LXR) regulate epidermal protein and lipid production, leading to superior barrier function. Additionally, some of these activators exhibit potent antihyperplastic and anti-inflammatory activity in irritant contact dermatitis and acute allergic contact dermatitis murine models. OBJECTIVE We evaluated the efficacy of PPAR/LXR activation in a hapten (oxazolone [Ox])-induced AD-like model (Ox-AD) in hairless mice. METHODS Ox-AD was established with 10 Ox challenges (every other day) on the flank. After the establishment of Ox-AD, twice-daily topical application with individual PPAR/LXR activators was then performed for 4 days, with continued Ox challenges every other day. The efficacy of topical PPAR/LXR activators to reduce parameters of Ox-AD was assessed physiologically, morphologically, and immunologically. RESULTS Certain topical activators of PPARalpha, PPARbeta/delta, and LXR, but not activators of PPARgamma, reversed the clinical dermatosis, significantly improved barrier function, and increased stratum corneum hydration in Ox-AD mice. In addition, the same activators, but again not PPARgamma, largely reversed the immunologic abnormalities in Ox-AD mice, including the increased T(H)2 markers, such as tissue eosinophil/mast cell density, serum thymus and activation-related chemokine levels, the density of chemoattractant receptor-homologous molecule expressed on T(H)2-positive lymphocytes (but not serum IgE levels), and reduced IL-1alpha and TNF-alpha activation, despite ongoing hapten challenges. CONCLUSION These results suggest that topical applications of certain activators/ligands of PPARalpha, PPARbeta/delta, and LXR could be useful for the treatment of AD in human subjects.

Epidermal Vascular Endothelial Growth Factor Production Is Required for Permeability Barrier Homeostasis, Dermal Angiogenesis, and the Development of Epidermal Hyperplasia : Implications for the Pathogenesis of Psoriasis

Primary abnormalities in permeability barrier function appear to underlie atopic dermatitis and epidermal trauma; a concomitant barrier dysfunction could also drive other inflammatory dermatoses, including psoriasis. Central to this outside-inside view of disease pathogenesis is the epidermal generation of cytokines/growth factors, which in turn signal downstream epidermal repair mechanisms. Yet, this cascade, if sustained, signals downstream epidermal hyperplasia and inflammation. We found here that acute barrier disruption rapidly stimulates mRNA and protein expression of epidermal vascular endothelial growth factor-A (VEGF-A) in normal hairless mice, a specific response to permeability barrier requirements because up-regulation is blocked by application of a vapor-impermeable membrane. Moreover, epidermal vegf(-/-) mice display abnormal permeability barrier homeostasis, attributable to decreased VEGF signaling of epidermal lamellar body production; a paucity of dermal capillaries with reduced vascular permeability; and neither angiogenesis nor epidermal hyperplasia in response to repeated tape stripping (a model of psoriasiform hyperplasia). These results support a central role for epidermal VEGF in the maintenance of epidermal permeability barrier homeostasis and a link between epidermal VEGF production and both dermal angiogenesis and the development of epidermal hyperplasia. Because psoriasis is commonly induced by external trauma [isomorphic (Koebner) phenomenon] and is associated with a prominent permeability barrier abnormality, excess VEGF production, prominent angiogenesis, and epidermal hyperplasia, these results could provide a potential outside-inside mechanistic basis for the development of psoriasis.

Ablation of the Calcium-Sensing Receptor in Keratinocytes Impairs Epidermal Differentiation and Barrier Function

The calcium-sensing receptor (CaR) plays an essential role in mediating Ca2+-induced keratinocyte differentiation in vitro. In this study, we generated keratinocyte-specific CaR knockout (EpidCaR-/-) mice to investigate the function of the CaR in epidermal development in vivo. EpidCaR-/- mice exhibited a delay in permeability barrier formation during embryonic development. Ion capture cytochemistry detected the loss of the epidermal Ca2+ gradient in the EpidCaR-/- mice. The expression of terminal differentiation markers and key enzymes mediating epidermal sphingolipid transport and processing in the EpidCaR-/- epidermis was significantly reduced. The EpidCaR-/- epidermis displayed a marked decrease in the number of lamellar bodies and lamellar body secretion, thinner lipid-bound cornified envelopes and a defective permeability barrier. Consistent with in vivo results, epidermal keratinocytes cultured from EpidCaR-/- mice demonstrated abnormal Ca2+I handling and diminished differentiation. The impairment in epidermal differentiation and permeability barrier in EpidCaR-/- mice maintained on a low calcium (0.02%) diet is more profound and persistent with age then in EpidCaR-/- mice maintained on a normal calcium (1.3%) diet. Deleting CaR perturbs the epidermal Ca2+ gradient and impairs keratinocyte differentiation and permeability barrier homeostasis, indicating a key role for the CaR in normal epidermal development.

Regulation of ABCG1 expression in human keratinocytes and murine epidermis

ABCG1, a member of the ATP binding cassette superfamily, facilitates the efflux of cholesterol from cells to HDL. In this study, we demonstrate that ABCG1 is expressed in cultured human keratinocytes and murine epidermis, and induced during keratinocyte differentiation, with increased levels in the outer epidermis. ABCG1 is regulated by liver X receptor (LXR) and peroxisome proliferator-activated receptor-δ (PPAR-δ) activators, cellular sterol levels, and acute barrier disruption. Both LXR and PPAR-δ activators markedly stimulate ABCG1 expression in a dose- and time-dependent fashion. PPAR-γ activators also increase ABCG1 expression, but to a lesser degree. In contrast, activators of PPAR-α, retinoic acid receptor, retinoid X receptor, and vitamin D receptor do not alter ABCG1 expression. In response to increased intracellular sterol levels, ABCG1 expression increases, whereas inhibition of cholesterol biosynthesis decreases ABCG1 expression. In vivo, ABCG1 is stimulated 3–6 h after acute barrier disruption by either tape stripping or acetone treatment, an increase that can be inhibited by occlusion, suggesting a potential role of ABCG1 in permeability barrier homeostasis. Although Abcg1-null mice display normal epidermal permeability barrier function and gross morphology, abnormal lamellar body (LB) contents and secretion leading to impaired lamellar bilayer formation could be demonstrated by electron microscopy, indicating a potential role of ABCG1 in normal LB formation and secretion.

Is the Filaggrin–Histidine–Urocanic Acid Pathway Essential for Stratum Corneum Acidification?

TO THE EDITOR Acidification of the surface of the stratum corneum (SC), the acid mantle, was initially thought to be important in the defense against infection. The growth of pathogenic microorganisms, such as Staphylococcus aureus and Streptococcus pyogenes, is inhibited by an acidic skin pH whereas the growth of resident (normal) skin flora is stimulated (Puhvel et al., 1975; Korting et al., 1990, 1992). However, recent studies have shown that acidification of the SC has additional functions, including regulating several key SC functions. A major function of the skin is to form a permeability barrier between the dry external environment and the moist interior of the body (Elias, 2007). This permeability barrier resides in the extracellular lipid membranes of the SC, and studies have shown that an acidified SC is required for the formation of a functionally competent permeability barrier (Mauro et al., 1998; Fluhr et al., 2001; Hachem et al., 2003). Specifically, in the SC b-glucocerebrosidase and acid sphingomyelinase metabolize glucosylceramides and sphingomyelin, respectively, to ceramides, which is the major family of lipids in the extracellular membranes that mediate permeability barrier function (Feingold, 2007). Both the enzymes require an acidic milieu for optimal enzymatic activity; hence, when the pH of the SC increases, the metabolism of glucosylceramides and sphingomyelin to ceramides is impaired, resulting in abnormal permeability barrier homeostasis (Holleran et al., 1992, 1993; Feingold, 2007). In addition, an acidic SC pH inhibits the activity of serine proteases thereby maintaining the cohesiveness and integrity of the SC (Hachem et al., 2005). With an increase in SC pH, the activities of these serine proteases are stimulated resulting in the degradation of corneodesmosomes and a decrease in SC integrity and cohesion (Fluhr et al., 2004b; Hachem et al., 2005). Thus, an acidic SC is important in regulating the metabolism and function of the SC, and alterations in SC pH could have numerous adverse effects. A variety of different pathways are postulated to contribute to the acid mantle Abbreviation: SC, stratum corneum

Topical calcineurin inhibitors compromise stratum corneum integrity, epidermal permeability and antimicrobial barrier function.

Please cite this paper as: Topical calcineurin inhibitors compromise stratum corneum integrity, epidermal permeability and antimicrobial barrier function. Experimental Dermatology 2010; 19: 501–510.