Truus Roelandt

Proteolytically active allergens cause barrier breakdown.

Two major allergens--the house dust mite Dermatophagoides pteronyssinus (Der p 1) and cockroach allergens--are proteolytically active and stimulate the protease-activated receptor 2 (PAR-2). Jeong et al. (2008, this issue) exposed mouse and human epidermis to both allergens and correlated the observed delay in permeability barrier recovery to PAR-2 activation/signaling. This article exposes the secretive boundaries between barrier homeostasis and immunity.

Cellular Changes that Accompany Shedding of Human Corneocytes

Corneocyte desquamation has been ascribed to either: 1) proteolytic degradation of corneodesmosomes (CD); 2) disorganization of extracellular lamellar bilayers; and/or 3) ‘swell-shrinkage-slough’ (SSS) from hydration/dehydration. To address the cellular basis for normal exfoliation, we compared changes in lamellar bilayer architecture and CD structure in DSquame® strips from the 1st vs. 5th stripping (‘outer’ vs. ‘mid’-stratum corneum [SC], respectively) from 9 normal adult forearms. Strippings were either processed for standard EM or for ruthenium (Ru-V)- or osmium-tetroxide (Os-V) vapor fixation, followed by immediate epoxy embedment, an artifact-free protocol that to our knowledge is previously unreported. CDs are largely intact in the mid-SC, but replaced by electron-dense (hydrophilic) clefts (lacunae) that expand laterally, splitting lamellar arrays in the outer SC. Some undegraded DSG1/DSC1 redistribute uniformly into corneocyte envelopes (CEs) in the outer SC (shown by proteomics, Z-stack confocal imaging and immunoEM). CEs then thicken, likely facilitating exfoliation by increasing corneocyte rigidity. In vapor-fixed images, hydration only altered the volume of the extracellular compartment, expanding lacunae further separating membrane arrays. During dehydration, air replaced water, maintaining the expanded extracellular compartment. Hydration also provoked degradation of membranes by activating contiguous acidic ceramidase activity. Together, these studies identify several parallel mechanisms that orchestrate exfoliation from the surface of normal human skin.

Topical antihistamines display potent anti-inflammatory activity linked in part to enhanced permeability barrier function.

Systemic antagonists of the histamine type 1 and 2 receptors (H1/2r) are widely used as anti-pruritics and central sedatives, but demonstrate only modest anti-inflammatory activity. Because many inflammatory dermatoses result from defects in cutaneous barrier function, and because keratinocytes express both Hr1 and Hr2, we hypothesized that H1/2r antagonists might be more effective, if they were used topically to treat inflammatory dermatoses. Topical H1/2r antagonists additively enhanced permeability barrier homeostasis in normal mouse skin by: i) stimulation of epidermal differentiation, leading to thickened cornified envelopes; and ii) enhanced epidermal lipid synthesis and secretion. Since barrier homeostasis was enhanced to a comparable extent in mast cell-deficient mice, with no further improvement following application of topical H1/2r antagonists, H1/2r antagonists likely oppose mast cell-derived histamine. In four immunologically-diverse, murine disease models, characterized by either inflammation alone (acute irritant contact dermatitis, acute allergic contact dermatitis), or by prominent barrier abnormalities (subacute allergic contact dermatitis, atopic dermatitis), topical H1/2r agonists aggravated, while H1/2r antagonists improved inflammation and/or barrier function. The apparent ability of topical H1r/2r antagonists to target epidermal H1/2r could translate into increased efficacy in the treatment of inflammatory dermatoses, likely due to decreased inflammation and enhanced barrier function. These results could shift current paradigms of antihistamine utilization from a predominantly-systemic to a topical approach.

Basis for Enhanced Barrier Function of Pigmented Skin

Humans with darkly-pigmented skin display superior permeability barrier function in comparison to humans with lightly-pigmented skin. The reduced pH of the stratum corneum (SC) of darkly-pigmented skin could account for enhanced function, because acidifying lightly-pigmented human SC resets barrier function to darkly-pigmented levels. In SKH1 (non-pigmented) vs. SKH2/J (pigmented) hairless mice, we evaluated how a pigment-dependent reduction in pH could influence epidermal barrier function. Permeability barrier homeostasis is enhanced in SKH2/J vs. SKH1 mice, correlating with a reduced pH in the lower SC that co-localizes with the extrusion of melanin granules. Darkly-pigmented human epidermis also shows substantial melanin extrusion in the outer epidermis. Both acute barrier disruption and topical basic pH challenges accelerate re-acidification of SKH2/J (but not SKH1) SC, while inducing melanin extrusion. SKH2/J mice also display enhanced expression of the SC acidifying enzyme, secretory phospholipase A2f (sPLA2f). Enhanced barrier function of SKH2/J mice could be attributed to enhanced activity of two acidic pH-dependent, ceramide-generating enzymes, β-glucocerebrosidase and acidic sphingomyelinase, leading to accelerated maturation of SC lamellar bilayers. Finally, organotypic cultures of darkly-pigmented-bearing human keratinocytes display enhanced barrier function in comparison to lightly-pigmented cultures. Together, these results suggest that the superior barrier function of pigmented epidermis can be largely attributed to the pH-lowering impact of melanin persistence/extrusion and enhanced sPLA2f expression.

Cannabinoid receptors 1 and 2 oppositely regulate epidermal permeability barrier status and differentiation

Cannabinoid receptors (CBR) 1 and 2 have been implicated in keratinocyte differentiation/proliferation. How CB receptors affect epidermal permeability barrier and stratum corneum structure and function remains unclear. Permeability barrier abrogation was induced by sequential tape‐stripping of the SC and assessed in both CB1R and CB2R knockout (−/−) mice in comparison with wild‐type (+/+) littermates. Absence of CB1R delays permeability barrier recovery, while the latter was found to be accelerated in CB2R −/− mice. While increased lamellar body (LB) secretion is observed in CB2R −/− mice accounting for the enhanced recovery, CB1R −/− animals display strong alterations in lipid bilayer structures. Markers for epidermal differentiation (i.e. filaggrin, loricrin and involucrin) and terminal differentiation (i.e. TUNEL assay and caspase‐14 activation) were respectively decreased and increased in CB1R and CB2R −/− mice. Surprisingly, CB1R agonist treatment of human cultured keratinocytes increases mRNA of p21 and cytokeratin 1 and 10 and decreases cyclin D1 but protein levels remained unchanged. Such paradox could partially be explained by the increase in non‐phosphorylated‐4E‐BP1, an inhibitor of mRNA translation, following CB1R agonist treatment. Altogether, these observations put forward the importance and the complexity of cannabinoid signalling for the regulation of permeability barrier and epidermal differentiation.

Actin dynamics regulate immediate PAR-2-dependent responses to acute epidermal permeability barrier abrogation

BACKGROUND Lamellar body (LB) secretion and terminal differentiation of stratum granulosum (SG) cells are signaled by both protease activated receptor-2 (PAR-2) and caveolin-1 (cav-1). OBJECTIVE To address the early dynamics of LB secretion, we examined cytoskeletal remodeling of keratinocytes in 3 mouse models following acute barrier abrogation: hairless mice, PAR-2 knockout (-/-) and cav-1 -/-. METHODS AND RESULTS Under basal conditions, globular (G)-actin accumulates in SG cells cytosol, while filamentous (F)-actin is restricted to peri-membrane domains. Barrier abrogation induces the apical movement of F-actin and the retreat of the SG-G-actin front, paralleled by upstream cytoskeletal kinases activation. This phenomenon was both enhanced by PAR-2 agonist, and inhibited by cytochalasin-D and in PAR-2 knockout mice. We found that plasma membrane conformational changes causing LB secretion are controlled by PAR-2-dependent cytoskeletal rearrangements. We next addressed the interaction dynamics between cytoskeleton and plasma membrane following PAR-2-induced actin stress fiber formation in both cav-1 -/- and wildtype cells. Actin stress fiber formation is increased in cav-1 -/- cells prior to and following PAR-2 agonist peptide-treatment, while absence of cav-1 inhibits E-cadherin-mediated cell-to-cell adhesion. CONCLUSION PAR-2 drives cytoskeletal/plasma membrane dynamics that regulate early LB secretion following barrier abrogation, stress fiber formation and keratinocyte adhesion.

Practical Use and Significance of Transepidermal Water Loss Measurements

Transepidermal water loss (TEWL) is universally recognized to be a measure of skin barrier function, either at baseline, after experimentally induced barrier abrogation or following topical treatments. In mammals, it is also known as “insensible water loss” as it is a process over which organisms have little physiological control. Measurements of TEWL (grams per square meter per hour) is useful for identifying skin damage caused by certain chemicals, physical insult (such as “tape stripping”), or pathological conditions such as eczema [11], as rates of TEWL increase in proportion to the level of damage even before the damage is clinically visible. It may thus be considered as the tool that evaluates the water barrier function of the epidermis.

Hemorrhagic regression of melanoma metastases during therapeutic vaccination: a report of three cases.

Melanoma metastases are characterized by pronounced neo-angiogenesis and spontaneous bleeding frequently occurring within central nervous system metastases. Clinically apparent spontaneous hemorrhage within subcutaneous melanoma metastases, however, is a rare event that coincides with progression of such metastases. We report, to our knowledge the first observation, on regression of subcutaneous metastases with hemorrhage of the overlying skin in three patients with stage IV melanoma who participated in clinical trials on therapeutic vaccination. In two patients, loss of arterial flow on Doppler ultrasound imaging was documented in the metastasis at the time of hematoma formation. One patient suffered from an intracranial hemorrhage in a subcentimetric brain metastasis coincident with the hemorrhagic regression of some of his skin metastases.