Both exposure of stratum corneum to neutral pH buffers and blockade of acidification mechanisms disturb cutaneous permeability barrier homeostasis and stratum corneum integrity/cohesion, but these approaches all introduce potentially confounding variables. To study the consequences of stratum corneum neutralization, independent of hydration, we applied two chemically unrelated superbases, 1,1,3,3-tetramethylguanidine or 1,8-diazabicyclo [5,4,0] undec-7-ene, in propylene glycol:ethanol (7:3) to hairless mouse skin and assessed whether discrete pH changes alone regulate cutaneous permeability barrier function and stratum corneum integrity/cohesion, as well as the responsible mechanisms. Both 1,1,3,3-tetramethylguanidine and 1,8-diazabicyclo [5,4,0] undec-7-ene applications increased skin surface pH in parallel with abnormalities in both barrier homeostasis and stratum corneum integrity/cohesion. The latter was attributable to rapid activation (<20 min) of serine proteases, assessed by in situ zymography, followed by serine-protease-mediated degradation of corneodesmosomes. Western blotting revealed degradation of desmoglein 1, a key corneodesmosome structural protein, in parallel with loss of corneodesmosomes. Coapplication of serine protease inhibitors with the superbase normalized stratum corneum integrity/cohesion. The superbases also delayed permeability barrier recovery, attributable to decreased beta-glucocerebrosidase activity, assessed zymographically, resulting in a lipid-processing defect on electron microscopy. These studies demonstrate unequivocally that stratum corneum neutralization alone provokes stratum corneum functional abnormalities, including aberrant permeability barrier homeostasis and decreased stratum corneum integrity/cohesion, as well as the mechanisms responsible for these abnormalities.

There is evidence that the "acid mantle" of the stratum corneum is important for both permeability barrier formation and cutaneous antimicrobial defense. The origin of the acidic pH of the stratum corneum remains conjectural, however. Both passive (e.g., eccrine/sebaceous secretions, proteolytic) and active (e.g., proton pumps) mechanisms have been proposed. We assessed here whether the free fatty acid pool, which is derived from phospholipase-mediated hydrolysis of phospholipids during cornification, contributes to stratum corneum acidification and function. Topical applications of two chemically unrelated secretory phospholipase sPLA2 inhibitors, bromphenacylbromide and 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol, for 3 d produced an increase in the pH of murine skin surface that was paralleled not only by a permeability barrier abnormality but also altered stratum corneum integrity (number of strippings required to break the barrier) and decreased stratum corneum cohesion (protein weight removed per stripping). Not only stratum corneum pH but also all of the functional abnormalities normalized when either palmitic, stearic, or linoleic acids were coapplied with the inhibitors. Moreover, exposure of intact murine stratum corneum to a neutral pH for as little as 3 h produced comparable abnormalities in stratum corneum integrity and cohesion, and further amplified the inhibitor-induced functional alterations. Furthermore, short-term applications of an acidic pH buffer to inhibitor-treated skin also reversed the abnormalities in stratum corneum integrity and cohesion, despite the ongoing decrease in free fatty acid levels. Finally, the secretory-phospholipase-inhibitor-induced alterations in integrity/cohesion were in accordance with premature dissolution of desmosomes, demonstrated both by electron microscopy and by reduced desmoglein 1 levels in the stratum corneum (shown by immunofluorescence staining and visualized by confocal microscopy). Together, these results demonstrate: (i) the importance of phospholipid-to-free-fatty-acid processing for normal stratum corneum acidification; and (ii) the potentially important role of this pathway not only for barrier homeostasis but also for the dual functions of stratum corneum integrity and cohesion.

Prolonged exposure of human epidermis to excess endogenous or exogenous glucocorticoids can result in well-recognized cutaneous abnormalities. Here, we determined whether short-term glucocorticoid treatment would also display adverse effects, specifically on two key epidermal functions, permeability barrier homeostasis and stratum corneum integrity and cohesion, and the basis for such changes. In humans 3 d of treatment with a potent, commonly employed topical glucocorticoid (clobetasol), applied topically, produced a deterioration in barrier homeostasis, characterized by delayed barrier recovery and abnormal stratum corneum integrity (rate of barrier disruption with tape strippings) and stratum corneum cohesion (microg protein removed per stripping). Short-term systemic and topical glucocorticoid produced similar functional defects in mice, where the basis for these abnormalities was explored further. Both the production and secretion of lamellar bodies were profoundly decreased in topical glucocorticoid-treated mice resulting in decreased extracellular lamellar bilayers. These structural changes, in turn, were attributable to a profound global inhibition of lipid synthesis, demonstrated both in epidermis and in cultured human keratinocytes. The basis for the abnormality in stratum corneum integrity and cohesion was a diminution in the density of corneodesmosomes in the lower stratum corneum. We next performed topical replacement studies to determine whether lipid deficiency accounts for the glucocorticoid-induced functional abnormalities. The abnormalities in both permeability barrier homeostasis and stratum corneum integrity were corrected by topical applications of an equimolar distribution of free fatty acids, cholesterol, and ceramides, indicating that glucocorticoid-induced inhibition of epidermal lipid synthesis accounts for the derangements in both cutaneous barrier function and stratum corneum integrity/cohesion. These studies indicate that even short-term exposure to potent glucocorticosteroids can exert profound negative effects on cutaneous structure and function. Finally, topical replenishment with epidermal physiologic lipids could represent a potential method to reduce the adverse cutaneous effects of both topical glucocorticoid treatment and Cushing's syndrome.