Gopinathan K. Menon

Processing of epidermal glucosylceramides is required for optimal mammalian cutaneous permeability barrier function.

The interstices of the mammalian stratum corneum contain lipids in a system of continuous membrane bilayers critical for the epidermal permeability barrier. During the transition from inner to outer stratum corneum, the content of polar lipids including glucosylceramides, decreases while ceramide content increases. We investigated whether inhibition of glucosylceramide hydrolysis would alter epidermal permeability barrier function. Daily topical applications of bromoconduritol B epoxide (BrCBE) to intact murine skin selectively inhibited beta-glucocerebrosidase, increased glucosylceramide content of stratum corneum with ceramide content remaining largely unchanged, and caused a progressive, reversible decrease in barrier function. Histochemistry of inhibitor-treated epidermis revealed persistence of periodic acid-Schiff-positive staining in stratum corneum cell membranes, consistent with retention of hexose moieties. Electron microscopy of inhibitor-treated samples revealed no evidence of toxicity or changes in the epidermal lipid delivery system. However, immature membrane structures persisted in the intercellular spaces throughout the stratum corneum, with reappearance of mature membrane structures progressing outward from the lower stratum corneum upon termination of BrCBE. Finally, the induced barrier abnormality was not reversed by coapplications of ceramide. These data demonstrate that glucosylceramide hydrolysis is important in the formation of the epidermal permeability barrier, and suggest that accumulation of glucosylceramides in stratum corneum intercellular membrane domains leads to abnormal barrier function.

Consequences of beta-glucocerebrosidase deficiency in epidermis. Ultrastructure and permeability barrier alterations in Gaucher disease.

Hydrolysis of glucosylceramide by beta-glucocerebrosidase results in ceramide, a critical component of the intercellular lamellae that mediate the epidermal permeability barrier. A subset of type 2 Gaucher patients displays ichthyosiform skin abnormalities, as do transgenic Gaucher mice homozygous for a null allele. To investigate the relationship between glucocerebrosidase deficiency and epidermal permeability barrier function, we compared the stratum corneum (SC) ultrastructure, lipid content, and barrier function of Gaucher mice to carrier and normal mice, and to hairless mice treated topically with bromoconduritol B epoxide (BrCBE), an irreversible inhibitor of glucocerebrosidase. Both Gaucher mice and BrCBE-treated mice revealed abnormal, incompletely processed, lamellar body-derived sheets throughout the SC interstices, while transgenic carrier mice displayed normal bilayers. The SC of a severely affected type 2 Gauchers disease infant revealed similarly abnormal ultrastructure. Furthermore, the Gaucher mice demonstrated markedly elevated transepidermal water loss (4.2 +/- 0.6 vs < 0.10 g/m2 per h). The electron-dense tracer, colloidal lanthanum, percolated between the incompletely processed lamellar body-derived sheets in the SC interstices of Gaucher mice only, demonstrating altered permeability barrier function. Gaucher and BrCBE-treated mice showed < 1% and < 5% of normal epidermal glucocerebrosidase activity, respectively, and the epidermis/SC of Gaucher mice demonstrated elevated glucosylceramide (5- to 10-fold), with diminished ceramide content. Thus, the skin changes observed in Gaucher mice and infants may result from the formation of incompetent intercellular lamellar bilayers due to a decreased hydrolysis of glucosylceramide to ceramide. Glucocerebrosidase therefore appears necessary for the generation of membranes of sufficient functional competence for epidermal barrier function.

Barrier function regulates epidermal lipid and DNA synthesis

The stratum corneum, the permeability barrier between the internal milieu and the environment, is composed of fibrous protein‐enriched corneocytes and a lipid‐enriched intercellular matrix. The lipids are a mixture of sphingolipids, cholesterol and free fatty acids, which form intercellular membrane bilayers. Lipid synthesis occurs in the keratinocytes in all nucleated layers of the epidermis, and the newly synthesized lipids are delivered by lamellar bodies to the interstices of the stratum corneum during epidermal differentiation. Disruption of barrier function by topical acetone treatment results in an increase in the synthesis of free fatty acids, sphingolipids and cholesterol in the living layers of the epidermis, leading to barrier repair. Cholesterol and sphingolipid synthesis are regulated by the rate‐limiting enzymes HMG CoA reductase and serine palmitoyi transferase (SPT). respectively. Acute barrier disruption leads to an increase in both enzymes, but with a different time curve: increase in HMG CoA reductase activity begins at 1.5 h, whereas the increase in SPT activity occurs 6 h after barrier impairment. Topical application of HMG CoA reductase or SPT inhibitors after acetone treatment delays barrier repair, providing further evidence for a role of cholesterol and sphingolipids in epidermal barrier function. Repeated application of lovastatin to untreated skin results in disturbed barrier function accompanied by increased DNA synthesis and epidermal hyperplasia. Therefore, we have examined the specific relationship between barrier function and epidermal DNA synthesis. After acute and chronic disturbances not only lipid, but also DNA synthesis, is stimulated. Thus, stimulation of DNA synthesis leading to epidermal hyperplasia may be a second mechanism by which the epidermis repairs defects in barrier function. The link between barrier function and both lipid and DNA synthesis is supported further by occlusion studies. Artificial barrier repair by latex occlusion prevents an increase in both lipid and DNA synthesis. In addition, increased epidermal lipid and DNA synthesis in essential fatty‐acid deficiency can be reversed by topical applications of the n‐6 unsaturated fatty acids, linoleic or columbinic acid. These studies may be of relevance in understanding the pathogenesis of hyperproliferative skin diseases, such as ichthyosis, psoriasis, atopic dermatitis, and irritant contact dermatitis.

Sphingolipids are required for mammalian epidermal barrier function. Inhibition of sphingolipid synthesis delays barrier recovery after acute perturbation.

Stratum corneum lipids comprise an approximately equimolar mixture of sphingolipids, cholesterol, and free fatty acids, arranged as intercellular membrane bilayers that are presumed to mediate the epidermal permeability barrier. Prior studies have shown that alterations in epidermal barrier function lead to a rapid increase in cholesterol and fatty acid synthesis which parallels the early stages of the repair process. Despite an abundance of indirect evidence for their role in the barrier, the importance of sphingolipids has yet to be demonstrated directly. Whereas sphingolipid synthesis also increases during barrier repair, this response is delayed in comparison to cholesterol and fatty acid synthesis (Holleran, W.M., et al. 1991. J. Lipid Res. 32:1151-1158). To further delineate the role of sphingolipids in barrier homeostasis, we assessed the impact of inhibition of sphingolipid synthesis on epidermal barrier recovery. A single topical application of beta-chloro-L-alanine (beta-CA), an irreversible inhibitor of serine-palmitoyl transferase (SPT), applied to acetone-treated skin of hairless mice resulted in: (a) greater than 75% inhibition of SPT activity at 30 min (P less than 0.001); (b) a global decrease in sphingolipid synthesis between 1 and 3 h (P less than 0.02); (c) reduction of epidermal sphingolipid content at 18 h (P less than 0.01); (d) delayed reaccumulation of histochemical staining for sphingolipids in the stratum corneum; and (e) reduced numbers and contents of lamellar bodies in the stratum granulosum. Finally, despite its immediate, marked diminution of sphingolipid synthesis, beta-CA slowed barrier recovery only at late time points (greater than 6 h) after acetone treatment. This inhibition was overridden by coapplications of ceramides (the distal SPT product), indicating that the delay in repair was not due to non-specific toxicity. These studies demonstrate a distinctive role for epidermal sphingolipids in permeability barrier homeostasis.

Calcium and potassium are important regulators of barrier homeostasis in murine epidermis.

Topical solvent treatment removes lipids from the stratum corneum leading to a marked increase in transepidermal water loss (TEWL). This disturbance stimulates a variety of metabolic changes in the epidermis leading to rapid repair of the barrier defect. Using an immersion system we explored the nature of the signal leading to barrier repair in intact mice. Initial experiments using hypotonic to hypertonic solutions showed that water transit per se was not the crucial signal. However, addition of calcium at concentrations as low as 0.01 mM inhibited barrier repair. Moreover, both verapamil and nifedipine, which block calcium transport into cells, prevented the calcium-induced inhibition of TEWL recovery. Additionally, trifluoroperazine or N-6-aminohexyl-5-chloro-1-naphthalenesulfonamide, which inhibit calmodulin, prevented the calcium-induced inhibition of TEWL recovery. Although these results suggest an important role for calcium in barrier homeostasis, calcium alone was only modestly effective in inhibiting TEWL recovery. Potassium alone (10 mM) and phosphate alone (5 mM) also produced a modest inhibition of barrier repair. Together, however, calcium and potassium produced a synergistic inhibition of barrier repair (control 50% recovery vs. calcium + potassium 0-11% recovery in 2.5 h). Furthermore, in addition to inhibiting TEWL recovery, calcium and potassium also prevented the characteristic increase in 3-hydroxy-3-glutaryl CoA reductase activity that occurs after barrier disruption. Finally, the return of lipids to the stratum corneum was also blocked by calcium and potassium. These results demonstrate that the repair of the epidermal permeability barrier after solvent disruption can be prevented by calcium, potassium, and phosphate. The repair process may be signalled by a decrease in the concentrations of these ions in the upper epidermis resulting from increased water flux leading to passive loss of these ions.

Cholesterol synthesis is required for cutaneous barrier function in mice.

Previous studies have shown that topical acetone treatment results in the removal of stratum corneum lipids and disruption of the permeability barrier. This disruption stimulates epidermal lipid synthesis which is associated with the rapid restoration of stratum corneum lipids and barrier function. The aim of this study was to determine the role of cutaneous cholesterol synthesis in the barrier recovery. Here we show that topical lovastatin, a competitive inhibitor of HMG CoA reductase, inhibits cholesterol synthesis. After acetone disruption of the barrier, the normal rapid return of cholesterol to the stratum corneum and recovery of barrier function is impaired in animals treated topically with lovastatin. When lovastatin animals are simultaneously treated topically with either mevalonate, the immediate product of HMG CoA reductase, or cholesterol, the final end product of the pathway, the recovery of the barrier is normalized. Lovastatin resulted in the delayed secretion and abnormal appearance of lamellar bodies. These results provide the first evidence demonstrating that cholesterol synthesis is required for the maintenance of barrier structure and function and suggests a crucial role for cholesterol synthesis in allowing for terrestrial existence.

Sonophoresis. II. Examination of the Mechanism(s) of Ultrasound-Enhanced Transdermal Drug Delivery

We have shown previously that high-frequency ultrasound (sonophoresis) can significantly enhance the transdermal delivery of a topically applied drug in vivo and that the augmentation of transport was caused by the action of the ultrasound on the skin. However, these earlier experiments did not reveal (i) the mechanism of sonophoresis, (ii) the pathway of drug permeation under the influence of ultrasound, and (iii) any potentially detrimental effects of the enhancement procedure on skin structure and morphology. In the study reported here, these three key issues have been addressed using electron microscopy to follow the penetration of an electron-dense, colloidal tracer (lanthanum hydroxide; LH). Experiments have again been performed using the hairless guinea pig animal model. Colloidal LH suspensions were applied to skin sites, which were then immediately exposed to ultrasound (at 10 or 16 MHz) for 5 or 20 min. Passive transport of LH under identical conditions (but without ultrasound) provided the control measurements. Tissue processing after the treatment periods utilized standard electron microscopy staining procedures. We found the following: (1) LH does not permeate the skin by passive diffusion; under the influence of ultrasound, on the other hand, it penetrates through the stratum corneum (SC) and the underlying viable epidermal cell layers via an apparently intercellular route. (2) LH transports through the epidermis to the upper dermis, even after only 5 min of ultrasound treatment, a remarkable and unexpected finding. (3) The SC and the cells of the epidermis do not appear to be adversely affected by either (a) ultrasound treatment at 10-MHz frequency (5- or 20-min exposure) or (b) 5 min of sonophoresis at 16 MHz. However, a 20-min treatment with ultrasound at 16 MHz resulted in altered cellular morphology compared to the passive control. The distribution of the tracer in the latter experiments was nonuniform and suggested that cavitational effects may have contributed to the adverse observations. Overall, the results demonstrate that exposure of the skin to ultrasound can induce the considerable and rapid facilitation of LH transport via an intercellular route. Prolonged exposures at high frequencies, however, can alter epidermal morphology, leading us to pose further questions pertaining to the duration and reversibility of ultrasound action on skin.

Lamellar bodies as delivery systems of hydrolytic enzymes: Implications for normal and abnormal desquamation

Lamellar body secretion results in the delivery of a selected array of hydrolytic enzymes to the extracellular domains of stratum corneum (SC). Deposition and activation of these enzymes in the interstices presumably is associated with the transformation of lamellar body‐derived lipids from a relatively polar to a non‐polar mixture, as well as the degradation of other non‐lipid intercellular substrates. To determine whether abnormal desquamation might result from failure of hydrolytic enzyme delivery to the SC interstices, we localized one catabolic enzyme, acid lipase, previously shown to be a reproducible marker for the lamellar body secretory system, by cytochemical methods within the epidermis of selected human (congenital ichthyosiform erythroderma, CIE) and animal (essential fatty‐acid deficient (EFAD) mouse epidermis and mouse tail epidermis) models associated with abnormal scaling or unusual SC retention. In addition, we compared the persistence of desmosomes within normal SC vs. the various models. Normal human and murine epidermis displayed abundant lipase activity both in lamellar bodies (LB) and in association with secreted lamellar body contents in the SC interstices. Despite normal quantities of LB in CIE, EFAD, and mouse tail epidermis, lipase activity was markedly deficient both in LB and in the SC intercellular domains. These studies support the hypothesis that normal desquamation is mediated by enzymatic modulations in lipid and/or protein content of the SC interstices, and that some forms of pathological or excessive scaling may be due to desmosomal persistence that results from defective or limited delivery of lamellar body‐derived, hydrolytic enzymes to the SC intercellular domains

Morphologic basis for a pore-pathway in mammalian stratum corneum

Although prior morphologic studies have shown that both polar and nonpolar materials permeate across the stratum corneum (SC) via a paracellular route, the actual pathway through these heterogeneous domains is unknown. We applied hydrophilic and hydrophobic tracers in vivo to murine skin under basal conditions and/or after permeation enhancement with occlusion, vehicle enhancers, a lipid synthesis inhibitor, sonophoresis, and iontophoresis. Ruthenium tetroxide, ruthenium red plus osmium tetroxide, in situ precipitation with osmium vapor, and microwave postfixation methods were used to visualize penetration pathways. Tracers invariably localized to discrete lacunar domains embedded within the extracellular lamellar membrane system, regardless of their polarity or the enhancement method. Moreover, while the lacunar domains remained discontinuous under basal conditions, they appeared to gain structural continuity with permeation enhancement. These results indicate that extracellular lacunar domains comprise a pore pathway for penetration of polar and nonpolar molecules across the SC.

Ultrastructural organization of avian stratum corneum lipids as the basis for facultative cutaneous waterproofing

The ultrastructure of naked neck epidermis from the ostrich (Struthio camelus) and ventral apterium from watered, and water‐deprived, Zebra finches (Taeniopygia [Poephila] guttata castanotis) is presented. The form and distribution of the fully differentiated products of the lipid‐enriched multigranular bodies are compared in biopsies post‐fixed with osmium tetroxide or ruthenium tetroxide. The fine structure of ostrich epidermis suggests it is a relatively poor barrier to cutaneous water loss (CWL). The fine structure from watered, and 16‐hr water‐deprived Zebra finches, considered in conjunction with measurements of CWL, confirms previous reports of “facultative waterproofing,” and emphasizes the rapidity of tissue response to dehydration. The seemingly counterintuitive facts that one xerophilic avian species, the ostrich, lacks a “good barrier” to CWL, whereas another, the Zebra finch, is capable of forming a good barrier, but does not always express this capability, are discussed. An explanation of these data in comparison to mammals centers on the dual roles of the integument of homeotherms in thermoregulation and conserving body water. It is concluded that birds, whose homeothermic control depends so much on CWL, cannot possess a permanent “good barrier,” as such would compromise the heat loss mechanism. Facultative waterproofing (also documented in lizards) protects the organism against sudden reductions in water availability. In birds, and probably in snakes and lizards, facultative waterproofing involves qualititative changes in epidermal cell differentiation. Possible control mechanisms are discussed.