Lamellar Granule Extrusion and Stratum Corneum Intercellular Lamellae in Murine Keratinocyte Cultures

Lamellar granules are specialized epidermal organelles containing stacks of membranous disks that are extruded into the intercellular spaces in the upper portion of the granular layer. The extruded disks are believed to undergo biochemical and biophysical changes to form the stratum corneum intercellular lipid sheets that constitute the epidermal permeability barrier. Little is known about this important component of epidermal differentiation, in part due to lack of a suitable in vitro model. We have demonstrated microscopically the presence of characteristic lipid membrane structures in a primary keratinocyte culture system which shown morphologic differentiation comparable to that seen in vivo. A basal cell-enriched fraction of isolated neonatal mouse keratinocytes was plated into Vitrogen-coated 30mm Millicell (Millipore, Bedford, Massachusetts) wells, fed daily with Medium 199 containing 10% fetal bovine serum, 10 μg/ml each of insulin and hydrocortisone, and kept at 32°C in a 5% CO2/95% air atmosphere in a humidified incubator. Three days after plating, cultures were placed on living, epidermis-free mouse dermis at the air/liquid interface. At 2 wk, histologic examination showed multiple well-organized cell layers, including a distinct granular layer and a well-developed stratum corneum. Transmission electron microscopy demonstrated numerous lamellar granules and extrusion of their contents into the intercellular space. After fixation with ruthenium tetroxide, stacked intercellular lamellae in the stratum corneum were seen. Both the presence of dermis and growth at the air/liquid interface were necessary to achieve complete differentiation. This system conclusively demonstrates the formation of complex epidermal lipid structures in vitro and should allow the mechanisms and regulation of their synthesis to be elucidated

The Role of the Corneocyte Lipid Envelopes in Cohesion of the Stratum Corneum

Treatment of isolated stratum corneum with certain detergents results in complete disaggregation of the corneocytes within hours at 45°C without agitation. This is prevented by prior heating of the tissue to 80° C or by solvent extraction of the intercellular lipids. In the present study, electron micros- copy revealed that the heated or solvent-extracted tissue was characterized by cell-to-cell contacts that appeared to involve the chemically bound hydroxyceramides which constitute the corneocyte lipid envelope. It is proposed that the irreversible bonding between corneocytes that results from heating or lipid extraction results from interdigitation of the sphingosine chains belonging to those hydroxyceramides that are bound to the corneocyte protein envelope by the ω-hydroxyl function of the 30- and 32-carbon hydroxyacid moieties. Similar interdigitaion of adjacent envelopes might be involved in natural stratum corneum cohesion, limited mostly to the periphery of corneocytes where the absence of inter-cellular lamellae allows the appropriate cell-to-cell contact

Molecular models of the Intercellular Lipid Lamellae in Mammalian Stratum Corneum

Intercellular lipid lamellae in the stratum corneum constitute the barrier to water diffusion and may also play a role in cohesion between corneocytes. The lamellae arise from stacks of lamellar disks that are extruded from the granular cells and then fuse edge-to-edge to form sheets. It has been proposed that each lamellar disk is formed from a flattened vesicle, and therefore consists of two lipid bilayers in close apposition. In the present study, electron microscopic examination of ruthenium-tetroxide-fixed stratum corneum from mouse, pig, and human skin revealed that the double bilayer pattern persists in the intercellular lamellae. In addition, distinctive patterning of the intercellular lamellae has led us to propose novel molecular arrangements of the intercellular lipids. These include interlamellar sharing of lipid chains to produce lipid monolayers between pairs of bilayers. The pattern reflects the provenance of the intercellular lamellae from lamellar granule disks and the nonrandom orientation of the lamellar lipids