The role of ceramides 1 and 2 in the stratum corneum lipid organisation.

A mixture of ceramide 1 and ceramide 2 (CER(1 + 2)) was isolated from pig stratum corneum and mixed in various molar ratios with cholesterol (CHOL) or with CHOL and palmitic acid (PA). The mixtures were hydrated in a buffer solution of pH 5.0 and their phase behaviour was studied by wide- and small-angle X-ray diffraction. The small-angle diffraction curve of the CHOL/CER(1 + 2) mixture at a molar ratio of 0.4 revealed the presence of only one peak at a spacing of 6.7 nm. Increasing the amount of CHOL to a molar ratio of 0.6 was accompanied by a shift of this peak to a smaller spacing (5.7 nm) and the appearance of two weak peaks at 11.8 and 4.1 nm spacings. Increasing the CHOL content to an equimolar ratio resulted in the appearance of two lamellar phases with periodicities of 5.5 and 12 nm, respectively. In a CHOL/CER(1 + 2) mixture at a molar ratio of 2 the periodicities of the two phases were 5.6 and 12 nm, respectively. From these observations it was concluded that the CHOL/CER(1 + 2) mixtures exerted similar phase behaviour, as reported earlier for intact SC (Bouwstra et al. (1995) J. Lipid Res. 36, 496-504) and for mixtures (Bouwstra et al. (1996) J. Lipid Res., in press) prepared from CHOL and total ceramide fraction (CER) isolated from pig stratum corneum. However, in the CHOL/CER mixtures a lower relative amount of CHOL was required to acquire these lamellar phases, indicating that at low CHOL contents, CER 3, 4, 5 and 6 play a crucial role in the formation of the lamellar phases. Furthermore, the solubility of CHOL in the mixtures increased in the presence of CER 1, suggesting its important role for the barrier function of the skin. When palmitic acid (PA) was included, the phase behaviour of the CHOL/CER(1 + 2)/PA mixture was more complex. Next to two lamellar phases, an additional phase with a spacing of 3.77 nm was observed, never seen in intact stratum corneum. In the absence of CHOL, the wide-angle diffraction pattern of the CER(1 + 2) revealed one sharp reflection at 0.456 nm and two diffuse reflections at 0.430, 0.417 nm and 0.395 nm, indicating the presence of a crystalline sublattice. In an equimolar mixture of CHOL/CER(1 + 2) no sharp 0.456 nm reflection was observed indicating a more disordered packing. Furthermore, phase separation of CHOL occurred, this conclusion is based on the presence of reflections corresponding to polycrystalline cholesterol monohydrate. These findings indicate that the lateral packing of mixtures of CHOL/CER(1 + 2) is more complex than that of the CHOL/CER mixtures that reveals a hexagonal lateral packing.

The role of ceramide composition in the lipid organisation of the skin barrier.

The lipid lamellae in the stratum corneum (SC) play a key role in the barrier function of the skin. The major lipids are ceramides (CER), cholesterol (CHOL) and free fatty acids (FFA). In pig SC at least six subclasses of ceramides (referred to as CER 1, 2-6) are present. Recently it was shown that in mixtures of isolated pig SC ceramides (referred to as CER(1-6)) and CHOL two lamellar phases are formed, which mimic SC lipid organisation very closely [J.A. Bouwstra et al., 1996, J. Lipid Res. 37, 999-1011] [1]. Since the CER composition in SC originating from different sources/donors often varies, information on the effect of variations in CER composition on the SC lipid organisation is important. The results of the present study with mixtures of CHOL including two different CER mixtures that lack CER 6 (CER(1-5) mixtures) revealed that at an equimolar molar ratio their lipid organisation was similar to that of the equimolar CHOL:CER(1-6) and CHOL:CER(1,2) mixtures, described previously. These observations suggest that at an equimolar CHOL:CER ratio the lipid organisation is remarkably insensitive toward a change in the CER composition. Similar observations have been made with equimolar CHOL:CER:FFA mixtures. The situation is different when the CHOL:CER molar ratio varies. While in the CHOL:CER(1-6) mixture the lamellar organisation hardly changed with varying molar ratio from 0.4 to 2, the lamellar organisation in the CHOL:CER(1-5) mixtures appeared to be more sensitive to a change in the relative CHOL content, especially concerning the changes in the periodicities of the lamellar phases. In summary, these findings clearly indicate that at an equimolar CHOL:CER molar ratio the lamellar organisation is least sensitive to a variation in CER composition, while at a reduced CHOL:CER molar ratio the CER composition plays a more prominent role in the lamellar phases. This observation may have an implication for the in vivo situation when both the CER composition and the CHOL:CER molar ratio change simultaneously.

Freeze-fracture electron microscopy of in vitro reconstructed human epidermis.

Epidermis has been reconstructed in vitro by seeding human keratinocytes on a human dermal substrate in an air-exposed culture. The end product has been examined by freeze-fracture electron microscopy, transmission electron microscopy (TEM) of thin sections, light microscopy, and lipid analysis using thin-layer chromatography. Light microscopic observation of hematoxylin-eosin stained, paraffin embedded cross-sections of the cell culture revealed a strong resemblance to its intact human counterpart, especially with respect to the morphologic organization in basal, spinous, granular, and horny layers. Freeze-fracture electron microscopy and TEM of thin sections generally confirmed the observed resemblances and additionally suggested the presence of lamellar bodies in the stratum granulosum, and of lamellar (lipid) structures between the corneocytes. However, some imperfections were also observed, including some anomalous lipid structures in the intercellular space. Lipid analyses in conjunction with essential fatty acid enrichment studies suggested that the structural anomalies observed in the cultured system may be caused by a lack of linoleyl-ceramides resulting from "immobilization" of linoleyl moieties in the form of triglycerides and phospholipids. In its present form, the air-exposed cell culture already looks very promising as a model for studies of, e.g., skin differentiation disorders such as psoriasis or ichthyosis, studies of the percutaneous penetration and intra(epi)dermal biotransformation of drugs, and skin toxicity screenings. It is furthermore expected that the aforementioned imperfections in the air-exposed cell culture should be avoidable by changing culture conditions such as the relative humidity and the pH, the composition of the medium, or both.

The formation of competent barrier lipids in reconstructed human epidermis requires the presence of vitamin C.

Our analysis of epidermal lipids revealed that (glucosyl)ceramide profiles in various human skin equivalents are different from those of native tissue. The main difference is the reduced content in skin equivalents of ceramides 4-7 and especially the very low content of the most polar ceramides 6 and 7, which contain hydroxylated sphingoid base and/or fatty acid. To facilitate hydroxylation, the culture medium was supplemented with vitamins C and E. Although in vitamin E-supplemented medium lipogenesis was not affected, in vitamin C-supplemented medium the content of glucosylceramides and of ceramides 6 and 7 was markedly increased, both in the presence and absence of serum and irrespective the substrate used (inert or natural, populated or not with fibroblasts). The improvement of the lipid profile was accompanied by a marked improvement of the barrier formation as judged from extensive production of lamellar bodies, their complete extrusion at the stratum granulosum/stratum corneum interface, and the formation of multiple broad lipid lamellar structures in the intercorneocyte space. The presence of well-ordered lipid lamellar phases was confirmed by small-angle x-ray diffraction. Some differences between native and reconstructed epidermis, however, were noticed. Although the long-range lipid lamellar phase was present in both the native and the reconstructed epidermis, the short lamellar phase was present only in native tissue. It remains to be established whether these differences can be ascribed to small differences in relative amounts of individual ceramides, to differences in fatty acid profiles, or to differences in cholesterol sulfate, pH, or calcium gradients. The results indicate the key role vitamin C plays in the formation of stratum corneum barrier lipids.

Reduced skin barrier function parallels abnormal stratum corneum lipid organization in patients with lamellar ichthyosis.

Most patients with autosomal recessive lamellar ichthyosis are known to have markedly impaired skin barrier function. We hypothesize that this may be due to imperfections in the composition and fine structure of the intercellular stratum corneum lipids. The aim of the present study was to test this hypothesis. To characterize the barrier properties in three female patients with lamellar ichthyosis, the following parameters were used and compared with those of healthy volunteers: transepidermal water loss, stratum corneum lipid profiles after topical acetone/ether extraction on the flexure side of the forearm, and small-angle x-ray diffraction. The extracted lipids were separated using high performance thin-layer chromatography and quantified, and the ceramide profile was determined. Small-angle x-ray diffraction was used to obtain information on the molecular structure and organization of the intercellular lipid domains of stratum corneum using stratum corneum scales collected by scraping. Transepidermal water loss was significantly increased in all three patients. Lipid analysis showed significant differences in the relative amounts of ceramide fractions 2-3a-3b-4-5, free fatty acid-ceramide ratio, and free fatty acid-cholesterol ratio. Small-angle x-ray diffraction showed smaller repeated distances of lipid bilayers in stratum corneum samples of the patients compared with the healthy volunteers. An additional diffraction peak was found in the patients compared with the healthy volunteers, which can be ascribed to crystalline cholesterol. These data suggest that there might be a relation between the impaired barrier function and stratum corneum lipid structural and composition changes.

Determination of stratum corneum lipid profile by tape stripping in combination with high-performance thin-layer chromatography.

Intercellular lipids in the stratum corneum (SC) are responsible for the barrier function of mammalian skin. The main components of the SC lipids are ceramides, cholesterol, and free fatty acids, as established by thin-layer chromatographic analysis of lipids extracted from the human and mammalian SC. Up to now, for lipid analysis the extracts of the entire SC has been used and information on whether the lipid composition changes with the depth in the SC is scarce. Tape stripping is a technique which removes corneocyte layers step by step with an adhesive film. The use of this technique for lipid analysis was hampered by the contamination of lipid extracts with compounds co-extracted from the tape with organic solvents used for the extraction of SC lipids. The aim of the present study was to establish a suitable analytical method for the determination of the local SC lipid composition. For this purpose, the SC samples were collected by sequential stripping with Leukoplex tape in five healthy volunteers. The lipids were extracted with ethyl acetate:methanol mixture (20:80) and separated by means of HPTLC. The results of this study revealed that the free fatty acid level is highest and the cholesterol and ceramide levels lowest in the uppermost SC layers (about 4 strippings). The levels remained unchanged in the underlying SC layers. In these layers, the ceramide level was about 60 wt% and the free fatty acid and cholesterol levels were about 20 wt% each. Ceramides could be separated into seven different fractions and the relative amounts of individual ceramide fractions did not significantly change with the SC depth. Cholesterol sulfate levels were about 5% of total cholesterol and did not change with the SC depth, except for the for the first strip where the level was about 1%. The method developed makes it possible to study the differences in the SC lipid profile in healthy and diseased human skin with relation to the SC lipid organization and to the skin barrier function in vivo.

Incorporation of linoleic acid by cultured human keratinocytes.

Linoleic acid is required for the formation and maintenance of the epidermal barrier, but most of the current in vitro keratinocyte culture systems are linoleic acid-deficient. The aim of the present study was to examine the efficiency of linoleic acid uptake in human keratinocyte cultures grown under submerged and air-exposed conditions in serum-free medium. The water-insoluble linoleic acid was bound to carrier molecules (cyclodextrin or bovine serum albumin). Comparable results were obtained with home-made and commercially available linoleic acid complexes. In the submerged cultures, the increase of the linoleic acid medium concentration (ranging from 0 to 20 microg/ml) resulted in a gradual increase in the linoleic acid cellular content, which exceeded 1.4 times the value found in native epidermis when the highest concentration of linoleic acid was used. The addition of linoleic acid did not alter the profile of the other epidermal fatty acids, with the exception of oleic acid, which decreased in parallel with the increasing linoleic acid content. While the content of linoleic acid found in phospholipids was similar to that in native epidermis, a large excess of linoleic acid was detected in triglycerides, the synthesis of which was markedly increased in cultures grown submerged in medium containing higher concentrations of linoleic acid. Under air-exposed conditions, the dermal substrate used seemed to be the most limiting factor for efficient linoleic acid supplementation. A low linoleic acid cellular content was detected when an inert filter was used. De-epidermized dermis was found to be the most permeable substrate for linoleic acid complexes. The cellular linoleic acid content increased in a parallel with the increasing linoleic acid concentration (ranging from 4 to 30 microg/ml), but the overall amount incorporated was lower than that in submerged cultures. The content of linoleic acid in the phospholipid and ceramide fractions isolated from reconstructed epidermis grown under air-exposed conditions was close to that of native epidermis, but the triglycerides remained abnormally enriched in linoleic acid, indicating persistence of some anomalies in epidermal lipogenesis in vitro.

Epidermal growth factor and temperature regulate keratinocyte differentiation.

The limited life-span and irregularities in epidermal differentiation and barrier function that have restricted the utility of presently available skin culture models for pharmacological and toxicological studies indicate that further modifications of culture conditions are required for optimization of these models. In the present study epidermis reconstructed on de-epidermized dermis was used to investigate the effects of temperature and epidermal growth factor (EGF) on epidermal differentiation and lipogenesis. When cultured at 37 degrees C, keratinocytes formed a well-differentiated epidermis whether EGF was present or not. However, the thickness of the epidermis, particularly of the stratum corneum, was higher in the presence of EGF. Both the differentiation-specific protein markers (keratins 1 and 10, involucrin and transglutaminase) and lipid markers (ceramides) were synthesized. EGF-induced increases in triglyceride content caused accumulation of lipid droplets within the stratum corneum which is indicative of a hyperproliferative effect of EGF. In the absence of EGF, a well-differentiated epidermis was generated at 33 degrees C with a morphology showing a higher resemblance to native epidermis than cultures grown at 37 degrees C. The stratum corneum was less compact and with practically no lipid droplets, irregularly shaped keratohyalin granules were abundant in the stratum granulosum, lamellar body extrusion was improved and the number of stratum corneum layers was reduced to normal levels. However, EGF supplementation had a deleterious effect on epidermal morphogenesis and differentiation of cultures grown at 33 degrees C. The epidermis lacked a stratum granulosum and the stratum corneum contained a high number of nuclear remnants. The synthesis of the early specific protein differentiation markers (keratins 1 and 10) was suppressed on both the protein and mRNA levels without significant interference with the synthesis of late differentiation lipid markers, such as ceramides. From this observation it can be concluded that the synthesis of keratins associated with terminal differentiation is profoundly affected by the presence of EGF and is sensitive to temperature and that of ceramides is not. The finding that TGF alpha did not modulate the morphogenesis and synthesis of keratins 1 and 10 in cultures grown at 33 degrees C indicates possible differences between the postreceptor binding processes of these EGF receptor ligands.

Triglyceride metabolism in human keratinocytes cultured at the air-liquid interface.

Although epidermis reconstructed in vitro histologically demonstrates the presence of fully differentiated tissue with cornified strata, it does not synthesize or release epidermal barrier lipids in the same proportions as does native skin, causing the barrier function to be impaired. Lipids, the content of which deviates the most, include triglycerides that are present in high amounts and stored as lipid droplets. Our recent studies have revealed that a high triglyceride content may be a reflection of a high synthetic rate and a low turnover. Therefore, the present study was undertaken to examine whether the triglyceride accumulation in the air-exposed cultures may be a result of insufficient supplementation of cells with oxygen, an excessive supplementation of cells with glucose, dysregulation of lipogenesis, or an impaired catabolism of triglycerides caused either by insufficient activity of triglyceride lipase and/or accumulation of free fatty acids due to insufficient activity of beta-oxidase. When keratinocytes were cultured at the air-liquid interface in medium containing a standard glucose concentration, both the lactate and triglyceride production was high. Lowering glucose content in the medium resulted in a decrease in both lactate production and triglyceride synthesis. However, even when grown at a low glucose concentration the triglyceride content remained higher than found in vivo and synthesized triglycerides were stored in the cells as a stable pool, suggesting that the catabolism of triglycerides was impaired. Since both lipase and beta-oxidase were found to be active in cultured keratinocytes, another factor or other factors are probably implicated in the regulation of triglyceride metabolism.

Validation of an in vivo extraction method for human stratum corneum ceramides.

A topical acetone/diethylether (A/E) lipid extraction method was evaluated for its suitability for use in the study of stratum corneum lipids in various skin disorders. Its efficiency was compared in vitro with topical chloroform/methanol (C/M) extraction and with the classical 'integral' C/M extraction (submerged tissue) of stratum corneum or whole epidermis. To estimate the depth of lipid removal by A/E extraction, light microscopic and freeze-fracture electron microscopic studies were carried out on A/E and C/M topically treated skin samples. The in vivo experiments consisted of topical A/E extraction and of classical C/M extraction of scrapings of the stratum corneum. Transepidermal water loss (TEWL) was measured before and after topical A/E extraction and after every scraping procedure, and correlated with TEWL values found after stripping of the stratum corneum. The total amount of lipid found with both topical extraction procedures was lower than that found with the integral extraction of the stratum corneum. Light microscopy showed that topical C/M extraction induced cell damage in the living epidermal cell layers. Great interindividual variation in overall lipid composition was shown in the in vitro experiments irrespective of the extraction protocol used. However, the ceramide (CER) profiles in a single skin sample from the same subject were similar irrespective of the protocol used, and a uniformity in the CER profiles was found in skin samples from different subjects. Similar results were obtained with in vivo topical A/E extractions: marked interindividual variation was seen in overall lipid composition, but not in the CER profile.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid and ultrastructural characterization of reconstructed skin models.

The study aimed at evaluating tissue architecture and quality of the permeability barrier in commercially available reconstructed human skin models; EpiDerm, SkinEthic and Episkin in comparison to native tissue. For this purpose, tissue architecture was examined by electron microscopy and epidermal lipid composition was analyzed by HPTLC. Stratum corneum lipid organization was investigated by electron microscopy in combination with RuO(4) post-fixation and by SAXD. Ultrastructurally, the overall tissue architecture showed high similarities with native epidermis. In the stratum corneum extracellular space, lipid lamellae consisting of multiple alternating electron-dense and electron-lucent bands were present. This regular pattern was not seen throughout the whole stratum corneum probably due to the observed irregular lamellar body extrusion in some areas. Lipid analyses revealed the presence of all major epidermal lipid classes. Compared with native epidermis the content of polar ceramides 5 and 6 was lower, ceramide 7 was absent, and the content of free fatty acids was very low. These differences in lipid composition may account for differences observed in SAXD pattern of Episkin and EpiDerm penetration models. In the latter only the long-distance periodicity unit of about 12 nm was observed and the short periodicity unit was missing. In conclusion, all three skin models provide a promising means for studying the effects of topically applied chemicals, although the observed deviations in tissue homeostasis and barrier properties need to be optimized.

Lack of desquamation - the Achilles heel of the reconstructed epidermis.

The use of human skin equivalents for screening tests aiming to assess repetitive application of various test agents is hampered by the lack of desquamation in vitro. The present study was undertaken to examine whether the desquamation can be induced by various treatments including mechanical stress, application of various agents that should decrease the surface pH and calcium level, activate the enzymes involved in desquamation process or UV irradiation. In addition, the effect of alpha-hydroxyacids, known to enhance desquamation and to improve the stratum corneum barrier function in vivo, was examined as well. Human epidermis reconstructed on de-epidermized dermis or on fibroblast-populated collagen matrices during a 2-week culture at the air-liquid interface underwent various treatments during an additional 3-week period. The effects of treatments were evaluated on the basis of tissue morphology and lipid composition. The results of the present study revealed that cell shedding could only be induced by a mild repetitive mechanical treatment. The lack of desquamation, under most in vitro conditions, has a practical consequence, since it may hamper the use of reconstructed epidermis for various screening studies aiming to examine the repetitive exposure to topical agents or UV irradiation. The gradual thickening of the stratum corneum will lead to its higher resistance to the environmental stimuli and in this way affect the outcome of the tests. Furthermore, from the results obtained in the present study, it became evident that one should be careful in selecting endpoints when, for example, the effects of agents known to modulate melanogenesis are examined.

Covalently bound lipids in reconstructed human epithelia.

The composition of free and covalently bound lipids in reconstructed epithelia generated with normal human keratinocytes, HaCaT cells and squamous carcinoma cells was investigated and compared with native skin. Stratum corneum isolated from native human and reconstructed epidermis was subjected to extensive extraction with chloroform-methanol mixtures followed by alkaline hydrolysis to release covalently bound lipids. High-performance thin layer chromatography was used for analysis of solvent-extractable and non-extractable lipids and gas liquid chromatography was performed to assess the fatty acid profile in extractable lipids. In both native and reconstructed tissue covalently bound lipids consisted of omega-hydroxyceramides, omega-hydroxyacids and free fatty acids. Small amounts of omega-hydroxyacids could already be detected in solvent-extractable fractions. omega-Hydroxyceramides consisted of Ceramide A, Ceramide B and a small fraction of unknown ceramides with intermediate polarity. The relative proportions of individual omega-hydroxyceramides were similar in both native and reconstructed stratum corneum. In contrast, differences were found in profiles of both solvent-extractable and non-extractable lipids isolated from epithelia reconstructed with transformed cell lines (HaCaT, SCC-12F2 and SCC-13 cells). Compared with native or reconstructed epidermis, in epithelia reconstructed with transformed cell lines the ceramide content was low, the most polar ceramides were missing and the content of free fatty acids was low. The same holds true for covalently bound lipids that were virtually absent in these epithelia. Marked similarities were demonstrated in the overall lipid composition of free and bound stratum corneum lipids in native epidermis and in epidermis reconstructed with normal human keratinocytes. The observed imbalance in fatty acid profile may account for differences in phase behaviour of stratum corneum lipids.

Effects of retinoids on differentiation, lipid metabolism, epidermal growth factor, and low-density lipoprotein binding in squamous carcinoma cells.

The relationship among keratinocyte differentiation capacity, lipid synthesis, low-density lipoprotein (LDL) metabolism, plasma membrane composition, and epidermal growth factor (EGF) binding has been studied in SCC-12F2 cells. The differentiation capacity of the cells, i.e., ionophore-induced cornified envelope formation, was inhibited by various retinoids and stimulated by hydrocortisone. Retinoids that caused a significant reduction of cornified envelope formation, i.e., retinoic acid and 13-cis-retinoic acid, caused only minor changes in lipid synthesis and plasma membrane composition. Arotinoid ethylsulfone, having a minor effect on cornified envelope formation, caused a drastic inhibition of cholesterol synthesis, resulting in changes in the plasma membrane composition. Hydrocortisone stimulated cornified envelope formation but had only minor effects on lipid synthesis and plasma membrane composition. Of all retinoids tested, only arotinoid ethylsulfone caused a drastic increase in EGF binding, while hydrocortisone had no effect. Retinoic acid, arotinoid ethylsulfone, and hydrocortisone had no effects on LDL binding and only minor effects on LDL degradation. These results clearly demonstrate that the plasma membrane composition is not related to keratinocyte differentiation capacity, but most likely does determine EGF binding. Furthermore, EGF binding does not determine keratinocyte differentiation capacity.

Differentiation of human keratinocytes: changes in lipid synthesis, plasma membrane lipid composition, and 125I-EGF binding upon administration of 25-hydroxycholesterol and mevinolin.

We have studied the relationship between differentiation capacity, plasma membrane composition, and epidermal growth factor (EGF) receptor expression of normal keratinocytes in vitro. The plasma membrane composition of the cells was modulated experimentally by cholesterol depletion, using specific inhibitors of cholesterol synthesis, such as 25-hydroxycholesterol and mevinolin. Exposure of the cells towards these inhibitors resulted in a drastic decrease of cholesterol biosynthesis, as determined from 14C-acetate incorporation into the various lipid fractions. This effect on cholesterol biosynthesis was reflected by changes in plasma membrane composition, as determined by lipid analysis of isolated plasma membrane fractions, these resulting in a decreased cholesterol-phospholipid ratio. The experimental modulation of plasma membrane composition by 25-hydroxycholesterol or mevinolin were accompanied by a decreased cornified envelope formation and by high expression of EGF binding sites. These phenomena were more pronounced in cells induced to differentiate by exposure of cells grown under low Ca2+ to normal Ca2+ concentrations, as compared to cells grown persistently under low Ca2+ concentrations. These results suggest a close correlation between plasma membrane composition, differentiation capacity, and EGF receptor expression.

Proliferation and differentiation of human squamous carcinoma cell lines and normal keratinocytes: effects of epidermal growth factor, retinoids, and hydrocortisone.

Exposure of squamous carcinoma cell (SCC) lines, exhibiting high levels of epidermal growth factor (EGF) receptors, to EGF for 6 d caused a dose-dependent inhibition of cell proliferation. This EGF-induced inhibition of cell proliferation occurred under both low (0.06 mM) and normal (1.6 mM) Ca2+ concentrations. Furthermore, the extent of EGF-induced inhibition of cell proliferation seemed to be independent of the number of EGF-receptors. This conclusion is based on the notion that the various SCC lines exhibited an increasing number of EGF receptors accompanied by a decreasing ability to differentiate, whereas no relationship was observed with the EGF-induced inhibition of cell proliferation in these cell lines. Retinoids caused also a dose-dependent inhibition of cell proliferation. The effects of EGF and retinoids were additive, indicating that different regulatory mechanisms are involved. On the other hand, hydrocortisone caused a stimulation of SCC-proliferation, also independent of EGF. In contrast to SCC cells, EGF did not affect significantly the rate of proliferation of normal keratinocytes. However, the simultaneous addition of EGF and hydrocortisone resulted in a significant increase in the rate of keratinocyte proliferation only in cells grown under normal calcium conditions. Differentiation capacity of normal keratinocytes and SCC lines was not affected by EGF. Furthermore, the retinoid-induced decrease and hydrocortisone-induced increase of competence of cells to form cornified envelopes was not affected by EGF. These observations suggest that the action of retinoids and hydrocortisone on both cell proliferation and cell differentiation occurs independently of EGF receptors.

Analysis of epidermal lipids of the healthy human skin: factors affecting the design of a control population.

The intervariability of studies on the lipids of human epidermis and stratum corneum is high because of the different origin of the skin samples and the variety of extraction methods used. In the present work, a high-performance thin-layer chromatographic technique has been used to study the parameters age, sex, and anatomical site for their effects on the lipid profiles recovered from healthy epidermal skin biopsy specimens. It was found that sex-related differences were seen at the level of the total ceramide concentration. Observed decreases in lipid concentration, due to ageing, depended on the anatomical site. Therefore, these variables should be controlled in a reproducible and standardized way in order to be able to study the direct relationship between skin condition and barrier lipid composition. Only when this relation is established, results of topical treatment can be scientifically evaluated.

Differentiation of cultured human keratinocytes: effect of culture conditions on lipid composition of normal vs. malignant cells.

Differentiation in keratinocytes can be experimentally modulated by changing the culture conditions. When cultured under conventional, submerged conditions, the extent of cellular differentiation is reduced in the presence of low calcium medium and is enhanced in medium containing physiologic calcium concentrations. Moreover, cultures grown at the air-medium interface or on a dermal substrate, or both, differentiate even further. Herein we report the effect of culture conditions on lipid composition in normal human keratinocytes and three squamous carcinoma cell (SCC) lines that vary in their capacity to differentiate as assessed by cornified envelope formation. Under submerged conditions, the total phospholipid content was lower, triglyceride content higher, and phospholipid:neutral lipid ratio lower in direct correlation to the degree of differentiation in these cultures. When grown at the air-medium interface on de-epidermized dermis, evidence of further morphologic differentiation was found only for well-differentiated SCC cells and normal keratinocytes. Similarly, the phospholipid content remained high in poorly differentiated SCC cells and it decreased modestly in well-differentiated SCC cells and markedly in normal keratinocytes. In all cell lines the triglyceride content was increased and cholesterol content decreased when compared to parallel submerged cultures, but these differences were most pronounced in well-differentiated cell lines. Acylceramides and acylglucosylceramides were found only in normal keratinocytes and only under the most differentiation-enhancing conditions. These studies demonstrate differentiation-related changes in the lipid content of both normal and neoplastic keratinocytes.