Susceptibility of human male keratinocytes to MHC-restricted H-Y-specific lysis.

We studied the expression of the male-specific mH antigen H-Y on cultured human skin cells by investigating susceptibility to H-Y-specific cytolysis using conventional class I-restricted CTL clones in a modified cell-mediated cytotoxicity assay. In contrast to what was found in the rodent system, we observed H-Y-specific lysis of human male keratinocytes. Susceptibility for H-Y-specific lysis was efficiently enhanced by exposure of the keratinocytes to IFN-gamma. Our results demonstrate that human skin cells are equally sensitive for the activity of H-Y-specific CTLs as target cells of lymphoid origin. Finally, the cellular recognition of the H-Y mH antigen in the skin further supports its possible target function in the local graft versus host attack.

Modulation of the production of a parathyroid hormone-like protein in human squamous carcinoma cell lines by interaction with fibroblasts.

Normal human keratinocytes as well as human squamous cell carcinomas produce a parathyroid hormone-like protein (PLP). However, PLP production by these cells is not a constant phenomenon. Since nothing is known about factors which regulate the production of PLP, in vitro studies were performed with normal keratinocytes and squamous carcinoma cell lines in order to establish conditions under which PLP production may vary. PLP was measured as cyclic AMP production in parathyroid hormone target cells (osteoblasts) which could be inhibited by a parathyroid hormone antagonist. The presence of PLP was confirmed using a radioimmunoassay specific for PLP. Results from the bioassay correlated very well with the data obtained by radioimmunoassay for PLP. The results confirm that human squamous carcinoma cells and normal keratinocytes produce PLP. PLP production appeared to be very sensitive to modulation of coculture of squamous carcinoma cells with fibroblasts. The effect of fibroblasts was not mediated by an effect on squamous carcinoma cell viability. Murine transformed fibroblasts (3T3 cells) as well as human normal foreskin fibroblasts were equally effective in inducing PLP production in these cells. The fibroblastic factor was apparently present in a soluble form in the coculture system which prevented direct cell-cell contact but allowed communication through the medium. Nevertheless, conditioned medium from 3T3 cells failed to induce PLP production by squamous carcinoma cells. This suggests a more complicated interaction between the two cell types than a one way message from fibroblasts to keratinocytes. Production of PLP by a number of squamous carcinoma cell lines was variable and not evidently correlated with the ability of these carcinoma cells to differentiate. Production of parathyroid hormone-like protein not only is the expression of a disturbed metabolism of a specific cell type but also reflects the cell-cell interaction in tumor tissue.

Regulation of lipid synthesis in relation to keratinocyte differentiation capacity.

Cultured keratinocytes and squamous carcinoma cells provide a useful model system for studying the processes involved in the regulation of differentiation, as the differentiation capacity of the cells can be modulated experimentally by changing the extracellular calcium concentration. Furthermore, the squamous carcinoma cell lines exhibit a defect in their differentiation capacity which they express to different extents. In this paper, the effect of external lipoproteins has been studied on lipid synthesis in normal keratinocytes and three squamous carcinoma cell (SCC) lines which showed a decreasing capacity to differentiate in the order of normal keratinocytes greater than SCC-12F2 greater than SCC-15 greater than SCC-4. The ability of the cells to form cornified envelopes was taken as a measure of differentiation capacity. The rate of total lipid synthesis as well as the phospholipid-neutral lipid ratio decreased in the order SCC-4 greater than SCC-15 greater than SCC-12F2 greater than or equal to normal keratinocytes, clearly correlating with the differentiation capacity of the cells. Because of the high rate of phospholipid synthesis and the low rate of ceramide synthesis, it is concluded that, under these in vitro conditions used, the maturation of keratinocytes proceeds to a lesser extent than that seen under in vivo conditions. In proliferating cells, in which the low-density lipoprotein (LDL) receptor is operative to a high extent, the rate of lipogenesis, especially that of neutral lipids, responded dramatically to changes of extracellular lipoprotein concentration. In the presence of lipoproteins a marked decrease of cholesterol and triacylglycerol synthesis and an increase of cholesterol ester synthesis has been observed. On the other hand, in differentiating cells lipogenesis appeared to be independent of extracellular lipoproteins, due to the absence of the LDL uptake mechanism, the only exception being the synthesis of triacylglycerols, the rate of which could be modulated to a certain extent by extracellular lipoproteins. The results presented here demonstrate a close inverse relationship between the regulation of lipogenesis by extracellular lipoproteins and the ability of the cells to differentiate.

Role of extracellular calcium in the regulation of 1,25-dihydroxyvitamin D3 formation in cultured human keratinocytes.

Cultured normal human keratinocytes (NHK) provide a useful experimental model for studies of processes occurring during terminal differentiation, since the extent of keratinocyte maturation can be manipulated experimentally by modulation of extracellular calcium concentration. When NHK are maintained in low calcium (0.06 mM) medium they proliferate but do not stratify. Raising the level of calcium to 1-2 mM results within a few hours in induction of keratinocyte differentiation. Results of the present study show that formation of 1,25-(OH)2D3 is higher in NHK grown at 0.06 mM than in NHK grown at 1.6 mM calcium concentration. After 2 h exposure of low calcium cultures to 1.6 mM calcium the 1,25-(OH)2D3 production starts to decrease. On the other hand, exposure of cells cultured in 1.6 mM calcium medium to 0.06 mM calcium concentration induced already within 4 h an increase in 1,25-(OH)2D3 formation which was not accompanied by a decrease in cornified envelope formation. Thereby, the present study demonstrated that calcium can regulate 1,25-(OH)2D3 formation independently of changes in keratinocyte differentiation.

Effects of LDL, HDL and their combination on the endogenous cholesterol synthesis in monocyte macrophages.

It is commonly assumed that the low density lipoprotein (LDL) degradation in extra-hepatic cells proceeds partly by way of the high-affinity LDL uptake process. In addition, it has been suggested that extra-hepatic LDL catabolism proceeds partly by way of preferential uptake and subsequent degradation of LDL by phagocytic scavenger cells. In order to study the effect of LDL and high density lipoprotein (HDL) on cholesterol metabolism in such scavenger cells, cultured human or pig monocytes were incubated with varying amounts of human or pig LDL, HDL and LDL/HDL mixtures, and the incorporation of [14C]acetate into cholesterol was measured. The addition of LDL suppressed endogenous cholesterol synthesis. Incubation of monocytes with LDL/HDL mixtures reduced the total amount of de novo synthesized cholesterol to the same extent as incubation with LDL alone. Our results suggest that in cultured monocyte macrophages HDL does not influence endogenous cholesterol synthesis, even in the presence of LDL.

Corticoids and cultured human epidermal keratinocytes: specific intracellular binding and clinical efficacy.

Cytosol of cultured human epidermal keratinocytes contains macromolecules, that bind corticoids with high affinity. Binding constants were in the same range for cultured keratinocytes originating from different individuals and did not change during serial cell cultivation. At 0 degree C and using 3H-triamcinolone acetonide as ligand, we obtained the following values; apparent Bmax = 160- 250 fmoles/mg protein and Kdiss = 3.1-5.0 nM; with 3H-dexamethasone Bmax = 200-350 fmoles/mg protein, Kdiss = 6.0-11.1 nM, and with 3H-hydrocortisone Bmax = 140-220 fmoles/mg protein, Kdiss = 17-25 nM. There was an indication that the binding capacity of the receptor system is higher the younger the age of the skin donor. A number of steroids and corticoids commonly used in dermatological practice were tested with respect to displacement of 3H-triamcinolone acetonide, 3H-dexamethasone, and 3H-hydrocortisone from binding sites in cytosol. Good correlation between clinical efficacy and specific binding was observed for all 3 ligands. Other steroids such as 17-beta estradiol and nandrolone did not show any affinity for the corticoid binding system. Progesterone displace 3H-corticoids from their binding sites, but the IC50 was of one order of magnitude larger.

Cultured human skin fibroblasts and keratinocytes: differences in the regulation of cholesterol synthesis.

The regulation of cholesterol synthesis in cultured human skin fibroblasts and keratinocytes was compared, the incorporation of [14C]-acetate or [14C]-octanoate into [14C]-cholesterol being taken as a measure of de novo cholesterol synthesis. The two types of cultured cells differed in the following features of the regulation of cholesterol synthesis: (1) Keratinocytes synthesized 10-fold more cholesterol/mg cell protein. (2) Keratinocytes retained a greater amount of the de novo synthesized cholesterol intracellularly, and fibroblasts released it to a much higher degree into the culture medium. (3) When the extracellular environment was deprived of cholesterol, the intracellular synthesis remained virtually unchanged in keratinocytes but increased markedly in fibroblasts. (4) The low-density lipoproteins (LDL) that enter the cells by receptor-mediated endocytosis and are then degraded in lysosomes, liberate cholesterol, which in turn interferes with the intracellular cholesterol synthesis. The lipoproteins strongly suppress cholesterol synthesis in fibroblasts, but do not have this effect in keratinocytes. (5) When added to the culture medium, nonlipoprotein cholesterol produced no effect on cholesterol synthesis in keratinocytes, whereas fibroblasts showed a marked suppression of this synthesis. The addition of 25-hydroxycholesterol to the culture medium led to a strong suppression of cholesterol synthesis in both fibroblasts and keratinocytes. These findings suggest that in both cell types the 3-hydroxy-3-methylglutaryl coenzyme A reductase activity can be suppressed by a sterol delivered to the cell in artificial nonlipoprotein form. (6) The amount of [125I]-LDL bound specifically to the cell membrane receptor and particularly the amount internalized and degraded by the cells is much lower in keratinocytes than in fibroblasts, as shown biochemically. In the ultrastructural studies no binding of LDL to keratinocytes was observed.

SV 40-transformed (SVK14) and normal keratinocytes: similarity in the expression of low-density lipoprotein, epidermal growth factor, glucocorticoid receptors, and the regulation of lipid metabolism.

Transformation of normal keratinocytes by simian virus 40 (SV 40) leads to the establishment of epithelial cell lines that can be cultured in the absence of the feeder layer and do not become senescent in culture. The SVK14 cell line developed by Taylor-Papadimitriou et al can serve as a model for study of the modification of various cellular processes by certain pharmacologic and physiologic agents, because these cells resemble normal keratinocytes with respect to a variety of parameters related to proliferation and differentiation, as follows: The SVK14 cells show the same ability to form ionophore-induced cross-linked envelopes that is strongly suppressed when the calcium level in the culture medium is reduced. When cultured in a high-calcium medium, both cell types showed a high rate of de novo cholesterol synthesis that was independent of the extracellular lipoprotein concentration. Cells cultured in a low-calcium medium had a much lower rate of cholesterol synthesis, but this rate increased markedly in cells preincubated in lipoprotein-deficient (LPDS) medium and decreased again with the addition of increasing amounts of low-density lipoprotein (LDL). Both types of cell showed decreased ability to bind epidermal growth factor (EGF) and LDL during calcium-induced differentiation, the expression of LDL and/or EGF receptors being high in low-calcium and low in high-calcium cells. Addition of etretinate (0.05-5.0 microM) suppressed cholesterol synthesis and strongly stimulated triglyceride synthesis in both cell types without significantly affecting the rate of protein synthesis. The addition of small doses of glucocorticoids (10(-9) to 10(-6) M) led to stimulation and higher doses (up to 5 X 10(-5) M) to inhibition of cell proliferation.

Defective low-density lipoprotein metabolism in cultured, normal, transformed, and malignant keratinocytes.

To obtain more information on differences in cellular behavior during the differentiation process, a number of types of epithelial cells with and without a defect in cornified envelope formation were compared as to the regulation of intracellular cholesterol synthesis by low-density lipoprotein (LDL) and the uptake and degradation of [125I]LDL. The following cells were cultured: normal skin fibroblasts (F), normal (K) and SV 40 transformed (SVK14) keratinocytes, and a number of squamous carcinoma cell (SCC) lines in which the defective terminal differentiation was found to occur in the following order of intensity: SCC-12F2 less than SCC-25 approximately equal to SCC-15 less than SCC-12B2 less than SCC-4. Compared with normal human fibroblasts, most of the cells under study showed a defective response to changes of the extracellular serum LDL concentration. The degree of inducibility of cholesterol synthesis after the cells were deprived of extracellular sources of cholesterol as well as the degree of the LDL-induced suppression of the intracellular cholesterol synthesis in cells preincubated in medium supplemented with lipoprotein-deficient serum decreased in the following order: F greater than SCC-4 greater than SCC-15 approximately equal to SCC-25 greater than SCC-12B2 congruent to SCC-12F2 greater than SVK14 approximately equal to K. A defect in LDL metabolism was found to be responsible for the partial or complete failure of LDL to regulate the cholesterol metabolism, because when sterol was delivered to all cell types in artificial nonlipoprotein form (i.e., as 25-hydroxycholesterol) a marked suppression of cholesterol synthesis was observed. For all SCC lines tested except SCC-12B2 good correlation was found between the degree of LDL-induced suppression of cholesterol synthesis and the decreasing ability of cells to differentiate into squames or cornified envelope-forming cells. The transformation of keratinocytes by SV 40 virus did not lead to any change in the response of the cells to changes in the extracellular LDL concentration since both the normal and the transformed keratinocytes showed the same response to LDL (i.e., no response).

Corticoids and human skin fibroblasts: intracellular specific binding in relation to growth inhibition.

The binding of 3H-triamcinolone acetonide to soluble macromolecules of cultured human skin fibroblasts was studied in an attempt to explain the mechanism underlying the inhibitory effects of glucocorticoids on cell growth. The results were as follows: Cultured human skin fibroblasts contain in cytosol a high affinity binding system for glucocorticoids. Various glucocorticoid derivatives competed for specific binding of 3H-triamcinolone acetonide. In some but not all instances this competition was related to the clinical efficacy of the derivatives under study and to their potency for the inhibition of cell growth. A specific glucocorticoid binding system was detectable in steroid-sensitive, low-density cell cultures (apparent Bmax = 200 fmoles/mg protein). The number of steroid binding sites was lower in high-density cell cultures (apparent Bmax = 125 fmoles/mg protein). The sensitivity to growth inhibition by glucocorticoids was markedly decreased in the high-density cell cultures. There were no differences in the affinity constants between these cell cultures (Kdiss. = 3.3 X 10-9 M). When cells were grown in medium containing glucocorticoid, renewal of the incubation medium led to disappearance of the growth-inhibitory effects, whereas specific binding was not affected. Nandrolone, an inhibitor of cell growth, abolished the growth-inhibitory effects of glucocorticoids but did not displace 3H-triamcinolone acetonide from its binding sites. The results suggest that in addition to a mechanism mediated by a glucocorticoid binding system with receptor like properties also other factors as well appear of relevance for the control of cell growth. These factors may be beyond the actual binding process of steroid and involve the action at the level of genomic expression of the cell.

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.

Normalization of epidermal calcium distribution profile in reconstructed human epidermis is related to improvement of terminal differentiation and stratum corneum barrier formation.

Calcium plays an important role in the regulation of cellular differentiation and desquamation of epidermal keratinocytes. In this study, we examined the calcium distribution in reconstructed epidermis in an attempt to understand the physiology of keratinocyte differentiation and desquamation in vitro. Ion capture cytochemistry (the potassium oxalate-pyroantimonate method) was employed to localize ionic calcium in reconstructed epidermis generated under three different culture conditions (in serum-containing medium, serum-free medium, and serum-free medium supplemented with retinoic acid), allowing a comparison of the physiology of incompletely and well-differentiated keratinocytes. The reconstructed epidermis generated in serum-containing medium showed features of incomplete differentiation, and compared with the native skin, a high calcium content within incompletely differentiated cells in the stratum corneum. Use of serum-free medium containing vitamin and lipid supplements led to a marked improvement of the stratum corneum ultrastructure and penetration pathway across the stratum corneum, indicating improved barrier formation of the reconstructed epidermis. In parallel, the calcium distribution pattern was normalized showing the highest levels of calcium in the stratum granulosum and low levels in the inner stratum corneum. Addition of retinoic acid to the serum-free medium resulted in an altered keratinocyte differentiation and re-appearance of large quantities of calcium precipitates in the stratum corneum. Proton probe X-ray microanalysis was applied to investigate the calcium distribution quantitatively in native and reconstructed epidermis generated in serum-free medium, and verified the calcium distribution demonstrated by the precipitation technique. Regardless of the presence or absence of calcium in the stratum corneum, all examined culture systems exhibited insufficient desquamation, which correlates with the finding that stratum corneum chymotryptic enzyme was present predominantly as an inactive precursor. This study demonstrates that improvement of the stratum corneum barrier properties in vitro is concurrent with the normalization of the epidermal calcium gradient, whereas deregulation of terminal differentiation correlates with an accumulation of calcium ions within incompletely differentiated corneocytes.

A new sensitive assay for measurement of cell-mediated cytotoxicity to intact layers of cultured human keratinocytes.

A cytotoxicity assay for sensitive measurement of cell-mediated lympholysis (CML) of human cultured keratinocytes (cK) is described. The usage of 51Cr-labeled keratinocytes in intact layers as target cells in this assay allows objective and accurate determination of lysis of keratinocytes which have not undergone trypsin- and suspension-induced membrane changes. Furthermore, the problem of high spontaneous 51Cr release values encountered with suspended keratinocytes is overcome. The assay was applied to study antigen-specific CML of cK by cloned cytotoxic T cells (CTL) and to determine the effect of IFN-gamma on the susceptibility of cK to lysis. The results showed that HLA-A2 specific CTLs could reproducibly lyse cK of HLA-A2 positive healthy skin donors both with and without incubation of cK with IFN-gamma. Applications of this keratinocyte cytotoxicity assay lie in determining the antigenic expression of human cK, in analysis of effector cell/keratinocyte interactions in CML and of the modulatory effects of cytokines on these mechanisms. The assay thus may provide a helpful tool in gaining insight into the role of CML of keratinocytes in the destruction of inflamed skin.

Skin equivalent: an attractive model to evaluate early melanoma metastasis.

The growth patterns and morphological phenotype of four human melanoma cell lines with different metastatic potentials were investigated in submerged and in air-exposed (skin equivalent) keratinocyte-melanoma cell co-cultures. In contrast to the submerged co-cultures, all four cell lines formed sharply demarcated tumour cell nests within the epidermal compartment of the skin equivalent model, with the morphology highly mimicking the in vivo situation. Differences among the melanoma cell lines tested were observed with respect to the number of clusters formed and the ability to exhibit invasive growth. Only the two metastatic cell lines were able to invade the dermal compartment. Screening of cellular adhesion molecules revealed that the expression patterns in different cell lines were heterogeneous and remained unchanged during the whole culture period, irrespective of whether the melanoma cells were located in the epidermal or dermal compartment. A correlation was found between expression of a lower number of different cellular adhesion molecules and the ability to acquire invasive growth capability. Our results indicate that melanoma cells exhibit a heterogeneous growth behaviour when co-cultured with human keratinocytes, and the air-exposed skin equivalent model was shown to be suitable for studying differences in growth patterns and potential invasive behaviour.

The involvement of protein kinase C in proliferation and differentiation of human keratinocytes--an investigation using inhibitors of protein kinase C.

Protein kinase C, the major cellular receptor for tumour-promoting phorbol esters, has been suggested as playing a key role in the regulation of proliferation and differentiation of epidermal cells. In the present study, we investigated the effects of various well-characterized inhibitors of protein kinase C on proliferation and differentiation of SV 40-transformed and normal human keratinocytes. The drugs were found to inhibit cell proliferation in a dose-dependent manner, displaying similar effects in both cell types and reflecting their potencies in inhibiting purified protein kinase C. In contrast, keratinocyte differentiation induced by treatment with a calcium ionophore or spontaneously, i.e. by exposure of cells grown in the presence of low calcium concentration (0.06 mM) to normal calcium concentration (1.6 mM), was not inhibited by the compounds tested. The potent protein kinase C inhibitor, staurosporine, was found even to enhance cell differentiation. Therefore, the present study provides evidence that the classical protein kinase C pathway plays a critical role in the regulation of keratinocyte proliferation rather than in calcium-induced differentiation.

Biphasic entry of glucocorticoids into cultured human skin keratinocytes and fibroblasts.

The uptake of glucocorticoids by cultured human skin keratinocytes and fibroblasts was found to be a rapid, temperature-sensitive process. All glucocorticoids tested accumulated in the cells, and the ratio between the intracellular and extracellular concentrations (ci/co ratio) was higher than 1. For most of the glucocorticoids under study there was good correlation between the lipophilicity and the rate of uptake. Since the uptake of glucocorticoids seems to be unsaturable in the concentration range used and no competition was observed between these compounds for entry into the cells, it may be assumed that the uptake of glucocorticoids is essentially a simple diffusion process based on a distribution of glucocorticoids between a lipid-rich phase and water. The analysis of the uptake process revealed that the entry of glucocorticoids into cultured human skin fibroblasts and keratinocytes is a non-mediated passive diffusion process that involves two distinct steps: a rapid, non-specific, high-capacity association to the cell membrane followed by a slower internalization process associated with a stronger binding of glucocorticoids within the cell.

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.

Effects of glucocorticosteroids on cultured human skin fibroblasts. V. Influence of anabolic steroids on the inhibitory effects of clobetasol-17-propionate on cell proliferation and collagen synthesis.

As previously found, the glucocorticosteroid clobetasol-17-propionate inhibits cell proliferation during the early growth stage of normal baby foreskin fibroblasts and collagen synthesis in confluent cultures of these cells. The degree of inhibition of cell proliferation decreases with increasing cell density and, moreover, is transient. The anabolic steroids nandrolone and nandrolone-phenyl-propionate have similar effects on these cells. Likewise the magnitude of the inhibition is dose-dependent. When present together the two types of drug do not act in an additive manner. Even at low concentrations the anabolic steroids abolish the inhibitory effect of the glucocorticosteroid on cell proliferation. Furthermore, in this case only the inhibitory effect of the glucocorticosteroid on collagen synthesis is found and there is no further increase in this effect due to the presence of the anabolic steroids. Our results imply that the use of low concentrations of anabolic steroids combined with glucocorticosteroids in topical application to the skin may abolish some of the undesirable side effects of the glucocorticosteroids.

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.