Binding of unmodified low-density lipoproteins to human fibroblasts. An investigation by immunoelectron microscopy.

The bindinf of unmodified low density lipoproteins to the plasma membrane of fibroblasts was studied at the ultrastructural level. The bound low density lipoprotein was visualized by an indirect immunoperoxidase technique, with the use of an antiserum against apoprotein B. Immunoreactive regions representing bound apoprotein B were found on the plasma membrane, in indented regions with a diameter of 0.15--0.30 micrometer and a fuzzy coat on the cytoplasmic side. Fibroblasts from a patient homozygous for hyperlipoproteinaemia type IIa showed no immunoreactive material in the indented regions. The specific 125I-labelled low density lipoprotein binding to these homozygous fibroblasts was 7% compared to control fibroblasts.

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.

Prevention of skin cancer and reduction of keratotic skin lesions during acitretin therapy in renal transplant recipients: a double-blind, placebo-controlled study.

PURPOSE: The purpose of this study was to investigate the effect of acitretin on the development of keratotic skin lesions, and on squamous cell carcinomas and basal cell carcinomas in a group of renal transplant recipients. PATIENTS AND METHODS: Forty-four renal transplant recipients with more than 10 keratotic skin lesions on the hands and forearms were enrolled onto a randomized, double-blind, placebo-controlled trial to test the possible skin cancer-preventing effect of a 6-month treatment with acitretin 30 mg/d. RESULTS: No deterioration in renal function occurred in any of the 38 assessable patients treated. During the 6-month treatment period, two of 19 patients (11%) in the acitretin group reported a total of two new squamous cell carcinomas, compared with nine of 19 patients (47%) in the placebo group who developed a total of 18 new carcinomas (chi 2 = 6.27, P = .01). The relative decrease in the number of keratotic skin lesions in the acitretin group was 13.4%, as compared with a relative increase in the placebo group of 28.2% (difference, 41.6%; 95% confidence interval, 11.5 to 71.7). Most patients treated with acitretin had mild mucocutaneous side effects, but these were easily manageable. Some patients experienced mild hair loss. With the exception of three patients, no increase in serum cholesterol or triglyceride above pretreatment levels was observed, and liver function remained unchanged in all patients. CONCLUSION: Acitretin 30 mg/d over 6 months had significantly more effect than placebo in the prevention of squamous cell carcinomas and reduced the occurrence of keratotic skin lesions in a group of renal transplant recipients with severe lesions. This effect was most pronounced in patients with a history of squamous cell carcinomas and basal cell carcinomas.

Relation between smoking and skin cancer.

PURPOSE: Tobacco smoking is a risk factor for several cancers. The risk of cutaneous malignancies related to smoking, however, is relatively unknown. We investigated the possible association between smoking and skin cancer. PATIENTS AND METHODS: A hospital-based case-control study was performed that included 161 patients with squamous cell carcinoma, 301 with nodular basal cell carcinoma, 153 with superficial multifocal basal cell carcinoma, 125 with malignant melanoma, and 386 controls. Information on smoking history was collected in personal interviews. Relative risks were estimated using exposure odds ratios from cross-tabulation and logistic regression. RESULTS: An association between smoking and squamous cell carcinoma of the skin was found (relative risk, 2.3; 95% confidence interval, 1.5 to 3.6; P: = .0001), with a higher risk for current smokers (relative risk, 3.3; 95% confidence interval, 1.9 to 5.5) than for former smokers (relative risk, 1.9; 95% confidence interval, 1.2 to 3.0). After adjustment for age, sex, and sun exposure, the relative risk of squamous cell carcinoma was 2.0 (95% confidence interval, 1.2 to 3.2; P: = .008). There was a dose-response relationship with number of cigarettes and pipes smoked. No significant association was found between smoking and nodular basal cell carcinoma, superficial multifocal basal cell carcinoma, or malignant melanoma. CONCLUSION: Tobacco smoking is an independent risk factor for cutaneous squamous cell carcinoma.

Modulation of low density lipoprotein receptor activity in squamous carcinoma cells by variation in cell density.

Morphological and biochemical studies on low density lipoprotein (LDL) receptor metabolism were performed in squamous carcinoma cells (SCC-15 and SCC-12F2). Modulation of terminal differentiation was achieved by culturing these cells at different cell densities. Studies on these cells cultured at low density (hardly any terminal differentiation) showed the following results: High affinity binding of LDL was excessive; LDL binding to SCC-15 cells was twice as high as that in SCC-12F2 cells and in fibroblasts. The distribution of the LDL binding visualized by LDL receptor antibodies was non-linear. There was no contact inhibition of LDL binding. LDL-gold particles were mainly bound to the plasma membrane outside coated pits. LDL-gold particles were internalized and delivered to dense bodies (= lysosomes). Degradation of LDL took place after a lag period of 10 min. Dissociation of LDL from the plasma membrane was substantial (more than 40% after a 120 min chase period). The same experiments on the cells cultured at high density (terminal differentiation present) showed several differences: A sharp decrease in high affinity LDL binding in both cell types. The internalization of surface bound LDL was defective in most of the squamous carcinoma cells. Dissociation of LDL from the plasma membrane was substantial, and after a chase period of 120 min at 37 degrees C still more than 20% of LDL remained intracellular and was not degraded. We postulate that LDL receptor-mediated endocytosis and degradation take place in squamous carcinoma cells but that during the process of terminal differentiation modulation of LDL-receptor metabolism occurs.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoelectron microscopic visualization of the transcytosis of low density lipoproteins in perfused rat arteries.

A postembedding labeling technique was employed to visualize human native low density lipoproteins (LDL) during transcytosis in rat arterial endothelium. For this purpose human LDL was perfused through rat vasculature before fixation and processing for immunoelectron microscopy. The LDL particles were located on sections by anti-human apolipoprotein B-100 (LDL) antibodies and secondary antibodies or protein-A conjugated to 10-nm colloidal gold. LDL molecules were seen in plasmalemmal vesicles as well as in the subendothelial space. No colloidal gold was found in the intercellular junctions. Perfusion with reductively methylated LDL, which cannot bind to the LDL receptor, gave a similar labeling pattern, indicating that transcytosis of LDL via plasmalemmal vesicles is most likely receptor independent. Furthermore, the passage of LDL through intact vascular endothelium is a vesicular transport rather than an intercellular diffusion process.

Visualization of binding and receptor-mediated uptake of low density lipoproteins by human endothelial cells.

Low density lipoproteins (LDL) were conjugated to colloidal gold for investigation of the ultrastructural aspects of binding and receptor-mediated internalization of LDL by cultured endothelial cells from the human umbilical artery and vein. The number of LDL receptors was increased by preincubation in lipoprotein-depleted serum. When the cells were incubated with LDL-gold particles for 2 h at 4 degrees C, the complexes were found in coated pits as well as in clusters attached to the plasma membrane. Small vesicles containing a few LDL-gold complexes appeared in the cytoplasm close to the plasma membrane when the cells were incubated with the conjugate for 5 min at 37 degrees C. After 15 min at 37 degrees C, larger vesicles with a pale matrix and membrane-orientated LDL-gold complexes were seen. After incubation for 30 min at 37 degrees C, colloidal gold particles were present in dense bodies. Quantification of the binding of LDL-gold complexes to the plasma membrane at 4 degrees C showed no differences between arterial and venous endothelial cells.

Visualization of the interaction of native and modified low density lipoproteins with isolated rat liver cells.

Morphological characteristics of the interaction of low density lipoproteins (LDL) and acetylated low density lipoproteins (AcLDL) with rat liver cells are described. These liver cell types are mainly responsible for the catabolism of these lipoproteins in vivo. Isolated rat liver Kupffer, endothelial, and parenchymal cells were incubated with LDL or AcLDL conjugated to 20 nm colloidal gold. LDL was mainly internalized by Kupffer cells, whereas AcLDL was predominantly found in endothelial cells. Kupffer and endothelial cells displayed different morphological characteristics in the processing of these lipoproteins. Kupffer cells bound LDL at uncoated regions of the plasma membrane often at the base of pseudopodia, and internalized the particles via small smooth vesicles. These uptake characteristics differ from the classical LDL uptake pathway, as described for other cell types, and may be related to the unique recognition properties of the receptor of Kupffer cells as observed in biochemical studies. Liver endothelial cells bound AcLDL in coated pits, followed by rapid uptake. Uptake proceeded through small coated vesicles, and after 5 min of incubation large (600-1200 nm) electron-lucent vacuoles (endosomes) with AcLDL-gold particles arranged along the membrane region were present. The endosomes were often associated closely with the cell membrane which might enable direct recycling of AcLDL receptors. These observations might explain the high efficiency of these cells in the processing of modified LDL in vivo.

Functional human epidermal Langerhans cells that lack Birbeck granules.

Birbeck granules (BG) are cytoplasmic organelles that are only found in Langerhans cells (LC). The function of BG is still unclear, although it has been claimed that they are actively involved in receptor-mediated endocytosis and participate in the antigen-processing/presenting function of LC. We have identified a healthy white 29-year-old man whose LC completely lack the presence of BG as determined by electronmicroscopic studies. This was observed repeatedly using skin biopsy specimens taken from several places on the body during a period of 2.5 years. The absence of BG in these LG was documented further by the lack of staining with a BG-specific monoclonal antibody. Despite the complete lack of BG, LC were present in normal numbers, had all the usual morphologic characteristics, and were CD1a and human leukocyte antigen (HLA) class II positive. Two observations indicate that these BG-negative LC display normal antigen-presenting capacity. First, the individual could be sensitized by the hapten diphenylcyclopropenone. This was accompanied by a strong increase in the cell surface expression of HLA class II antigens on his LC, suggesting LC activation. Second, his epidermal cells elicited a normal positive response in an allogeneic mixed epidermal cell lymphocyte reaction. Together these observations strongly suggest that BG are not a prerequisite for normal LC function in vivo and in vitro.

Familial dysbetalipoproteinemia: a genetically heterogenous disease caused by mutations of the ligand apolipoprotein E.

Apolipoprotein E is present on the surface of very-low-density lipoprotein (VLDL) and chylomicron-remnants and is essential for the receptor mediated endocytosis of these particles via hepatic receptors. Several types of mutations of the apoE can cause a deficiency in the clearance of these remnant particles. An accumulation of lipoprotein-remnant particles may occur and familial dysbetalipoproteinemia (FD) develops. Genotyping of the various apoE variants and relation of these mutations with their effect on the lipoprotein-remnant removal have provided more insight in the structure-relationship of apoE ligand-receptor interactions. It is postulated that the apoE2 (Arg158----Cys) mutation is just outside the binding domain and that its deficient binding can be stimulated by exogenous factors. This hypothesis can explain why apoE2/E2 homozygosity can only induce FD under certain circumstances. ApoE mutations that occur in the binding domain, e.g., apoE2 (Lys146----Gln) have a direct effect on the ligand-receptor binding and, in these individuals, FD is inherited in an autosomal dominant way. Finally, apoE3-Leiden has an arginine residue at 112 and has a repeat of seven extra amino acid residues just outside the binding domain. Because of this repeat, conformational changes of the binding domain can ensue. Due to the fact that in apo E3-Leiden the arginine residue is present at 112, apoE3 Leiden is predominantly present on chylomicron and VLDL remnants. In these persons FD is also inherited in an autosomal dominant way.

Complement-mediated endothelial cell damage in immune complex vasculitis of the skin: ultrastructural localization of the membrane attack complex.

Activation of the complement system is an important element in our concept of the pathomechanism of immune complex (IC) vasculitis. Both deposition of IC and attraction of polymorphonuclear leukocytes (PMN) are effected by products of complement activation. Actual tissue damage, however, is believed to be caused by PMN penetrating the vessel wall. Our former finding that deposits of membrane attack complex of complement (MAC) are found predominantly in skin lesions of patients with IC vasculitis and not in perilesional skin, has raised the question whether the complement system itself (by way of the MAC) contributes to tissue damage. Our present study shows the ultrastructural localization of MAC in lesional and clinically uninvolved skin in two patients with a cutaneous IC vasculitis. Lesional skin deposits of MAC were found on endothelial cells (EC) of upper dermal vessels and on infiltrating PMN. Uninvolved skin deposits of MAC were found on some EC, but clearly to a lesser extent than on EC of the lesional skin. In the skin of two healthy controls MAC was only found sporadically on EC. Deposits of MAC on EC in the lesional skin were often associated with a typical form of local cell swelling. This local form of endothelial cell swelling was incidentally seen in vessels of clinically uninvolved skin, but not in the skin of the two controls. The association of the endothelial cell swelling with deposits of MAC suggests that the complement system can have a direct damaging effect on EC in IC vasculitis by the assembly of MAC on the endothelial cell membrane.

The determination of lipids and proteins in suction blister fluid.

The concentration of 5 different proteins in suction blister fluid and serum was determined by immunotechniques. These proteins, varying in size and molecular weight (6,600-2,300,000) were insulin, albumin, high density lipoprotein determined as apoprotein A-I, alpha 2-macroglobulin and low density lipoprotein measured as apoprotein B. The difference in the blister fluid/serum concentration ratio of the proteins was dependent on the molecular weight and followed mainly the law of diffusion. Moreover, the amounts of insulin, albumin and apoproteins A-I and B in suction blister fluid were the same as those reported in peripheral lymph. The results indicate that the sieve function of the capillary basement membrane remains intact during the formation of the suction blisters. Suction blister fluid might therefore be regarded as representative of interstitial fluid. The concentrations of 4 different lipids (cholesterol, cholesterolesters, triglycerides and phospholipids) were also determined and their blister fluid/serum concentration ratio proved to have a fairly constant value of 0.25.

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).

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.

Effect of topical sunscreens on the UV-radiation-induced suppression of the alloactivating capacity in human skin in vivo.

Exposure of mice or humans to solar or artificial ultraviolet radiation (UV) has been shown to induce a number of changes in the immune system that may influence their susceptibility to skin tumors. The protective effect of sunscreens on these changes is not clear. Thirty-two patients with a variety of dermatoses routinely undergoing treatment with standard UVB (n = 19) or PUVA (n = 13) therapy were studied. One of the two tested sunscreens or its vehicle was applied to the right flexor forearm immediately prior to each total-body UV exposure. Epidermal sheets were obtained by the suction-blister method from the left flexor forearm before treatment and from both flexor forearms after 4 weeks of photo- or photochemotherapy and used as stimulator epidermal cells (EC) in the mixed epidermal cell-lymphocyte reaction (MECLR). After 4 weeks of either UVB or PUVA therapy the MECLR responses on EC from both arms were markedly decreased. Neither the tested sunscreens nor their vehicles prevented the UV-induced suppression of the alloactivating capacity. The failure of sunscreens to protect against the UV-induced suppression of the alloactivating capacity could be explained in two ways. First, the energy not absorbed by the sunscreen could be sufficient to induce suppression of the alloactivating capacity. An alternative explanation could be systemic immune suppression by UV. In order to discriminate between these possibilities only the right forearms of 10 healthy volunteers, treated with a sunscreen or its vehicle, were irradiated with UVB during 4 weeks. In this manner systemic immune suppression by UVB could be excluded. This experiment resulted in a similar suppression of the MECLR responses, as induced by total body UVB irradiation, without any protection by the sunscreen. Apparently, the UV dose not absorbed by the sunscreen was capable to induce suppression of the alloactivating capacity. Our results indicate that people protected from sunburn by sunscreens may be exposed to UV for a long period of time, and thereby subject themselves to its immunosuppressive action.

Low density lipoprotein receptor expression on keratinocytes in normal and psoriatic epidermis.

Biochemical and morphologic studies on the interaction of low density lipoprotein (LDL) with cultured normal keratinocytes and squamous carcinoma cells have shown a negative correlation between LDL receptor activity and terminal differentiation of the epidermal cells [Ponec M et al, J Invest Dermatol 83:436-440, 1984 and Vermeer, BJ et al, J Invest Dermatol 86:195-200, 1986]. Whether such in vitro studies pertain to the epidermis in vivo is not known. To obtain information on the distribution of LDL receptors in the epidermis in situ, morphologic studies were performed using LDL-gold as an ultrastructural marker. When freshly isolated mouse and human epidermal cells were incubated with LDL-gold complexes, only keratinocytes with the morphologic characteristics of basal cells showed binding and uptake of LDL-gold. No LDL receptor activity was found on Langerhans cells, melanocytes or highly differentiated keratinocytes. Since cell separation techniques can destroy receptors, the staphylococcal epidermolytic toxin was utilized to produce intercellular and intra-epithelial splitting of the epidermis. In preparations of both normal mouse and human epidermis, LDL-gold binding was restricted to basal cells and a few suprabasal keratinocytes. In contrast, in psoriatic epidermis, and to a lesser extent, essential fatty acid-deficient mouse epidermis, cells in the stratum spinosum showed abundant LDL-gold binding. Thus LDL-gold may be a useful marker for epidermal differentiation.

Comparison of melanocytes and keratinocytes in ultraviolet-induced DNA damage per minimum erythema dose sunlight: applicability of ultraviolet action spectra for risk estimates.

Action spectra are being used in risk estimates for ultraviolet (UV) damage. The purpose of our investigation was to compare the susceptibility of cultured melanocytes and keratinocytes to UV-induced DNA damage per minimum erythema dose (MED) and to determine whether the predictions made with action spectra agree with the damage actually induced. Genetic damage was measured as the number of T4-endonuclease V-sensitive sites (ESS). Predictions made with the action spectrum for the induction of DNA damage in melanocytes after irradiation with sunlight and a solar simulator were 15.9 and 13.2 ESS per 10(8) daltons per MED, respectively; with the action spectrum for the induction of DNA damage in keratinocytes the predictions were 12.1 and 9.8 ESS per 10(8) daltons per MED, respectively. To determine the actual damage per MED, cultured cells were irradiated with sunlight or a solar simulator, and MED was determined with an erythema UV meter. The induction of DNA damage in melanocytes after sunlight and solar simulator irradiation was 8.01 and 6.7 ESS per 10(8) daltons per MED, respectively, and in keratinocytes 7.49 and 7.12 ESS per 10(8) daltons per MED, respectively. This was considered to be in agreement with the predicted data. The use of action spectra for risk estimates in melanocytes appears justified.

LDL receptors in keratinocytes.

The presence of sufficient amounts of cholesterol in the epidermis is necessary for proper functioning of plasma membranes in the viable epidermal cell layers and also for the barrier quality of lipid intercellular bilayers of the stratum corneum. Cholesterol can be generated by local epidermal synthesis, or imported from the circulation as low-density lipoprotein (LDL), which is internalized by the cells by receptor-mediated endocytosis. Because of the complex structure of the skin, a model consisting of cultured human keratinocytes has been used to study in detail the regulation of epidermal sterologenesis in relation to keratinocyte differentiation. Experimental modulation of the differentiation of normal human keratinocytes has been achieved by varying extracellular calcium concentration or by comparison of a number of human squamous carcinoma cell lines and normal keratinocytes. These studies have clearly demonstrated a reciprocal correlation between the ability of cells to differentiate and LDL receptor activity. Regulation of LDL receptor expression has been found to occur at the DNA, mRNA, and protein levels, depending on the cell line studied. In normal but not malignant keratinocytes, the induction of keratinocyte differentiation was associated not only with a decrease of functional LDL receptors but also with changes in their cellular distribution. This conclusion is drawn from the observations that only in normal human keratinocytes, cultured at physiologic calcium concentrations, high levels of intracellular, cytoskeleton-associated receptors were found. Differentiation-related modulations of the LDL-receptor expression and of the cellular LDL-receptor distribution found in cultured keratinocytes were in agreement with observations made in the epidermis in situ.

Binding and internalization of low-density lipoproteins in SCC25 cells and SV40 transformed keratinocytes. A morphologic study.

Binding of low-density lipoproteins (LDL) to the plasma membrane and internalization of low-density lipoprotein receptor complexes were investigated in an epithelial tumor cell derived from the tongue (SCC25) and in SV40-transformed keratinocytes (SVK14 cells). For light microscopic studies an immunofluorescence technique with antiapoprotein B as well as conjugation procedure by which a fluorochrome 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanide (DIL) was conjugated with LDL (LDL-DIL) was used. Binding of LDL to the plasma membrane at 4 degrees C was observed in most SCC25 cells but not in SVK14 cells. The internalization of LDL-DIL was absent in SVK14 cells and was excessive in SCC25 cells. In SCC25 cells, internalization of the LDL-DIL particles was heterogeneously distributed over various cells. When a pulse-chase experiment was performed with LDL-DIL, less LDL was internalized into the SCC25 cells in comparison with a continuous label experiment. For the ultrastructural studies LDL conjugated with colloidal gold was used. In the binding experiments at 4 degrees C most LDL-gold particles were attached to the plasma membrane outside coated pits. During internalization experiments with LDL-gold particles it was observed that within 5-15 min at 37 degrees C several LDL-gold particles were seen in electron-dense structures near the plasma membrane. The electron-dense structures containing LDL-gold, as observed after an internalization period of 5-15 min, may represent the first endosomal compartment as described for transferrin receptors in A431 cells. After a period of 30 min at 37 degrees C the LDL-gold particles were observed in electron-lucent vesicles (multivesicular bodies) and dense bodies. However coated vesicles containing LDL-gold particles were seen sporadically. It is concluded that the route of internalization of LDL into the SCC25 cells differs from that of other cell types. No internalization of LDL gold was found in SVK14 cells, thus, in this respect, the SVK14 cells resemble normal keratinocytes. The morphologic data are in good agreement with biochemical studies published earlier (Ponec M et al, J Invest Dermatol 83:436-440, 1984). Both investigations suggest that LDL receptor activity is modulated during the process of terminal differentiation.

Differential effects of interleukin 1-alpha (IL-1 alpha) or tumor necrosis factor-alpha (TNF-alpha) on motility of human melanoma cell lines on fibronectin.

Interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha) induce a motogenic response in a number of benign and malignant cells. We examined the chemokinetic effects of these cytokines on the cell migration of four melanoma cell lines on fibronectin using modified Boyden chambers and video-time lapse analysis. Flow cytometry analysis of IL-1 receptors, TNF receptors, and shifts in beta 1 integrin expression were correlated with the effects of these cytokines on cell migration on fibronectin. The four melanoma cell lines exhibited heterogeneous expression of types I and II IL-1 receptors as well as p60 TNF receptors. Scant p80 TNF receptor expression was detected on only one cell line. Three of four melanoma cell lines demonstrated type I IL-1 receptors by Western blotting. IL-1 alpha and TNF-alpha induced heterogeneous modulation of beta 1 integrin expression in the four melanoma cell lines tested; downward shift of the alpha 2, alpha 3, alpha 4, and beta 1 integrin subunits was detected among three of the melanoma cell lines as were upward shifts of the alpha 4, alpha 5, and alpha 6 integrin subunits among three of the melanoma cell lines. IL-1 alpha and TNF-alpha induced enhanced migration on fibronectin in one of the melanoma cell lines and were related to an upward shift in the alpha 4 and alpha 5 integrin subunit expression. Taken together, the findings indicate that expression of a particular receptor for IL-1 or TNF does not necessarily signal a motogenic response in melanoma cells, but induces heterogeneous shifts in beta 1 integrin expression. However, upregulation in alpha 4 and alpha 5 integrin subunits appears to relate to enhanced migration on fibronectin.