Liposome uptake by cultured macrophages mediated by modified low-density lipoproteins.

We have investigated effects of native low-density lipoproteins (LDL) and malondialdehyde-treated LDL on the interaction of 5(6)-carboxyfluorescein-labeled liposomes bearing antibodies to LDL with cultured J774 macrophages. It was found that an addition of modified LDL to the incubation medium resulted in 15-20-fold increase of carboxyfluorescein binding to cells, whereas native LDL did not produce such effect. The increase of carboxyfluorescein binding to macrophages in the presence of modified LDL was not due to an enhanced leakage of the label from liposomes. The modified-LDL-mediated binding of carboxyfluorescein to cells was reduced to 20-30% of the initial level in the presence of cell-respiration inhibitors (NaF and antimycin A). Fluorescent microscopy data also indicate the modified-LDL-induced incorporation of liposome contents into cells. The results obtained in this study make it possible to assume that in the presence of malondialdehyde-treated LDL, liposomes with antibodies to LDL may be incorporated into macrophages via the receptor-mediated pathway for modified LDL.

Cholesterol can stimulate secretion of apolipoprotein B by cultured human hepatocytes.

During a 5 day cultivation of human hepatocytes in a primary culture the secretion of apolipoprotein B was measured by enzyme-linked immunosorbent assay. Density-gradient ultracentrifugation demonstrated that the majority of the secreted apolipoprotein B was associated with the very-low-density lipoprotein fraction. Exposure of the cells to cholesterol (5-100 micrograms/ml) resulted in a dose-dependent increase in apolipoprotein B secretion rate.

Effect of cell cholesterol content on apolipoprotein B secretion and LDL receptor activity in the human hepatoma cell line, HepG2.

Human hepatoma HepG2 cells were used to study the effects of cholesterol loading and depletion on apolipoprotein B (apoB) secretion and low-density lipoprotein (LDL) receptor activity. Exposure of HepG2 cells to cholesterol and oleic acid, which elevated intracellular cholesterol levels, stimulated apoB secretion and reduced receptor-mediated uptake of LDL, whereas recombinant complexes of apolipoprotein A-I with dimyristoylphosphatidylcholine, which depleted the cellular cholesterol pool, inhibited apoB secretion and up-regulated LDL receptors. Significant negative correlation (r = -0.92, P less than 0.001) between the levels of apoB secretion and LDL uptake was found. These data suggest that the cholesterol content of the cells may induce concomitant changes in apoB secretion and LDL receptor activity.

Low-density lipoproteins interact with liposome-binding sites on the cell surface.

Under physiological conditions significant amounts of low-density lipoprotein LDL particles ar taken up by cells independently of specific high-affinity LDL receptors (apo-B receptors). Previously it was established that some cells contain surface sites capable of binding liposomes. We proposed that liposome-binding sites could contribute to LDL interaction with the cell surface via phospholipid molecules of LDL particles. To check this hypothesis we studied the competitive interaction of human LDL and DPPC liposomes with mouse embryo fibroblasts depleted of apo-B receptors by preliminary incubation with LDL. We have found that after removal of the liposome-binding sites from cell lamellae these areas of the cell surface lose their ability to bind LDL.

High-affinity association and degradation of 125I-labelled low density lipoproteins by human hepatocytes in primary culture.

Catabolism of homologous low density lipoproteins (LDL) was studied in primary culture of human hepatocytes (HH). The cell association and degradation of 125I-labeled LDL (125I-LDL) were curvilinear functions of substrate concentration. Cell association and degradation of 125I-LDL were inhibited by excess unlabeled LDL. Reductive methylation of unlabeled LDL abolished its ability to complete with 125I-LDL for cell association and degradation. Preincubation of HH with unlabeled LDL caused a 63% inhibition of the 125I-LDL degradation. It is concluded that the catabolism of LDL by HH proceeds in part through a receptor-mediated pathway similar to that demonstrated on extrahepatic cells.

[125I]LDL uptake in rabbit arteries perfused in situ. Effect of HDL on intact and de-endothelialized vessels.

The uptake of [125I]LDL by rabbit abdominal and thoracic aortae, and carotid arteries perfused in situ was studied. [125I]LDL uptake in vessels with intact endothelial covering was saturable, Ca2+, Mg2+-dependent and was inhibited by a 100-fold excess of LDL and HDL, on the average by 30% and 60%, respectively. In de-endothelialized vessels [125I]LDL uptake increased by approximately 3-fold, and was not reduced in the presence of excess of unlabeled LDL and HDL.

Apolipoprotein B release from activated human platelets.

Apolipoprotein B (apoB) release from activated washed human platelets was measured by enzyme-linked immunosorbent assay (ELISA) using monospecific rabbit antibodies to human low density lipoprotein (LDL). Activation of platelets with thrombin, Ca2+-ionophore A23187 or stable analogue of prostaglandin endoperoxides U46619 stimulated release of approximately 20 ng apoB/10(8) platelets. Thrombin-induced apoB release was inhibited by the prostacyclin analogue carbacyclin. Dose-response curves of thrombin stimulation and carbacyclin inhibition of apoB and beta-thromboglobulin (beta-TG) release were very similar. Treatment of platelets with heparin did not remove significant amounts of apoB or affect the subsequent release of apoB induced by thrombin. The results of density gradient ultracentrifugation indicated that most of the apoB was released in the LDL density range. These data suggest that human platelets contain immunoreactive apoB, which can be released during platelet activation.

Immunoenzyme assessment of human apoB-lipoprotein binding to immobilized receptor of low density lipoproteins. 2. Binding of isolated lipoproteins.

The receptor of low density lipoproteins (LDL-receptor) from bovine adrenal cortex membranes was immobilized in standard 96-well polystyrene plates using monoclonal V5-antibodies to the LDL-receptor. The binding of the immobilized LDL-receptor with human low density lipoproteins (LDL) and very low density lipoproteins (VLDL) was determined using peroxidase-labelled antibodies to human apoB. The value of Kd for the interaction of LDL with the immobilized LDL-receptor for 40 samples of LDL was found to be from 5 to 20 micrograms apoB per ml. The immobilized LDL-receptor failed to bind LDL modified by acetylation or malonic dialdehyde, while the binding of non-modified LDL to the immobilized LDL-receptor was inhibited in the presence of EDTA, which is known to be specific for the interaction of LDL with the LDL-receptor. Unlike LDL, VLDL were more variable in the binding to the LDL-receptor. The value of Kd for the interaction of VLDL with the LDL-receptor for 40 samples of VLDL was found to be from 0.5 to 10 micrograms apoB per ml. Thus, the described method is suggested to study the interaction of apoB-containing lipoproteins with the LDL-receptor.

Immunoenzyme assessment of human apoB-lipoprotein binding to immobilized receptor of low density lipoproteins. 1. Preparation of anti-receptor monoclonal antibodies.

The preparation and properties of V5 monoclonal antibody to low density lipoprotein receptor (LDL-receptor) from bovine adrenal cortex membranes are described. The monospecific V5 antibody recognizes the LDL-receptor (the only protein with molecular mass of 140 kD) in bovine adrenal cortex membranes. V5 antibody fails to compete with human low density lipoproteins (LDL) for binding to the LDL-receptor. After absorption in standard 96-well polystyrene plates, V5 antibody efficiently binds the affinity-purified LDL-receptor from the solution and the subsequent binding of the LDL-receptor with human LDL was determined using peroxidase-labelled antibodies to apolipoprotein B. The LDL-receptor immobilized by V5 antibodies is suggested for use in studies on the binding of human lipoproteins to the LDL-receptor.

Corticosteroidal regulation of the glucocorticoid-binding capacity of rat heart cytoplasmic fraction.

The effects were studied of variations in blood corticosteroid levels on the glucocorticoid-binding capacities of two types of complex-forming substances, whose presence in the myocardium had been established in previous studies. The hormone binding was assessed by the method of adsorption of free forms of steroids on activated dextrancoated charcoal with application of Scatchard's analysis. The binding capacity was tested both in the presence of endogenous steroids and in cytoplasmic fraction made free of the hormones. After adrenalectomy, experimental stress, and administration of exogenous glucocorticoids the concentration of the complex-forming substance I changed in parallel with changing blood transcortin levels. This finding corroborates the concept that the complex-forming substance I is an intercellular (and possibly partially also intracellular) pool of transcortin or a transcortin-like protein; this pool regulates the accessibility of glucocorticoids to the cytoreceptor proper. The intracellular concentration of the complex-forming substance II (performing the function of receptor) changed only little with varying corticosteroid levels, and variations in its binding capacity evidently resulted from shifts in the equilibrium between the "occupied" and "free" findings sites as well as in the distribution of this protein between the cytoplasmic and nuclear fractions.

Enzyme immunoassay of the receptors for modified low density lipoprotein.

Mouse macrophages (line J 774) were incubated with monospecific goat anti-low density lipoprotein antibodies, which were conjugated to horseradish peroxidase (AB-HRP). Addition of low density lipoprotein (LDL) modified by treatment with malondialdehyde to cultures of these cells resulted in a dose-dependent increase in the amount of cell-associated enzyme activity. The concentration curve was hyperbolic with half-saturation of modified LDL at a concentration of about 3 micrograms/ml. This effect was completely blocked by polyinosinic acid and was not observed in experiments with human fibroblasts, which do not exhibit high affinity binding sites that recognize chemically modified LDL. Our data indicate that receptor-mediated endocytosis of AB-HRP in the presence of native or modified LDL may be used as very simple, efficient, and sensitive assay for investigation of the scavenger receptors for modified LDL.

Enzyme-linked immunoreceptor assay of low-density-lipoprotein receptors.

A new modification of enzyme immunoassay-enzyme-linked-immunoreceptor assay (ELIRA) was used to study low-density lipoprotein (LDL) binding by cultured human skin fibroblasts. Time-course and concentration curves obtained by this method were typical of LDL binding by cultured fibroblasts. According to ELIRA, fibroblasts bound native LDL 10-20 times more effectively than reductively methylated LDL or native LDL in the presence of heparin. Receptor-specific Km and maximum binding capacity calculated from these data were 5.4 micrograms/ml and 177.5 ng/mg of cell protein, respectively. Receptor-specific Km and maximum binding capacity for surface 125I-LDL binding measured in the same experiment were 11.0 micrograms/ml and 138.0 ng/mg of cell protein. Incubation of cells with isolated LDL or with unfractionated serum containing the same amount of apoB yielded similar concentration curves for lipoprotein binding. These data indicate that the ELIRA can be used for investigation of receptor-mediated lipoprotein binding without purification of lipoproteins.

Flow cytofluorometric study of lipoprotein interactions with cultured endothelial cells.

Accumulation of rhodamine B isothyocyanate-conjugated low density lipoproteins (R-LDL) in cultured endothelial cells from human umbilical cord was studied with a fluorescence activated cell sorter. R-LDL uptake was blocked at 0 degrees C, inhibited by addition of excess of nonlabeled low density lipoprotein, high density lipoprotein-2 or high density lipoprotein-3. High density lipoprotein-2 was about twice as effective in inhibition of R-LDL uptake as high density lipoprotein-3. Endothelial cells that formed a contact-inhibited monolayer lost the ability to incorporate R-LDL via a receptor-mediated pathway. Using R-LDL, it was possible to distinguish cells with different levels of R-LDL incorporation.