The interaction of apolipoprotein (apo) E-free high-density lipoprotein (HDL) with parenchymal, endothelial and Kupffer cells from liver was characterized. At 10 min after injection of radiolabelled HDL into rats, 1.0 +/- 0.1% of the radioactivity was associated with the liver. Subfractionation of the liver into parenchymal, endothelial and Kupffer cells, by a low-temperature cell-isolation procedure, indicated that 77.8 +/- 2.4% of the total liver-associated radioactivity was recovered with parenchymal cells, 10.8 +/- 0.8% with endothelial cells and 11.3 +/- 1.7% with Kupffer cells. It can be concluded that inside the liver a substantial part of HDL becomes associated with endothelial and Kupffer cells in addition to parenchymal cells. With freshly isolated parenchymal, endothelial and Kupffer cells the binding properties for apo E-free HDL were determined. For parenchymal, endothelial and Kupffer cells, evidence was obtained for a saturable, specific, high-affinity binding site with Kd and Bmax. values respectively in the ranges 10-20 micrograms of HDL/ml and 25-50 ng of HDL/mg of cell protein. In all three cell types nitrosylated HDL and low-density lipoproteins did not compete for the binding of native HDL, indicating that lipids and apo B are not involved in specific apo E-free HDL binding. Very-low-density lipoproteins (VLDL), however, did compete for HDL binding. The competition of VLDL with apo E-free HDL could not be explained by label exchange or by transfer of radioactive lipids or apolipoproteins between HDL and VLDL, and it is therefore suggested that competition is exerted by the presence of apo Cs in VLDL. The results presented here provide evidence for a high-affinity recognition site for HDL on parenchymal, liver endothelial and Kupffer cells, with identical recognition properties on the three cell types. HDL is expected to deliver cholesterol from peripheral cells, including endothelial and Kupffer cells, to the liver hepatocytes, where cholesterol can be converted into bile acids and thereby irreversibly removed from the circulation. The observed identical recognition properties of the HDL high-affinity site on liver parenchymal, endothelial and Kupffer cells suggest that one receptor may mediate both cholesterol efflux and cholesterol influx, and that the regulation of this bidirectional cholesterol (ester) flux lies beyond the initial binding of HDL to the receptor.

The interaction of low density lipoprotein, acetylated low density lipoprotein and apolipoprotein E—free high density lipoprotein with parenchymal, endothelial and Kupffer cells of human liver was visualized. For this purpose, the fluorescent phospholipid analog 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethyl indocarbocyanine perchlorate was used to label the lipoproteins. The involvement of both parenchymal and nonparenchymal cells in the uptake of 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethyl indocarbocyanine perchlorate—labeled low density lipoprotein and acetylated low density lipoprotein was studied using in vitro perfusion of human liver tissue blocks. In addition, primary hepatocyte cultures were used to visualize the interaction with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethyl indocarbocyanine perchlorate‐labeled apolipoprotein E—free high density lipoprotein and (modified) low density lipoprotein. 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethyl indocarbocyanine perchlorate‐low density lipoprotein showed a time‐dependent and concentration‐dependent interaction with both hepatocytes and Kupffer cells, although the intensity of the interaction with parenchymal cells varied strongly among the liver donors. Uptake of 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethyl indocarbocyanine perchlorate‐low density lipoprotein by both cell types was strongly inhibited by the presence of excess unlabeled low density lipoprotein in the (perfusion) medium. Methylation and hydroxyac‐etaldehyde treatment of low density lipoprotein prevented the uptake of low density lipoprotein. This indicated that the uptake of low density lipoprotein by Kupffer and parenchymal cells was mediated by the low density lipoprotein receptor. 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethyl indocarbocyanine perchlorate‐acetylated low density lipoprotein was mainly taken up in situ by liver endothelial cells and by a minor population of Kupffer cells. Polyinosinic acid, a known inhibitor of the scavenger receptor, prevented the uptake by liver endothelial cells. Therefore human liver endothelial cells express active scavenger receptors on their surface. Apolipoprotein E—free 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethyl indocarbocyanine perchlorate‐high density lipoprotein was found to be associated with the membrane of cultured liver parenchymal cells but was not taken up intracellularly, indicating a cholesterol exchange process occurring extracellularly at the plasma membrane. The cellular localization of lipoprotein receptors and uptake of the various classes of lipoproteins are comparable with the situation in rats. (HEPATOLOGY 1991;13:79–90.)

Characterization of the interaction of iodinated apolipoprotein (apo) E-free high density lipoprotein (HDL) with cultured human hepatocytes provides evidence for a saturable, Ca 2+ -independent, high affinity binding site with an apparent k m value of 20 fig/ml of apolipoprotein. Nitrated HDL and low density lipoprotein (LDL) did not compete for the binding of HDL, in contrast to very low density lipoprotein (VLDL). It is suggested that VLDL competition is exerted by the presence of apo Cs. Degradation of HDL was relatively low and in some cases not detectable. In cases where degradation was found, inhibitors of the lysosomal pathway of protein degradation had no effect, while LDL degradation was inhibited more than 80%. In the presence of 10 fiM of monensin, the cell-association of HDL was unaffected, but the degradation was inhibited by 30%. Under similar conditions, LDL association was inhibited by 40% and LDL degradation, by 90%. Incubation of human hepatocytes with fluorescently labeled HDL (Dil-HDL) revealed (in contrast to Dil-LDL) mainly strong membranebound fluorescence and hardly any labeling of small intracellular vesicles. It is concluded that human hepatocytes possess a specific high affinity site for human HDL with recognition properties similar to those described earlier on rat hepatocytes. No evidence that the binding of HDL is actively coupled to uptake and lysosomal degradation could be obtained, indicating that binding of LDL and HDL to human hepatocytes is coupled differently to intracellular pathways.

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