SEQUESTRATION OF ACETYLATED LDL AND CHOLESTEROL CRYSTALS BY HUMAN MONOCYTE-DERIVED MACROPHAGES

Monocyte-derived macrophages accumulate and process cholesterol in ath erosclerotic lesions. Because of the importance of this process, we ex amined the interaction of cholesterol crystals and acetylated low dens ity lipoprotein (AcLDL) with human monocyte-macrophages in a combined chemical and morphological study. These two forms of cholesterol induc ed extensive compartmentalization of the macrophage cytoplasm, Unexpec tedly, the compartments maintained a physical connection to the extrac ellular space as demonstrated with ruthenium red staining. The compart ments formed through invagination of the top surface of the macrophage plasma membrane. Some cholesterol crystals and AcLDL were sequestered within these surface-connected compartments for up to five days in th e case of the crystals and for one day in the case of AcLDL. Pulse-cha se studies of fractionated macrophages indicated that [H-3]cholesterol redistributed from the surface-connected compartments into lysosomes (where the cholesterol remained unesterified) and into lipid droplets (where the cholesterol was stored as cholesteryl ester). Intracellular uptake and esterification of cholesterol was blocked by cytochalasin D. However, once cholesterol was sequestered in the surface-connected compartments, subsequent esterification of the cholesterol could not b e inhibited by cytochalasin D. Apolipoprotein E was localized within t he surface-connected compartments by immunogold labeling suggesting a possible function for this protein in the processing of lipid taken up through the sequestration pathway. Removal of microcrystalline choles terol from the medium resulted in release of most of the accumulated c holesterol microcrystals from the macrophages, as well as disappearanc e of the surface-connected compartments. Thus, sequestration is a nove l endocytic mechanism in which endocytic compartments remain connected to the extracellular space. This differs from phagocytosis where endo cytic vacuoles rapidly pinch off from the plasma membrane. Sequestrati on provides a means for macrophages to remove substances from the extr acellular space and later release them.