Dual pathways for the secretion of lysosomal cholesterol into a medium from cultured macrophages

The removal of cholesterol from macrophages is important for reversing foam cell formation. In a previous study, we demonstrated that mouse peritoneal macrophages in culture secrete significant amounts of unesterified cholest erol from the lysosomes into the medium during endocytosis and subsequent m etabolism of cholesterol-containing liposomes [Furuchi, T., Aikawa, K,, Ara i, H,, and Inoue, K, (1993) J, Biol, Chem, 268, 27345-27348], In this study , we found that at least two distinct mechanisms are involved in this proce ss, The efflux of unesterified cholesterol into the medium was greatly supp ressed by pregnenolone, an inhibitor of lysosomal cholesterol transport, bu t an appreciable proportion of the unesterified cholesterol was still relea sed into the medium. Analysis of the medium containing the secreted cholest erol by NaBr density gradient ultracentrifugation revealed that the unester ified cholesterol was distributed in two different density peaks (bottom an d d = similar to 1.1). The d = similar to 1.1 peak material formed high-den sity lipoprotein (HDL)-like particles that were produced and secreted by th e macrophages, The lipid components of these particles were phosphatidylcho line and sphingomyelin, while the sole protein component was apolipoprotein E (apo E). Treatment with pregnenolone completely abolished the production of these HDL-like particles but had little effect on the bottom fractions. These data indicate that macrophages release lysosomal cholesterol via bot h pregnenolone-sensitive and -insensitive pathways, and that only the chole sterol secreted through the pregnenolone-sensitive pathway is associated wi th endogenously synthesized apo E-containing HDL-like particles. Moreover, we found that the pregnenolone-sensitive pathway operated independently of the presence or absence of exogenous HDL, whereas secretion via the pregnen olone-insensitive pathway was greatly stimulated by exogenously added HDL.