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 cholesterol from the lysosomes into the medium during endocytosis and subsequent metabolism of cholesterol-containing liposomes [Furuchi, T., Aikawa, K., Arai, 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 process. The efflux of unesterified cholesterol into the medium was greatly suppressed by pregnenolone, an inhibitor of lysosomal cholesterol transport, but an appreciable proportion of the unesterified cholesterol was still released into the medium. Analysis of the medium containing the secreted cholesterol by NaBr density gradient ultracentrifugation revealed that the unesterified cholesterol was distributed in two different density peaks (bottom and d =/ approximately 1.1). The d =/ approximately 1.1 peak material formed high-density lipoprotein (HDL)-like particles that were produced and secreted by the macrophages. The lipid components of these particles were phosphatidylcholine 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 both pregnenolone-sensitive and -insensitive pathways, and that only the cholesterol secreted through the pregnenolone-sensitive pathway is associated with 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 pregnenolone-insensitive pathway was greatly stimulated by exogenously added HDL.