Acid sphingomyelinase-deficient macrophages have defective cholesterol trafficking and efflux

Cholesterol efflux from macrophage foam cells, a key step in reverse choles terol transport, requires trafficking of cholesterol from intracellular sit es to the plasma membrane. Sphingomyelin is a cholesterol-binding molecule that transiently exists with cholesterol in endosomes and lysosomes but is rapidly hydrolyzed by lysosomal sphingomyelinase (L-SMase), a product of th e acid sphingomyelinase (ASM) gene. We therefore hypothesized that sphingom yelin hydrolysis by L-SMase enables cholesterol efflux by preventing choles terol sequestration by sphingomyelin. Macrophages from wildtype and ASM kno ckout mice were incubated with [H-3]cholesteryl ester-labeled acetyl-LDL an d then exposed to apolipoprotein A-I or high density lipoprotein. In both c ases, [H-3]cholesterol efflux was decreased substantially in the ASM knocko ut macrophages. Similar results were shown for ASM knockout macrophages lab eled long-term with [H-3]cholesterol added directly to medium, but not for those labeled for a short period, suggesting defective efflux from intracel lular stores but not from the plasma membrane. Cholesterol trafficking to a cyl-coenzyme A.-cholesterol acyltransferase (ACAT) was also defective in AS M knockout macrophages. Using filipin to probe cholesterol in macrophages i ncubated with acetyl-LDL, we found there was modest staining in the plasma membrane of wild-type macrophages but bright, perinuclear fluorescence in A SM knockout macrophages. Last, when wild-type macrophages were incubated wi th excess sphingomyelin to "saturate" L-SMase, [H-3]cholesterol efflux was decreased. Thus, sphingomyelin accumulation due to L-SMase deficiency leads to defective cholesterol trafficking and efflux, which we propose is due t o sequestration of cholesterol by sphingomyelin and possibly other mechanis ms. This model may explain the low plasma high density lipoprotein found in ASM-deficient humans and may implicate L-SMase deficiency and/or sphingomy elin enrichment of lipoproteins as novel atherosclerosis risk factors.