FORMATION OF CHOLESTEROL MONOHYDRATE CRYSTALS IN MACROPHAGE-DERIVED FOAM CELLS

In a previous study using the J774 macrophage foam cells, we quantitat ed the accumulation of unesterified (free) cholesterol derived from ch olesteryl ester hydrolysis in lysosomes, after phagocytic uptake of ch olesteryl ester droplets. In the present study, we examined whether th e accumulation of free cholesterol in lysosomes leads to the formation of cholesterol monohydrate crystals by analyzing the lipid compositio n of low density lysosome fractions isolated from cholesteryl ester-lo aded macrophages after a 24-h incubation. Phase diagrams of the consti tuent lipids in the lipid-filled lysosomes predicted the formation of cholesterol monohydrate crystals. The formation of cholesterol monohyd rate crystals was observed in cholesteryl ester-loaded macrophages aft er a 48-h incubation by polarizing light microscopy. The crystals had a density of 1.04 g/ml and the morphology of cholesterol monohydrate c rystals with an acute edge angle of about 80 degrees The crystals appe ared as needles as well as plates and melted only when heated to great er than 85 degrees C. The physical properties of these crystals are ch aracteristic of cholesterol monohydrate. In our studies, crystal forma tion was observed even when cells had active acyl-CoA:cholesterol acyl transferase or when cholesterol effiux was stimulated. Electron micros copy and acid phosphatase cytochemistry of lysosomes in cholesteryl es ter-loaded cells confirmed that cholesterol crystal formation occurred within lipid-loaded lysosomes. Time-lapse video microscopic studies r evealed that most of the cells containing cholesterol monohydrate crys tals not only remain viable but also have the capacity to translocate single crystals within cells. The data demonstrate that lysosomal accu mulation of free cholesterol in macrophages after phagocytic uptake an d hydrolysis of cholesteryl ester droplets leads to the formation of c holesterol monohydrate crystals within lipid-filled lysosomes. Such a process may lead to deposition of free cholesterol and cholesterol mon ohydrate crystals in macrophage foam cells during the progression of a therosclerosis.