Morphological characterization of the cholesteryl ester cycle in cultured mouse macrophage foam cells

Mouse peritoneal macrophages can be induced to accumulate cholesteryl esters by incubating them in the presence of acetylated low density lipoprotein. The cholesteryl esters are sequestered in neutral lipid droplets that remain in the cell even when the acetylated low density lipoprotein is removed from the culture media. Previous biochemical studies have determined that the cholesterol component of cholesteryl ester droplets constantly turns over with a half time of 24 h by a cyclic process of de-esterification and re-esterification. We have used morphologic techniques to determine the spatial relationship of cholesteryl ester, free cholesterol, and lipase activity during normal turnover and when turnover is disrupted. Lipid droplets were surrounded by numerous 7.5-10.0-nm filaments; moreover, at focal sites on the margin of each droplet there were whorles of concentrically arranged membrane that penetrated the matrix. Histochemically detectable lipase activity was associated with these stacks of membrane. Using filipin as a light and electron microscopic probe for free cholesterol, we determined that a pool of free cholesterol was associated with each lipid droplet. Following incubation in the presence of the exogenous cholesterol acceptor, high density lipoprotein, the cholesteryl ester droplets disappeared and were replaced with lipid droplets of a different lipid composition. Inhibition of cholesterol esterification caused cholesteryl ester droplets to disappear and free cholesterol to accumulate in numerous myelin-like structures in the body of the cell. Cholesterol homeostasis is important for normal cell function. The activities of the enzymes involved in the endogenous synthesis of cholesterol are in balance with cell surface receptors that mediate the internalization of exogenous cholesterol transport proteins such as low density lipoprotein (LDL') (1). Because of this tightly regulated process, cholesterol ordinarily does not accumulate in cells. However, when cells do accumulate more cholesterol than is required to meet metabolic needs, it is stored as cholesteryl ester lipid droplets, which are nonmembrane bound inclusions that are distributed in the cytoplasm of the ceil (2). These lipid droplets are found in some steroid secreting cells (3, 4). Moreover, in certain disease processes, abnormal numbers of cholesteryl ester lipid droplets accumulate in cells, and as a result these cells have a foamy appearance (5). Heretofore, it has been difficult to study the metabolic activity of these lipid droplets in either normal or diseased cells of the body. However, cultured ~Abbreviations used in this paper: ACAT, acyl-CoA:cholesterol acyltransferase; acetyl-LDL acetylated low density lipoprotein; DME, Dulbecco's modified Eagle medium: HDL, high density lipoprotein. peritoneal macrophages can be induced to accumulate large amounts of cholesteryl esters (2, 6) and this in vitro cell system can be used to study cholesterol metabolism in cells that have large amounts of stored cholesterol (7). Previous biochemical and morphologic studies have established several important features about cholesteryl ester accumulation in cultured macrophages. (a) These cells accumulate large amounts of cholesteryl esters when incubated with chemically modified LDL (acetylated LDL [acetylLDL]) but not with normal LDL (6). (b) Cholesteryl estercontaining lipid droplets, which are birefringent under polarized light, appear by electron microscopy as nonmembrane bound inclusions that are free in the cytoplasm of the cell (2). (c) When macrophages that have accumulated large numbers of cholesteryl ester lipid droplets are incubated in the absence of acetyl-LDL, the cholesteryl ester level remains constant. However, when these cells are incubated in the presence of an extracellular cholesterol acceptor such as high density lipoprotein (HDL), there is a rapid loss of cholesteryl esters in response to the net egress of cholesterol from the cell (7). Because the cholesteryl ester-laden macrophages resemble THE JOURNAL OF CELL BIOLOGY . VOLUME 97 OCTOBER 1983 1156-1168 1156 © The Rockefeller University Press 0021-9525183110/1156/13 $1.00 on O cber 0, 2017 jcb.rress.org D ow nladed fom in many respects the foam cells found in atherosclerotic lesions (5, 8), this culture system has been used to study how cholesterol acceptors, such as HDL, can induce the removal of cholesterol. These studies have established the important concept that the cholesteryl ester lipid droplets are in a state of dynamic flux (7). They are continually being broken down by a nonlysosomal cholesteryl esterase into free cholesterol and fatty acids. When an extracellular cholesterol acceptor is not present, the free cholesterol generated by the esterase is re-esterified by the enzyme acyl-CoA: cholesterol acyltransferase (ACAT) in an energy-dependent reaction. These cholesteryl esters are sequestered into neutral lipid droplets. If, however, an extracellular cholesterol acceptor is present, the free cholesterol generated by the esterase leaves the cell and becomes associated with the cholesterol acceptor. Finally, if the ACAT enzyme is inhibited by pharmacological concentrations of progesterone, the de-esterification reaction continues and, in the absence of an extracellular cholesterol acceptor, the cell accumulates free cholesterol. This process of deesterification and re-esterification of cholesterol has been termed the cholesteryl ester cycle (7). In the present study we have utilized morphological and cytochemical techniques to study the cholesteryl ester cycle in cholesteryl ester-loaded macrophages. Using filipin as a specific cytochemical probe for free cholesterol, we have determined the location of the free cholesterol pool using both fluorescence and electron microscopy. In addition, this probe has been used to identify the cellular compartment that accumulates free cholesterol in the presence of progesterone. Using cytochemical methods, we have localized a lipase that has the properties of the cholesteryl esterase involved in the de-esterification reaction. Finally, we have studied the morphologic changes that take place when cholesterol leaves the cell in response to the presence of an exogenous cholesterol acceptor. These studies provide new and important information about the spatial arrangement of the enzymes and substrates involved in the cholesteryl ester cycle. MATERIALS AND METHODS Male Swiss Webster mice (25-30 g) were obtained from Simon Laboratories (Gilroy, CA). Dulbecco's modified Eagle medium (DME) and Dulbecco's PBS were purchased from Gibco Laboratories (Grand Island, NY). All other tissue culture supplies were obtained as previously reported (7). Glutaraldehyde was obtained from Electron Microscopy Sciences (Fort Washington, PA). Araldite was obtained from Ladd Research Industries, Inc. (Burlington, VT). Filipin was generously provided by Dr. Joseph Grady at Upjohn Co. (Kalamazoo, MI). Taurocholate was purchased from P.-L. Biochemicals, Inc. (Milwaukee, WI). Tween 80 and quinine hydrochloride were obtained from Sigma Chemical Co. (St. Louis, MO). Triolein was purchased from NuChek Prep (Elysian, MN). Lipoproteins: Human LDL (d 1.019-1.063 g/ml) and HDL3 (d 1.1251.215 g/ml) were isolated from the plasma of individual healthy subjects by ultracentrifugation (9). LDL was acetylated with repeated additions of acetic anhydride as previously described (6). The concentrations of acetyl-LDL and HDL are given in terms of the protein content of the lipoproteins. Reconstituted Acetyl-LDL: Acetyl-LDL was reconstituted with triglycerides by extracting the endogenous cholesteryl esters with heptane and reconstituting with triolein as previously described (10). The reconstituted lipoprotein was designated r[triolein] acetyl-LDL. Preparation of Mouse Macrophage Monolayers: Resident peritoneal macrophages were harvested from mice in PBS by a previously described modification (2, 6) of the procedure developed by Edelson and Cohn ( l l ). The peritoneal fluid of 5-30 mice was pooled (6l 0 x 106 cells/mouse) and the cells were collected by centrifugation (400 g, min, room temperature) and washed once with 30 ml of DME. The cells were resuspended in DME containing 20% (vol/vol) fetal calf serum, penicillin (100 U/ml), and streptomycin (100/~g/ml) at a final concentration of 2--4 x l06 ceUs/ml. Aliquots of this suspension (0.5-1 ml) were dispensed into plastic Petri dishes (35 mm x 10 mm) and incubated in a humidified CO2 (5%) incubator at 37"C. After 2 h, each dish was washed three times with 2 ml of DME to remove nonadherent cells, and then used in the experimental protocol described. Standard Protocol for Loading Macrophages with Cholesteryl Esters: On day 0, each dish ofmacrophages received 1 ml of DME containing I mg/ml of human albumin, 50 #g/ml of acetyl-LDL, penicillin (100 U/ml), and streptomycin (100 #g/ml) and was incubated at 37"(7 for 48 h with a change of media at 24 h. On day 2, each monolayer was washed twice with 2 ml of DME containing 5% fetal calf serum, penicillin (100 U/ml) and streptomycin (100 #g/ml) and then washed once with 2 ml of DME alone. Each monolayer was then incubated at 37 ° for 24 h with 1 ml of DME containing 1 mg/ml of human albumin, penicillin (100 U/ml), and streptomycin (100 #g/ml). On day 3, the media was discarded and each dish was treated as described. Polarized Light Microscopy: Monolayers of macrophages were grown on glass coversli~ as described above. After the indicated incubation, the coverslips were removed and mounted on glass slides in a drop of media. To enhance birefringence, the slides were first warmed to 40-45°C for l0 min, then cooled to 10°C for 10 min before examination at 23°C (2). The cells were photographed with phase and polarized optics using a Zeiss Photomicroscope Ill (Carl Zeiss, Inc. NY.). Localization of Eilipin-binding by Fluorescence Microscopy: Monolayers of macrophages were grown on gla