Cholesterol-loaded Macrophages Research Articles

Regulation of phospholipid biosynthesis during cholesterol influx and high density lipoprotein-mediated cholesterol efflux in macrophages

We have studied the rate of phospholipid synthesis and turnover in mouse peritoneal macrophages in reaction to cholesterol influx and high density lipoprotein (HDL)-mediated cholesterol efflux, using three different radioactive precursors, 32PO4(3-), [3H]choline, and [14C]oleic acid. The cells were loaded with cholesterol for up to 18 h with acetyl-low density lipoprotein (LDL), and phospholipid synthesis was measured at various time intervals and compared with nonloaded macrophages. In the first 2 h of cholesterol loading, a twofold increase in the rate of synthesis for sphingomyelin, phosphatidylcholine, phosphatidylserine-inositol, and phosphatidylethanolamine was observed. After this initial up-regulation, the rate of phospholipid synthesis continuously declined upon further cholesterol loading, while the turnover rate of cellular phospholipids was not affected under the same conditions. The lysosomal inhibitor chloroquine abolished the down-regulation, revealing a strong correlation between phospholipid synthesis and lysosomal enzyme activity which was presumably dependent on the release of cholesterol from the lysosome. The reduction in phospholipid synthesis induced by cholesterol loading is reversible by the addition of HDL3 to the cells. When HDL3 was added to the culture medium, a two- to threefold increase in phosphatidylcholine synthesis and a twofold increase in sphingomyelin formation was observed after 3 h. Ca2+ antagonists of the dihydropyridine type, which down-regulate HDL-receptor activity and promote the formation and cellular release of lamellar bodies derived from the lysosomal compartment (Schmitz, G., et al. 1988. Arteriosclerosis. 8: 46-56, and Robenek, H., and G. Schmitz. 1988. Arteriosclerosis. 8: 57-67), specifically enhance the synthesis of sphingomyelin in cholesterol-loaded macrophages. Inhibitors of acyl-CoA:cholesterol acyltransferase (Octimibate, progesterone) increase both the synthesis of sphingomyelin and phosphatidylcholine, and enhance HDL-receptor activity. The results indicate that cholesterol and phospholipid metabolism are coordinately regulated in macrophages. Moreover, the formation of phosphatidylcholine and sphingomyelin seems to be an important factor for the promotion of HDL-receptor-mediated cellular cholesterol efflux.

Expression of elastase activity by human monocyte-macrophages is modulated by cellular cholesterol content, inflammatory mediators, and phorbol myristate acetate.

We investigated the effects of a number of stimulatory agents on the production of both cell-associated and extracellular elastase-type enzymes on human monocyte-macrophages in vitro and of the modulation of such effects by modification of cellular cholesterol content. The stimulatory agents included phorbol myristate acetate (PMA) and the inflammatory mediators, lipopolysaccharide (LPS), opsonized zymosan (OZ), and platelet activating factor (PAF). Using the synthetic substrate, N-succinyl-trialanyl-paranitroanilide (SANA), we detected cell-associated elastase-like activity in monocyte-derived macrophages. Such activity increased markedly with cell maturation over the period from 5 to 15 days of adherence culture. While PAF (10 micrograms/ml) and LPS (10 micrograms/ml) were without effect on cell-associated elastase-like activity in macrophages, PMA (100 ng/ml) and OZ (1 mg/ml) markedly stimulated such activity in cells cultured for 15 days. Furthermore, a fivefold increase in the cell-associated elastase-like activity of macrophages occurred upon cholesterol loading of the cells with acetylated low density lipoprotein (AcLDL). By contrast, this activity was markedly diminished upon depletion of cellular cholesterol content after incubation with high density lipoprotein (HDL3). Latent elastinolytic activity in the culture medium was detected by use of a radioactive substrate, insoluble 3H-elastin, after initial tryptic treatment of the medium. Such latent elastase activity was secreted only by activated macrophages; the relative potency of stimulation was: PMA greater than LPS = PAF greater than OZ. Increase in cellular cholesterol content alone markedly enhanced the secretion of elastase (from undetectable levels to 28 ng of 3H-elastin degraded/hr/micrograms DNA). In all cases, both the cell-associated and secreted latent elastinolytic activities were due to metalloproteases, in view of their 90% inhibition by 2 mM EDTA. Cholesterol-loaded macrophages, which displayed an approximately 40-fold increase in total cholesterol content as compared to control cells, remained sensitive to the action of activators of OZ and PMA, while LPS and PAF exerted only weak effects. Our data indicate that cellular cholesterol content and inflammatory mediators are effective stimulants of the production and secretion of elastase-type enzymes by human monocyte-macrophages. Among these factors, cellular cholesterol content, OZ, PAF, and LPS may represent factors of relevance to the inflammatory role of the macrophage in atherogenesis and more specifically to the alteration of elastin structure in the extracellular matrix of the vessel wall.

Receptor-mediated uptake and ‘retroendocytosis’ of high-density lipoproteins by cholesterol-loaded human monocyte-derived macrophages: possible role in enhancing reverse cholesterol transport

Human monocyte-derived macrophages (MDM) are cholesterol-loaded, and the rates of uptake, degradation and resecretion of high-density lipoproteins are measured and compared to the rates in control cells. Results show the binding activity of these lipoproteins is upregulated in cholesterol-loaded cells; the bound and internalized lipoproteins are not degraded to any appreciable extent but primarily resecreted as a larger particle. The enhancement of binding activity for high-density lipoproteins is arrested when cycloheximide is added to the medium, suggesting that protein synthesis is involved. Preliminary evidence also indicates that HDL 3 (without apoE) after internalisation is converted intracellularly to a larger apoE-containing HDL 2-like particles. Thus, MDM appears to possess specific receptors for HDL 3 without apoE that may function to facilitate HDL-mediated removal of excess cholesterol from cells.

Low-density lipoprotein and scavenger receptor activities are modulated by secretory products derived from cells of the arterial wall

The atherosclerotic lesion consists of cholesterol-loaded macrophages, smooth muscle cells, and other cells of the arterial wall. Conditioned medium from human monocyte-derived macrophages (HMDM) stimulated both acetylated and native low-density lipoprotein (Ac-LDL and LDL, respectively) degradation in autologous cells by 25% and 90%, respectively, and this was due to an increase in the number of both LDL and Ac-LDL receptors. Macrophage conditioned medium also resulted in an approximate doubling of LDL degradation by human arterial smooth muscle cells (HASMC), endothelial cells (HEC), and skin fibroblasts (HSF). Macrophage degradation of both Ac-LDL and LDL was enhanced 15% to 45% by conditioned medium derived from HASMC and HSF, respectively, but not by HEC-conditioned medium. Conditioned medium from HASMC, like that from macrophages, could also enhance LDL degradation by smooth muscle cells, fibroblasts, endothelial cells, and macrophages. Thus, the current study demonstrated that arterial wall cells secretory products can affect cellular lipoprotein receptor activities. This phenomenon could lead to increased cellular cholesterol accumulation and foam cell formation.

A macrophage receptor that recognizes oxidized low density lipoprotein but not acetylated low density lipoprotein

The formation of cholesterol-loaded macrophage foam cells in arterial tissue may occur by the uptake of modified lipoproteins via the scavenger receptor pathway. The macrophage scavenger receptor, also called the acetylated low density lipoprotein (Ac-LDL) receptor, has been reported to recognize Ac-LDL as well as oxidized LDL species such as endothelial cell-modified LDL (EC-LDL). We now report that there is another class of macrophage receptors that recognizes EC-LDL but not Ac-LDL. We performed assays of 0 degrees C binding and 37 degrees C degradation of 125I-Ac-LDL and 125I-EC-LDL by mouse peritoneal macrophages. Competition studies showed that unlabeled Ac-LDL could compete for only 25% of the binding and only 50% of the degradation of 125I-EC-LDL. Unlabeled EC-LDL, however, competed for greater than 90% of 125I-EC-LDL binding and degradation. Unlabeled Ac-LDL was greater than 90% effective against 125I-Ac-LDL; EC-LDL competed for about 80% of 125I-Ac-LDL binding and degradation. Copper-oxidized LDL behaved the same as EC-LDL in all the competition studies. Copper-mediated oxidation of Ac-LDL produced a superior competitor which could now displace 90% of 125I-EC-LDL binding. After 5 h at 37 degrees C in the presence of ligand, macrophages accumulated six times more cell-associated radioactivity from 125I-EC-LDL than from 125I-Ac-LDL, despite approximately equal amounts of degradation to trichloroacetic acid-soluble products, which may imply different intracellular processing of the two lipoproteins. Our results suggest that 1) there is more than one macrophage "scavenger receptor" for modified lipoproteins; and 2) oxidized LDL and Ac-LDL are not identical ligands with respect to macrophage recognition and uptake.

High density lipoprotein stimulates sterol translocation between intracellular and plasma membrane pools in human monocyte-derived macrophages.

Binding of high density lipoprotein (HDL) to its receptor on cultured fibroblasts and aortic endothelial cells was previously shown to facilitate sterol efflux by initiation of translocation of intracellular sterol to the plasma membrane. After cholesterol-loaded human monocyte-derived macrophages were incubated with either [3H]mevalonolactone or lipoprotein-associated [3H]cholesteryl ester to radiolabel intracellular pools of sterol, incubation with HDL3 led to stimulation of 3H-labeled sterol translocation from intracellular sites to the cell surface which preceeded maximum 3H-labeled sterol efflux. A similar pattern was demonstrated for macrophages that were preloaded with cholesterol derived from either low density lipoprotein (LDL), acetyl-LDL, or phospholipase C-modified LDL. However, in macrophages that were not loaded with cholesterol, HDL3 stimulated net movement of 3H-labeled sterol from the plasma membrane into intracellular compartments, the opposite direction from that seen for cholesterol-loaded cells. A similar influx pattern was found in nonloaded macrophages and fibroblasts that were labeled with trace amounts of exogenous [3H]cholesterol. Cholesterol translocation from intracellular pools to the cell surface of cholesterol-loaded macrophages appeared to be stimulated by receptor binding of HDL, since chemical modification of HDL with tetranitromethane (TNM), which abolishes its receptor binding, reduced its ability to stimulate 3H-labeled sterol translocation and efflux. In nonloaded cells, however, the ability of HDL3 to stimulate sterol efflux and movement of sterol from the plasma membrane into intracellular pools was unaffected by TNM modification. Thus, binding of HDL to its receptor on cholesterol-loaded macrophages appears to promote translocation of intracellular cholesterol to the plasma membrane followed by cholesterol efflux into the medium. However, in nonloaded macrophages, HDL stimulates sterol movement from the plasma membrane into intracellular pools by a receptor-independent process.

Platelet-enhanced apolipoprotein E production by human macrophages: a possible role in atherosclerosis

Cholesterol-loaded human monocyte-derived macrophages increase their production of apolipoprotein E (apoE). Although cholesterol loading is often achieved with modified plasma lipoproteins, macrophages can be loaded also by coculture with platelets. Therefore, the relationship between platelet-mediated cholesteryl ester accumulation and apoE secretion was examined. Macrophages were isolated by adherence and cultured for 6 days in serum-free medium. Secreted apoE was measured with a sensitive solid-phase radioimmunoassay. Maximum apoE secretion by the adherent macrophages from 5 x 10(6) peripheral blood mononuclear cells was obtained with 3 x 10(8) platelets and was ten-fold greater than control cells cultured in the absence of platelets. Platelet-mediated apoE secretion was consistently greater than that obtained by culture with either native or acetylated low density lipoproteins. Whereas the 1000 g supernatants of unstimulated platelets were poor inducers of apoE secretion, substances rich in cholesterol that were shed from thrombin-stimulated platelets and recovered in the 1000 g supernatants were almost as active as intact platelets. In all studies, platelet-induced secretion of apoE paralleled the capacity of platelets to induce macrophage cholesterol accumulation, indicating that macrophage apoE secretion was readily influenced by macrophage cholesterol metabolism.

Ca++ antagonists and ACAT inhibitors promote cholesterol efflux from macrophages by different mechanisms. II. Characterization of intracellular morphologic changes.

The effects of the slow Ca++ channel blocker, nifedipine, and ACAT inhibitor, octimibate, on cholesterol-loaded macrophages were compared at the ultrastructural level. The Ca++ antagonist induced the formation of membrane-surrounded "lamellar bodies" originating from lysosomes. The macrophages secreted these lamellar bodies, which were rich in phospholipids and cholesterol, into the culture medium even in the absence of cholesterol acceptors. In contrast, the ACAT inhibitor induced the formation of lamellar bodies originating from lipid droplets, which were also surrounded by membranes. There is strong evidence that these latter membranes were newly synthesized at the margin of the lipid droplets by the endoplasmic reticulum. The lamellar bodies descending from lipid droplets after ACAT inhibitor treatment were not secreted by the cells. They were stored in the cytoplasmic compartment in the absence of high density lipoproteins (HDL). When HDL were added to the medium, the lamellar bodies specifically interacted with endosomes containing the internalized HDL particles and disappeared concomitantly with an enhanced HDL-mediated cholesterol efflux. From the biochemical data in the accompanying article and the morphological data here, we conclude that macrophages release cholesterol by two major pathways: 1) an HDL-independent secretion of lamellar bodies containing cholesterol, which originate from lysosomes, and 2) an HDL-dependent release of cholesterol via the formation of lamellar bodies descending from lipid droplets, which intracellularly fuse with HDL-containing endosomes. The cholesterol-enriched HDL are then secreted as intact lipoproteins.

Ca++ antagonists and ACAT inhibitors promote cholesterol efflux from macrophages by different mechanisms. I. Characterization of cellular lipid metabolism.

The effects of the slow Ca++ channel blocker, nifedipine, and ACAT inhibitor, octimibate, on the cholesterol metabolism of cholesterol-loaded macrophages were compared. We demonstrated that apolipoprotein A-I containing high density lipoproteins (HDL) bind to specific receptor sites on macrophages, are internalized, take up cholesterol, and are then released from the cells as native lipoproteins. The ACAT inhibitor enhances HDL receptor activity and promotes HDL-mediated cholesterol efflux from cultured mouse peritoneal macrophages. In contrast, the Ca++ antagonist increases acetyl LDL-mediated cholesterol influx, abolishes the increase in HDL binding induced by cholesterol accumulation, enhances apo E synthesis, and promotes cholesterol efflux by a mechanism independent of the presence of HDL in the surrounding medium. Concomitantly, a decrease in nucleoside transporter activity, an increase in intracellular ATP hydrolysis, adenosine and cyclic AMP concentration, and a stimulation of the activities of acid and neutral cholesteryl ester hydrolase and ACAT indicated that protein kinase A-catalyzed phosphorylation reactions might be involved in the increase in cholesterol efflux. The Ca++ antagonist-induced efflux occurred only with lysosomal-associated cholesterol, while the ACAT inhibitor acted on the formation of cytoplasmic lipid droplets. The secreted lipoprotein particles contained 68% unesterified cholesterol and 21% phospholipids, 8% esterified cholesterol, and 3% triglycerides. The phospholipid components were: 72% phosphatidylcholine, 22% sphingomyelin, and 6% phosphatidylserine, phosphatidylinositol, and phosphatidylethanolamine. We conclude that macrophages release cholesterol in two ways: 1) an HDL-mediated release of unesterified cholesterol increasing upon ACAT inhibition, and 2) an HDL-independent secretion of cholesterol which can be amplified by Ca++ antagonists.

Obligatory role of cholesterol and apolipoprotein E in the formation of large cholesterol-enriched and receptor-active high density lipoproteins.

The formation of large cholesterol-enriched high density lipoproteins (HDL1/HDLc) from typical HDL3 requires lecithin:cholesterol acyltransferase activity, additional cholesterol, and a source of apolipoprotein (apo-) E. The present study explores the role of apo-E in promoting HDL1/HDLc formation and in imparting to these lipoprotein particles the ability to interact with the apo-B,E(low density lipoprotein (LDL] receptor. Incubation of normal canine serum with cholesterol-loaded mouse peritoneal macrophages resulted in the formation of HDL1/HDLc that competed with 125I-LDL for binding to the apo-B,E(LDL) receptors on cultured human fibroblasts. Cholesterol efflux from macrophages was necessary because incubation of normal canine serum with nonloaded macrophages did not cause HDL1/HDLc formation. However, cholesterol delivery to the serum was not sufficient to result in HDL1/HDLc formation. Apolipoprotein E had to be available. Incubation of apo-E-depleted canine serum with cholesterol-loaded J774 cells, a macrophage cell line that does not synthesize apo-E, demonstrated that no HDL1/HDLc formation was detected even in the presence of significant cholesterol efflux. However, addition of exogenous apo-E to the serum during the incubation with cholesterol-loaded J744 cells promoted the formation of large receptor-active HDL1/HDLc. The receptor binding activity of these particles produced in vitro correlated with the amount of apo-E incorporated into the HDL1/HDLc. Apolipoproteins A-I and C-III were ineffective in promoting HDL1/HDLc formation; thus, apo-E was unique in allowing HDL1/HDLc formation. These results demonstrate that when lecithin:cholesterol acyltransferase activity, cholesterol, and apo-E are present in serum, typical HDL can be transformed in vitro into large cholesterol-rich HDL1/HDLc that are capable of binding to lipoprotein receptors.

Receptor-mediated uptake of remnant lipoproteins by cholesterol-loaded human monocyte-macrophages.

Normal human monocyte-macrophages were cholesterol-loaded, and the rates of uptake and degradation of several lipoproteins were measured and compared to rates in control cells. Receptor activities for 125I-rabbit beta-very low density lipoproteins (beta-VLDL), 125I-human low density lipoprotein, and 125I-human chylomicrons were down-regulated in cholesterol-loaded cells; however, the rate of uptake and degradation of 125I-human chylomicron remnants was unchanged from control cells. Cholesterol-loaded alveolar macrophages from a Watanabe heritable hyperlipidemic rabbit, which lack low density lipoprotein receptors, showed receptor down-regulation for 125I-beta-VLDL but not for 125I-human chylomicron remnants. In addition to chylomicron remnants, apo-E-phospholipid complexes competed for 125I-chylomicron remnant uptake, but apo-A-I-phospholipid complexes did not. Chylomicrons competed for lipoprotein uptake in control cells but were not recognized under conditions of cholesterol loading. Chylomicron remnants and beta-VLDL were equally effective in competing for 125I-beta-VLDL and 125I-chylomicron remnant uptake in cholesterol-loaded macrophages. When normal human monocyte-macrophages were incubated in serum supplemented with chylomicron remnants, the cholesteryl ester content increased 4-fold over cells incubated in serum with low density lipoprotein added. We conclude: 1) specific lipoprotein receptor activity persists in cholesterol-loaded cells; 2) this receptor activity recognizes lipo-proteins (at least in part) by their apo-E content; and 3) cholesteryl ester accumulation can occur in monocyte-macrophages incubated with chylomicron remnants.

Formation of cholesterol- and apoprotein E-enriched high density lipoproteins in vitro.

The delivery of cholesterol to canine serum or plasma altered the distribution of cholesterol and apoproteins in subclasses of high density lipoproteins (HDL). In these experiments, two in vitro systems were employed. The first system used cholesterol-celite particles to deliver cholesterol to canine plasma during 4-h incubations. When the cholesterol distribution in the lipoproteins was analyzed by Geon-Pevikon electrophoresis, an increase in cholesterol content was found in the slower migrating subclasses of HDL (HDL1 and HDLc). A large increase in apoprotein E (apo-E) was also observed in the lipoproteins. Densitometric analysis of lipid-stained, 4 to 30% gradient acrylamide gels of canine plasma after incubation with cholesterol-celite revealed that the concentration of the major high density lipoproteins (HDL3) decreased, and the concentration of subclasses of HDL-with apo-E (HDL1 and HDLc) increased 2- to 5-fold. In the second system, cholesterol-loaded mouse peritoneal macrophages released cholesterol to HDL in an incubation medium containing 10 to 20% canine serum. The HDL1 and HDLc, which demonstrated slower electrophoretic mobility as determined by Geon-Pevikon block electrophoresis, became enriched in cholesterol and cholesteryl esters. Gradient gel electrophoresis showed substantial increases in these subclasses of HDL-with apo-E. The cholesterol-loaded mouse peritoneal macrophages synthesized and secreted apo-E into the medium. When L-[35S]methionine was used as a precursor, 65 to 90% of the 35S-labeled protein associated with the lipoproteins in the 1.02 to 1.09 density range was immunoprecipitated with antibody directed against rat apo-E. Gradient gel electrophoresis of density fractions demonstrated the presence of HDL1 and HDLc as the major lipoproteins. In addition, when canine 125I-HDL3 (primarily apo-A-I-containing HDL) were added to canine serum and incubated with cholesterol-loaded macrophages, the appearance of HDL1 and HDLc was associated with a marked increase in the 125I label in these newly formed, cholesteryl ester-rich lipoproteins. There was a corresponding marked reduction in the 125I-HDL3 in the serum. Similar results were observed using human HDL3 and human serum.

Independent pathways for secretion of cholesterol and apolipoprotein E by macrophages.

Cholesterol-loaded macrophages secrete cholesterol and apolipoprotein E. The current studies show that this secretion occurs by two independent pathways. In the absence of serum, the cells secrete apolipoprotein E, but not cholesterol. In the presence of monensin (an inhibitor of protein secretion), the cells secrete cholesterol, but little apolipoprotein E. After secretion, apolipoprotein E and cholesterol associate with high-density lipoprotein to form a particle that can deliver cholesterol to the liver by receptor-mediated endocytosis. We conclude that apolipoprotein E does not function to remove cholesterol from macrophages but rather to participate in "reverse cholesterol transport."

Mouse macrophages synthesize and secrete a protein resembling apolipoprotein E.

Monolayers of mouse peritoneal macrophages were shown to synthesize and secrete a protein that resembles apoprotein E (apoE), a normal constituent of plasma lipoproteins. Synthesis and secretion were studied by incubation of macrophages with L-[35S]methionine and analysis of the 35S-labeled proteins secreted into the culture medium. The 35S-labeled protein resembling apoE showed the following properties: (i) it floated in the ultracentrifuge at a density less than 1.215 g/ml, indicating that it was associated with lipid; (ii) by NaDodSO4/polyacrylamide gel electrophoresis, its Mr of 35,000 was identical to that of authentic apoE obtained from mouse plasma very low density lipoprotein; (iii) its isoelectric point of 5.4 was the same as that of authentic mouse apoE; (iv) it comigrated with authentic mouse apoE after two-dimensional isoelectric focusing/NaDodSO4/polyacrylamide gel electrophoresis; and (v) it was quantitatively precipitated by a monospecific antibody directed against rat apoE. Synthesis and secretion of the apoE-like protein was stimulated 3- to 8-fold when the macrophages were loaded with cholesterol by incubation with either acetylated low density lipoprotein (acetyl-LDL) or beta-migrating very low density lipoprotein from cholesterol-fed rabbits. When the cells were incubated with acetyl-LDL, the apoE-like protein composed approximately 2% of the total 35S-labeled protein synthesized by the cells and approximately 10% of the total 35S-labeled protein secreted into the medium. The current findings suggest a role for apoE in the plasma transport of cholesterol excreted from cholesterol-loaded macrophages.