Apolipoprotein E produced by human monocyte-derived macrophages mediates cholesterol efflux that occurs in the absence of added cholesterol acceptors.

Human monocyte-derived macrophages can efflux accumulated cholesterol without exogenously added cholesterol acceptors (Kruth, H. S., Skarlatos, S. I., Gaynor, P. M., and Gamble, W. (1994) J. Biol. Chem. 269, 24511-24518). Most of the effluxed cholesterol accumulates in the medium as apolipoprotein E-discoidal lipid particles. In the current study, we determined whether and to what degree cholesterol efflux from human monocyte-macrophages depended on apolipoprotein E secretion. Unexpectedly, 2-week-old differentiated monocyte-macrophages secreted similar amounts of apolipoprotein E without or with cholesterol enrichment. Apolipoprotein E mRNA levels in these macrophages were not increased by cholesterol enrichment and were comparable with levels in HepG2 cells. Without cholesterol enrichment, monocyte-macrophages secreted lipid-poor apolipoprotein E with a density >1.21 g/ml. By contrast, cholesterol enrichment of monocyte-macrophages induced the association of apoE with phospholipid and cholesterol to form discoidal particles that floated at densities of 1.08-1.10 g/ml. An anti-apolipoprotein E monoclonal antibody added to the culture medium significantly inhibited cholesterol and phospholipid efflux from the monocyte-macrophages. This showed that apolipoprotein E was required for most of the cholesterol efflux, and that apolipoprotein E did not leave macrophages with lipid but rather associated with lipid after it was secreted. Thus, 1) apolipoprotein E was constitutively secreted by differentiated human monocyte-macrophages, 2) apolipoprotein E only formed discoidal particles following macrophage cholesterol enrichment, 3) apolipoprotein E was necessary for cholesterol efflux to occur in the absence of added cholesterol acceptors and, in addition 4) the level of macrophage unesterified cholesterol was not rate-limiting for this cholesterol efflux, and 5) net phospholipid synthesis occurred in macrophages secondary to apoE-mediated loss of macrophage phospholipid. In conclusion, apolipoprotein E functions in an autocrine pathway that mediates cholesterol efflux from human monocyte-derived macrophages.

facilitating clearance of atherogenic remnant lipoproteins from the plasma (1,2). Recent studies suggest that apoE may be anti-atherogenic by additional mechanisms. Transgenic expression of apoE restricted to the blood vessel wall (3) or to macrophages (including those in the vessel wall) (4) inhibits development of atherosclerotic lesions in mice, even without altering plasma cholesterol levels. This suggests that expression of apoE locally within the vessel wall is sufficient to produce an anti-atherogenic effect.
One mechanism by which locally produced apoE could be anti-atherogenic is by promoting reverse cholesterol transport from the vessel wall. This could occur through the association of apoE with plasma-derived high density lipoprotein (HDL) which increases HDL's capacity to carry cholesterol (5,6). On the other hand, it is possible that apoE could affect reverse cholesterol transport independent of plasma-derived HDL.
We have observed that differentiated human monocyte-macrophages efflux accumulated cholesterol even when these macrophages are incubated in basal medium without any added serum components or HDL that could function as cholesterol acceptors (7). Most of this effluxed cholesterol accumulates in the medium within apoE-discoidal lipoprotein particles. However, it has not been determined whether and to what degree cholesterol efflux depends on apoE secretion by human monocyte-macrophages. Would this macrophage self-generated cholesterol efflux continue normally if apoE function was impaired?
The cholesterol efflux that occurs from human monocytemacrophages in the absence of serum or added lipoproteins could be independent of apoE. Some cholesterol effluxed by human monocyte-macrophages is contained within vesicular lipoproteins. These vesicular lipoproteins have a 22,000-Da protein that is not related to apoE (7). In addition, under certain conditions, release of multilamellar lipid particles can be a pathway for cholesterol excretion from macrophages (7)(8)(9). Because of the presence of other possible pathways for macrophage cholesterol excretion, it is not clear to what extent apoE secretion is necessary for cholesterol excretion from human monocyte-macrophages. Also, the relationship between apoE secretion and discoidal particle production in human monocytemacrophages has not been examined. Thus, the purpose of this study was 1) to determine the importance of apoE in mediating cholesterol efflux from these cells, and 2) to determine the relationship between apoE secretion and production of apoEdiscoidal particles.

MATERIALS AND METHODS
Incubation of Monocyte-derived Macrophages with AcLDL and Microcrystalline Cholesterol-Human monocyte-derived macrophages * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
were cultured and incubated with human acetylated low density lipoprotein (AcLDL) or microcrystalline cholesterol, and analyzed for lipids as described previously (7).
Density Gradient Analysis of ApoE in Culture Medium-Following incubations, a total of 12 ml of culture medium was collected from a six-well cluster dish for each condition and centrifuged at 1000 ϫ g for 25 min. This and subsequent procedures were carried out at 4°C. Centrifugation removed any floating cells and some of the microcrystalline cholesterol that remained after incubations. The supernatant was then passed through low protein-binding polysulfone 0.45-m (pore size) filters (Gelman Sciences, Ann Arbor, MI) to remove any remaining cells or microcrystalline cholesterol. The filtrate was concentrated to 4 ml in an Amicon (Danvers, MA) stirred cell with a 10,000 molecular weight cut-off cellulosic filter. Then, the concentrated filtrates were dialyzed 36 h against two changes of 4 liters of 0.15 M NaCl solution containing 0.02% sodium azide and 0.1% disodium-EDTA (pH 7.4). Recovery of cholesterol and apoE following concentration and dialysis was Ͼ80%.
Isopycnic density gradient centrifugation of the filtered and concentrated culture medium was carried out as described previously (7).
Quantification of ApoE-ApoE in culture media and gradient fractions was quantified using a sandwich-type ELISA. Wells of microtiter plates (96-well Immulon, Dynatech, Chantilly, VA) were coated overnight at 4°C with 1 g/ml anti-apoE monoclonal capture antibody in 100 l of 0.2 M carbonate-bicarbonate coating buffer, pH 9.4 (Pierce). The monoclonal capture antibody was the same as that used for immunoblotting. After removal of the coating buffer, wells were incubated for 2 h at room temperature with a blocking buffer (phosphate-buffered saline containing 3% bovine serum albumin, pH 7.4) and then rinsed four times with a wash buffer (phosphate-buffered saline containing 0.05% Tween 20). One hundred l of samples (diluted 1:20 in blocking buffer) or apoE standards (also in blocking buffer) was added to wells and incubated 1 h at 37°C. Then, wells were rinsed four times with wash buffer, and incubated at 37°C for 1 h with 100 l of polyclonal goat anti-human apoE antibody diluted in blocking buffer (3.3 g of IgG/ml, Biogenesis, Sandown, NH). Next, wells were rinsed another four times with wash buffer and incubated at 37°C for 1 h with 100 l of affinity-purified polyclonal rabbit anti-goat IgG-peroxidase conjugate (30 units/ml, Sigma) diluted 1:10,000 with blocking buffer. Wells were rinsed six times with wash buffer and then incubated for 15 min at room temperature with 100 l of 0.2 g/liter TMB (3,3Ј,5,5Ј-tetramethylbenzidine) peroxidase substrate system (Kirkegaard and Perry, Gaithersburg, MD). Finally, 100 l of 2 M sulfonic acid was added to each well, and the absorbance at 450 nm was determined.
Northern Blot Analysis of ApoE mRNA-Total RNA was isolated from cell cultures with TRIzol reagent (Life Technologies, Inc.) according to the manufacturer's instructions. The RNA was treated with RNase-free DNase (Promega, Madison, WI). Samples containing 15 g of total RNA were precipitated with ethanol, dried, and resuspended in 20 l of buffer containing 50% formamide, 6% formaldehyde, 0.05% bromphenol blue, 1 mM EDTA, and 50 mM MOPS (pH 7.0). The RNA samples were heated at 65°C for 15 min and immediately cooled on ice. Next, the RNA samples were electrophoresed in a 1.5% formaldehyde agarose gel run at 4 volts/cm for 5 h. The separated RNAs were transferred overnight to a positively charged nylon membrane by capillary elution and cross-linked with UV light (1200 microjoules) to the membrane.
A full-length apoE cDNA clone was used as a template to prepare sense and antisense apoE riboprobes labeled by in vitro transcription with digoxigenin-11-UTP using SP6 and T7 RNA polymerases (Boehringer Mannheim). A riboprobe to detect glyceraldehyde-3-phosphate dehydrogenase mRNA was prepared from a 1.0-kilobase pair cDNA fragment of glyceraldehyde-3-phosphate dehydrogenase attached to a T7 RNA polymerase promoter (Clontech, Palo Alto, CA). The RNA blot was prehybridized for 3 h at 68°C in 20 ml of prehybridization solution (formulated as specified in the Genius System User's Guide for filter hybridization, Boehringer Mannheim). The blot was hybridized overnight in the prehybridization solution (68°C) containing probes at 30 ng/ml. Then, chemiluminescent detection of the RNA blot was carried out according to the user's guide provided by Boehringer Mannheim.

Monocyte-derived Macrophages Constitutively Secreted ApoE-Previous investigation has
shown that cholesterol accumulation by macrophages can induce synthesis and secretion of apoE (12)(13)(14). However, human monocyte-macrophages (2-week-old cultures) incubated with AcLDL (100 g/ml) for 3 days showed no significant difference in their secretion of apoE compared with monocyte-macrophages that were not incubated with AcLDL (Table I). Monocyte-macrophages incubated with microcrystalline cholesterol (130 nmol/ml) secreted only slightly more apoE (a 15% increase) into the culture medium over a 3-day incubation period than did control monocytemacrophages. ApoE secretion following enrichment of monocyte-macrophages with cholesterol was also examined (as opposed to apoE secretion during cholesterol enrichment). Monocyte-macrophages incubated with AcLDL (25 g/ml) for 2 days showed equivalent amounts of apoE secretion during a 4-day postincubation period in RPMI 1640 medium (Table II). During this period of postincubation, cholesterol-enriched macrophages excreted over 3-fold more cholesterol than control macrophages excreted.
It was previously reported that an increase in apoE secretion induced in mouse macrophages by cholesterol enrichment was mediated in part by an increase in apoE mRNA (13,14). Therefore, we examined whether cholesterol enrichment may have substantially increased apoE mRNA without producing any large increase in apoE secretion. Cholesterol enrichment of monocyte-macrophages did not increase apoE mRNA levels compared with those levels in control monocyte-macrophages ( Fig. 1). Furthermore, the apparent level of apoE mRNA in monocyte-macrophages was as high as the level detected in HepG2 cells. Thus, apoE mRNA and apoE secretion were almost maximally expressed in differentiated human monocytemacrophages. The difference between these results and those of previous studies (12)(13)(14) is likely due to the extent of differentiation of the monocyte-macrophages (differentiation in-  15) in our studies and our use of culture serum selected for low levels of endotoxin (endotoxin inhibits apoE secretion; Ref. 16).
Cholesterol and Phospholipid Secretion from Macrophages Was Linked to Secretion of ApoE-Our previous study (7) shows that cholesterol-enriched human monocyte-macrophages secrete cholesterol and phospholipid and lower their cholesterol content without the addition of an exogenous cholesterol acceptor such as HDL. About two-thirds of the effluxed cholesterol and phospholipid accumulated in the medium as part of apoE-containing discoidal particles, while about onethird accumulated in the medium as part of vesicles that lacked apoE. However, in those studies it was not determined whether apoE was necessary for cholesterol efflux to occur. To examine whether apoE secreted by monocyte-macrophages functioned to promote cholesterol efflux from the macrophages, lipid efflux was examined in the presence of an anti-apoE monoclonal antibody.
The anti-apoE monoclonal antibody significantly inhibited cholesterol efflux from monocyte-macrophages (Table III). Monocyte-macrophages incubated with a control mouse monoclonal antibody excreted 63 nmol of cholesterol/mg of cell protein into the medium and showed a similar decrease (57 nmol of cholesterol/mg) in their cholesterol content. In contrast, monocyte-macrophages incubated with the anti-apoE monoclonal antibody excreted about one-third the cholesterol that control monocyte-macrophages excreted, and showed less decrease in their cholesterol content.
Similar to the effect on cholesterol excretion, monocyte-macrophages incubated with the anti-apoE monoclonal antibody excreted about one-third the phospholipid that control monocyte-macrophages excreted. Although control monocyte-macrophages excreted 23 nmol of phospholipid/mg of cell protein, they showed no decrease in their phospholipid content. This could have only occurred through synthesis by the macrophages of the same amount of phospholipid lost from the cells. Incubation of monocyte-macrophages with the control antibody did not affect cholesterol and phospholipid levels in the cells and medium.
Exogenous ApoE Further Stimulated Monocyte-derived Macrophage Cholesterol Excretion-Next, we determined whether the amount of apoE produced by monocyte-macrophages was rate-limiting for cholesterol efflux from these cells. Monocytemacrophage cultures were first enriched with cholesterol by a 2-day incubation with AcLDL. Then, cholesterol content during a 3-day period of efflux into basal medium was monitored in the absence and presence of 10 g/ml recombinant human apoE. This amount of apoE was more than double the concentration of apoE that we had observed in the medium of macrophages following 3 days of efflux. During the 3 days of efflux, macrophages decreased their cholesterol content by 47 nmol of cholesterol/mg of cell protein compared with a decrease of 75 nmol of cholesterol/mg of cell protein, in the presence of added apoE (Table IV). Thus, how much apoE that the macrophages secreted into the medium was rate-limiting with respect to decreasing macrophage cholesterol content. Additional exogenous apoE decreased the cholesterol content of macrophages beyond the decrease in cholesterol content that occurred with the level of apoE secreted by macrophages.
Macrophage Unesterified Cholesterol Content Was Rate-limiting for Cholesterol Efflux Only in the Presence of Additional ApoE-It was next determined whether monocyte-macrophage  1. Comparison of apoE mRNA content of normal and cholesterol-enriched human monocyte-macrophages. Two-week-old monocyte-macrophage cultures were incubated for 3 days in 2 ml of RPMI 1640 medium either without or with 130 nmol/ml of microcrystalline cholesterol as indicated. HepG2 cells were incubated for 6 days with 2 ml of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Fifteen g of total RNA for each sample was electrophoresed, blotted onto a nylon membrane, hybridized to digoxigeninlabeled antisense and sense riboprobes (apoE or glyceraldehyde-3-phosphate dehydrogenase (G3PDH)), and detected by chemiluminescence. Shown are the reactions of the antisense riboprobes. No reaction was observed with the control sense probes (data not shown).

TABLE III Effect of an apoE monoclonal antibody on cholesterol efflux from
monocyte-macrophages Two-week-old monocyte-macrophage cultures were incubated for 2 days in 2 ml of RPMI 1640 medium with 25 g/ml AcLDL. Then, cultures were rinsed and incubated for 4 days in 2 ml of RPMI 1640 medium containing 0.1% bovine serum albumin and 50 g/ml of either an anti-apo E mouse monoclonal antibody (clone 21-F3-D2, Biogenesis, Sandown, NH) (17) or an irrelevant (MOPC 21, Cappel, Durham, NC) mouse monoclonal antibody. Both monoclonal antibodies were IgG fractions purified from ascites fluid. Following incubations, the cholesterol and phospholipid contents of cells and media were determined. Shown are the averages Ϯ S.E. of triplicate cultures.  (19). Incubation of macrophages during the efflux period with an ACAT inhibitor decreased cellular cholesteryl esters and increased cellular unesterified cholesterol. This was because cholesteryl esters hydrolyzed during this period could not undergo re-esterification and, thus, accumulated in the cells as unesterified cholesterol (Table V, Experiment 1). The decrease in cholesteryl ester content of the monocyte-macrophages compared with controls was balanced by an equivalent increase in unesterified cholesterol content. Although more unesterified cholesterol was available for potential efflux in the monocyte-macrophages treated with ACAT inhibitor, the total cholesterol content of these macrophages did not decrease more than the total cholesterol content of control macrophages incubated without the ACAT inhibitor (Table V, Experiment 1). This showed that the macrophage level of unesterified cholesterol was not rate-limiting for cholesterol efflux from the macrophages.
However, a second experiment showed that this was because secretion of apoE by the macrophages treated with ACAT was not sufficient to remove the accumulating unesterified cholesterol (Table V, Experiment 2). Although much less cholesterol accumulated in monocyte-macrophages incubated with AcLDL in experiment 2, nevertheless, ACAT inhibition still failed to enhance cholesterol efflux from the macrophages. However, in the presence of added exogenous apoE, ACAT inhibition promoted a greater decrease in the cholesterol content of the macrophages than occurred without the ACAT inhibitor.
Cholesterol Enrichment of Monocyte-macrophages Induced Lipid Association of ApoE-We initially thought that cholesterol efflux from human monocyte-macrophages would be regulated by cholesterol inducing increased synthesis of apoE. Increased apoE synthesis could then cause increased production of apoE-phospholipid discs that could transport cholesterol from the macrophages. However, as shown above, apoE was constitutively secreted by 2-week-old human monocyte-macrophages, but monocyte-macrophages produce apoE-containing discoidal particles only following cholesterol enrichment (7). It was not previously determined (7) how much apoE secreted from monocyte-macrophages was lipid-associated in the absence and presence of cholesterol-enrichment of these cells. Only about 20% of apoE secreted from transfected mouse mam-mary-derived cells, and about 10% of apoE secreted from transfected Chinese hamster ovary cells is lipid-associated (20,21). On the other hand, most apoE secreted from transfected L cells, as well as apoE secreted from cholesterol-enriched mouse peritoneal macrophages, is lipid-associated (22,23). Therefore, we examined whether cholesterol enrichment of human monocyte-derived macrophages influenced the association of macrophage secreted apoE with lipid. The density was determined for apoE in medium from monocyte-macrophages incubated for 3 days either in serum-free RPMI 1640 medium or this medium supplemented with 130 nmol/ml microcrystalline cholesterol. Density gradient centrifugation showed that apoE remained at d Ͼ 1.21 g/ml (i.e. remained lipid-poor) when monocyte-macrophages were not enriched with cholesterol (Fig. 2a). By contrast, about half the apoE from medium of cholesterol-enriched monocyte-macrophages floated to d 1.08 -1.10 g/ml (Fig. 2b). We showed previously that apoE in this fraction is associated with discoidal lipid particles (7). Thus, although monocyte-macrophages constitutively produce apoE, the apoE becomes lipid-associated forming discoidal particles only after monocyte-macrophages become cholesterol-enriched.
Western blot analysis of apoE was carried out to assess whether any change in apoE molecular weight could have been associated with induction of discoidal particle formation. Lower and higher molecular weight forms of apoE were observed. Varying molecular weight forms occur due to different degrees of glycosylation of apoE (24). The higher molecular weight form of apoE accumulated in medium of macrophages incubated without or with cholesterol (Fig. 3, c and d). Cellular apoE was composed of predominantly the lower molecular weight apoE with a small amount of higher molecular weight apoE (Fig. 3, a  and b). The patterns of cellular apoE were similar for macrophages incubated without or with cholesterol. Thus, the degree of glycosylation of apoE did not appear to be a factor in cholesterol-induced association of apoE with lipid. Also, Western blot analysis showed equal amounts of immunodetectable apoE in equal portions of media (and cells) from macrophages incubated without or with cholesterol. This finding confirmed the quantitative ELISA determinations of relatively similar levels of apoE in media from macrophages incubated without or with cholesterol presented above. DISCUSSION The goal of this investigation was to learn about the function of apoE in mediating monocyte-derived macrophage cholesterol efflux that occurs in the absence of added cholesterol acceptors such as HDL or apolipoproteins. We have found that 1) apoE produced by monocyte-macrophages mediates most of the cholesterol efflux from the macrophages; 2) cholesterol regulated the association of apoE with phospholipid; 3) apoE associated with lipid after apoE was secreted from macrophages (not during secretion); 4) the level of macrophage unesterified cholesterol was not rate-limiting for cholesterol efflux, but the level of secreted apoE was rate-limiting; and 5) net phospholipid synthesis occurred in macrophages secondary to apoEmediated loss of macrophage phospholipid (net phospholipid synthesis was not due to cholesterol enrichment).
Previously, it was shown that apoE secretion from mouse peritoneal macrophages does not decrease the cholesterol content of these cells, although apoE-containing discoidal particles could be produced by these macrophages (25,26). Hara and Yokoyama (27) concluded that this was because mouse peritoneal macrophages do not secrete sufficient levels of apoE to stimulate significant cholesterol efflux. They showed that cholesterol efflux does occur from mouse peritoneal macrophages when sufficient exogenous apoE is added to the culture media. Expression of the human apoE gene in J774 macrophages (a   TABLE IV  Effect of exogenous apoE on monocyte-macrophage cholesterol content during efflux Two-week-old human monocyte-macrophage cultures were incubated for 2 days in RPMI 1640 medium with 100 g/ml AcLDL. Then cultures were rinsed and allowed to efflux into RPMI 1640 medium containing 0.1% bovine serum albumin without or with added recombinant human apoE (10 g/ml) (18). Following incubations, the cholesterol and protein contents of cells were determined. mouse macrophage cell line that does not express its endogenous apoE gene) stimulated efflux of radiolabeled cholesterol in the presence of cAMP or an ACAT inhibitor (28). However, the level of efflux was not sufficient to decrease the cholesterol content of these cells. In contrast to these earlier studies with other types of macrophages, we found that apoE secreted from human monocyte-macrophages did mediate cholesterol efflux from these cells and also decreased their cholesterol content. This was shown from results in which an anti-apoE antibody decreased (about two-thirds) the efflux of cholesterol from monocyte-macrophages. In our earlier work (7), we showed that a minor portion of cholesterol released from monocyte-macrophages was present in vesicles that contained a 22,000-Da protein unrelated to apoE. It is possible that residual cholesterol efflux that was not blocked by the anti-apoE antibody occurred in these vesicles. Previous studies have shown that cholesterol accumulation by macrophages can induce the synthesis and secretion of apoE (12)(13)(14). Therefore, we thought that cholesterol enrichment of the monocyte-macrophages would induce secretion of apoE that would then be available to form apoE-lipid discoidal particles. However, this turned out not to be the case. The monocytemacrophages in our study constitutively secreted apoE essentially unaffected by cholesterol accumulation in the macrophages. Although apoE was constitutively secreted, it became lipid-rich and floated to density 1.08 -1.10 g/ml only when macrophages had accumulated cholesterol. Thus, cholesterol did not stimulate formation of apoE-discoidal particles by stimulating apoE secretion; instead, cholesterol induced the complexing of apoE with phospholipid and cholesterol. This finding is consistent with earlier proposals that cholesterol alters the physical chemical properties of the plasma membrane in a way  2. Density gradient analysis of apoE from media of normal and cholesterol-enriched monocyte-macrophages. Twoweek-old monocyte-macrophage cultures were incubated for 3 days in 2 ml of RPMI 1640 medium either without (a) or with 130 nmol/ml microcrystalline cholesterol (b). Then, media were concentrated and subjected to isopycnic density gradient centrifugation. ApoE was determined in gradient fractions by ELISA. ApoE floated to Хd 1.08 -1.10 g/ml only when macrophages were incubated with cholesterol. The arrows indicate higher and lower molecular weight isoforms of apoE. Cells contained predominantly a lower molecular weight isoform of apoE with a small amount of higher molecular weight isoform. Media contained only the higher molecular weight isoform of apoE. that favors interaction of amphipathic apolipoproteins with phospholipid (29).
The fact that anti-apoE antibody added to the medium blocked cholesterol and phospholipid efflux is consistent with the idea that apoE did not leave the macrophage with lipid (otherwise, the antibody should not have blocked lipid efflux). The finding suggests that apoE picked up lipid after the apoE was released from the macrophage (30). This was considered an unlikely possibility for apoE-transfected L cells because, when incubated with the transfected L cells, exogenously added apoE did not become associated with lipid (21).
Net phospholipid synthesis occurred during cholesterol efflux from cholesterol-enriched human monocyte-macrophages. This was shown by the fact that the phospholipid content of monocyte-macrophages did not decrease, while phospholipid accumulated in culture medium during efflux. Cholesterol stimulation of net phospholipid synthesis in mouse macrophages has been observed (31,32). However, cholesterol could not have directly stimulated net phospholipid synthesis in the human monocyte-macrophages as appears to be the case in mouse macrophages. This was because the content of macrophage phospholipid did not increase in the presence of the anti-apoE monoclonal antibody, although this antibody inhibited phospholipid excretion. Thus, removal of phospholipid from the macrophage (rather than cholesterol enrichment of the macrophage) must have stimulated net phospholipid synthesis. If cholesterol had directly stimulated phospholipid synthesis, then in the presence of the anti-apoE monoclonal antibody, macrophage phospholipid content should have increased an amount equivalent to the decrease in phospholipid excretion (16 nmol of phospholipid/mg of cell protein, calculated from data in Table III). However, this did not occur.
The level of macrophage unesterified cholesterol was not rate-limiting for cholesterol efflux that occurred in basal medium without serum or added lipoproteins. The lack of effect of ACAT described for this in vitro situation may not apply to the in vivo situation within the blood vessel wall. ApoE concentrations could be higher in the tissue spaces of the blood vessel wall compared with the apoE concentrations achieved in cell culture where a monolayer of monocyte-macrophages secretes apoE into a large volume of culture fluid relative to the number of cells. At higher apoE concentrations produced by adding exogenous apoE to the culture medium, the level of macrophage unesterified cholesterol was rate-limiting for cholesterol efflux. With added apoE, inhibition of ACAT did stimulate a greater decrease in the cholesterol content of monocyte-macrophages compared with the decrease that occurred without added apoE. This was similar to what has been reported previously for plasma HDL-mediated cholesterol efflux from macrophages (33). We attempted to decrease the volume of culture medium to determine the effect on apoE secretion and cholesterol efflux. However, decreasing the volume of culture medium resulted in detachment of monocyte-macrophages from the culture dish.
The accessibility of all regions of atherosclerotic lesions to sufficient levels of plasma-derived HDL could be a factor that limits reverse cholesterol transport from lesions. Given that apoE secretion by human monocyte-macrophages can mediate cholesterol efflux from these cells in vitro, apoE secretion by these cells in human atherosclerotic lesions has the potential to mediate cholesterol efflux in lesions. The significance of cho-lesterol efflux from macrophages mediated by production of its own nascent HDL is that this efflux would not depend on the availability of plasma HDL. In conclusion, apoE has antiatherogenic potential not only by accelerating removal of atherogenic lipoproteins from the blood, but also by removing cholesterol from monocyte-macrophages through an autocrine mechanism of cellular cholesterol efflux.