Sterol 27-Hydroxylase– and ApoAI/Phospholipid–Mediated Efflux of Cholesterol From Cholesterol-Laden Macrophages

Evidence for an Inverse Relation Between the Two Mechanisms

From King Gustaf V Research Institute, Karolinska Hospital, Stockholm (J.W., B.K., J.N.); and Department of Medical Laboratory Science and Technology, Division of Clinical Chemistry, Karolinska Institute, Huddinge (I.B., U.D.), Sweden.

Correspondence to Jan Westman, King Gustaf V Research Institute, Karolinska Hospital, S-171 76 Stockholm, Sweden.

	Abstract

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Abstract—Cholesterol-laden, human monocyte–derived macrophages were found to contain 27-hydroxycholesterol in proportion to their content of cholesterol ester. In accordance with previous work with human lung alveolar macrophages, there was a significant efflux of 27-hydroxycholesterol and 3ß-hydroxy-5-cholestenoic acid from the cultured cells. The efflux of 27-hydroxycholesterol was proportional to the cellular content of this steroid. Incubation of cholesterol-laden macrophages with reconstituted discoidal complexes made from apolipoprotein A-I and phospholipids resulted in a decrease in total cellular cholesterol, an increase in the efflux of free cholesterol, and a concomitant decrease in the total production and efflux of 27-oxygenated steroids, in particular, 3ß-hydroxy-5-cholestenoic acid. Reconstituted discoidal complexes with the Milano variant of apolipoprotein A-I gave virtually identical results, whereas high density lipoprotein was less efficient. These results suggest that cultured cholesterol-laden cells can export some of their excess cholesterol in the form of 27-hydroxycholesterol, 3ß-hydroxy-5-cholestenoic acid, and free cholesterol. In the presence of exogenous cholesterol acceptors, export of free cholesterol becomes more effective, resulting in less cholesterol exported via the 27-hydroxylase pathway. The balance between the two mechanisms for removal of cholesterol from macrophages may be of importance for formation of foam cells and development of atherosclerosis.

Key Words: foam cell macrophages • 27-hydroxylase • 27-oxygenated sterols • HDL • cholesterol acceptor

	Introduction

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The most important mechanism for elimination of intracellular cholesterol involves the lipoprotein HDL. Recently, an additional mechanism has been described in human alveolar macrophages utilizing the enzyme 27-hydroxylase^*.¹ Cholesterol is 27-hydroxylated to yield 27-hydroxycholesterol or further hydroxylated by the same enzyme² to give the more polar product 3ß-hydroxy-5-cholestenoic acid. The latter steroids are easily secreted from the cells.^{1 3}

Sterol 27-hydroxylase is widely distributed in different tissues, such as the liver, brain, and kidney.^{5 6 7} In the liver this enzyme has an important function in the biosynthesis of bile acids.^{2 4} In extrahepatic tissues the role of sterol 27-hydroxylase may be to convert cholesterol into 27-hydroxycholesterol and 3ß-hydroxy-5-cholestenoic acid. The latter compounds are efficiently transported to the liver and converted into bile acids. We have recently shown that as much as 4% of the total formation of bile acids in humans may occur by this mechanism.³ In this context it is of interest that a genetic deficiency of sterol 27-hydroxylase is characterized by reduced synthesis of bile acids, in particular, chenodeoxycholic acid, accumulation of cholestanol, and development of xanthomas.⁸ In addition, these subjects develop premature atherosclerosis despite normal circulating levels of cholesterol.

The relative importance of the sterol 27-hydroxylase–mediated mechanism and the HDL-mediated mechanism for cholesterol removal is not known. When exposed to sufficient amounts of HDL, the HDL mechanism is considerably more important than the sterol 27-hydroxylase mechanism in cultured human alveolar macrophages.³ The exposure of tissue cells to HDL may, however, differ, and the relative importance of the sterol 27-hydroxylase mechanism may thus differ depending on the type and localization of the cell.

In the present work we measured the cellular content of 27-oxygenated sterols in cholesterol-laden human macrophages and studied the importance of such oxygenated products for efflux of cholesterol under different conditions. The efflux was studied both in the absence and presence of reconstituted HDL particles made from phospholipids and native apoAI or recombinant apoAI-M, a mutant form of apoAI in which one amino acid is substituted for cysteine.⁹ The present results suggest that in the presence of very effective exogenous cholesterol acceptors, export of free cholesterol becomes more effective, resulting in less cholesterol being exported via the 27-hydroxylase pathway. Thus, there seems to be an inverse relation between the two mechanisms under some experimental conditions.

	Methods

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Materials
Tissue culture media and supplements were obtained from GIBCO BRL (Life Technologies Ltd). Human apoAI was obtained from Organon Teknika Corp and recombinant apoAI-M from Pharmacia. Ficol-Paque was a product of Pharmacia Biotech Norden AB. Enzymes for cholesterol analyses were obtained from Boehringer Mannheim and other chemicals from Sigma Chemical Co. Phospholipids were analyzed using a kit from Wako Chemicals GmbH.

Cells and Cell Culture
Buffy coats from healthy donors were obtained from the blood bank. Mononuclear cells were isolated from buffy coats according to the method of Boyum¹⁰ by using dextran and Ficol-Paque sedimentation. The mononuclear cells obtained were resuspended in RPMI 1640 with 15% heat-inactivated human serum from healthy donors, seeded into tissue culture dishes, and incubated in a humidified atmosphere with 5% CO₂ at 37°C. After 2 hours, the dishes were rinsed with warm PBS, and fresh medium with 15% human serum was added. The next day, the dishes were again rinsed with PBS and fresh medium with 15% human serum was added. Thereafter the medium was changed every third day. When serum-free medium was used in the experiments, RPMI 1640 was always supplemented with 10 mg/mL BSA, 5 µg/mL insulin, and 5 µg/mL transferrin (unless otherwise stated).

Cell Viability
Cell viability was checked by trypan blue exclusion and was usually very close to 100% and never below 95% at the end of the experiments.

AcLDL
LDL (d=1.019 to 1.063 g/mL) was isolated from human plasma by ultracentrifugation. Acetylation of LDL was performed according to the method of Basu et al¹¹ based on the method by Fraenkel-Conrat.¹² These particles were found to have a greater negative charge than native LDL by agarose electrophoresis in barbital buffer. Protein concentrations were determined according to Lowry et al¹³ with BSA as the standard.

Cholesterol Loading
Human monocytes were incubated for 7 days in medium with 15% human serum. The medium was then changed to serum-free medium consisting of RPMI 1640, 10 mg/mL BSA (unless stated otherwise), 5 µg/mL insulin, and 5 µg/mL transferrin. Cholesterol loading of the cells was performed by incubation with 100 µg protein per milliliter of AcLDL for 48 hours followed by a 24-hour incubation in RPMI 1640 with 10 mg/mL BSA without AcLDL to ensure equilibration of the different cellular cholesterol pools. Cells not incubated with AcLDL are referred to as "unladen," in contrast to "cholesterol-laden" cells.

Extraction of Lipids and Total Protein From the Cells
Lipid extraction was done essentially according to Brown et al¹⁴ and Hara and Radin.¹⁵ The cell culture dishes containing the attached cells were rinsed several times with PBS, followed by extraction with hexane/isopropanol (3:2, vol/vol). The hexane/isopropanol extract was shaken with half its volume of aqueous Na₂SO₄ (1 g/15 mL), and the mixture was left to separate into two phases. The upper phase was aspirated, 50 µg/mL BHT (final concentration) was added, and the vials were stored at -20°C. After removal of the hexane/isopropanol NaOH (0.2 mol/L) was added, and the dishes were left for 1 hour before they were scraped. The NaOH extracts were kept at 4°C until protein determinations were made according to Lowry et al.¹³ The total amount of protein per tissue culture dish varied between 0.1 and 0.7 mg protein.

Extraction of Lipids From Incubation Medium
The incubation medium was aspirated and centrifuged at 1000 rpm for 10 minutes. The supernatant was aspirated, 0.3 mmol/L EDTA (final concentration) and 50 µg/mL BHT (final concentration) were added, and the samples were stored at -20°C until analysis.

For cholesterol analyses 1 mL of the medium was added to a Folch tube, 5 mL of methanol was added, and finally 10 mL of trichloromethane was added; afterward, 15 mL of 0.9% NaCl was layered on top and the tube was left at room temperature for 24 hours. Eight milliliters of the lower phase was removed, dried with N₂, dissolved in 200 µL isopropanol, and immediately used for cholesterol analyses.

Analysis of 27-Oxygenated Sterols
Analysis of 27-Oxygenated Sterols in the Incubation Medium
To 2 mL of incubation medium, 600 ng norcholestenoic acid and 400 ng [²H₅]27-hydroxycholesterol (internal standards for cholestenoic acid and 27-hydroxycholesterol, respectively) were added. The pH of the incubation medium was adjusted to 3 with addition of HCl, and lipids were extracted with 10 mL diethyl ether. The ether extract was washed with water, and the solvent was evaporated under a gentle stream of N₂. The residue was dissolved in 0.5 mL chloroform.

A 100-mg Bond Elut LRC NH₂ solid-phase extraction column (Varian) was equilibrated with 4 mL hexane. The lipid extract in 0.5 mL chloroform was applied to the solid-phase extraction column. The column was eluted with 4 mL chloroform/isopropanol (2:1, vol/vol) and subsequently with 4 mL of 2% acetic acid in diethyl ether.

The chloroform/isopropanol eluate, containing the 27-hydroxycholesterol, was dried and treated with pyridine/hexamethyldisilazane/trimethylchlorosilane (3:2:1, vol/vol/vol) at 60°C for 30 minutes to convert the hydroxyl groups to trimethylsilyl ethers. The amount of 27-hydroxycholesterol in the eluate was determined by gas chromatography–mass spectrometry.

The ether eluate from the LRC NH₂ column was dried and redissolved in 1 mL methanol. 2,2-Dimethoxypropane (0.7 mL) and concentrated HCl (11 µL) were added, and the mixture was incubated at 55°C for 30 minutes to convert the carboxyl groups to methyl esters. The reaction mixture was dried and treated with pyridine/hexamethyldisilazane/trimethylchlorosilane as described above. The amount of cholestenoic acid in the eluate was determined by gas chromatography–mass spectrometry using a Hewlett-Packard 5890 series II gas chromatograph equipped with an HP-5MS capillary column (30 mx0.25 mm) and connected to an HP 5972 mass selective detector. The mass spectrometer was operated in the selected ion monitoring mode using the ions m/z 461 and m/z 456 for [²H₅]27-hydroxycholesterol and 27-hydroxycholesterol, respectively, and the sum of the ions m/z 488, 398, and 359 and m/z 502, 412, and 373 for norcholestenoic acid and 3ß-hydroxy-5-cholestenoic acid, respectively.

Analysis of 27-Oxygenated Sterols in the Cell Lipid Extract
Internal standards for 27-hydroxycholesterol and cholestenoic acid were added to the hexane/isopropanol extract of cell lipids, and the solvent was evaporated under a gentle stream of N₂. The residue was dissolved in 0.5 mL chloroform and subsequently applied to a solid-phase extraction column (Bond Elut LRC NH₂) as described above. 27-Hydroxycholesterol and cholestenoic acid were analyzed by gas chromatography–mass spectrometry as described above.

Analysis of Esterified and Free Cholesterol
Cholesterol measurements were performed with a Perkin-Elmer LS 30 luminescence spectrometer essentially according to the methods outlined by Heider and Boyett¹⁶ and Gamble et al.¹⁷ An aliquot of the hexane/isopropanol extract was dried with N₂, resuspended in isopropanol, and analyzed for free and total cholesterol. Cholesterol ester was calculated as the difference between total and free cholesterol. There is a possibility that the amount of free cholesterol measured by this enzymatic analysis also includes small amounts of 27-hydroxycholesterol and 3ß-hydroxy-5-cholestenoic acid present in the sample. Because we have not been able to test this possibility, we have not made any corrections by reducing the free cholesterol value with the amount of oxysterols obtained by analysis. In some of the experiments described below, wherein the amounts of free cholesterol and oxysterols were of comparable magnitude, the amount of free cholesterol could thus be lower than the values presented here. This fact does not violate any of the major conclusions drawn.

HDL
HDL (d=1.063 to 1.21 g/mL) was isolated from human plasma by ultracentrifugation.¹⁸

RDCs
Complexes of apoAI (or apoAI-M) and EYL were made as described by Matz and Jonas.¹⁹ In brief, 36 mg of EYL in ethanol was dried in a glass tube under N₂, and 2 mL of sodium cholate (10 mg/mL) was added. The tube was vigorously vortexed and then left under agitation at 4°C for at least 2 hours. A solution of 1.5 mg/mL apoAI (or apoAI-M) in PBS was prepared, the EYL/cholate solution was added until a final ratio of EYL to apoAI of 2.5:1 (wk/w) was obtained, and the tube was left under agitation overnight at 4°C. Finally, the solution was dialyzed 10 times over several days at 4°C against 1 L PBS.

The concentration of apoAI or apoAI-M in the complexes was determined by protein analysis according to the method of Lowry et al,¹³ and phospholipid concentration was determined with an enzymatic kit. Concentrations of free apoAI, free apoAI-M, or RDCs were always expressed as micrograms of protein per milliliter. The size of the complexes was determined by gel electrophoresis as described below. By size-exclusion high-performance liquid chromatography, apoAI was found to be monomeric to 98%, whereas apoAI-M was found to be dimeric to 85% to 90%.

Gel Electrophoresis Under Nondenaturing Conditions
Hydrated Stokes' diameters were determined by means of electrophoresis under nondenaturing conditions as described by Cheung²⁰ by using commercially available 4% to 20% linear polyacrylamide gradients gels according to the supplier's instructions. The gel was stained by incubation in 0.1% (wk/v) Coomassie brilliant blue R-250 in methanol/acetic acid/water(5:1:4, vol/vol/vol) for 16 hours at room temperature and destained in methanol/acetic acid/water (5:1:4, vol/vol/vol) until the background staining had completely disappeared. Gels were scanned in an LKB Ultrascan XL scanner (LKB), and particle sizes were determined from a standard curve made from thyroglobulin, ferritin, catalase, lactate dehydrogenase, and BSA. The hydrated Stokes' diameter of free apoAI and apoAI-M was 7.0 nm, whereas that of RDCs of apoAI or apoAI-M was 8.0 to 8.5 nm. Gel electrophoresis was used mainly as a method to ensure that RDCs had been formed, as the difference in size between free apoAI/apoAI-M and RDCs was clearly visualized by this method.

Incubation of Cholesterol-Laden Cells With HDL or RDCs
The dishes with cholesterol-laden cells were washed, and fresh medium with RPMI 1640 containing 10 mg/mL BSA, insulin (5 µg/mL), and transferrin (5 µg/mL) was added to all dishes. Various concentrations of HDL or RDCs made of apoAI/EYL or apoA I-M/EYL were added, and an equal volume of PBS was added to control dishes. Usually, three tissue culture dishes were used for each type of incubation, which lasted for 20 hours.

Statistical Analysis and Presentation of Results
Numerical results of experiments were given as mean±SD or mean±SEM calculated from separate incubations. In Tables 1 and 2, Wilcoxon's test was used to analyze differences in mean values. Linear regressions in Fig 2a and 2b were performed with the least-squares fit method. In Figs 3a, 3b, 5a, and 5b, differences in mean values were analyzed by one-way ANOVA followed by Duncan's multiple range test. In the presentation of the results, the cellular content refers to the cellular content at the end of the last incubation, whereas the efflux into the medium refers to the total (integrated) efflux into the medium during the last incubation.

View larger version (13K): [in this window] [in a new window]   Figure 2. Correlation, in cholesterol-laden HMDMs, between (a) intracellular 27-hydroxycholesterol content and intracellular cholesterol ester content (slope=.0081, r=.57, P=.0001) and (b) 27-hydroxycholesterol in the medium and intracellular 27-hydroxycholesterol content (slope=2.63, r=.89, P=8.2x10-9). These data were derived both from unladen and cholesterol-laden cells after incubation with or without RDCs.

View larger version (24K): [in this window] [in a new window]   Figure 3. Cholesterol content in cholesterol-laden HMDMs after incubation for 20 hours with varying concentrations of (a) apoAI/EYL or apoAI-M/EYL or (b) HDL or apoAI-M/EYL. Values for free cholesterol (filled bars), total cholesterol (open bars), and cholesterol ester (striped bars) are shown. Mean values±SD are given (n=3). ** and * Denote that the indicated means were significantly different at P=.01 and P=.05, respectively, from the corresponding mean for cholesterol-laden cells in the absence of RDCs or HDL.

View larger version (32K): [in this window] [in a new window]   Figure 5. Total 27-oxygenated sterols (diagonally striped bars), 3ß-hydroxy-5-cholestenoic acid in the medium (open bars), 27-hydroxycholesterol in the medium (horizontally striped bars), and 27-hydroxycholesterol in cell extract (filled bars) from a 20-hour incubation of cholesterol-laden cells with varying concentrations of (a) apoAI/EYL or apoAI-M/EYL or (b) HDL or apoAI-M/EYL. Mean values±SD are shown (n=3). ** and * Denote that the indicated means were significantly different at P=.01 and P=.05, respectively, from the corresponding mean for cholesterol-laden cells in the absence of HDL or RDCs.

	Results

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Cellular Content of Cholesterol and 27-Oxygenated Sterols in Unladen and Cholesterol-Laden Cells
Incubation of monocyte-derived macrophages with 100 µg/mL AcLDL resulted in a substantial increase in intracellular cholesterol ester content (as much as 50% of total cholesterol) and also in free cholesterol (Fig 1).

View larger version (16K): [in this window] [in a new window]   Figure 1. Cholesterol content in HMDMs that were incubated for 48 hours in medium with varying concentrations of AcLDL and then for 24+20 hours in medium without AcLDL (to create a situation comparable with the control incubations in the efflux experiments). Free cholesterol (open circles), total cholesterol (filled circles), and ester cholesterol (open triangles) are shown. Values shown are mean±SD (n=3).

Cholesterol loading caused an increase in intracellular 27-hydroxycholesterol, and there was a positive correlation between the content of 27-hydroxycholesterol and that of cholesterol ester in the cells (Fig 2a, r=.57, P=.0011). In three separate experiments, the slopes of the curves, ie, the molar ratio of 27-hydroxycholesterol to that of cholesterol ester, varied between 0.0009 and 0.008 (ie, 0.1% to 0.8%), and the correlation coefficients varied between .57 and .89, suggesting that there exists an intracellular pool of 27-hydroxycholesterol that is 0.5% (mol/mol) of the size of the cholesterol ester pool. No significant amounts of 3ß-hydroxy-5-cholestenoic acid could be found in the cells. The AcLDL used to load the cells with cholesterol typically contained 9.0x10^-5 mole 27-hydroxycholesterol per mole cholesterol; thus, 1% to 10% of the 27-hydroxycholesterol in the cells could have originated from the AcLDL.

Efflux of Free Cholesterol and 27-Oxygenated Sterols Into the Medium: Effects of BSA
Cholesterol-laden HMDMs released more free cholesterol into the medium containing BSA than did unladen HMDMs. When no BSA was present in the medium, efflux of free cholesterol from cholesterol-laden cells was considerably lower than when BSA was present in the medium (Table 1).

There was a substantial efflux of 3ß-hydroxy-5-cholestenoic acid and 27-hydroxycholesterol from cholesterol-laden HMDMs (ie, with AcLDL) into the medium with BSA, whereas unladen cells (without AcLDL) caused a small (if any) efflux of these substances (Table 2). From cholesterol-laden cells the efflux of 3ß-hydroxy-5-cholestenoic acid was on the order of 5 nmol/mg cell protein, whereas efflux of 27-hydroxycholesterol was on the order of 1 to 2 nmol/mg protein (Table 2). These effluxes should be compared with the efflux of free cholesterol, which was 10 nmol/mg protein under the same conditions (Table 1). Thus, under these conditions, the molar ratio of the efflux of free cholesterol to that of 27-hydroxycholesterol to that of 3ß-hydroxy-5-cholestenoic acid was 10:1:5. Table 2 also suggests that efflux of 27-oxygenated sterols (especially 27-hydroxycholesterol) into the medium was lower in the absence of BSA.

There was a close correlation between the efflux of 27-hydroxycholesterol into the medium and the amount of 27-hydroxycholesterol in the cells (Fig 2b, r=.89, P<.0001). Approximately three fourths of the molar mass of 27-hydroxycholesterol was found to be present in the medium and roughly one fourth in the cells at the end of the last incubation. The molar mass of 3ß-hydroxy-5-cholestenoic acid in the medium was 10 to 40 times higher than the molar mass of 27-hydroxycholesterol in the cells at the end of the last 20 hours of incubation.

Efflux of Free Cholesterol and 27-Oxygenated Sterols Into the Medium: Effects of RDCs Made From ApoAI/EYL or ApoAI-M/EYL
Incubation with increasing amounts of cholesterol acceptors like RDCs of apoAI-M or apoAI (Fig 3a and 3b) and HDL (Fig 3b) caused a substantial decrease in total cell cholesterol. RDCs with apoAI or apoAI-M appeared to be equally effective (Fig 3a), whereas HDL seemed to be less effective (Fig 3b) when plotted as micrograms of protein per milliliter. The cholesterol content of the added HDL was 0.28 µmol free cholesterol per milligram protein and 0.91 µmol cholesterol ester per milligram protein, whereas the added RDCs of apoAI or apoAI-M were free of cholesterol.

Incubation with increasing amounts of RDCs of apoAI and apoAI-M caused an increased efflux of free cholesterol into the medium (Fig 4). Again, RDCs of apoAI and apoAI-M seemed to be equally effective. Efflux of cholesterol ester into the medium was very small or absent (not shown). Unladen cells caused little efflux of cholesterol (at least in the absence of a cholesterol acceptor). Efflux of free cholesterol stimulated by HDL could not be measured due to the high cholesterol content in the HDL added. The total amount of 27-oxygenated sterols was calculated as the sum of 3ß-hydroxy-5-cholestenoic acid and 27-hydroxycholesterol in the medium and of 27-hydroxycholesterol in the cell extract (no 3ß-hydroxy-5-cholestenoic acid could be found in the cell extract).

View larger version (26K): [in this window] [in a new window]   Figure 4. Free cholesterol content in the medium after a 20-hour incubation of unladen HMDMs in the medium without RDCs (diagonally striped bars), cholesterol-laden HMDMs in medium without RDCs (horizontally striped bars), or cholesterol-laden HMDMs in the medium with RDCs of apoAI/EYL (open bars) or in medium with RDCs of apoAI-M/EYL (filled bars). Mean values±SD are given (n=3).

Incubation with RDCs of apoAI and apoAI-M (Fig 5a) caused a substantial decrease and HDL a somewhat smaller decrease (Fig 5b) in the total production of 27-oxygenated sterols as well as in the efflux of these metabolites into the medium. This decrease was parallel by an increased efflux of free cholesterol (Fig 4). In Fig 6 some of the data from Fig 5a have been plotted to better illustrate the pronounced decrease in efflux of 3ß-hydroxy-5-cholestenoic acid and the much smaller decrease in efflux of 27-hydroxycholesterol with increasing concentrations of RDCs. Thus, increasing amounts of RDCs in the medium caused a decrease in the fraction of 27-oxygenated sterols of the total efflux of cholesterol. At 50 µg/mL of RDCs, the molar ratio of free cholesterol to 27-hydroxycholesterol to 3ß-hydroxy-5-cholestenoic acid was found to be 60:1:1 in the medium after the end of the incubation.

View larger version (18K): [in this window] [in a new window]   Figure 6. 3ß-Hydroxy-5-cholestenoic acid (circles) and 27-hydroxycholesterol (triangles) in the medium after a 20-hour incubation of cholesterol-laden HMDMs with varying concentrations of apoAI/EYL (open symbols) or apoAI-M/EYL (filled symbols). Mean values±SD are shown (n=3). ** and * Denote that for 3ß-hydroxy-5-cholestenoic acid, the indicated means were significantly different at P=.01 and P=.05, respectively, from the corresponding mean for cholesterol-laden cells in the absence of RDCs.

Cholesterol Efflux–Promoting Properties of Free ApoAI or ApoAI-M Compared With RDCs
The results in Fig 7 suggest that free apoAI and free apoAI-M have some cholesterol efflux–promoting potential, but it is substantially smaller than that of apoAI in the form of RDCs. No difference in efflux-promoting capacity between free apoAI and free apoAI-M was found.

View larger version (25K): [in this window] [in a new window]   Figure 7. Free cholesterol (filled bars), total cholesterol (open bars), and cholesterol ester (striped bars) content in cholesterol-laden HMDMs after incubation for 20 hours with free apoAI, free apoAI-M, or apoAI/EYL. Mean values±SD are given (n=3). ** and * Denote that the indicated means were significantly different at P=.01 and P=.05, respectively, from the corresponding mean for cholesterol-laden cells in the absence of RDCs or free protein.

The conclusion above is strengthened by the results presented in Fig 8, which show that efflux of free cholesterol from cholesterol-laden macrophages into the surrounding medium is small after incubation with free apoAI or apoAI-M compared with incubation with apoAI in the form of RDCs. Fig 8 also shows that incubation with free apoAI or apoAI-M has only a minor effect on the efflux of 3ß-hydroxy-5-cholestenoic acid into the medium, whereas this efflux is markedly reduced by apoAI in the form of RDCs. No significant difference was found between free apoAI and free apoAI-M in this respect.

View larger version (24K): [in this window] [in a new window]   Figure 8. 3ß-Hydroxy-5-cholestenoic acid in the medium (open bars), 27-hydroxycholesterol in the medium (filled bars), and free cholesterol in the medium (striped bars) after a 20-hour incubation of cholesterol-laden cells with free apoAI, free apoAI-M, or apoAI/EYL. Mean values±SD are shown (n=6 for cholestenoic acid n=3 for other cases). **Denotes that the indicated means were significantly different at P=.01 from the corresponding mean for cholesterol-laden cells in the absence of RDCs or free protein.

	Discussion

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The present finding that 27-hydroxycholesterol accumulated in the cholesterol-laden cells in a quantity proportional to the intracellular cholesterol ester content is interesting. The possibility that the synthesis of 27-hydroxycholesterol is limited by cholesterol availability has been discussed previously,^{3 21} but no clear evidence for this has hitherto been presented.

Because it is known that side-chain oxidized oxysterols are transported through lipophilic membranes much faster than is cholesterol,²² flux of 27-hydroxycholesterol and 3ß-hydroxy-5-cholestenoic acid from the plasma membrane into the medium in the absence of an extracellular cholesterol acceptor can be expected to be considerably faster than that of free cholesterol.

In the present work, a substantial efflux of cholesterol in the form of free cholesterol, 27-hydroxycholesterol, and 3ß-hydroxy-5-cholestenoic acid was observed from cholesterol-laden cells when the medium contained BSA, whereas unladen cells showed a smaller (if any) efflux of free cholesterol and these steroids. In medium without BSA or an extracellular cholesterol acceptor, efflux of free cholesterol and 27-oxygenated sterols (especially 27-hydroxycholesterol) from cholesterol-laden cells was reduced, suggesting that part of the flux of these sterols into the medium with BSA is due to an association with BSA. In accordance with this, it has recently been shown that 25-hydroxycholesterol associates with albumin.²³

We have previously reported that the classic reverse cholesterol-transport mechanism is considerably more effective than the sterol 27-hydroxylase–mediated mechanism in lung macrophages exposed to optimal amounts of HDL.³ The present observations demonstrated a highly significant decrease in the flux of 27-oxygenated steroids from cholesterol-laden macrophages when the latter were exposed to HDL or, even more effectively, RDCs made from apoAI and phospholipids. Thus, there was an inverse relation between the two mechanisms under the experimental conditions employed. It should be emphasized that this inverse relationship was obtained in macrophages heavily loaded with cholesterol. The situation may thus be different in cells containing less cholesterol. The most obvious explanation is that there may be competition for free cholesterol between the intramitochondrial sterol 27-hydroxylase and the lipophilic extracellular acceptor. The flux of free cholesterol from the inner mitochondrial membrane to the plasma membrane and then to the extracellular acceptor would then decrease the substrate saturation of the enzyme.

It has previously been demonstrated that oxysterols may have a direct inhibitory effect on cholesterol efflux.^{24 25} However, the intracellular concentration of 7-oxocholesterol needed to depress the efflux of cholesterol from cholesterol-laden macrophages was reported to be 50 nmol/mg protein, which is 50-fold higher than the concentration of 27-hydroxycholesterol in the cells studied herein. In our previous study on human cultured alveolar macrophages, inhibition of sterol 27-hydroxylase by cyclosporin had no significant effect on cholesterol efflux.³ Because this inhibition reduces the intracellular concentration of 27-oxygenated steroids by 90%, it is evident that physiological concentrations of these steroids must have little or no effect on reverse cholesterol transport.

In previous work with human alveolar macrophages, substrate availability was found to be a critical factor for enzyme activity.³ It was found that increasing the intracellular concentration of cholesterol in such cells by a factor of 10 was followed by a twofold increase in the secretion of 27-oxygenated steroids into the medium, without an increase in enzyme protein concentration. Substrate availability was thus clearly more important than increased synthesis of enzyme protein under the conditions employed. At present we cannot exclude the possibility that part of the HDL- or RDC-induced decrease in total 27-oxygenated sterols in the cholesterol-laden macrophages may have been due to effects on enzyme synthesis. Preliminary studies in which sterol 27-hydroxylase mRNA was measured in these cells by Northern blotting did not show significant effects of these cholesterol acceptors.

The HDL- or RDC-mediated decrease in the flux of 27-oxygenated steroids from the cholesterol-laden macrophages was mainly a decrease in the efflux of 3ß-hydroxy-5-cholestenoic acid. Production of this acid requires three subsequent steps by sterol 27-hydroxylase, with 27-hydroxycholesterol as an intermediate.² If cholesterol is rapidly extracted from the cells by the extracellular acceptor, this might explain the markedly reduced flux of 3ß-hydroxy-5-cholestenoic acid from cells exposed to HDL or RDCs. Regardless of the mechanism, the inverse relation between HDL-mediated reverse cholesterol transport and the sterol 27-hydroxylase–dependent transport of cholesterol metabolites is of interest in relation to the function of macrophage. Macrophages in tissues are normally exposed to considerably lower concentrations of lipoproteins than are cells in the bloodstream. An alternative to the classic reverse cholesterol-transport mechanism may thus be required under some conditions. It seems likely that the balance between the two mechanisms for removal of cholesterol from macrophages might be of importance for formation of foam cells and development of atherosclerosis.

The present results suggest that apoAI or apoAI-M in the form of free protein has far less cholesterol efflux–promoting properties than do RDCs made from apoAI (or ApoAI-M) and EYL. In an earlier work²⁶ we have shown that EYL liposomes have a much smaller cholesterol efflux–promoting capacity than do RDCs made of apoAI and EYL. Together these results suggest that apoAI (or apoAI-M) in complex with an appropriate phospholipid forms a particle that has a much larger cholesterol efflux–promoting potential than do either of these particle components alone.

Finally, this work also suggests that there are no marked differences in the cholesterol efflux–promoting properties of "synthetic HDL" made of apoAI or apoAI-M. Thus, if apoAI-M has a stronger antiatherogenic potential than apoAI, it is probably related to processes other than the initial steps of reverse cholesterol transport.

	Selected Abbreviations and Acronyms

 

AcLDL = acetylated LDL ApoAI = apolipoprotein A-I ApoAI-M = apolipoprotein A-I Milano EYL = egg yolk lecithin HMDM = human monocyte–derived macrophages RDC = reconstituted discoidal complex

	Acknowledgments

 
This work was supported by grants from the Foundation of Old Servants (to J.W. and J.N.), Professor Nanna Svartz' Fund (to J.N.), King Gustaf V 80th Anniversary Foundation (to J.W. and J.N.), the Swedish Heart-Lung Foundation (to I.B. and J.N.), and the Swedish Medical Research Council (to I.B. and J.N.). Financial support was also obtained from Pharmacia AB (Stockholm, Sweden). The skillful technical assistance of Annelie Olsson and Karin Husman is much appreciated.

	Footnotes

 
¹ According to recent nomenclature, the mitochondrial stereospecific -hydroxylation of one of the ultimate methyl groups of the steroid side chain should be denoted "27-hyroxylase." For a review, see Reference 4.

Received April 15, 1997; accepted November 11, 1997.

	References

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