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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1421-1431

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1421-1431.)
© 1997 American Heart Association, Inc.

Articles

Evidence for Prolonged Cell-Surface Contact of Acetyl-LDL Before Entry Into Macrophages

Xiaohui Zha; Ira Tabas; Philip L. Leopold; Nancy L. Jones; ; Frederick R. Maxfield

From the Departments of Pathology (X.Z., P.L.L.), and Medicine, Anatomy and Cell Biology (I.T.), Columbia University, College of Physicians and Surgeons; the Department of Biochemistry, Cornell University Medical College (F.R.M.), New York, NY; and the Department of Pathology, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC (N.L.J.).

Correspondence to Frederick R. Maxfield, Department of Biochemistry, Cornell University Medical College, 1300 York Ave, New York, NY 10021. E-mail frmaxfie{at}mail.med.cornell.edu


*    Abstract
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Abstract Acetyl-LDL stimulates acyl-CoA:cholesterol acyltransferase (ACAT) much more effectively than LDL in mouse peritoneal macrophages. Previous work with another potent ACAT stimulator, ß-VLDL, suggested that atherogenic lipoproteins may use internalization pathways distinct from that of LDL. Brief incubation of fluorescently labeled acetyl-LDL and LDL followed by a short chase period without lipoproteins was used to compare endocytic pathways. LDL was delivered rapidly to perinuclear vesicles, corresponding to late endosomes and lysosomes. A substantial fraction (>40%) of acetyl-LDL was initially retained in the cell periphery, while the rest was rapidly delivered to late endosomes that also contained LDL. Fluorescence of peripheral 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)–acetyl-LDL could be quenched by TNBS, indicating accessibility of the peripheral acetyl-LDL to the extracellular space. Quantification of fluorescence intensities demonstrated that >40% of the cell-associated DiI–acetyl-LDL but only about 10% of DiI-LDL fluorescence was quenchable by TNBS after a 3-minute chase. Fucoidin can efficiently displace DiI–acetyl-LDL bound to cells at 0°C. DiI–acetyl-LDL in the TNBS-quenchable peripheral compartments, however, was resistant to fucoidin. Electron microscopy of colloidal gold–acetyl-LDL showed that acetyl-LDL on the cell surface was often associated with microvilli or ruffles. After clearance from the surface, the peripheral acetyl-LDL was also delivered to the late endosomes and lysosomes. These results indicate that a substantial portion of acetyl-LDL enters macrophages through a pathway that initially differs from that of LDL. This pathway involves a prolonged retention of acetyl-LDL on the plasma membrane. This surface retention may affect ACAT activation in macrophages.


Key Words: scavenger receptors • endocytosis • microvilli


*    Introduction
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Cholesteryl ester–loaded macrophages, or foam cells, are a prominent feature of atherosclerotic lesions.1 2 In cultured mouse peritoneal macrophages, CE accumulation can be induced by incubating with ß-VLDL or modified forms of LDL, such as acetyl-LDL.3 4 This CE accumulation is catalyzed by an enzyme, ACAT, whose activity is regulated by cholesterol level in cells.5 6 Cholesterol delivered by native LDL is a rather poor stimulator of ACAT in macrophages.3

The regulation of ACAT has been shown to be posttranslational7 8 9 and closely related to intracellular transport of free cholesterol to the ACAT-containing compartments.10 Cholesterol derived from lipoproteins appears to mix with endogenous cellular cholesterol pools before being transported to ACAT.11 12 13 The exact mechanism by which lipoprotein-derived cholesterol stimulates ACAT, however, remains to be elucidated.

The endocytic pathway by which lipoproteins enter macrophages appears to play an important role in stimulating ACAT.14 15 16 LDL is internalized via clathrin-coated pits and delivered rapidly to late endosomes and lysosomes.3 CEs in LDL are then hydrolyzed, and free cholesterol leaves lysosomes.17 In mouse peritoneal macrophages, ß-VLDL and LDL enter macrophages by distinct endocytic pathways15 despite the fact that they bind to the same LDL receptor. In contrast to LDL, a large portion of ß-VLDL remains in the cell periphery for several minutes before it eventually reaches the same late endosomes and lysosomes as LDL.14 The ability of ß-VLDL to stimulate ACAT is correlated positively with the extent of ß-VLDL retention in the cell periphery when different size fractions of ß-VLDL are compared.16 The peripheral compartments containing ß-VLDL are tubular plasma-membrane-connected invaginations called STEMs , and ß-VLDL undergoes catabolic processing while in these structures.14 This factor suggests that the retention of ß-VLDL in STEMs may be related to the differential effects of LDL and ß-VLDL on ACAT stimulation. It is possible that prolonged contact of lipoproteins with the plasma membrane and their catabolism in these tubular structures facilitates the direct delivery of cholesterol to the plasma membrane. Since plasma membrane cholesterol is likely to be the main source of substrate for ACAT13 and ACAT activity is believed to be substrate limited, this prolonged retention could be critical for enhanced cholesterol esterification.

Acetyl-LDL is another lipoprotein that potently stimulates ACAT in macrophages.18 In several respects, acetyl-LDL mimics oxidized LDL found in atherosclerotic lesions.3 19 20 21 22 Produced by chemical modification of LDL, acetyl-LDL no longer binds to LDL receptors and is instead taken into cells by scavenger receptors.21 22 Macrophages express a high level of scavenger receptors, which mediate uptake of a wide variety of ligands, including biologically oxidized LDL.20 21 22 23 Unlike LDL receptors, scavenger receptors in macrophages are notsubject to downregulation by the level of cellular cholesterol.20 22 This leads to massive cholesterol accumulation in the presence of acetyl-LDL and the eventual conversion of macrophages into foam cells.

In the present study, we investigated the endocytic pathway of acetyl-LDL in mouse peritoneal macrophages by fluorescence microscopy and electron microscopy. Results presented here show that a significant portion of acetyl-LDL enters macrophages via a pathway different from that of LDL. This pathway involves a transient retention of acetyl-LDL on the plasma membrane before endocytosis and transport to lysosomes, but the surface localization is different from the STEM structures that take up ß-VLDL.


*    Methods
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Cells
Peritoneal macrophages from unstimulated female ICR mice (15 to 25 g, Harlan Sprague Dawley, Inc, Indianapolis, Ind) were plated onto poly-D-lysine–coated coverslip-bottom dishes and cultured for 2 days in DMEM plus 10% (vol/vol) lipoprotein-deficient serum containing penicillin (100 U/mL) and streptomycin (100 µg/mL) as previously described.15 All experiments were performed in DHB (DMEM, 10 mmol/L HEPES, 0.2% fatty acid–free BSA, pH 7.4) on day 3 after plating. CHO-mSRAII24 and CHO-haSR-BI cell lines were generously provided by Dr Monty Krieger (Massachusetts Institute of Technology). These cells were grown in monolayer culture in Ham's F-12 medium containing 5% fetal bovine serum.

Lipoproteins and Reagents
Human LDL was prepared as previously described.15 Acetyl-LDL was prepared by acetylation of LDL with acetic anhydride as described previously.22 ß-VLDL from cholesterol-fed rabbits was prepared by ultracentrifugation at a density of 1.006 g/mL.25 The d<1.006 lipoproteins, which contain mostly ß-VLDL but also some pre–ß-VLDL, will be referred to as simply "ß-VLDL." {alpha}2M was purified, converted to the receptor-binding form, and conjugated to fluorescein isothiocyanate as previously described.26 Acetyl-LDL or LDL were labeled with DiI (all fluorescence probes from Molecular Probes) by the method of Pitas et al27 and stored at 4°C under argon. LDL was also labeled with cholesteryl 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoate (cholesteryl BODIPY FL C12) to yield BODIPY-LDL by reconstitution using the method of Krieger et al.28 BODIPY–ß-VLDL was made by transferring cholesteryl BODIPY FL C12 from BODIPY-LDL to ß-VLDL using purified cholesteryl ester transfer protein (a gift from Drs Paul Kussie and Alan Tall, Columbia University). DiI-labeled lipoproteins had similar cholesterol/protein ratios to the respective unlabeled lipoproteins from which they were derived.15 DiI–acetyl-LDL uptake by macrophages was inhibited by the addition of excess unlabeled acetyl-LDL (50-fold excess) but not by LDL (50-fold excess). Similarly, BODIPY-LDL uptake was inhibited by excess unlabeled LDL (50-fold excess) but not by acetyl-LDL.

Fucoidin and dextran sulfate (500 000 kD) were from Sigma Chemical Company. TNBS (Sigma) was prepared as a 100 mmol/L stock solution in PBS and stored at 4°C. Maleylated BSA was a gift from Drs S. Silverstein and J.B. El-Khoury (Columbia University).

Fluorescence Microscopy
Cells were labeled with fluorescent probes and viewed using a Leitz Diavert microscope (Leitz Wetzlar, Germany) with a 63x, NA 1.4 objective and filter sets described previously.15 Fluorescence images were acquired and digitized using a cooled CCD camera (Photometrics, Inc). Fluorescence quantification in competition experiments was carried out with a 25x, NA 0.8 objective to collect a large number of cells per field. Quantification of fluorescence intensity was performed using ISee software (Inovision Corporation) running on a SPARC station 4/330 computer system (Sun Microsystems Inc). Some images were also recorded with a Videoscope KS 1381 image intensifier, a VS 2000N video camera, and a JVC 6650 US-VHS videocassette recorder.

DiI Fluorescence Quenching by TNBS
To measure TNBS quenching, macrophages were incubated with DiI–acetyl-LDL or DiI-LDL for 3 minutes at 37°C and chased in the absence of the ligands for various periods of time. DiI remaining on the cell surface was then quenched by addition of TNBS (final concentration 5 mmol/L) in medium 1 (mmol/L: 150 NaCl, 20 HEPES, 5 KCl, 1 CaCl2, 1 MgCl2, pH 7.4). DiI images were acquired before and immediately after TNBS addition to each field of cells. Each image, after background subtraction and bleaching correction, was integrated to give the total fluorescence intensity in that field. The difference in total fluorescence intensities between the two images, taken from the same field of cells before and after TNBS addition, represents the amount of DiI fluorescence quenched by TNBS in that field. After correction for DiI quenching efficiency (60%, see discussion below), this difference (quenched DiI) was then divided by the total initial fluorescence intensity of the field (total DiI) to give the fraction of DiI-labeled lipoproteins accessible to TNBS quenching.

Electron Microscopy
DiI–acetyl-LDL (830 µg/mL) was first diluted to 100 µg/mL with 0.05 mol/L EDTA buffer, pH 5.5. The lipoprotein suspension (40 µL) was injected with rapid mixing into 0.5 mL of 17 nm colloidal gold suspension in a glass tube. The mixture was vortexed gently for an additional 30 seconds to ensure complete adsorption of the lipoprotein to the gold particles.29 BSA from a stock solution (100 mg/mL in 0.05 mol/L EDTA, pH 5.5) was then added to a final concentration of 2 mg/mL. This mixture was centrifuged at 1000g for 1 hour over a 40% sucrose cushion to separate excess colloidal gold. The supernatant was then centrifuged at 9000g over a 40% sucrose cushion to recover the conjugates in the sucrose cushion, as the excess unlabeled acetyl-LDL remained in the supernatant. Recovered gold–acetyl-LDL from the sucrose cushion was dialyzed against PBS and then DMEM to yield purified conjugates. Conjugates were used within 24 hours. Gold-LDL conjugates were made by a similar protocol.

Mouse peritoneal macrophages were cultured as described above. On day 3, cells were incubated with gold-labeled acetyl-LDL for 3 minutes at 37°C and chased with DHB in the absence of ligand for 3 minutes at 37°C. Cells were then prepared for electron microscopy as described previously.30 Briefly, cells were first fixed with 3% paraformaldehyde for 30 minutes at room temperature. After rinsing with 0.1 mol/L sodium cacodylate buffer, pH 7.2, cells were fixed with 2.5% glutaraldehyde plus 1% tannic acid, pH 7.0, for another 30 minutes at room temperature. This was followed by a fixation with 1% OsO4 and 1.5% potassium ferricyanide in 0.1 mol/L cacodylate for 60 minutes at room temperature. Cells were then dehydrated and embedded in epoxy resin (EM-bed 812, Electron Microscopy Sciences). Thin sections (50 to 60 nm) were cut and visualized on a JEOL JEM-1200 EX II electron microscope operating at 80 keV. Thick sections (300 to 500 nm) were visualized at 300 keV with a Philips CM-30 IVEM.

For whole-mount IVEM studies, samples were prepared in a manner similar to that of Jones et al.31 Freshly isolated mouse peritoneal macrophages were plated on glass coverslips coated with poly-D-lysine and cultured for 2 days. On day 3, cells were labeled with gold–acetyl-LDL or gold-LDL for 3 minutes at 37°C and chased in the absence of ligands for 3 minutes. Cells were then fixed in 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer, pH 7.4, for 10 minutes at room temperature, processed for whole-mount electron microscopy,32 and visualized at 300 keV in the Philips CM-30 IVEM.


*    Results
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Morphological Analysis of the Endocytic Pathway of Fluorescently Labeled Acetyl-LDL, LDL, {alpha}2M, and ß-VLDL
The primary goal of this study was to compare the endocytic pathway of acetyl-LDL with other proteins in macrophages. Acetyl-LDL and LDL were labeled with DiI and BODIPY, respectively. Macrophages were incubated with both DiI–acetyl-LDL and BODIPY-LDL for 3 minutes at 37°C and then rinsed and chased in the absence of lipoproteins at 37°C for 3, 5, and 10 minutes. The cells were then fixed and observed by digital fluorescence microscopy. A representative result is shown in Fig 1Down. After 3 minutes' chase, LDL was concentrated in perinuclear compartments, mainly late endosomes or lysosomes15 (Fig 1DDown, arrows). Much of the acetyl-LDL was colocalized with LDL in these perinuclear vesicular structures at the end of the 3-minute chase (Fig 1ADown, arrows). There was, however, a significant portion of the acetyl-LDL that remained in the cell periphery (Fig 1ADown, arrowheads). The amount of peripheral acetyl-LDL gradually decreased at longer chase times (Fig 1BDown and 1CDown), while LDL remained in the perinuclear vesicles. By the end of a 10-minute chase, there was only a small amount of acetyl-LDL visible in the cell periphery (Fig 1CDown).



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  		Figure 1. Double-label fluorescence microscopy of macrophages incubated with DiI–acetyl-LDL and BODIPY-LDL. Macrophages were incubated with DiI–acetyl-LDL (10 µg/mL) and BODIPY-LDL (10 µg/mL) for 3 minutes at 37°C and chased in the absence of lipoproteins at 37°C for 3, 5, and 10 minutes, as indicated. Cells were then fixed with 4% paraformaldehyde in PBS for 5 minutes at room temperature and viewed by fluorescence microscopy. A substantial portion of acetyl-LDL (top row) appeared to be retained in the cell periphery (arrowheads in A), while LDL (bottom row) from the same field of cells showed only localization in perinuclear organelles (arrows in D). Bar=10 µm.

Like LDL, {alpha}2M is internalized into cells through the clathrin-coated-pit pathway33 although it binds to a different receptor, the lipoprotein receptor–related lipoprotein.34 Macrophages were double labeled with DiI–acetyl-LDL and fluorescein-{alpha}2M for 3 minutes at 37°C and chased at 37°C for another 3 minutes in the absence of ligands. As was the case with LDL, {alpha}2M was concentrated in the perinuclear region after 3 minutes' chase, colocalizing with part of acetyl-LDL (Fig 2ADown and 2BDown, arrowhead), and there was again a significant portion of acetyl-LDL in the cell periphery (Fig 2ADown, arrow). Labeling of several processes at the edge of the cells is clearly visible in the DiI–acetyl-LDL fluorescence image (Fig 2ADown).



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  		Figure 2. Fluorescence microscopy of macrophages double labeled with DiI–acetyl-LDL and fluorescein-{alpha}2M. Cells were incubated with DiI–acetyl-LDL (10 µg/mL) and fluorescein-{alpha}2M (100 µg/mL) for 3 minutes and chased in the absence of ligands for 3 minutes. Cells were then fixed with 4% paraformaldehyde in PBS and viewed by fluorescence microscopy. A portion of the DiI–acetyl-LDL colocalized with {alpha}2M in perinuclear organelles (arrowheads). However, a significant part of acetyl-LDL was not colocalized with {alpha}2M and appeared to be in the cell periphery near the plasma membrane (arrows). Bar=10 µm.

ß-VLDL has been shown in previous studies to be retained in STEMs at the cell surface for several minutes before being endocytosed and delivered to lysosomal compartments.15 To determine whether ß-VLDL and acetyl-LDL share the same pathway, cells were incubated with DiI–acetyl-LDL and BODIPY–ß-VLDL for 3 minutes at 37°C and chased for 3 minutes in ligand-free medium. As shown in Fig 3Down, most of the ß-VLDL was seen in the cell periphery after 3 minutes' chase (Fig 3BDown), and little appeared in the perinuclear region. Acetyl-LDL, on the other hand, was located in both the cell periphery (Fig 3ADown, arrows) and the perinuclear region (Fig 3ADown, arrowheads). Moreover, within the cell periphery, acetyl-LDL appeared as a large number of fine dots (Fig 3ADown, arrows), while ß-VLDL was in relatively fewer but brighter peripheral compartments (Fig 3BDown). There was very limited colocalization between these two lipoproteins. These data indicate that although both acetyl-LDL and ß-VLDL are retained in the cell periphery longer than LDL, most of the peripheral acetyl-LDL is in compartments that do not contain ß-VLDL. As described in a later section, electron microscopy confirmed that the peripheral distribution of acetyl-LDL was different from the STEM localization of ß-VLDL.



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  		Figure 3. Fluorescence microscopy of macrophages double labeled with DiI–acetyl-LDL and BODIPY–ß-VLDL. Cells were incubated with DiI–acetyl-LDL (10 µg/mL) and BODIPY–ß-VLDL (5 µg/mL) for 3 minutes at 37°C. Cells were then chased in ligand-free medium at 37°C for 3 minutes and fixed with paraformaldehyde. A portion of the acetyl-LDL was in perinuclear organelles (arrowheads), which contained only a small amount of ß-VLDL. Both acetyl-LDL and ß-VLDL have a significant portion remaining in the cell periphery (arrows). There was very limited colocalization between the two lipoproteins even in the periphery of the cell. Bar=10 µm.

Accessibility of Acetyl-LDL Compartments to a Membrane-Impermeant Fluorescence Quencher
To determine whether peripheral acetyl-LDL was in surface-connected structures, a small highly charged fluorescence quencher, TNBS, was used. TNBS is a membrane impermeant agent that rapidly quenches DiI fluorescence.35 To measure the quenching efficiency of DiI–acetyl-LDL bound only to the cell surface, cells were labeled with DiI–acetyl-LDL at 0°C and then exposed to TNBS. TNBS was able to rapidly quench surface-bound DiI fluorescence to about 40% of the initial intensity (data not shown).

To examine the peripheral compartments seen at 37°C, macrophages were incubated with DiI–acetyl-LDL for 3 minutes at 37°C and chased in the absence of ligands for 3 minutes before being exposed to TNBS. Fluorescence images of DiI–acetyl-LDL were recorded from the same field of cells before (Fig 4ADown) and immediately after (Fig 4DDown) addition of TNBS. DiI–acetyl-LDL was located in both peripheral and perinuclear regions before adding TNBS (Fig 4ADown). After exposure to TNBS, however, the fine dots of peripheral DiI–acetyl-LDL fluorescence (Fig 4ADown, arrows) were heavily quenched (Fig 4DDown, arrows) showing the presence of DiI on the outside of the cell. As expected, TNBS had little effect on perinuclear DiI-containing compartments (Fig 4ADown and 4DDown, arrowheads). Macrophages were also incubated with DiI–acetyl-LDL for 3 minutes at 37°C but chased in the absence of ligands for 10 minutes (Fig 4BDown and 4EDown) or 20 minutes (Fig 4CDown and 4FDown). The fraction of DiI fluorescence quenchable by TNBS was sharply decreased as the chase time increased. TNBS caused little change by the end of 20 minutes' chase.



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  		Figure 4. Quenching of DiI–acetyl-LDL by TNBS in living macrophages. Macrophages were incubated with DiI–acetyl-LDL (10 µg/mL) for 3 minutes at 37°C and chased in the absence of ligand for 3, 10, and 20 minutes. Fluorescence images of DiI–acetyl-LDL were recorded before (top row) and immediately after (bottom row) addition of TNBS (final concentration, 5 mmol/L in medium 1). The peripheral fluorescence of DiI–acetyl-LDL was quenched within a few seconds, whereas perinuclear compartments were not affected (A and D). When cells were chased for 10 minutes (B and E) and 20 minutes (C and F), respectively, after 3 minutes' labeling, less and less DiI–acetyl-LDL remained accessible to TNBS.

Live cells are constantly taking up extracellular fluid through endocytosis, and newly formed endosomes fuse rapidly with existing endosomes within a few minutes.26 To ensure that TNBS quenching in live cells was not due to vesicle-fusion events immediately after endocytosis, the quenching process was also recorded using an image-intensified video camera. DiI fluorescence in the cell periphery was quenched within a second after addition of TNBS, indicating that the quenching process is endocytosis independent (data not shown).

For comparison, the same quenching experiments were also carried out with DiI-LDL. Cells were incubated with DiI-LDL for 3 minutes at 37°C and chased another 3 minutes before exposure to TNBS. As shown in Fig 5Down, little DiI-LDL fluorescence was quenched by TNBS (Fig 5ADown and 5BDown), indicating that most of the DiI-LDL–containing compartments had sealed off from the plasma membrane after 3 minutes' chase. Surface-labeled DiI-LDL at 0°C showed a quenching efficiency similar to that of DiI–acetyl-LDL (data not shown).



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  		Figure 5. Quenching of DiI-LDL by TNBS in living macrophages. Cells were incubated with DiI-LDL (10 µg/mL) for 3 minutes at 37°C and chased for 3 minutes in the absence of ligand. Fluorescence images of DiI-LDL were taken before (A) and immediately after (B) addition of TNBS (final concentration, 5 mmol/L in medium 1). Little DiI-LDL fluorescence was quenched by TNBS, indicating that DiI-LDL–containing compartments had sealed off from the plasma membrane after 3 minutes' chase. Some changes in fluorescence pattern are due to motion of organelles and cells between the two exposures.

Kinetics of LDL and Acetyl-LDL Clearance From the Plasma Membrane
Results presented above showed that peripheral DiI–acetyl-LDL containing structures were plasma membrane connected, and DiI–acetyl-LDL becomes sealed off from the plasma membrane more slowly than DiI-LDL. To measure the kinetics of the sequestration process, total fluorescence intensities from several fields of cells were measured before and after TNBS addition. The fraction of surface-accessible DiI-lipoproteins was determined from the relative fluorescence quenching due to TNBS, corrected for the efficiency of TNBS quenching (see "Methods"). As shown in Fig 6Down, the decreased accessibility to TNBS with time indicates that both acetyl-LDL and LDL were increasingly sequestered away from the cell surface. DiI-LDL, however, was sequestered into cells much more rapidly than acetyl-LDL. There was less quenchable DiI-LDL than quenchable DiI–acetyl-LDL at all time points examined. For example, about 50% of DiI-LDL was surface accessible after a 1-minute chase, and about 10% remained on the plasma membrane at the end of 3 minutes' chase. DiI–acetyl-LDL had about 80% remaining accessible to TNBS at 1 minute, and more than 40% remained on the cell surface after 3 minutes. Only after 20 minutes' chase in the absence of ligands did the percentage of quenchable DiI–acetyl-LDL drop to near zero.



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  		Figure 6. Clearance of DiI–acetyl-LDL and DiI-LDL from the plasma membrane. Macrophages were incubated with DiI–acetyl-LDL ({bullet}) or DiI-LDL ({diamondsuit}) for 3 minutes and then chased for various periods of time before quenching surface fluorescence by TNBS. DiI fluorescence images were acquired before and immediately after TNBS addition (final concentration, 5 mmol/L) in each field of cells. The percentage surface accessible represents the percentage of total cellular fluorescence lost due to TNBS multiplied by 1.67 to account for the partial quenching of DiI by TNBS. Each data point represents an average of three or four fields in one individual experiment, and error bars represent SDs among fields. Curves are computer fittings: LDL (dashed line), y=74.6xe-0.53t; acetyl-LDL, y=71.4xe-0.57t+37.1xe-0.083t (solid line) and y=86.7xe-0.207t (dotted line).

The kinetics of LDL clearance from the cell surface can be fit well by a single exponential decay (Figure 6Up, dashed line) with t1/2=1.3 minutes. Acetyl-LDL clearance from the cell surface can be fit with a single exponential curve (dotted line: t1/2=3.3 minutes) or somewhat better with a double exponential curve (solid line) with fast (t1/2=1.2 minutes) and slow (t1/2=8.4 minutes) components. The precision of the experiments does not allow us to distinguish whether the process is described by one or two exponential decays. In either case, however, it is clear that a large fraction of acetyl-LDL enters cells more slowly than LDL.

Accessibility of Peripheral Acetyl-LDL Compartments to Fucoidin
These results indicated that the peripherally retained acetyl-LDL was located on the cell surface and accessible to small molecules. ß-VLDL has been shown to be retained in a surface invagination that had poor accessibility to large molecules.14 We investigated whether acetyl-LDL might be in a surface structure with similar inaccessibility. DiI–acetyl-LDL–containing compartments were therefore tested for their accessibility to fucoidin, a high-molecular-weight polysaccharide (40 to 100 kD) with a high affinity for the scavenger receptors.22 The ability of fucoidin to displace acetyl-LDL bound to scavenger receptors on macrophages was first demonstrated in an experiment carried out on ice: cells were incubated on ice with DiI–acetyl-LDL for 30 minutes and then chased in culture medium with or without fucoidin for 2 hours on ice as well. After 2 hours' incubation on ice in the absence of fucoidin, DiI–acetyl-LDL remained on the cell surface (Fig 7ADown). Its distribution was indistinguishable from the surface labeling of cells without a 2-hour chase on ice (not shown). Incubation in the presence of fucoidin for 2 hours on ice, however, displaced most acetyl-LDL from the cell surface (Fig 7BDown). Fucoidin, therefore, was able to release DiI–acetyl-LDL bound to scavenger receptors on the cell surface.



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  		Figure 7. Acetyl-LDL bound to the surface of macrophages at 0°C can be displaced by fucoidin. Cells were labeled with DiI–acetyl-LDL (10 µg/mL) for 30 minutes at 0°C and then incubated with either DMEM (A) or fucoidin/DMEM (500 µg/mL, B) for 2 hours at 0°C. Cells were then fixed with 4% paraformaldehyde/PBS at 0°C for 10 minutes and viewed by fluorescence microscopy. Cells without fucoidin treatment (A) show bright DiI surface labeling. Fucoidin, however, displaced most of the DiI surface labeling (B). Bar=10 µm.

To determine whether fucoidin was able to displace acetyl-LDL associated with macrophages at 37°C, cells were incubated with DiI–acetyl-LDL for 3 minutes and chased in the absence of ligands for another 3 minutes at 37°C. Cells were then rapidly cooled to 0°C and incubated with fucoidin for 2 hours on ice. After 2 hours' incubation in the absence of fucoidin on ice, macrophages demonstrated the characteristic distribution of DiI–acetyl-LDL (Fig 8ADown). The peripheral DiI remained quenchable by TNBS (Fig 8BDown). When fucoidin was added to the medium during 2 hours of incubation on ice, there was little if any change in the DiI–acetyl-LDL distribution compared with control. A large number of fine peripheral dots were observed, in addition to perinuclear lysosomal compartments (Fig 8CDown). When the same cells shown in Fig 8CDown were exposed to TNBS, the peripheral fine dots of DiI fluorescence (Fig 8CDown, arrow), which were not displaceable by fucoidin, were quenched immediately (Fig 8DDown). This showed that fucoidin was not able to displace acetyl-LDL from these plasma membrane structures after an incubation at 37°C.



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  		Figure 8. Peripheral acetyl-LDL incubated at 37°C cannot be displaced by fucoidin but remains accessible to TNBS. Macrophages were labeled with DiI–acetyl-LDL for 3 minutes and chased for 3 minutes at 37°C. Cells were then incubated with only medium (A) or fucoidin+medium (500 µg/mL) (C) for 2 hours at 0°C and fixed in 4% paraformaldehyde/PBS for 10 minutes at 0°C. DiI–acetyl-LDL kept its typical distribution after 2 hours' incubation at 0°C with medium alone (A), and incubation with fucoidin did not change the DiI–acetyl-LDL pattern (C). After adding TNBS (B and D) the peripheral DiI–acetyl-LDL in both sets of cells could be quenched by TNBS (B and D).

Electron Microscopic Characterization of Acetyl-LDL Compartments
To characterize acetyl-LDL–containing compartments at the ultrastructural level, macrophages were incubated with gold-labeled acetyl-LDL, and these cells were examined by electron microscopy. The specificity of gold conjugates was confirmed ultrastructurally by a competition with 50-fold excess of unlabeled acetyl-LDL. When cells were labeled with gold–DiI–acetyl-LDL and observed by fluorescence microscopy, gold conjugates showed the characteristic distribution of DiI–acetyl-LDL in macrophages.

Cells were incubated with gold–acetyl-LDL for 3 minutes at 37°C and chased for 3 minutes in the absence of ligands. Cells were then fixed and sectioned for transmission electron microscopy. Consistent with our fluorescence observations, some of the acetyl-LDL was in endosomes (Fig 9ADown, large open arrow) and lysosomes (Fig 9ADown, long thin arrow) after 3 minutes' chase. Importantly, some of the acetyl-LDL was observed to be retained at the plasma membrane. Surface-retained acetyl-LDL was frequently associated with surface projections (ruffles or microvilli), especially at the base of these projections (curved arrows, Fig 9ADown, 9BDown, and 9CDown). Some of the surface-retained acetyl-LDL was in areas that did not show a definite specialization (Fig 9BDown and 9CDown, small, straight arrows). When macrophages were incubated with gold-LDL for 3 minutes and chased for 3 minutes at 37°C in the absence of ligands, LDL was mainly found in multivesicular bodies, presumably late endosomes (Fig 9DDown). Little LDL remained on the plasma membrane.



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  		Figure 9. Thin-section electron microscopy of colloidal gold–labeled acetyl-LDL and LDL in macrophages. Cells were incubated with 5 µg/mL colloidal gold–acetyl-LDL (A, B, and C) or 10 µg/mL gold-LDL (D) for 3 minutes at 37°C and then chased for 3 minutes in the absence of ligand at 37°C. Cells were then fixed and processed for thin-section electron microscopy. Acetyl-LDL was in both the plasma membrane (B and C; small, straight arrows) and in endosomes and lysosomes (A, open arrows and long thin arrows), respectively. LDL was mainly in multivesicular bodies (D). Acetyl-LDL was often associated with surface projections such as ruffles or microvilli (A, B, and C; curved arrows). Bar=1 µm.

Acetyl-LDL–containing compartments were also examined by IVEM. Macrophages were again incubated with gold–acetyl-LDL for 3 minutes and chased at 37°C for 3 minutes. Cells were then fixed, and whole cells or thick sections were processed for IVEM. Stereo images of whole-cell and thick-section IVEM (Fig 10ADown and 10BDown) provide a more complete view of acetyl-LDL–containing compartments. Many gold–acetyl-LDL particles were apparently concentrated in vesicles, probably early and late endosomes (Fig 10ADown, large arrow). There was, however, a significant amount of acetyl-LDL distributed on the cell surface and along the microvilli (eg, the microvillus just below and to the right of the letter A). The association of surface acetyl-LDL with microvilli and ruffles is shown clearly in Fig 10BDown, a stereo pair of a thick section (0.3 to 0.5 µm) viewed by IVEM. Several of these surface projections have dense labeling with gold particles along their surface. It seems likely that acetyl-LDL associated with microvilli and other surface structures is what was observed at the cell surface in our fluorescence experiments. We did not observe acetyl-LDL in the deep surface invaginations (STEMs) that contain ß-VLDL.



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  		Figure 10. IVEM of colloidal gold–labeled acetyl-LDL and LDL interactions with macrophages. Cells were labeled with colloidal gold conjugates as described in the legend to Fig 9Up. Panel A shows a stereo pair of whole-mount IVEM on macrophages labeled with colloidal gold–acetyl-LDL. There is dense labeling of a microvillus just below the letter A. Acetyl-LDL was seen in late endosomes (open arrow), but a large portion of acetyl-LDL was diffusely distributed on the plasma membrane and along the microvilli. Panel B shows a stereo pair of thick-section IVEM, illustrating association of acetyl-LDL with microvilli and ruffles. Bar=10 µm.

Characterization of Scavenger Receptors in Macrophages
The class A macrophage scavenger receptors bind a wide variety of polyanions, such as fucoidin and dextran sulfate, in addition to modified lipoproteins.21 Recently, a new set of receptors, cataloged as class B, has been cloned.36 These receptors bind to acetyl-LDL, oxidized LDL, and maleylated BSA with high affinity, but not to polyanions.36 Class B receptors may exhibit different endocytosis kinetics than class A receptors. In that case, acetyl-LDL might bind to more than one class of scavenger receptors and therefore display a more complex endocytosis kinetics than LDL in mouse peritoneal macrophages. The inhibition of acetyl-LDL internalization in mouse peritoneal macrophages by fucoidin, dextran sulfate, and maleylated BSA was investigated. In addition to the broad polyanion binding, class A receptors can be distinguished from class B by their weak binding of maleylated BSA. Ligand binding on class B scavenger receptors can be competitively inhibited by a very low concentration of maleylated BSA (4 to 10 µg/mL).36

Macrophages were incubated with DiI–acetyl-LDL alone or in the presence of blocking agents at 37°C for 3 minutes and chased for 3 minutes in ligand-free medium. Cell-associated DiI–acetyl-LDL was measured by quantitative fluorescence microscopy. As shown in Fig 11ADown, 50 µg/mL of fucoidin was able to inhibit more than 80% of acetyl-LDL uptake. Higher concentrations up to 1 mg/mL did not further block the uptake (data not shown). Dextran sulfate (500 kD) inhibited acetyl-LDL uptake in a dose-dependent manner similar to that of fucoidin. Acetyl-LDL uptake was not significantly inhibited by 10 µg/mL maleylated BSA (Fig 11BDown), a concentration that was able to effectively block acetyl-LDL uptake in haSR-BI cells, a CHO cell line transfected with class B hamster scavenger receptors36 (data not shown). Only at higher concentrations (50 to 500 µg/mL) was acetyl-LDL uptake progressively blocked by maleylated BSA. The inhibition by maleylated BSA plateaued at 200 µg/mL (data not shown). When the same competition experiment by maleylated BSA was performed on a CHO cell line (CHO-SRAII) transfected with a murine macrophage scavenger class A receptor, a similar dose-dependent pattern was seen. Maleylated BSA did not block acetyl-LDL uptake at 10 µg/mL in CHO-SRAII cells but inhibited uptake only at higher concentrations (50 to 500 µg/mL). This finding suggested that the receptors responsible for acetyl-LDL uptake in mouse peritoneal macrophages were mainly class A. To rule out the possibility that class B scavenger receptors may exist in a relatively small number on macrophages, a double competition experiment was also performed. Cells were incubated with acetyl-LDL, fucoidin (200 µg/mL), and maleylated BSA (10 µg/mL). Adding maleylated BSA to the competition assay did not cause any further inhibition of acetyl-LDL uptake relative to cells incubated with fucoidin alone, nor did maleylated BSA change the peripheral distribution of acetyl-LDL. It is therefore unlikely that class B scavenger receptors play a significant role in acetyl-LDL uptake in mouse peritoneal macrophages. Class A scavenger receptors were apparently responsible for acetyl-LDL endocytosis in mouse peritoneal macrophages, including the portion that is retained in the cell periphery.



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  		Figure 11. Inhibition of acetyl-LDL uptake by fucoidin or maleylated BSA in macrophages. Cells were labeled with DiI–acetyl-LDL for 3 minutes alone or in the presence of blocking agents and then chased for another 3 minutes at 37°C in ligand-free medium. Cell-associated acetyl-LDL was measured by quantifying total fluorescence intensity per cell. Fluorescence intensity of DiI–acetyl-LDL in the absence of blocking agent was normalized to 1.0. Fucoidin at 50 µg/mL inhibited more than 80% acetyl-LDL uptake (A), and maleylated BSA did not significantly inhibit any (B).


*    Discussion
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 *Discussion
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The results presented here demonstrate that the endocytic clearance of acetyl-LDL from the surface of mouse macrophages is slow compared with the clearance of LDL. Following this slowed clearance, acetyl-LDL joins LDL in the late endosomes/lysosomes. Surface-retained acetyl-LDL appears in fine dots in the cell periphery, and it is accessible from the extracellular space, as demonstrated by TNBS quenching. Quantitative fluorescence measurements further reveal that surface-retained acetyl-LDL (slow component of internalization) accounts for more than 40% of total cell-associated acetyl-LDL. Surface-retained acetyl-LDL is resistant to release by fucoidin. Electron microscopy studies confirmed the fluorescence microscopy results and showed that acetyl-LDL was frequently associated with microvilli or ruffles, especially at the base or intersections of these projections.

It is not immediately clear why fucoidin failed to release surface-retained acetyl-LDL. As shown in Figs 9Up and 10Up, it is not due to acetyl-LDL accumulation in deep invaginations, where it might be inaccessible to fucoidin. Fucoidin can successfully release acetyl-LDL bound to the cell surface when endocytosis is blocked at 0°C. However, after a short 37°C incubation (6 minutes total) fucoidin was unable to displace the surface-retained acetyl-LDL. It is possible that during the incubation at 37°C a conformational change occurred in receptor-ligand complexes, such that fucoidin could no longer displace acetyl-LDL from the receptor. It is also possible that the acetyl-LDL binding becomes multivalent at 37°C, and this could reduce displacement by fucoidin. Surface-retained acetyl-LDL is often associated with microvilli or ruffles (which are abundant in macrophages), especially at the base and intersections of these structures. These membrane deformations may facilitate multivalent binding by allowing more points of contact between an acetyl-LDL particle and receptors in the plasma membrane. The basis for association of acetyl-LDL with surface projections is not known. Some receptors, including unoccupied insulin receptors, preferentially associate with microvilli and are retained there.37 The sequence requirements for this association are not known, but it does depend on portions of the cytoplasmic domain of the insulin receptor. It is possible that class A scavenger receptors have a similar localization signal. Alternatively, the acetyl-LDL bound to the scavenger receptor may weakly interact with other proteins that give it a slight preference for localization on microvilli or ruffles.

In addition to constitutive particle uptake, this prolonged cell-surface retention of acetyl-LDL may contribute to its ability to stimulate ACAT in mouse peritoneal macrophages. Acetyl-LDL and LDL share many physical properties, such as similar cholesterol content and size, but only acetyl-LDL strongly triggers ACAT activity when an equal amount of cholesterol is delivered to cells.18 Despite the difference in uptake kinetics, both LDL and acetyl-LDL are eventually delivered to the lysosomes, where they are hydrolyzed to produce free cholesterol and other lipids. Since free cholesterol produced by lipoproteins in the lysosomes should function independent of its origin, it is reasonable to assume that the ability of atherogenic lipoproteins to stimulate ACAT may be a consequence of differences in endocytic processes before lipoproteins reach lysosomes. Acetyl-LDL does bind to different receptors in macrophages than LDL, and differential activation of signaling pathways could be involved. However, little evidence for signaling by either LDL or acetyl-LDL exists.38 Thus, transient retention in a plasma-membrane-connected structure may be the significant event distinguishing acetyl-LDL from LDL before reaching the lysosomes.

Recent results have indicated that other atherogenic lipoproteins enter macrophages through large surface-connected structures that differ from the classical clathrin-coated-pit endocytic pathway. ß-VLDL was shown to enter and be retained in surface-connected tubules (STEMs) in mouse peritoneal macrophages.14 More recently, Kruth et al39 reported that in human monocyte derived macrophages, acetyl-LDL was sequestered in very large plasma-membrane-connected compartments before its eventual delivery to lysosomes. In the case of ß-VLDL, the propensity to be retained in STEMs was correlated positively with the ability to stimulate ACAT when different size fractions of ß-VLDL were examined or when the effective valence of ß-VLDL was altered by treatment with apoE antibodies.16 This finding suggested that the retention of lipoproteins in plasma-membrane-connected structures may facilitate cholesterol esterification in macrophages.

How could prolonged cell-surface contact affect the ACAT stimulatory properties of lipoproteins? One possibility is that there is direct delivery of cholesterol or lipid from the lipoprotein particles to the plasma membrane. A similar surface exchange, in the reverse direction, has been proposed for HDL extraction of cholesterol from the plasma membrane.40 HDL is also able to transfer its cholesterol content to hepatic cells, steroidogenic tissue, and adipose tissue, apparently through its interaction with plasma membrane.41 42 Macrophages have been shown recently to secrete sphingomyelinase,43 and this enzyme could potentially act on sphingomyelin in the ß-VLDL or acetyl-LDL particles to destabilize the lipoprotein and facilitate release of its cholesterol to the plasma membrane. There is experimental evidence that ß-VLDL particles undergo structural disintegration while in STEMs.14 When ß-VLDL was double labeled with two fluorescence probes (DiI and DiO), the extent of energy transfer between the two probes decreased while ß-VLDL was in STEMs, indicating particle breakdown. In this hypothetical model, catabolism of lipoproteins at the cell surface may release cholesterol or other lipids directly into the plasma membrane, where they have a greater signaling effect than if delivered to lysosomes. It is noteworthy that prolonged interaction of lipoproteins with the plasma membrane may also be characteristic of the lipoprotein uptake by macrophages in the wall of blood vessels, since the lipoproteins become enmeshed in the extracellular matrix.

There is evidence that ACAT is stimulated by net expansion of cellular cholesterol pools above a threshold level.18 The lipid composition of the plasma membrane was shown to affect this threshold.44 Cholesterol delivered directly to the plasma membrane could affect cholesterol trafficking to ACAT.45 For instance, LDL-derived cholesterol effluxes rapidly out of cells and stimulates ACAT poorly, whereas cholesterol from ß-VLDL or acetyl-LDL is mainly converted to CE by ACAT.18 Although lysosome-derived free cholesterol appears rapidly in the plasma membrane11 and is required for ACAT activation,46 47 48 this cholesterol can either be retained or released from cells. A small fluctuation of cholesterol on the plasma membrane (due to direct cholesterol transfer from lipoproteins, for example) might send a signal for cells to retain lysosome-derived cholesterol, which consequently leads to ACAT activation. These speculations will require extensive experimental testing, but they suggest a possible mechanism for surface retention to affect ACAT stimulation.

In summary, we have investigated in detail the internalization pathway of acetyl-LDL in mouse peritoneal macrophages. We found that a significant amount of acetyl-LDL was retained on the plasma membrane before its eventual delivery to the lysosomes. Although a direct mechanistic link between the surface retention and ACAT activity is not yet established, our results, along with the earlier observations from ß-VLDL,14 provide a strong argument for the importance of lipoprotein/macrophage interaction at the plasma membrane level. Further studies on other atherogenic lipoproteins, such as LDL aggregates, should help to clarify the nature of this interaction.


*    Selected Abbreviations and Acronyms
 
{alpha}2M = {alpha}2-macroglobulin
ACAT = acyl-coenzyme A:cholesterol acyltransferase
CE = cholesteryl ester
DiI = 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate
DMEM = Dulbecco's modified Eagle's medium
IVEM = intermediate-voltage electron microscope
STEM = surface tubule for entry into macrophages


*    Acknowledgments
 
This work was supported by National Institutes of Health grants HL-39703 and HL-21006 (I. Tabas), HL-41990 (N.L. Jones), and DK-27083 (F.R. Maxfield), by the Aaron Diamond Foundation (F.R. Maxfield and P.L. Leopold), and by an American Heart Association New York City Affiliate Fellowship (X. Zha). The authors are grateful to Drs Paul Kussie and Alan Tall (Department of Medicine, Columbia University) for the cholesteryl ester transfer protein, Dr Monty Krieger (MIT) for the CHO-mSRAII and CHO-haSRBI cell lines, and Drs S. Silverstein and J.B. El-Khoury (Columbia University) for the maleylated BSA.

Received June 24, 1996; accepted September 6, 1996.


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