© 1997 American Heart Association, Inc.
Articles |
Evidence for Prolonged Cell-Surface Contact of Acetyl-LDL Before Entry Into Macrophages
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
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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
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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.
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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." 
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.
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Results |
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Morphological Analysis of the Endocytic Pathway of Fluorescently Labeled Acetyl-LDL, LDL,
2M,
and ß-VLDLThe 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 1
. After 3 minutes' chase, LDL was
concentrated in perinuclear compartments, mainly late endosomes or
lysosomes15 (Fig 1D, arrows). Much of the
acetyl-LDL was colocalized with LDL in these perinuclear vesicular
structures at the end of the 3-minute chase (Fig 1A, arrows). There
was, however, a significant portion of the acetyl-LDL that remained in
the cell periphery (Fig 1A, arrowheads). The amount of
peripheral acetyl-LDL gradually decreased at longer chase
times (Fig 1B and 1C), 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 1C).
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Like LDL, 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-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, 2M was
concentrated in the perinuclear region after 3 minutes' chase,
colocalizing with part of acetyl-LDL (Fig 2A and 2B, arrowhead), and there was again a
significant portion of acetyl-LDL in the cell periphery (Fig 2A, arrow). Labeling of several processes at the edge of the cells is
clearly visible in the DiI–acetyl-LDL fluorescence image (Fig 2A).
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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 3, most of the ß-VLDL was seen in the
cell periphery after 3 minutes' chase (Fig 3B), and little appeared in
the perinuclear region. Acetyl-LDL, on the other hand, was located in
both the cell periphery (Fig 3A, arrows) and the perinuclear region
(Fig 3A, arrowheads). Moreover, within the cell periphery, acetyl-LDL
appeared as a large number of fine dots (Fig 3A, arrows), while
ß-VLDL was in relatively fewer but brighter peripheral
compartments (Fig 3B). 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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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 4A) and immediately after (Fig 4D)
addition of TNBS. DiI–acetyl-LDL was located in both
peripheral and perinuclear regions before adding TNBS (Fig 4A). After exposure to TNBS, however, the fine dots of
peripheral DiI–acetyl-LDL fluorescence (Fig 4A, arrows) were heavily quenched (Fig 4D, arrows) showing the presence of
DiI on the outside of the cell. As expected, TNBS had little effect on
perinuclear DiI-containing compartments (Fig 4A and 4D, 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 4B and 4E) or 20 minutes (Fig 4C and 4F). 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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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 5, little DiI-LDL fluorescence
was quenched by TNBS (Fig 5A and 5B), 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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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 6, 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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) or DiI-LDL (
) 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 6
, 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 7A). 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 7B). Fucoidin, therefore, was able to release DiI–acetyl-LDL
bound to scavenger receptors on the cell surface.
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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 8A). The peripheral DiI
remained quenchable by TNBS (Fig 8B). 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 8C). When the same
cells shown in Fig 8C were exposed to TNBS, the peripheral
fine dots of DiI fluorescence (Fig 8C, arrow), which were not
displaceable by fucoidin, were quenched immediately (Fig 8D). 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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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 9A, large open arrow)
and lysosomes (Fig 9A, 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 9A, 9B, and 9C). Some of the surface-retained acetyl-LDL was in areas that
did not show a definite specialization (Fig 9B and 9C, 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 9D). Little LDL remained on the plasma membrane.
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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 10A and 10B) 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 10A, 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 10B, 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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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 11A, 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 11B), 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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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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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 9 and 10, 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.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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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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