Elevated Plasma Membrane Cholesterol Content Alters
Macrophage Signaling and Function
Chunbo Qin, Tomokazu Nagao, Inna Grosheva, Frederick R. Maxfield, Lynda M. Pierini
Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
Objective—During atherogenesis, macrophages migrate into the subendothelial space where they ingest deposited
lipoproteins, accumulate lipids, and transform into foam cells. It is unclear why these macrophages do not remove their
lipid loads from the region. This study was aimed at testing the hypothesis that macrophage behavior is altered when
membrane cholesterol levels are elevated, as might be the case for cells in contact with lipoproteins within
atherosclerotic lesions.
Methods and Results—We examined the effects of elevating membrane cholesterol on macrophage behavior. J774
macrophages were treated with either acetylated low-density lipoprotein (ac-LDL) and ACAT inhibitor or cholesterol-chelated methyl-␤-cyclodextrin (chol-M␤CD) to increase membrane cholesterol levels. Our results show that
elevating the membrane cholesterol of J774 macrophages induced dramatic ruffling, stimulated cell spreading, and
affected F-actin organization. Cellular adhesion was required for these effects, and Rac-mediated signaling pathways
were involved. Additionally, 3-dimensional transwell chemotaxis assays showed that migration of J774 macrophages
was significantly inhibited when membrane cholesterol levels were raised.
Conclusions—These findings indicate that increased membrane cholesterol causes dramatic effects on macrophage cellular
functions related to the actin cytoskeleton. They should provide new insights into the early steps of atherogenesis.
(Arterioscler Thromb Vasc Biol. 2006;26:372-378.)
Key Words: actin 䡲 atherosclerosis 䡲 cholesterol 䡲 macrophages 䡲 migration
A
generation, and inhibition of cell migration.5,6 These findings
suggest that the atherosclerotic environment, and specifically
interactions with modified lipoproteins, can explain the retention of macrophages in lesions. However, it is still unclear
how interactions with modified lipoproteins alter macrophage
function: Can the changes in actin organization and migration
be attributed to a signaling event initiated by binding of
modified lipoproteins to their receptor(s)? Or are they a result
of modulations in cellular cholesterol content after cholesterol transfer from the lipoproteins? One particularly interesting
observation is that as macrophages begin to engulf aggregated matrix-bound LDL particles, they maintain prolonged
contact with the LDL aggregates.7 During this prolonged
contact there can be rapid transfer of cholesterol and cholesteryl esters from the LDL aggregates to the macrophage,
and transient increases in the cholesterol content of macrophage plasma membranes may result. Because reduction in
membrane cholesterol levels has been shown to affect many
cellular processes,8 –14 presumably by altering membrane lipid
organization, we postulated that increasing membrane cholesterol content may likewise affect cell function. If elevated
membrane cholesterol does indeed alter cell function, then
this may explain why macrophages within atherosclerotic
lesions are unable to egress from the affected area.
lthough it has long been accepted that elevated serum
cholesterol levels correlate with development of atherosclerotic lesions, the reasons for this are imperfectly understood. It is known that the cholesterol carrier, low-density
lipoprotein (LDL), normally found circulating in the blood, is
deposited in the subendothelial space.1,2 These LDL deposits
undergo aggregation, association with extracellular matrix
materials, and oxidation.3,4 Monocytes that have traversed the
endothelium then engulf the matrix-bound aggregated lipoprotein particles and develop into lipid-filled macrophages
(known as foam cells). Macrophage lipid accumulation, and
subsequent foam cell formation, promotes growth of the
atherosclerotic plaque, whereas lesion regression, induced
pharmacologically or through diet, may be attributed partly to
foam cell migration out of the area. This synopsis illustrates
the critical role of cell migration in the progression of
atherosclerosis and suggests that understanding the causes of
macrophage accumulation within the atherosclerotic lesion is
important for developing strategies to curtail the progression
and/or promote the regression of the disease. Currently, it is
unclear why macrophages that enter atherosclerotic lesions
remain there rather than depart with their lipid loads.
In vitro, treatment of macrophages with modified LDL
caused alterations in actin organization, reduction of force
Original received August 23, 2005; final version accepted November 9, 2005.
From the Departments of Biochemistry (C.Q., T.N., I.G., F.R.M.) and Surgery (L.M.P.), Weill Medical College of Cornell University, New York, NY.
Correspondence to Lynda M. Pierini, Department of Surgery, Box 287, Weill Medical College of Cornell University, New York, NY 10021. E-mail
[email protected]; or Frederick R. Maxfield, Department of Biochemistry, Box 63, Weill Medical College of Cornell University, New York, NY
10021. E-mail [email protected]
© 2006 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
372
DOI: 10.1161/01.ATV.0000197848.67999.e1
Qin et al
Increasing Cholesterol Affects Macrophage Behavior
As a first step to test this hypothesis, we investigated how
global increases in membrane cholesterol modify macrophage biology, using cholesterol delivery to cells via methyl␤-cyclodextrin rather than delivery by lipoproteins. This
allowed us to examine the effects of cholesterol elevation in
the absence of possible effects from binding of apolipoproteins to their receptors. Interestingly, we found that elevated
levels of membrane cholesterol affect F-actin organization
and F-actin– dependent functions, but these changes in the
actin cytoskeleton can be inhibited by blocking scavenger
receptors even when cholesterol is delivered via
methyl-␤-cyclodextrin.
Methods
Cells and Cell Culture
Mouse macrophage cell line J774A.1 was obtained from the American Type Culture Collection. See http://atvb.ahajournals.org for
details on growth and maintenance of the cells.
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Lipoproteins and Reagents
Human LDL was prepared as previously described.15 Acetylated
LDL (Ac-LDL) was prepared by acetylation of LDL with acetic
anhydride as described previously.16 ACAT inhibitor 58035, originally from Sandoz Inc, was kindly provided by Dr Ira Tabas.
Methyl-␤-cyclodextrin (M␤CD) and cholesterol-chelated methyl-␤cyclodextrin (as “water-soluble cholesterol,” molar ratio 1:6 cholesterol/M␤CD) were purchased from Sigma. All treatment concentrations involving cholesterol-chelated-M␤CD (chol-M␤CD) were
based on the weight of M␤CD. Fucoidan and Clostridium difficile
toxin B were purchased from Sigma and Calbiochem, respectively.
Phalloidin Staining and Filipin Staining
To visualize F-actin, J774 cells were simultaneously fixed, permeabilized, and labeled by incubation with 3.3% paraformaldehyde,
0.05% glutaraldehyde, 0.25 mg/mL saponin, and 2 U/mL AlexaFluor
488 phalloidin (Molecular Probes) in phosphate-buffered saline for
15 minutes at room temperature. Images were then acquired with a
Zeiss LSM510 laser scanning confocal microscope, or a Leica
DMIRB widefield microscope equipped with a Princeton Instruments cooled charge-coupled device camera driven by MetaMorph
Imaging System software (Universal Imaging Corporation, Downingtown, Pa).
To quantify changes in cellular free cholesterol, cells were fixed
with 3.3% paraformaldehyde for 15 minutes and then incubated with
50 ␮g/mL filipin (Sigma) for 1.5 hours at room temperature.
Wide-field images were then obtained. Details on quantitative
analysis of phalloidin- or filipin-stained cells can be found in the
supplemental materials.
Macropinocytosis Assay
J774 cells, grown on polylysine-coated glass coverslip-bottom dishes
for 16 hours, were first treated with 5 mmol/L chol-M␤CD for
indicated times or left untreated, then incubated with 1 mg/mL
TRITC-conjugated Dextran (160 kDa; Sigma) for 15 minutes before
fixation.17 Wide-field fluorescence images of all samples were
acquired under identical conditions for quantitative analysis using
MetaMorph. The images were thresholded so that only dextranpositive endosomes were included, and all images were thresholded
to the same scale. The integrated fluorescence in the thresholded area
was measured and divided by the total number of cells to yield a
measure of fluorescence intensity per cell.
Scanning Electron Microscopy
J774 cells in suspension were left untreated or treated with 5 mmol/L
chol-M␤CD for 30 minutes at 37°C, and then fixed for 10 minutes by
addition of equal volume of 8% paraformaldehyde, 5% glutaralde-
373
hyde, and 0.04% picric acid in 0.1 mol/L sodium cacodylate buffer,
pH 7.3. Samples were then prepared for scanning electron microscopy as described in the supplemental materials.
Immunofluorescence
For costaining of Rac and F-actin, J774 cells were fixed with 3.3%
paraformaldehyde in the presence of 0.25 mg/mL saponin and 1
U/mL AlexaFluor 488 phalloidin for 15 minutes at room temperature. Excess AlexaFluor 488 phalloidin was included throughout the
remaining labeling steps. Rac was visualized with a mouse monoclonal antibody against Rac1 (clone 102; BD Biosciences Pharmingen), followed by an AlexaFluor 546-conjugated goat anti-mouse
secondary antibody (Molecular Probes). Images were then acquired
with a Zeiss LSM510 laser scanning confocal microscope using a
63⫻1.4 numeric aperture objective.
Determination of Activated Rac
The PAK pull-down assay was performed using a Rac activation
assay kit (Cytoskeleton) following the manufacturer’s instruction.
Briefly, cells were grown to ⬍ 80% confluence on 100 mm tissue
culture dishes (Falcon). After chol-M␤CD treatment, cells were
lysed in lysis buffer, clarified by centrifugation, and incubated with
GST-PAK beads for 1 hour at 4°C. Bound Rac was analyzed by
SDS-PAGE separation on a 4% to 12% polyacrylamide gel, and
visualized by Western blotting with monoclonal anti-Rac1 antibody
and HRP-conjugated goat anti-mouse IgG (Sigma) using the ECL
system (Pierce). For normalization, whole cell lysates were run in
parallel.
Macrophage Migration Assay
Migration of J774 cells was tested in a chemotaxis assay using
Transwell Inserts (Costar, Cambridge, Mass) with 5-␮m polycarbonate filter inserts. Further details regarding the conditions used for the
migration assay are provided in the supplemental materials.
Results
Cholesterol Loading by Ac-LDL and ACAT
Inhibitor Induces Membrane Ruffles and
Stimulates Cell Spreading
To load cell membranes with cholesterol, we treated J774
macrophages with 50 ␮g/mL ac-LDL and 10 ␮g/mL ACAT
inhibitor 58035 for 5 hours. Ac-LDL no longer binds to LDL
receptors, but it is taken into cells by scavenger receptors,
which are not subject to down regulation by the level of
cellular cholesterol.16,18 Ac-LDL uptake strongly triggers
ACAT activity when cellular free cholesterol reaches certain
threshold levels.19 Thus, cotreatment with ac-LDL and ACAT
inhibitor leads to the accumulation of free cholesterol in the
cells. Under the conditions used in these experiments, the
cellular free cholesterol level in treated cells was increased
⬇2-fold over control cells, as determined by filipin staining
(data not shown). To assess the validity of fluorescence image
analysis of filipin-stained cells as a quantitative measure of
cellular cholesterol content, the results from this technique
were compared with those obtained by gas chromatographic
analysis of cellular lysates.20 Both techniques yielded similar
results (Figure I, available online at http://atvb.ahajournals.
org), demonstrating that quantitative fluorescence imaging of
filipin-stained cells is an accurate method for evaluating
cellular free cholesterol levels under conditions used in this
study.
To observe the effects that treatment with ac-LDL and the
ACAT inhibitor had on macrophage biology, treated and
control cells were plated onto fibronectin-coated dishes for 10
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Figure 1. Loading plasma membranes of macrophages with
cholesterol by ac-LDL and ACAT inhibitor treatment stimulated
cell spreading and membrane ruffling. J774 macrophages were
treated with 50 ␮g/mL ac-LDL and 10 ␮g/mL 58035 for 5 hours
then plated onto fibronectin-coated dishes for 10 minutes
before fixation and staining for F-actin. Arrows indicate membrane ruffles. Scale bar, 10 ␮m.
minutes, fixed, and stained for F-actin. Figure 1 shows that
cells treated with ac-LDL and the ACAT inhibitor become
more spread and form more F-actin-rich membrane ruffles
than untreated cells, whereas neither ac-LDL nor the ACAT
inhibitor alone could cause these morphological changes
(data not shown). These results suggest that raising the
cholesterol content in the plasma membranes of macrophages
alters their biology.
Increased Cell Spreading and Membrane Ruffling
in Ac-LDL/ACAT Inhibitor-Treated Cells Can Be
Attributed to Increases in Free Cholesterol
Although treatment with ac-LDL and the ACAT inhibitor
induced a clear response in J774 macrophages, it was not
clear if the spreading and ruffling were caused by signals
initiated by ac-LDL, the ACAT inhibitor, or cholesterol
loading. To determine whether raising membrane cholesterol
levels, rather than signaling via the cholesterol delivery
system (either the ac-LDL or the ACAT inhibitor), caused the
observed effects, we used cholesterol-chelated methyl-␤cyclodextrin (chol-M␤CD) to acutely raise the cholesterol
content of the plasma membrane. Treatment with 5 mmol/L
chol-M␤CD for either 15 or 30 minutes increased total
cellular cholesterol levels by 1.5- to 2-fold (Figure 2A),
which results in levels comparable to those obtained after
treatment with ac-LDL and the ACAT inhibitor. These
changes in membrane cholesterol stimulated cell spreading,
with the average cell area for cells treated with chol-M␤CD
for 15 or 30 minutes increasing by 45% or 60%, respectively,
compared with control cells (Figure 2A). The effects of
cholesterol loading on cell spreading were readily reversed
when membrane cholesterol was returned to starting levels by
subsequent treatment with M␤CD, and there is a clear
correlation between the cellular cholesterol content and the
extent of cell spreading (Figure 2A). As for cells treated with
ac-LDL and the ACAT inhibitor (Figure 1), J774 cells that
were treated with chol-M␤CD for 15 minutes and then plated
Figure 2. Increasing membrane cholesterol reversibly induced
cell spreading and membrane ruffling. (A) Modulation of membrane cholesterol caused corresponding changes in cell area.
J774 cells, grown on polylysine-coated dishes overnight, were
left untreated (control), treated with 5 mmol/L chol-M␤CD (⫹), or
treated sequentially with 5 mmol/L Chol- M␤CD (⫹) and then
10 mmol/L M␤CD (⫺) for indicated times before fixation. Cells
were then stained with filipin to determine the relative free cholesterol levels per cell (gray bars) or labeled with a fluorescent
conjugate of phalloidin to delineate the cell periphery for cell
area measurements (white bars). Quantification of fluorescence
images is described in supplemental materials. Data represent
means⫾SD from at least 3 independent experiments. (B) Raising membrane cholesterol by treatment with chol-M␤CD
induced membrane ruffling (arrowheads). J774 macrophages
were left untreated (control) or treated with 5 mmol/L cholM␤CD for 15 minutes (cholesterol-loaded) in suspension, and
then plated onto fibronectin-coated dishes for 10 minutes
before fixation and staining for F-actin. Projected confocal
images formed from 20 0.7-␮m confocal slices are presented.
Scale bar, 10 ␮m.
onto fibronectin-coated dishes for 10 minutes exhibited
F-actin-rich ruffles (Figure 2B). Confocal fluorescence images of the lower adherent surfaces of control and cholesterol-loaded cells show that F-actin is accumulated at the edges
of the cholesterol-loaded J774 cells but not the control cells
(Figure IIA, available online at http://atvb.ahajournals.org),
and quantification of the F-actin at the cell edge shows that
cholesterol-loaded cells have approximately twice the amount
of F-actin near the cell edges compared with control cells
(Figure IIB). Interestingly, although cholesterol loading
caused increased membrane ruffling, there was no apparent
increase in the total F-actin content (compare the intensity of
the phalloidin staining in control versus treated cells in
Figures 1 and 2B). Quantification of the total F-actin content
in control and chol-M␤CD-treated J774 cells confirmed that
there was no increase in the total amount of F-actin after
chol-M␤CD treatment, implying that there was a change in
the organization and/or localization of F-actin but not an
increase in actin polymerization (Figure IIB). These results
show that increasing membrane cholesterol levels causes
morphological and cytoskeletal changes in macrophages.
Qin et al
Increasing Cholesterol Affects Macrophage Behavior
375
Figure 3. Increasing membrane cholesterol stimulated macropinocytosis. J774 cells were left untreated (control), or treated
with 5 mmol/L chol-M␤CD for indicated times (chol-loaded),
then incubated with 1 mg/mL TRITC-Dextran for 15 minutes
before fixation. Data shown are from 1 representative experiment of 3. Error bars represent SD.
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Cholesterol Loading Induced
Macropinocytosis Activity
Because one obvious effect of increasing membrane cholesterol levels on J774 macrophages was the induction of
membrane ruffles, we predicted that macropinocytosis activity of J774 cells might also be induced on cholesterol loading.
To test the effects of cholesterol loading on macropinocytosis, control or chol-M␤CD–treated cells were incubated with
TRITC-conjugated dextran for 15 minutes. The samples were
then fixed, and fluorescence images were taken for quantitative analysis. As shown in Figure 3, the amount of fluorescent
dextran taken up by the cells was dramatically increased
when the J774 cells were cholesterol-loaded, indicating that
increasing membrane cholesterol levels causes an increase in
membrane pinocytic activity.
Effects of Cholesterol Loading on Membrane
Ruffling and F-Actin Organization Require
Cell Adhesion
The results presented suggested that cholesterol itself might be
acting as a signaling molecule. However, because our experiments
were performed on adherent cells, it was also possible that cholesterol loading was potentiating signals initiated by adhesion molecules. To determine whether cell adhesion played a role in the
cellular changes induced by cholesterol loading, we altered membrane cholesterol levels of J774 cells in suspension, and then fixed
the samples for analysis by scanning electron microscopy or by
confocal fluorescence microscopy after staining with fluorescent
phalloidin (Figure 4A). We found no obvious differences in the
surface morphologies or F-actin organization of control and cholM␤CD–treated cells, indicating that the effects of increased membrane cholesterol on membrane ruffling and F-actin reorganization
required cell adhesion and increasing cellular cholesterol was not
sufficient to induce this effect by itself.
It has been shown that macrophage adhesion in vitro is
mediated in large part by ␤2 integrins and scavenger receptor
A.21 To test the role of these adhesion molecules, we used
specific inhibitors to block their contribution during cholesterol loading. Our data suggest that RGD-binding integrins
and ␤2 family integrins do not contribute to the cellular
changes induced by cholesterol loading (Figure III, available
Figure 4. Signaling via scavenger receptor-mediated adhesion
or ligation is required for cellular responses to cholesterol loading. (A) J774 macrophages in suspension did not show morphological changes when treated with chol-M␤CD. Cells were either
left untreated (control) or treated with 5 mmol/L chol-M␤CD for
30 minutes (cholesterol-loaded) before fixation. Scanning electron micrographs (SEM, top panels) and projected confocal
images of F-actin staining (lower panels) are presented. (B) A
general scavenger receptor inhibitor, fucoidan, blocked the
effects of cholesterol loading. J774 cells were grown on
polylysine-coated dishes overnight, in the presence (right panel)
or absence (left and middle panels) of 250 ␮g/mL fucoidan. The
cells were either left untreated (control), or treated with
5 mmol/L chol-M␤CD for 30 minutes (chol-loaded) with or without fucoidan. Projected confocal images of phalloidin-stained
cells are presented. Scale bar, 10 ␮m.
online at http://atvb.ahajournals.org). In contrast, the ruffling
and spreading induced by cholesterol loading were inhibited
by fucoidan (Figure 4B), indicating that cellular attachment
via receptors other than scavenger receptors coupled with
cholesterol-loading is not sufficient to mediate the observed
cellular changes. To determine whether ligation of scavenger
receptors and cholesterol loading would be sufficient to
induce ruffling in the absence of cell attachment, we treated
cells in suspension with fucoidan (a scavenger receptor
ligand) and loaded them with cholesterol. This treatment did
not induce any significant ruffling in suspended cells (Figure
IV, available online at http://atvb.ahajournals.org), further
indicating that signaling via scavenger receptor attachment
and/or ligation of scavenger receptors by an unknown molecule in the substratum is involved.
Rac Is Activated and Recruited to Membrane
Ruffles When Membrane Cholesterol Levels
Are Increased
To begin to understand which signaling steps elevated membrane cholesterol levels affect, we investigated the effects of
cholesterol modulation on Rac activation because it is wellestablished that Rho GTPases play important roles in actin
polymerization. Rac activation has been particularly associated with the formation of membrane ruffles.22,23
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First, we used indirect immunofluorescence to localize endogenous Rac1 in control and chol-M␤CD–treated macrophages
using both J774 cells (Figure 5A), as well as primary human
monocyte-derived macrophages (Figure V, available online at
http://atvb.ahajournals.org). In control J774 cells, Rac 1 (shown
in red) had a patchy distribution within the cell cytosol; Rac1
was found bounded within the F-actin (shown in green) and,
even within the few visible membrane extensions (arrows), it did
not colocalize with F-actin (note the lack of yellow color in
“control” panel). In contrast, after cells were treated with
chol-M␤CD, Rac1 was found almost entirely in membrane
ruffles (arrows), and it colocalized extensively with F-actin (note
yellow color in “cholesterol-loaded” panel.) Similar results were
observed in human monocyte-derived macrophages (Figure V).
The membrane recruitment of Rac1 after chol-M␤CD treatment
suggests that elevating membrane cholesterol levels leads to Rac
activation.
The activation of Rac was further confirmed using a pulldown assay with the Rac-GTP binding domain of PAK.24 Figure
5B shows that Rac1 was activated within 1 minute of cholM␤CD treatment, peaked at ⬇15 minutes, and started to
decrease at 30 minutes. In addition, treatment with Clostridium
difficile toxin B completely abolished the ruffling stimulated by
membrane cholesterol loading (Figure 5C). Toxin B specifically
monoglucosylates and inactivates Rho-family GTPases,25 and it
has been a commonly used inhibitor to study the cellular
function of Rac.26 –28 Taken together, these results suggest that
Rac-mediated signaling pathways, possibly in concert with other
Rho-family GTPases, are involved in the observed morphological changes induced by cholesterol loading.
Migration Is Inhibited in Macrophages With
Elevated Membrane Cholesterol Levels
These results demonstrate that raising membrane cholesterol
levels causes changes in cellular morphology and the organization of F-actin in macrophages. Because cell morphology and
F-actin organization are vital components of cell migration, and
because cell migration is an important factor in atherogenesis,
we investigated the effects of cholesterol loading on macrophage
migration. We used 3-dimensional Transwell migration assays
to study the effects of cholesterol loading on J774 macrophage
chemotaxis. J774 cells were first treated with 5 mmol/L cholM␤CD for 15 minutes, and then induced to migrate in response
to 10 nM recombinant C5a.29 After 2 hours, cells that had
migrated through the Transwell filter were fixed and counted.
Figure 6 shows that the migration of cholesterol-loaded J774
macrophages was inhibited by 60% compared with control cells,
and this inhibition could be completely reversed when the
membrane cholesterol of cholesterol-loaded cells was reduced
by subsequent treatment with M␤CD.
Discussion
Numerous studies have investigated the effects of reducing membrane cholesterol levels on signal transduction, but far fewer have
looked at the effects of the potentially more physiologically relevant
event of increasing membrane cholesterol levels. Here we have
investigated the effects of raising cellular cholesterol on cellular
functions related to the actin cytoskeleton. Specifically, we have
shown that raising the cholesterol content in macrophage mem-
Figure 5. Rac GTPase was activated upon membrane cholesterol
loading. (A) Rac (labeled in red) was recruited to ruffling membranes in cholesterol-loaded macrophages. J774 cells grown on
polylysine-coated dishes were left untreated (control) or treated
with 5 mmol/L chol-M␤CD for 30 minutes (chol-loaded) before fixation and immunofluorescence. Green color represents F-actin.
Projected confocal images are presented. (B) Rac was activated in
cholesterol-loaded cells. J774 cells grown on tissue culture dishes
(107 cells/dish) were left untreated (control) or treated with
5 mmol/L chol-M␤CD (chol-loaded) for indicated times before harvest. A PAK pull-down assay was used to detect the amount of
activated Rac under each treatment condition. A representative
Western blot is shown. (C) Toxin B treatment completely abolished
the ruffling stimulated by cholesterol-loading in J774 macrophages.
J774 cells, grown on polylysine-coated dishes overnight, were left
untreated (control), treated with 5 mmol/L chol-M␤CD for 30 minutes (chol-loaded), or treated with 5 mmol/L chol-M␤CD and 10
ng/mL toxin B for 30 minutes (chol-loaded⫹toxin B). The cells
were then fixed and stained for F-actin. Projected confocal images
are presented. Scale bar, 10 ␮m.
branes affects Rac-mediated F-actin organization, increases membrane pinocytic activity, and decreases cell migration. These
changes are similar to those observed when macrophages were
treated with modified lipoproteins,5,6 suggesting that the changes
induced by modified lipoproteins can be attributed, in part, to
alterations in cellular cholesterol levels.
A key pathogenic event in the development of atherosclerosis
is the retention of lipoprotein particles in the subintima.30 These
lipoprotein particles subsequently undergo aggregation, association with extracellular matrix proteoglycans, and oxidative
modification.31 The physiological significance of the findings
presented in this study becomes evident at the stage of atherosclerotic lesion formation when monocytes/macrophages first
contact aggregated matrix-bound LDL. Previous studies have
shown that macrophage interactions with aggregated, matrixbound LDL particles initiate specific cellular events that can lead
to increases in membrane cholesterol levels.7,32 Specifically,
macrophage uptake of aggregated matrix-bound LDL was found
to be slow compared with aggregated lipoproteins that are not
matrix-bound; during the slow internalization process, matrixbound LDL was found to reside for extended times within deep
Qin et al
Increasing Cholesterol Affects Macrophage Behavior
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Figure 6. Elevated membrane cholesterol inhibited the Transwell
migration of J774 cells. J774 cells were loaded with cholesterol
by incubation with 5 mmol/L chol-M␤CD for 15 minutes (cholloaded). To test the reversibility of this treatment, an aliquot of
cholesterol-loaded cells was subsequently treated with
10 mmol/L M␤CD for 10 minutes to lower cholesterol levels
(reversal). The migration of untreated, cholesterol-loaded, and
cholesterol-reversed cells was tested in a Transwell migration
assay in response to 10 nM C5a. Results were normalized to
the number of untreated (control) cells that had migrated across
the Transwell filter after incubation for 2 hours. Data shown are
from one representative experiment. Error bars represent SD.
invaginations in the macrophage cell surface.7 Because the rate
of cholesterol ester hydrolysis was found to greatly exceed the
rate of protein degradation during the uptake of aggregated
matrix-bound LDL, it is thought that the prolonged contact of
the aggregates with the macrophage cell surface allows selective
uptake of cholesterol ester from the extracellular lipoprotein.7 In
other words, there is transfer of cholesterol ester directly from
LDL aggregates to the macrophage plasma membrane, rapid
cholesterol ester hydrolysis, and a consequent increase in membrane cholesterol levels. Additional studies showed that uptake
of aggregated matrix-bound LDL requires the actin-myosin
cytoskeleton, presumably for extension of macrophage plasma
membrane around the matrix-retained aggregates in a process
reminiscent of phagocytosis.32 We show here that when membrane cholesterol levels are elevated, there is an increase in
actin-mediated membrane activities, including membrane ruffling and macropinocytosis. This observation is consistent with a
recent finding that ACAT1-deficient peritoneal macrophages,
which accumulate free cholesterol on ac-LDL treatment, appeared to have increased surface activities.33 Based on these
observations, one can propose a model for a pathogenic circle of
events: First, macrophages come into contact with matrix-bound
LDL aggregates, cholesterol ester transfer occurs, macrophage
membrane cholesterol levels increase, and actin-mediated membrane extensions are stimulated. This causes a broader area of
cell-surface contact with the aggregates, more cholesterol ester
transfer, a further increase in membrane cholesterol levels,
additional membrane extensions, greater cell-surface contact
with the aggregates, and so on. This scenario could explain, in
part, why macrophages within atherosclerotic lesions continue to
take up retained lipoproteins and become foam cells.
How Does Cholesterol Induce Cellular Changes?
Cholesterol loading does not increase actin-based ruffling in
suspended cells, and the ruffling induced by cholesterol loading
of attached cells is blocked by incubation with fucoidan, a ligand
for scavenger receptors. These data indicate that cholesterol
377
itself does not serve as a signaling molecule to induce the effects
observed in this study. Instead, increasing membrane cholesterol
levels may alter plasma membrane organization in a way that
potentiates signaling by adhesion molecules. Our data suggest
that scavenger receptors contribute to the cholesterol-sensitive
signals that lead to changes in cell function. In this regard it is
interesting to note that scavenger receptors can activate signaling
pathways leading to actin polymerization and focal adhesion
formation, presumably by binding ligands in the extracellular
matrix.34
The Rho family GTPases, Rho, cdc42, and Rac, are known to
be major mediators of signaling leading to actin reorganization.35,36 The Rho GTPases are thought to regulate the formation
of distinct actin filament-containing structures. Because the
morphological responses in macrophages induced by membrane
cholesterol loading resembled the effects of Rac activation, we
investigated the involvement of Rac signaling in cholesterolinduced membrane ruffling. By each of 3 different methods
(PAK pull-down assay, visualization of Rac recruitment to the
plasma membrane, and Toxin B inhibition), we found that Rac
was involved in the morphological and cytoskeletal changes
induced by cholesterol loading. These findings are consistent
with our previous ones in human neutrophils, namely, that when
the levels of cholesterol in plasma membranes of neutrophils are
reduced, both stimulated membrane ruffling and Rac recruitment
to the plasma membrane are inhibited.12
Membrane Cholesterol and Cell Migration
Despite having potentiating effects on membrane activity and
F-actin reorganization, cholesterol loading caused the inhibition of macrophage migration. Cell migration is a highly
integrated, multi-step process consisting of cell polarization,
membrane extension at the front of the cell, regulated
formation and release of adhesions along the length of the
cell, and retraction of the cell rear (uropod). These steps are
orchestrated in part by the interactive regulation of the
Rho-family GTPases.37–39 Our data clearly show that Rac
activity and localization are altered after cholesterol loading,
and it is likely that the activities of the other Rho family
members are also affected by changes in membrane cholesterol levels. Misregulation of these signaling molecules could
lead to the inability of the cells to correctly polarize, retract
their uropods, or regulate their attachments with the substratum. Several studies have shown that plasma membrane
organization is critical for cell migration,11,12,40 and our
previous work on neutrophils showed that depleting membrane cholesterol inhibited neutrophil migration because the
cells were unable to form membrane extensions and polarize.12 Further studies are required for understanding the exact
defect in the migration of cholesterol-loaded macrophages.
The observations reported here indicate that increased membrane
cholesterol causes dramatic effects on the actin cytoskeleton in
macrophages associating with extracellular matrix. It is possible that
after initial contact of a macrophage with lipoproteins in the vessel
wall, cholesterol transfer to the cell causes changes similar to those
observed in this study. The increased membrane ruffling and
extensions could increase the contact of the macrophage with the
lipoprotein deposit, leading to further cholesterol delivery. The
decreased motility of the cell would keep it in the region of the
378
Arterioscler Thromb Vasc Biol.
February 2006
lipoprotein deposit. Additional studies will be required to determine
to what extent contact with lipoprotein particles causes local
increases in membrane cholesterol levels and the consequences of
these local changes for cell function. These findings should provide
new insights into the early steps of atherogenesis and might
eventually lead to innovative methods of preventing lesion formation or promoting lesion regression.
Acknowledgments
This work was supported by National Institutes of Health grants
P01-HL072942. We thank Leona Cohen-Gould for technical help
with the scanning electron microscopy experiment. We also thank Dr
William Muller for helpful discussions.
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Elevated Plasma Membrane Cholesterol Content Alters Macrophage Signaling and
Function
Chunbo Qin, Tomokazu Nagao, Inna Grosheva, Frederick R. Maxfield and Lynda M. Pierini
Arterioscler Thromb Vasc Biol. 2006;26:372-378; originally published online November 23,
2005;
doi: 10.1161/01.ATV.0000197848.67999.e1
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Supplemental Materials
Elevated Plasma Membrane Cholesterol Content Alters Macrophage Signaling and
Function
Chunbo Qin, Tomokazu Nagao, Inna Grosheva, Frederick R. Maxfield, and Lynda M.
Pierini
From the Departments of Biochemistry (C.Q., T.N., I.G., F.R.M.) and Surgery (L.M.P.),
Weill Medical College of Cornell University, New York, NY
Methods
Cells and Cell Culture
J774 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM)
supplemented with 10% heat-inactivated fetal bovine serum, 50 units/ml penicillin and 50
µg/ml streptomycin in a humidified atmosphere (5% CO2 plus 95% air) at 37 °C. Stock
cultures were grown on polystyrene petri dishes (VWR) and split every 2-3 days. On the
day prior to experiments utilizing adherent J774 cells, the cells were plated onto
polylysine-coated glass-bottom dishes [made by affixing No.1 coverslips (Gold Seal)
beneath holes in the bottom of 35 mm petri dishes (Corning)],1 and incubated in complete
culture medium for 16 hrs. The next day, the cells were washed two times with serumfree HEPES-buffered DMEM (pH 7.4), and incubated with cholesterol-modifying
reagents in serum-free HEPES-buffered DMEM for indicated times at 37°C. For
experiments utilizing suspended cells, cells were grown to ~ 85% confluence in complete
culture medium, washed twice with serum-free HEPES-buffered DMEM, and then
suspended by trituration in the same buffer (2.5 x 105 cells/ml) for further treatment.
Human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient
sedimentation in Ficoll (Amersham Biosciences) as described previously.2 Lymphocytes
were washed away after PBMCs were plated onto fibronectin-coated dishes for 30 min.
Monocytes, which remained attached to the fibronectin-coated dishes, were differentiated
in vitro by incubation in DMEM containing 10% heat-inactivated FBS and 10 ng/ml
macrophage colony stimulating factor (M-CSF, R&D Systems) for 7 days.
Materials
RGD peptide (Gly-Arg-Gly-Asp-Ser) was from Sigma. Purified rat anti-mouse CD18
monoclonal antibody (GAME-46) was purchased from BD Pharmingen.
Lipid Extraction and Free Cholesterol Measurement
J774 cells, grown overnight on 12-well tissue culture plates (Becton Dickinson), were
washed with PBS after chol-MβCD treatment and then extracted with two 30 min
incubation in 0.5 ml hexane:isopropanol (3:2) at room temperature. Half of the cellular
lipid extract from each sample was analyzed for free cholesterol by gas-liquid
chromatography,3 while the other half was analyzed for phospholipid content.4
Fluorescence Image Analysis
For quantitative analysis of phalloidin- or filipin-stained J774 cells, wide-field
fluorescence images of all samples within one experiment were acquired with a 25 x 0.75
numeric aperture objective under identical conditions, and the images were then
quantified using MetaMorph image analysis software. Following background correction,
the average fluorescence intensity per cell was measured for more than 100 cells per
condition, and these measurements were normalized to the level of the unstimulated
control cells. In order to measure the area of the cells, fluorescence images, acquired at a
focal plane close to the adherent surface of phalloidin-labeled cells, were thresholded to
exclude all the cell-free regions. The total thresholded area was measured and divided by
the number of the cells. All data represent means +/- S.D. from at least three independent
experiments.
Scanning Election Microscopy
The fixed cells were suction-filtered onto polylysine-coated Nucleopore polycarbonate
filters (0.45 μm, Whatman). Filters were washed three times in the same sodium
cacodylate buffer and post-fixed in 1% osmium tetroxide and 1.5% potassium
ferricyanide (aqueous solution) for 1 hr. Samples were dehydrated through a graded
series of ethanol-water washes (50%, 70%, 85%, 95% ethanol, respectively, followed by
3 times of 100% ethanol), critical point dried through carbon dioxide, and lightly coated
with gold palladium. Samples were then examined at 20 kV using a JEOL 100 CX-II
electron microscope fitted with an ASID-scanning unit, and photographs were recorded
on Polaroid Type 55 P/N film.
Macrophage Migration Assay
J774 cells (105 cells/well) were plated on the transwell filters, and incubated for 2 h in
normal growth medium. The cells were treated with 5 mM chol-MβCD in 20 mM
HEPES-buffered DMEM for 15 min, and then washed once with DMEM containing
0.5% FBS. These filter inserts were placed in wells containing 600 μl of 10 nM human
recombinant C5a (Sigma) diluted in DMEM. Migration was allowed to proceed for 2 h
at 37° C in a CO2 incubator. After this incubation period, the filters were fixed with 1%
glutaraldehyde, and cells that had not migrated were removed from the upper surface of
the filter by scraping. The filters were then stained with 1 μM TO-PRO-3 iodide
(Molecular Probes), mounted onto coverslip dishes, and observed via confocal
microscopy (LSM510, Carl Zeiss). The numbers of cells that had migrated across the
filters were determined by counting the number of cells in at least 5 random fields of
confocal images acquired with a 10x objective at the bottom of each filter.
Figure Legends
Figure I. Quantification of cellular free cholesterol content by GC analysis of cellular
extracts and fluorescence imaging of filipin-stained cells. J774 cells were left untreated
(control) or treated with 5 mM chol-MβCD (chol-loaded) for the indicated times. For
GC analysis (hatched bar), free cholesterol levels are expressed as a fraction of the
phospholipid content in the same sample to normalize for variations in cell number. For
filipin-stained cells (white and dark bars), data represent integrated filipin fluorescence
per cell. GC analysis and quantitative imaging were performed on samples prepared on
the same day. Error bars represent s.d.
Figure II. Increasing membrane cholesterol reversibly induced membrane ruffling. A,
Treatment with chol-MβCD causes accumulation of F-actin along the cell edges. J774
cells, grown on polylysine-coated dishes overnight, were left untreated (control), treated
with 5 mM chol-MβCD for indicated times (cholesterol-loaded), or treated sequentially
with 5 mM chol-MβCD for 15 min and then 10 mM MβCD for 30 min (reversal) before
fixation and staining for F-actin. Projected confocal images of the lower adherent surface
(consisting of 3 bottom slices at the step size of 1.25 μm) are presented. Scale bar, 10
μm. B, Cholesterol-loaded cells show an increase in edge-accumulated, but not total
cellular, F-actin. Edge intensity (white bar) was measured by quantifying the integrated
fluorescence within a series of 5 x 5 pixel boxes created randomly along the edge of each
cell.
Figure III. Inhibitors of integrins did not block the effects of cholesterol loading. J774
cells were grown on Fn-coated dishes for 1 hr in the presence of 200 µg/ml RGD peptide
(c), 50 µg/ml anti-β2 function-blocking antibody (f), or without any inhibitors (a,b,d,e).
The cells were either left untreated (control), or treated with 5 mM chol-MβCD for 30
min (chol-loaded) with or without the indicated inhibitors. Wide-field fluorescence
images of phalloidin-stained cells are presented. Scale bar, 10 μm.
Figure IV. Cholesterol loading and fucoidan treatment did not induce ruffling in
suspended J774 macrophages. J774 cells, suspended in DMEM-HEPES, were left
untreated (a), treated with 250 μg/ml fucoidan alone (c), or treated with 5mM cholMβCD for 30 min in the absence (b) or presence (d) of 250 μg/ml fucoidan. The cells
were then fixed in suspension and stained with phalloidan. Projected confocal images are
presented. Scale bar, 10 μm.
Figure V. Cholesterol loading induced cell spreading and stimulated Rac recruitment to
membranes in primary human monocyte-derived macrophages. Human monocytederived macrophages grown on Fn-coated dishes were left untreated (control) or treated
with 5 mM chol-MβCD for 30 min (chol-loaded) before fixation and labeling by
immunofluorescence. Projected confocal images are presented. Rac is shown in red,
while F-actin is shown in green. Scale bar, 10 μm.
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Figure I
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Figure V